猪圆环病毒pVAX1-ORF2-ISS核酸疫苗及与猪细胞因子联合免疫研究
|
摘要
猪圆环病毒病由猪圆环病毒2型(Porcine circovirus type2,PCV2)所致,该病除表现为猪断乳后多系统衰竭综合征(Postweaning multisystemic wasting syndrome,PMWS)外,还与猪皮炎肾病综合征(porcine dermatitis and nephropathy syndrome,PDNS)、猪呼吸道疾病综合征(Porcine respiratory disease complex,PRDC)、增生性坏死性肺炎(Proliferative and necrotizing pneumonia,PNP)、先天性震颤((CongenitaltemorAll,CT-All)等猪圆环病毒相关疾病(PCVAD)有关。猪圆环病毒病在世界不同的国家与地区引致广泛流行与致病,也是我国规模式化猪场主要传染病之一,给世界养猪业造成巨大的经济损失,引起全世界高度关注。目前,国内还没有猪圆环病毒2型商品化疫苗,国外的疫苗研究也在近年来才开展。研究新型猪圆环病毒2型核酸疫苗提高疫苗免疫的效果,对于防制猪圆环病毒病有重要意义。
本研究分离鉴定了猪圆环病毒2型病毒株,构建pVAX1-ORF2-ISS核酸疫苗和制备核酸疫苗壳聚糖复合物,开展猪圆环病毒pVAX1-ORF2-ISS核酸疫苗及与猪细胞因子佐剂联合免疫猪,为研制新型、高效、安全的猪圆环病毒2型核酸疫苗及测定评价筛选猪细胞因子分子免疫佐剂提供理论依据和实验数据。
主要研究结果如下:
1.猪圆环病毒2型病的检测及病毒株的分离鉴定。采用ELISA技术对9个规模化猪场987头猪的血清进行了猪圆环病毒2型抗体检测,结果表明:受检未免疫猪血清中,圆环病毒2型抗体阳性率为74.3%;采用PCR检测了可疑病料104头,可疑病料中PCR检测阳性率为77.9%,从病料中分离鉴定了一株猪圆环病毒2型(PCV2whn),在透射电镜下观察到持续感染猪圆环病2型的PK15细胞胞浆及细胞核内有包涵体,以及呈晶格状排列的病毒粒子,直径18±2nm;测定了PCV2whn分离株的全基因组序列,猪圆环病毒2型分离株的基因组全长为1767bp,与我国不同地方的猪圆环病毒2型病毒的分离株基因组的同源性高达92.7-97.5%,与欧洲、美洲、南非分离株的同源率在94.2-96%之间。本研究自主分离鉴定获得新的猪圆环病毒2型分离株,为猪圆环病毒2型研究获取了新的材料。
2.pVAX1-ORF2-ISS核酸疫苗构建及核酸疫苗壳聚糖纳米颗粒制备。选用pVAX1为真核表达载体,以猪圆环病毒2型ORF2为构建目的基因,合成插入含11个CpG motif全长92bp的ISS序列,采用PCR扩增PCV20RF2片段,通过酶切及连接等操作将两段序列亚克隆到pVAX1载体中,构建了pVAX1-ORF2-ISS核酸疫苗,并采用酶切鉴定所构建的真核表达载体。结果显示两个片段正确插入到载体中。使用壳聚糖对pVAX1-ORF2-ISS核酸包被,通过透射电镜和胶阻滞分析,结果显示,pVAX1-ORF2-ISS与壳聚糖结合,成功构建与制备pVAX1-ORF2-ISS核酸疫苗壳聚糖复合物。本研究以pVAX1为优化载体,合成插入ISS序列,设计构建了含ISS与ORF2的pVAX1-ORF2-ISS新型核酸疫苗,此研究尚未见报道。
3.猪白细胞介素IL-4、IL-6、IL-6/4的鉴定及其分子佐剂壳聚糖纳米颗粒的制备。选用“四川大学“动物疫病防控与食品安全四川省重点实验室”提供的猪白细胞介素IL-4、IL-6、IL-6/4真核表达质粒,利用酶切方法对猪白细胞介素IL-4、IL-6、IL-6/4的真核表达载体进行了鉴定,结果证明3种白细胞介素的目的片段大小与预期大小符合。使用壳聚糖对猪白细胞介素IL-4、IL-6、IL-6/4的真核表达载体进行了包被,通过透射电镜和胶阻滞分析结果显示,真核表达质粒全部与壳聚糖结合,成功制备猪白细胞介素IL-4、IL-6、IL-6/4的分子免疫佐剂壳聚糖纳米颗粒。
本研究应用成功制备的猪白细胞介素IL-4、IL-6、IL-6/4真核表达分子免疫佐剂质粒与猪圆环病毒2型核酸疫苗进行的联合免疫实验,在国内外未见报道。
4.猪圆环病毒pVAX1-ORF2-ISS核酸疫苗免疫及与猪细胞因子联合免疫猪。使用构建和制备的猪圆环病毒pVAX1-ORF2-ISS核酸疫苗免疫及与猪细胞因子分子佐剂联合免疫实验猪,通过测定不同分组仔猪免疫后的特异性抗体滴度以及T淋巴细胞亚类CD3+、CD4+、CD8+细胞免疫变化情况,对pVAX1-ORF2-ISS核酸疫苗免疫及与猪细胞因子分子佐剂联合免疫效果作出了分析和评定。同时,使用pVAX1-ORF2-ISS核酸疫苗在小白鼠体上做安全性实验。结果显示,pVAX1-ORF2-ISS核酸疫苗能够诱导仔猪产生特异性抗体和T淋巴细胞亚类的免疫应答,免疫期持续60d以上,能诱导细胞免疫并产生特异性抗体,pVAX1-ORF2-ISS核酸疫苗与本课题组构建保存的pVAX1-ORF2比较,其免疫效果优于pVAX1-ORF2(P＜0.05),与空载体及空白对照组相比,差异极显著(P＜0.01);3种猪细胞因子分子佐剂与pVAX1--ORF2-ISS核酸疫苗同时免疫仔猪能显著提高pVAX1-ORF2-ISS核酸疫苗的免疫效果(P＜0.05),以IL-6、IL-6/4为佳,IL-4次之;核酸疫苗的安全性试验表明pVAX1-ORF2-ISS核酸疫苗安全。同类研究均以小鼠进行免疫实验,而本研究用猪圆环病毒2型新型核酸疫苗免疫及与猪细胞因子分子佐剂联合免疫猪未见报道。
本研究用构建pVAX1-ORF2-ISS核酸疫苗和制备核酸疫苗壳聚纳米颗粒,开展的猪圆环病毒pVAX1-ORF2-ISS核酸疫苗及与猪细胞因子佐剂联合免疫猪研究,为研制新型、高效、安全的猪圆环病毒2型核酸疫苗及测定评价猪细胞因子分子免疫佐剂提供了理论依据和实验数据。
Porcine circovirus virus(PCV2) is recognized as the essential infectious agent of postweaning multi-systemic wasting syndrome(PMWS),porcine dermatitis and nephropathy syndrome(PDNS),porcine respiratory disease complex(PRDC), proliferative and necrotizing pneumonia(PNP) and congenital temorall(CT-All).It is epidemic in many countries in the world and is also a major infectious disease in intensive pig farms of China.It caused a great economic loss to world's pig industry. Therefore,there are many scientists and other people engaging pig raising paying much more attention to PCV2.However,there has not effective commercial vaccine in China and only a few researches for PCV2 vaccine in foreign countries up to now.Thus,it is very important to develop novel PCV2 DNA vaccine and apply cytokine molecular adjuvant to improve immune effects of vaccine.
This research was carried out to construct novel PCV2 DNA vaccine,prepared vaccine with CNP complex and did combined immunization with porcine cytokine molecular adjuvants.The aim of this study was to improve the immunity of PCV2 DNA vaccine and evaluate the effects of porcine cytokine molecular adjuvants,which supply theoretical basis and experimental data for prevention of PCV2.
The main content of the research is as follows.
1.The isolation and identification of PCV2 Sichuan strain.Using ELISA to detected blood serum antibody of 987 pigs from 9 farms revealed that the PCV2 positive rate was 74.3%.104 specimen of dead pigs from 9 pig farms were identified as PCV2 by PCR and the virus were isolated.The results showed that the PCV2 positive rate of the specimen was 77.9%;A PCV2 whn strain was isolated,presented inclusion body in cell plasm and cell nuclear of PK15 cell,and the virion whose diameter was 12±2nm, presented crystal lattice arrangement by electron microscope;The complete genome of the virus was sequenced and the full-length was 1767nt,which showed 92.7-97.5% genetic homology with the isolates of China and 94.2-96%with European,American and South Africa isolates.We are successfully isolated and identificatied PCV2 in Sichuan province and got accession number in Genbank,which offered new materials to research of PCV2.
2.The construction of pVAXI-ORF2-ISS DNA vaccine and the preparation of the DNA vaccine-chitosan-nanoparticle(CNP) complex.The pVAX1-ORF2-ISS DNA vaccine was constructed through the following steps.ORF2 of PCV2 and 11 CpG motif as ISS sequence were inserted into pVX1 eukaryotic expression vector.Then identified by enzyme digestion analysis.The results showed that the two fragments were rightly inserted into vector.CNP was used to coating pVAX1-ORF2-ISS plasmid.The transmission electron microscope and glue blocking analysis indicated that vaccine plasmids combined with CNP and pVAX1-ORF2-ISS DNA vaccine were successfully constructed and prepared.We had successfully constructed a kind of pVAX1-ORF2-ISS novel DNA vaccine with pVAX1 vector and ISS,which was reported at the first time.
3.The identification of porcine cytokine molecular IL-4,IL-6 and IL-6/4 and the preparation of CNP as molecular adjuvant.The porcine cytokine molecular IL-4,IL-6 and IL-6/4 eukaryotic expression plasmid offered by ministry of education key laboratory for Sichuan university biological resource and ecological environment.Enzyme digestion analysis was applied to identify the three kinds of plasmid and the results showed that the length of target fragments accorded with expectations.CNP was used to coating IL-4, IL-6 and IL-6/4 plasmid.The transmission electron microscope and glue blocking analysis indicated that all the plasmids combined with CNP and cytokine molecular adjuvant were successfully prepared.We successfully prepared IL-4,IL-6 and IL-6/4 eukaryotic expression vector,which was reported at the first time.
4.The combined immunization experiment on pigs using PCV2 pVAX1-ORF2-ISS DNA vaccine and porcine cytokine molecular adjuvant.By testing piglets in every experimental group of specific antibody titers and the quantity of lymphocytes CD~(3+), CD~(4+) and CD~(8+),analysis and evaluation were achieved about the pVAX1-ORF2-ISS DNA vaccine and porcine cytokine molecular adjuvant.Moreover,safety experiment was performed using white mice.The results showed that pVAX1-ORF2-ISS DNA vaccine could induce the generation of specific antibody and cellular immunity responses,and the duration of immunity could last over 60 days.The effects of pVAX1-ORF2-ISS and pVAX1-ORF2 DNA vaccines were also compared.The result proved that pVAX1-ORF2-ISS was better than pVAX1-ORF2 DNA vaccine(P＜0.05) and the difference was extremely significant(P＜0.01) when compared with empty vector and blank control groups.Also,the three cytokine molecular adjuvant could extremely significantly improve the immune effects of pVAX1-ORF2-ISS DNA vaccine(P＜0.05). IL-6 was the best,followed by IL-6/4 and IL-4.DNA vaccine safety experiment indicated that the pVAX1-ORF2-ISS DNA vaceine was safe and reliable.White mice were common used to study PCV2 DNA vaccine of pVAX1-ORF2-ISS and combined immunization with porcine cytokine molecular adjuvant,and it was the first time using pigs as animal model,which could get more practical experimental data.
This research was carried out to construct novel PCV2 DNA vaccine,prepared vaccine with CNP complex and did combined immunization with porcine cytokine molecular adjuvants.It had improved the immunity of PCV2 DNA vaccine and evaluate the effects of porcine cytokine molecular adjuvants,which supply theoretical basis and experimental data for the research of novel,effective and safe vaccine of PCV2.
本研究分离鉴定了猪圆环病毒2型病毒株,构建pVAX1-ORF2-ISS核酸疫苗和制备核酸疫苗壳聚糖复合物,开展猪圆环病毒pVAX1-ORF2-ISS核酸疫苗及与猪细胞因子佐剂联合免疫猪,为研制新型、高效、安全的猪圆环病毒2型核酸疫苗及测定评价筛选猪细胞因子分子免疫佐剂提供理论依据和实验数据。
主要研究结果如下:
1.猪圆环病毒2型病的检测及病毒株的分离鉴定。采用ELISA技术对9个规模化猪场987头猪的血清进行了猪圆环病毒2型抗体检测,结果表明:受检未免疫猪血清中,圆环病毒2型抗体阳性率为74.3%;采用PCR检测了可疑病料104头,可疑病料中PCR检测阳性率为77.9%,从病料中分离鉴定了一株猪圆环病毒2型(PCV2whn),在透射电镜下观察到持续感染猪圆环病2型的PK15细胞胞浆及细胞核内有包涵体,以及呈晶格状排列的病毒粒子,直径18±2nm;测定了PCV2whn分离株的全基因组序列,猪圆环病毒2型分离株的基因组全长为1767bp,与我国不同地方的猪圆环病毒2型病毒的分离株基因组的同源性高达92.7-97.5%,与欧洲、美洲、南非分离株的同源率在94.2-96%之间。本研究自主分离鉴定获得新的猪圆环病毒2型分离株,为猪圆环病毒2型研究获取了新的材料。
2.pVAX1-ORF2-ISS核酸疫苗构建及核酸疫苗壳聚糖纳米颗粒制备。选用pVAX1为真核表达载体,以猪圆环病毒2型ORF2为构建目的基因,合成插入含11个CpG motif全长92bp的ISS序列,采用PCR扩增PCV20RF2片段,通过酶切及连接等操作将两段序列亚克隆到pVAX1载体中,构建了pVAX1-ORF2-ISS核酸疫苗,并采用酶切鉴定所构建的真核表达载体。结果显示两个片段正确插入到载体中。使用壳聚糖对pVAX1-ORF2-ISS核酸包被,通过透射电镜和胶阻滞分析,结果显示,pVAX1-ORF2-ISS与壳聚糖结合,成功构建与制备pVAX1-ORF2-ISS核酸疫苗壳聚糖复合物。本研究以pVAX1为优化载体,合成插入ISS序列,设计构建了含ISS与ORF2的pVAX1-ORF2-ISS新型核酸疫苗,此研究尚未见报道。
3.猪白细胞介素IL-4、IL-6、IL-6/4的鉴定及其分子佐剂壳聚糖纳米颗粒的制备。选用“四川大学“动物疫病防控与食品安全四川省重点实验室”提供的猪白细胞介素IL-4、IL-6、IL-6/4真核表达质粒,利用酶切方法对猪白细胞介素IL-4、IL-6、IL-6/4的真核表达载体进行了鉴定,结果证明3种白细胞介素的目的片段大小与预期大小符合。使用壳聚糖对猪白细胞介素IL-4、IL-6、IL-6/4的真核表达载体进行了包被,通过透射电镜和胶阻滞分析结果显示,真核表达质粒全部与壳聚糖结合,成功制备猪白细胞介素IL-4、IL-6、IL-6/4的分子免疫佐剂壳聚糖纳米颗粒。
本研究应用成功制备的猪白细胞介素IL-4、IL-6、IL-6/4真核表达分子免疫佐剂质粒与猪圆环病毒2型核酸疫苗进行的联合免疫实验,在国内外未见报道。
4.猪圆环病毒pVAX1-ORF2-ISS核酸疫苗免疫及与猪细胞因子联合免疫猪。使用构建和制备的猪圆环病毒pVAX1-ORF2-ISS核酸疫苗免疫及与猪细胞因子分子佐剂联合免疫实验猪,通过测定不同分组仔猪免疫后的特异性抗体滴度以及T淋巴细胞亚类CD3+、CD4+、CD8+细胞免疫变化情况,对pVAX1-ORF2-ISS核酸疫苗免疫及与猪细胞因子分子佐剂联合免疫效果作出了分析和评定。同时,使用pVAX1-ORF2-ISS核酸疫苗在小白鼠体上做安全性实验。结果显示,pVAX1-ORF2-ISS核酸疫苗能够诱导仔猪产生特异性抗体和T淋巴细胞亚类的免疫应答,免疫期持续60d以上,能诱导细胞免疫并产生特异性抗体,pVAX1-ORF2-ISS核酸疫苗与本课题组构建保存的pVAX1-ORF2比较,其免疫效果优于pVAX1-ORF2(P＜0.05),与空载体及空白对照组相比,差异极显著(P＜0.01);3种猪细胞因子分子佐剂与pVAX1--ORF2-ISS核酸疫苗同时免疫仔猪能显著提高pVAX1-ORF2-ISS核酸疫苗的免疫效果(P＜0.05),以IL-6、IL-6/4为佳,IL-4次之;核酸疫苗的安全性试验表明pVAX1-ORF2-ISS核酸疫苗安全。同类研究均以小鼠进行免疫实验,而本研究用猪圆环病毒2型新型核酸疫苗免疫及与猪细胞因子分子佐剂联合免疫猪未见报道。
本研究用构建pVAX1-ORF2-ISS核酸疫苗和制备核酸疫苗壳聚纳米颗粒,开展的猪圆环病毒pVAX1-ORF2-ISS核酸疫苗及与猪细胞因子佐剂联合免疫猪研究,为研制新型、高效、安全的猪圆环病毒2型核酸疫苗及测定评价猪细胞因子分子免疫佐剂提供了理论依据和实验数据。
Porcine circovirus virus(PCV2) is recognized as the essential infectious agent of postweaning multi-systemic wasting syndrome(PMWS),porcine dermatitis and nephropathy syndrome(PDNS),porcine respiratory disease complex(PRDC), proliferative and necrotizing pneumonia(PNP) and congenital temorall(CT-All).It is epidemic in many countries in the world and is also a major infectious disease in intensive pig farms of China.It caused a great economic loss to world's pig industry. Therefore,there are many scientists and other people engaging pig raising paying much more attention to PCV2.However,there has not effective commercial vaccine in China and only a few researches for PCV2 vaccine in foreign countries up to now.Thus,it is very important to develop novel PCV2 DNA vaccine and apply cytokine molecular adjuvant to improve immune effects of vaccine.
This research was carried out to construct novel PCV2 DNA vaccine,prepared vaccine with CNP complex and did combined immunization with porcine cytokine molecular adjuvants.The aim of this study was to improve the immunity of PCV2 DNA vaccine and evaluate the effects of porcine cytokine molecular adjuvants,which supply theoretical basis and experimental data for prevention of PCV2.
The main content of the research is as follows.
1.The isolation and identification of PCV2 Sichuan strain.Using ELISA to detected blood serum antibody of 987 pigs from 9 farms revealed that the PCV2 positive rate was 74.3%.104 specimen of dead pigs from 9 pig farms were identified as PCV2 by PCR and the virus were isolated.The results showed that the PCV2 positive rate of the specimen was 77.9%;A PCV2 whn strain was isolated,presented inclusion body in cell plasm and cell nuclear of PK15 cell,and the virion whose diameter was 12±2nm, presented crystal lattice arrangement by electron microscope;The complete genome of the virus was sequenced and the full-length was 1767nt,which showed 92.7-97.5% genetic homology with the isolates of China and 94.2-96%with European,American and South Africa isolates.We are successfully isolated and identificatied PCV2 in Sichuan province and got accession number in Genbank,which offered new materials to research of PCV2.
2.The construction of pVAXI-ORF2-ISS DNA vaccine and the preparation of the DNA vaccine-chitosan-nanoparticle(CNP) complex.The pVAX1-ORF2-ISS DNA vaccine was constructed through the following steps.ORF2 of PCV2 and 11 CpG motif as ISS sequence were inserted into pVX1 eukaryotic expression vector.Then identified by enzyme digestion analysis.The results showed that the two fragments were rightly inserted into vector.CNP was used to coating pVAX1-ORF2-ISS plasmid.The transmission electron microscope and glue blocking analysis indicated that vaccine plasmids combined with CNP and pVAX1-ORF2-ISS DNA vaccine were successfully constructed and prepared.We had successfully constructed a kind of pVAX1-ORF2-ISS novel DNA vaccine with pVAX1 vector and ISS,which was reported at the first time.
3.The identification of porcine cytokine molecular IL-4,IL-6 and IL-6/4 and the preparation of CNP as molecular adjuvant.The porcine cytokine molecular IL-4,IL-6 and IL-6/4 eukaryotic expression plasmid offered by ministry of education key laboratory for Sichuan university biological resource and ecological environment.Enzyme digestion analysis was applied to identify the three kinds of plasmid and the results showed that the length of target fragments accorded with expectations.CNP was used to coating IL-4, IL-6 and IL-6/4 plasmid.The transmission electron microscope and glue blocking analysis indicated that all the plasmids combined with CNP and cytokine molecular adjuvant were successfully prepared.We successfully prepared IL-4,IL-6 and IL-6/4 eukaryotic expression vector,which was reported at the first time.
4.The combined immunization experiment on pigs using PCV2 pVAX1-ORF2-ISS DNA vaccine and porcine cytokine molecular adjuvant.By testing piglets in every experimental group of specific antibody titers and the quantity of lymphocytes CD~(3+), CD~(4+) and CD~(8+),analysis and evaluation were achieved about the pVAX1-ORF2-ISS DNA vaccine and porcine cytokine molecular adjuvant.Moreover,safety experiment was performed using white mice.The results showed that pVAX1-ORF2-ISS DNA vaccine could induce the generation of specific antibody and cellular immunity responses,and the duration of immunity could last over 60 days.The effects of pVAX1-ORF2-ISS and pVAX1-ORF2 DNA vaccines were also compared.The result proved that pVAX1-ORF2-ISS was better than pVAX1-ORF2 DNA vaccine(P＜0.05) and the difference was extremely significant(P＜0.01) when compared with empty vector and blank control groups.Also,the three cytokine molecular adjuvant could extremely significantly improve the immune effects of pVAX1-ORF2-ISS DNA vaccine(P＜0.05). IL-6 was the best,followed by IL-6/4 and IL-4.DNA vaccine safety experiment indicated that the pVAX1-ORF2-ISS DNA vaceine was safe and reliable.White mice were common used to study PCV2 DNA vaccine of pVAX1-ORF2-ISS and combined immunization with porcine cytokine molecular adjuvant,and it was the first time using pigs as animal model,which could get more practical experimental data.
This research was carried out to construct novel PCV2 DNA vaccine,prepared vaccine with CNP complex and did combined immunization with porcine cytokine molecular adjuvants.It had improved the immunity of PCV2 DNA vaccine and evaluate the effects of porcine cytokine molecular adjuvants,which supply theoretical basis and experimental data for the research of novel,effective and safe vaccine of PCV2.
引文
1. Akeshita S, Gage JR. Kishimoto T, et al. Differential regulation of IL-6 gene transcrition and expression by IL-4 and IL-10 in human monocytic cell lines [J]. J Immunolt 1996, 2591-2598.
2. Allan G M. Ellis J A. Porcine circoviruses: a review[J]. J Vet Diagn Invest. 2000a, 12(5):3-14.
3. BallasZK, RasmussenW L, Krieg AM, et al. Induction of NK activity inmurine and human cells by CpG motifys in oligodoxynucleotides and bacterial DNA[J]. J Immunol, 1996, 157(5):1840-1845.
4. Barry MA, Johnston SA. Biological features of genetic immunization[J]. Vaccine, 1997, 15(8):788-791.
5. Blanchard P, Mahe D, Cariolet R, et al. Protection of swine a-gainst post-weaning multisystemic wasting syndrome (PMWS)by porcine circovirus type 2 (PCV2) proteins[J]. Vaccine, 2006, 21(31):4565-4575.
6. Blanchard P, Mahe D, Cariolet R, et al. Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins[J]. Vaccine, 2003, 21(3):4565-4575.
7. Bodmeier R, Chen HG, Paeratakul et al. A novel approach to the delivery of micro or nanoparticles [J]. Pharm Res, 1989, 6(5):413-417.
8. Bodmeier R, Chen HG, Paeratakul et al. A novel approach to the delivery of micro or nanoparticles [J]. PharmRes, 1989, 6(5):413-417.
9. Canals A, Grimm DR, Gasbrre LC, et al. Molecular cloningof cDNA encoding porcine interleukin-15[J]. Gene, 1997, 195:337-339.
10. Cencic A, LeFevre F, Koren S, et al. Tetracycline-controlled expression of glycosylated porcine interferon-gamma in mammlian cells [J]. Anim Biotechnol, 1999, 10(1-2):63-79.
11. CencicA, LeFevreF, Koren S, et al. Tetracycline-controlledexpression of glycosylated porcine interferon-gamma inmammlian cells[J]. Anim Biotechnol. 1999, 10 (1-2): 63-79.
12. Chakrabarti R, Chang Y, Song K. Plasmids encoding membrane-bound IL-4 or IL-12 strongly costimulates DNA vaccination against carcinoembryonic antigen (CEA). Vaccine, 2004, 22(9-10):1199-1205.
13. Choi C, Chae C. Colocalization of porcine reproductive and respiratory syndrome virus and porcine circovirus 2 in porcine dermatitis and nephrology syndrome by double-labeling technique[J]. Vet Pathol, 2001, 38(4): 436.
14. Choi C, Kwon D, Min K, et al. In-situ hybridization for thedetection of inflammatory cytokines (IL-1, TNF-and IL-6) inpigs naturally infected with Actinobacillus pleuropneumoniae[J]. J Comp Pathol, 1999, 121 (4): 349-356.
15. Crawley A, Raymond C, Wilkie BN. Control of immunoglob-ulin isotype by porcine B-cells cultured withcytokines [J]. Vet Immunol Immunopathol, 2003, 91:141-154.
16. Darwich L, Pi S, Rovira A, et al. Cytokine mRNA expression profiles in lymphoid tissues of pigs naturally affected by postweaning multisystemic wasting syndrome[J].Gen Virol, 2003, 84: 2117-2125.
17. Darwich L, Segalds J, Domingo M, et al. Changes in the CD~4+, CD~8+, CD~4/CD~8 double positive cells and postweaning multisystemic wasting syndrome afected pigs and age matched uninfected Wasted and healthy pigs[J]. Clin Diagn Lab Immunol, 2002, 9(4): 236-242.
18. Darwich L, Segales J, Domingo M, et al. Multisystemic wasting syndrome-affected pigs and age-matched unifected wasted and healthy pigs correlate with lesions and Porcine circovirus type 2 load in lympoid tissues american society for Microbiology[J]. Clinical and Diagnostic Laboratory Immunology, 2002, 9(2):236-242.
19. Davis HL, Brazolot Millan CL, Mancini M, et al. DNA-based immunization against hepatitis B surface antigen (HBsAg) in normal and HBsAg-transgenic mice [J]. Vaccine, 1997, 15(8):849-852.
20. Davis HL, Suparto II, Weeratna RR, et al. CpG DNA overcomes hypo-responsiveness to hepatitis B vaccine in orangutans[J].Vaccine, 2000, 18:1920-1924.
21. Davis HL. Use of CpG DNA for enhancing specific immune responses[J]. Cun Top Microbiol Immunol, 2000, 247:171-183.
22. DavisH L, Weeratna R, Waldschmidt TJ , et al. CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen[J]. J Immunol, 1998. 160(2):870-876.
23. Doolan D L, Segales M, Hedstrom R C, et al. Circumventing genetic restriction of protection against malaria with multigene DNA immunization: CD8+ cell-, interferon gamma-, and nitricoxide-dependent immunity [J]. J Exp Med, 1996. 183(4): 1739-1746.
24. Eberl M, Beck E, Coulson C P, et al. IL-18 potentiates the adjuvant properties of IL-12 in the induction of a strong Thl type immune response against a recombinant antigen[J]. Vaccine, 2000. 18:2002-2008.
25. Ellis J. Hassard L. Clark E, et al. Isolation of circovirus from lesions of pigs with postweaning multisyste ic wasting syndrome[J]. Can Vet J, 1998. 39(2):44-51.
26. Ellis J. Krakowka S, Larimore M, et al. Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic pigs[J]. J Vet Diagn Invest. 1999. 11(2):3-14.
27. Fenaux M, Halbur PG. Haqshenas G. et al. Cloned genomic DNA of type 2 porcine circovirus is infectious when injected directly into the liver and lymphnodes of pigs: characterization of clinical disease, virus distribution, and pathologic lesions[J]. J Virol. 2002, 76(2):541-551.
28. Fenaux M, Halbur PG; Haqshenas G et al. Cloned genomic DNA of type 2 porcine circovirus is infectious when injected directly into the liver and lymphnodes of pigs: characterization of clinical disease, virus distribution, and pathologic lesions[J]. J Virol. 2002, 76 (2):541-551.
29. Fenaux M, Opriessnig T, Halbur PG, et al.Two amino acidmutations in the capsid protein of type 2 porcine circovirus(PCV2) enhanced PCV2 replicationin vitroand attenuated thevirusin vivo[J].J Virol, 2004, 78:13440-13446.
30. Fenaux M, Opriessnig T, Halbur P G, et al.A chimeric porcinecircovirus (PCV) with the immunogenic capsid gene of thepathogenic PCV type 2 (PCV-2) cloned into the genomic back-bone of the nonpathogenic PCV1 induces protective immunityagainst PCV2 infection in pigs[J].J Virol, 2004, 78:6297-6303.
31. Foss DL, Zilliox MJ. Murtaugh MP. Bacterially induced actiation of interlerkin-18 in porcine intestinal mucosa[J]. Vet Immunol Immunopathol, 2001, 78: 263-277.
32. Foumout S, Dozois C M, Yerle M, et al. Cloning, chromoso-mal location, and tissue expression of the gene for piginterleukin-l8[J]. Immunogenetics, 2000, 51:358-365.
33. Francisco Ruiz-Fons, Joaqun Vicente, Dolo Vidal, et al. Seroprevalence of six reproductive pathogensin European wild boar(Sus scrofa)from Spain:The effect on wild boar female reproductive performance[J].Theriogenology. 2006, 65:731-743.
34. Gallichan WS, Woolstencroft RN, Guarasci T, et al. In tranasal immunization with CpG oligodeoxynucleotides as an adjuvant dramatically increases IgA and protection against herpess implex virus-2 in the genitaltract[J].J Immunol, 2001, 166:3451-3457.
35. Gregor SD, Kevin She, Luke Terret, et al. CpG stimulation of precursor B-lineage acute lymphoblastic leukemia induces adistinct change incostimulatory molecule expression and shift sallogeneic T cells toward a Th1 response[J]. Blood, 2005, 105:3641-3647.
36. Guo YJ, Wu D, Chen RW, et al. Cloning, high level expression and purification of porcine IFN gamma[J]. Sheng Wu Gong Cheng Xue Bao, 2001, 17(2):183-186.
37. Guo YJ, Wu D, Chen RW, et al. Cloning, high level expression and purification of porcine IFN gamma[J]. Sheng Wu GongCheng Xue Bao, 2001, 17 (2): 183-186.
38. Haddad D , Ramprakash J , Sedegah M , et al. Plasmid vaccine expressing granulocyte-macrophage colony-stimulating factor attracts infiltrates including immature dendritic cdeel intoinjected muscles[J]. J Immunol, 2000, 165: 3772-3781.
39. Hamel AL, Lin LL, Nayar GS. Nucleoitde sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs[J]. J Virol, 1998, 72(6):5262-5267.
40. Hartmann G, Krieg AM, et al. Mechanism and function of a newly identified CpG DNA motify in human primary Bcells[J].J Immunol, 2000, 164:944-952.
41. Hui G, Hashimoto C. 2007. Interleukin26 has differential influence onthe ability of adjuvant formulations to potentiate antibody reponses to aplasmodium falciparum blood2stage vaccine[ J ]. Vaccine, 25:6598-660.
42. Inumaru S, Takamatsu HC. Cloning of porcine granulocy-te-macrophage colony-stimulating factor [J]. Immunol CellBiol, 1995, 73: 474-476.
43. Ishii N, Fukushima J, Kaneko T, et al. Cationic liposomes area strong adjuvant for a DNA vaccine of human immunodeficiency virus type[J]. Aids Research in Human Retroviruses, 1997, 13:1421-1428.
44. Ju C, Fan H, Tan Y, et al. Immunogenicity of a recombinantpseudorabies expressing ORF1-ORF2 fusion protein of porcine circovirus type 2[J].Vet Microbiol , 2005 , 109(3-4):179-190.
45. Kamstrup S, Barfoed A M, Frimann T H, et al. Immunisation against PCV2 structural protein by DNA vaccination of mice[J]. Vaccine, 2004, 22:1358-1361.
46. Kamstrup S, Barfoed AM. Frimann TH, et al. Immunisation against PCV2 structural protein by DNA vaccination of mice[J]. Vaccine, 2004, 22(11-12):1358-1361.
47. Kamstrup S, Barfoed AM. Immunisation against PCV2 struc-tural protein by DNA vaccination of mice[J].Vaceine, 2004, 22:1358-1361.
48. Kiatipattanasakul-Banlunara W, Tantilertcharoen R, Suzuki K, et al. Detection of porcine circovirus 2 (PCV-2) DNA by nested PCR from formalin-fixed tissues of postweaning multi-systemic wasting syndrome (PMWS) pigs in Thailand [J]. J Vet Med Sci, 2002, 64(5): 449-452.
49. Kim JJ, Yang JS, Vancot TC. Modulation of Antigen-specific Humoral Responses in Rhasus Macaques by using cytokine cDNAs as DNA vaccine adjuvant[J]. J Virol, 2000, 74 (7):3427-3429.
50. KojimaY, Xin KQ, Ooki T, et al. Adjuvant effect of multi-CpG motif on an HIV-DNA vaccine[J]. Vaccine, 2002, 20:2857-2865.
51. Kokuho T, Watanabe S, Yokomizo Y, et al. Production ofbiologically active, heterodimeric porcine interleukin-12 using amonocistronic baculoviral expression system[J]. Vet Immunol Immunopathol, 1999, 72:289-302.
52. Krieg AM, Yi AK, Matson S, et al. CpG motifs in bacterial DNAtrigger direct B-cell activation[J]. Nature, 1995, 374(6522):546-549.
53. Ladekjaer-Mikkelsen AS, Nielsen J, Stadejek T, et al. Reproduction of postweaning multisystemic wasting syndrome(PMWS) in immunostimulated and non-immunostimulated 3-week-old piglets experimentally infected with porcine circovirus type2 ( PCV2) [J]. Vet Microbiol, 2002, 89(5):97-114.
54. Lagging LM, Meyer K, Hoft D, et al. Immune responses to plasmid DNA encoding the hepatitis C virus core protein [J]. J Virol, 1995, 69(9):5859-5863.
55. Lee SW, Youn JW, Seong BL, et al. IL-6 induces long-term protective immunity against a lethal challenge of influenza virus[J].Vaccine, 1999. 17:490-496.
56. Lefevre F, Guillomot MD, Andrea S, et al. Interferon-delta:the first member of a noval type I interferon family [J]. Biochimie, 1998, 80 (8-9): 779-788.
57. Levy F, Kristofic C, HeusseC, et al. Role of IL-13 in CD, T cell-dependent IgE productio in atopy[J]. Int Arch Alleray Immunol, 1997, 112(1): 49.
58. Lewis PJ. Babiuk LA. DNA vaccines: a review[J].Adv Res, 1999, 94:129-188.
59. Libin Wen, Xin Guo, Hanchun Yang. Genotyping of porcine circovirus type 2 from a variety of clinicalconditions in China[J].Veterinary Microbiology, 2005, 110:141-146.
60. Lillum. Chitosan and its use as pharmaceutical excipient[J]. Pharm Res. 1998, 15:1326-1331.
61. Mahe D, Blanchard P. Truong C, et al. Differential recognition of ORF2 protein from type I and type 2 porcine circovinises and identification of immunorelevant epitoes[J]. F Gen Virol, 2000, 81(5):1815-1824.
62. Manickan E, Rouse RJ, Yu Z, et al, Genetic immunization against herpes simplex virus. Protection is mediated by CD4+ T lymphocytes [J]. J Immunol, 1995, 155(1):259-265.
63. Mankertz A, Hillenbrand B. Analysis of transcription of porcine circovirus type 1[J]. J Gen Virol, 2002, 83:2743-2751.
64. Maroushek BK, Bosworth BT, Stabel TJ, et al. Cloning and expression of porcine recombinant soluble tumor necrosisfactor receptor 1[J]. Am J Vet Res, 1998. 59 (10): 1317-1322.
65. McNeilly F, McNair I, Mackie DP, et al. Production, characterization and applications of monoclonal antibodies to porcine cercovirus 2[J]. Arc Virol, 2001, 146(6):909-922.
66. Montgomery DL, Shiver JW, Leander KR, et al. Heterologous and homologous protection against influenza A by DNA vaccination: optimization of DNA vectors[J]. DNA Cell Biol, 1993, 12(9):777-783.
67. Muneta Y, Mori Y, Shimoji Y, et al. Porcineinterleukin-18:cloning, characterization of the cDNA and expression with the baculovirus system [J]. Cytokine, 2000, 12:566-572.
68. Muneta Y, Zhao HK, Inumaru S, et al. Large-scale product-ion of porcine mature interleukin-18 (IL-18) in silkworms using a hybrid baculovirus expression system[J]. J Vet Med Sci2003, 65 (2): 219-223.
69. Mwangi SM, Stabel TJ, Kehrli ME. Development of abaculovirus expression system for soluble porcine tumornecrosis factor receptor type I and soluble porcine tumornecrosis factor receptor type Ⅰ IgG fusion protein [J]. VetImmunol Immunopathol. 2002, 86 (3-4): 251-254.
70. Nawagitgul P, Haris P A, Morozov I, et al. Modified indirect porcine circovirus (PCV) type2-based and recombinant capsid protein (ORF2)-based enzyme-linked immunosorbent assays for detection of antibodies to PCV[J]. Clin Diag Lab Immunol, 2002, 9(2):33-40.
71. Nawagitgul P, Morozov I, Bolin SR, et al. Open reading frame 2 of porcine cercovirus type 2 encodes a major capsid protein[J]. J Gen Virol, 2000, 81(8):2281-2287.
72. Nishimura H, Hiromatsu K, Kobayashi N, et al. IL-15 is anovel growth factor for murinedγ T cells induced by Salmonellainfection[J]. J Immunol, 1996, 156: 663-669.
73. P.Blanchard, D.Mahe, R.Cariol et al. Protection of swine against posting- weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type2 (PCV2) proteins[J].Vaccine, 2003, 21:4565-4575.
74. P.Blanchard, D.Mahe, R.Cariolet. Protection of swine against posting- weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type2 (PCV2) pro teins [J].Vaccine, 2003, 21:4565-4575.
75. Pasquini S, Xiang Z, Wang Y, et al. 1997. Cytokine and costimulatorymolecules as genetic adjuvants[ J ]. Immunology and Cell Biology, 75:397-401.
76. Raz E, Watanabe A, Baird SM, et al. Systemic immunological effects of cytokine genes injected into skeletal muscle[J]. PricNalt Acad Sci USA, 1993, 90:45234534.
77. Raz E, Watanse A, Baird S M, et al. Systemic immunological effects of cytokine genes injected into skeletal muscle [J]. Proc Natl Acad Sci USA, 1993, 90(10):4523-4527.
78. Rosell C, Segales J, Ramos-Vara JA, et al. Identification of porcine circovirus in tissues of pigs withporcine dermatitis and nephropathy syndrome[J]. Vet Rec, 2006, 146(2):40-43.
79. Rotinghaus S T, Poland G A, Jacobson R M, et al. Hepatitis BDNA vaccine induces protective antibody responses in human non responders to conventional vaccination[J]. Vaccine, 2003, 21 (31):4604-4608.
80. Sanchez J r RE , Meerts P, Nauwynck HJ, et al. PCV2 replication in lymph nodes of pigs inoculated in lategestation or postnatally: Virus quantification, immunophenotyping of target cells and relation to clinical and pathological outcome of infection [A] . Proceedings of the 4th International Symposium on Emerging and Re-emerging Pig Diseases[C]. Rome: Palazzo dei Congressi, 2003:162-163.
81. Sato Y, Roman M, Tighe H. et al. Immunostimulatory DNA sequences necessary for effective intradermal gene immunization[J]. Science, 1996, 273:352-354.
82. Sebastien Racine, Ali Kheyar, Carl A. Gagnon, et al Eucaryotic Expression of the Nucleocapsid Protein Gene of Porcine Circovirus Type 2 and Use of the Protein in an Indirect Immunofluorescence Assay for Serological Diagnosis of Postweaning Multisystemic Wasting Syndrome in Pigs[J].Clin Diagn Lab Immunol, 2004, 11(4):736-741.
83. Sebastien Racine, Ali Kheyar, Carl A. Gagnon, et al. Eucaryotic Expression of the Nucleocapsid Protein Gene of Porcine Circovirus Type 2 and use of the Protein in an Indirect Immunofluorescence Assay for Serological Diagnosis of Postweaning Multisystemic Wasting Syndrome in Pigs[J]. Clin Diagn Lab Immunol, 2004, 11(4):736-741.
84. Segalds J, Rosell C, Domingo M .Pathological findings as sociated with naturally acquired porcine circovirus type 2 associated diseases[J]. Vet Microbiol, 2004, 98(7): 137-149.
85. Segales J, Alonso F, Rosell C, et al. Changes in peripheral blood leukocyte populations in pigs with natural postweaning multisystemic wasting syndrome[J]. Veterinary Immunology and Immunopathology, 2001, 81:37-44.
86. Sin B, MisirligilZ, AybayC, et al. Effect of allerge specific immunotherapy (IT) on natural killer cell actirity (NK), IgE, IFN-gamma levels and clinical response in patients with allergic rhinitis and asthma[J]. Investig Allgol Clin Immunol, 1999, 6(6):341.
87. Skjolaas KA, Grieger DM, Hill C M, et al. Glucocorticoidregulation type 1 and type 2 cytokines in cultured porcinesplenocytes[J]. Vet Immunol Immunopathol, 2002, 87:79-87.
88. Song Y, Jin M, Zhang S, et al. Generation and immunogenicityof a pseudorabies virus expressing cap protein ofporcine circovirus type 2[J].Vet Microbiol, 2007, 119(2-4):97-104.
89. Sorden SD. Update on porcine circovirus and postweaning multisystemic wasting syndrome (PMWS) [J]. Swine Health Prod, 2000, 8(6):133-136.
90．Spie R E,章建康．用于预防传染病的核酸疫苗及其管理[J]．国外医学：预防、诊断、治疗用生物制品分册, 1997, 20(3)：122—125．
91. Sruadhat S, Intrakamhaeng M, Damrongwatanapokin S. The correlation of virus specific interferon-gamma production and protection against classical swine fever virus infection[J]. Vet Immunol Immunopathol, 2001, 83:177-189.
92. Sruadhat S, Intrakamhaeng M, Damrongwatanapokin S. Thecorrelation of virus-specific interferon-gamma production andprotection against classical swine fever virus infection[J]. Vetlmmunol Immunopathol, 2001, 83:177-189.
93. Stevenson GW, Kiupel M, Mittal SK, et al. Tissue distribution and genetic typing of porcine circoviruses in pigs with naturally occurring congenital tremors [J]. J Vet Diagn Invest, 2001, 13(1): 57-62.
94. Sun JH, Yan YX, Jiang J. DNA immunization against very virulent infectious bursal disease virus with VP2-4-3 gene and chicken IL-6 gene[J]. J Vet Med B Infect Dis Vet Public Health, 2005, 52(1):1-7.
95. Suradhat S. Thanawongnuwech R, Poovorawan Y. Upregula-tion of IL-10 gene expression in porcine peripheral bloodmononuclear cells by porcine reproductive and respiratorysyndrome virus[J]. J Gen Virol, 2003, 84(2): 453-459.
96. T Tokunaga, O Yano, E Kuramoto, et al. Synthetic oligonucleotides with particular base sequences from the cDNA encoding proteins of Mycobacterium bovis BCG induce interferons and activate natural Killer cells[J]. Microbiol Immunol, 1992, 36(1):55-66.
97. Tang D C, DeVit M, Johnston SA. Genetic immunization is a simple method for eliciting an immune response[J]. Nature, 1992, 356(6365):152-154.
98. Tischer J, Gelderblom H, Vetermann W, et al.Avery small porcine virus with circular single -stranded DNA[J]. Nature, 1982, 295(2):64-66.
99. Ueha A, Kitazawa H, Tomioka Y, et al. cDNA cloning andexpression of swine IL-7 from neonatal intestinal epithelium[J]. Biochim Biophys Acta. 2001, 1517(3): 468-471.
100. Uenishi H, Hiraiwa H, Sawazaki T, et al. Genomic organiza-tion and assignment of the interleukin 7 gene (IL7) to porcinechromosome 4q11-q13 by FISH and by analysis of radiationhybrid panels[J]. Cytogenet Cell Genet, 2001, 93 (1-2): 65-72.
101. Wang XW, Jiang WM, Jiang P, et al. Construction and im-munogenicity of recombinant adenovirus expressing the capsidprotein of porcine circovirus 2(PCV2) in mice[J].Vaccine, 2006, 24:3374-338.
102. Wei Zhao, Hidenobu Soejima, Ken Higashimoto, et al. The Essential Role of Histone H3 Lys9 Di-Methylation and McCP2 Binding in MGMT Silencing with Poor DNA Methylation of the Promoter CpG Island[J]. J. Biochem. (Tokyo), 2005, 137:431 -440.
103. Wellenberg GJ, Pesch S, Berndsen FW. Isolation and characterization of porcine circovirus type 2 from pigs showing signs of postweaning multisystemic wasting syndrome in the Netherlands[J].Veterinary Quarterly, 2000, 22(3):167-172.
104. YamamotoS. YamamotoT, TokunagaT. et al. Unique palindromic sequence sinsynthetic oligonucleotides are required to induce IFN (correction of INF) and augment 1FN -mediated (correction of INF)natural killer activity[J].J Immounl,1992,148:4072-4076.
105.Yi A K,Yoon J G,Krieg AM.Convergence of CpG DNA and BCR mediated signals at the c-Jun N-terminal kinase and NF-kappaB activation pathways-regulation by mitigen activated protein kinase[J].Int Immunol,2003,15(5):577-591.
106.Zelphati O,Nguyen C,Ferrari M,et al.Stable and monodisperse lipoplex formulations for gene delivery[J].Gene Ther,1998,5(9):1272-1282.
107.Zhou J Y,Shang SB,Gong H,et al.In vitroexpression,mon-oclonal antibody and bioactivity for capsid protein of porcinecircovirus type Ⅱ without nuclear localization signal[J].J Bio-technol,2005,118(2):201-211.
108.陈创夫,余兴龙,乔军等.IL-2与猪瘟病毒E2基因真核双表达载体的构建及其免疫增强作用的研究[J].石河子大学学报,2001,5(2):90-94.
109.陈庆新,周继勇.猪圆环病毒2型感染的流行病学分析及其突变体致病性[D].浙江农业大学博士学位论文,浙江杭州,2006,7.
110.陈香美,于力方.白细胞介素对系膜细胞增殖的影响及系膜细胞产生白细胞介素的观察[J].中华微生物免疫学杂志.1991,11(2):109.
111.程凯慧,李俊.含CPG基序系列猪圆环病毒核酸疫苗的构建[J].中国兽医科技,2008,38(12):32.
112.樊惠英,陈焕春.猪圆环病毒2型基因工程疫苗的研究[D].华中农业大学博士学位论文,中国武汉,2007,6.
113.冯鹰,符林春,张奉学.AIDS鼠模型最新免疫学研究进展[J].微生物学免疫学进展,2005,33(2):87-88.
114.胡青海,焦新安,徐耀辉,等.鸡CD4和CD8基因cDNA的克隆及其真核表达质粒的构建[J]扬州大学学报(农业与生命科学版),2004,25(1):56-60.
115.胡青海,焦新安.鸡CD4和CD8分子研究进展[J].动物医学进展,2005,26(4):16-20.
116.姜新.核酸疫苗:一种新型疫苗[J].生物技术通报,1998,3:21-24.
117.金宁一,秦晓冰,郑敏,等.猪2型圆环病毒核酸疫苗的接种Balb/c小鼠实验免疫研究[J].中国生物工程杂志,2005,25(7):76-79.
118.金珍蜻,李东复等.慢性肝病患者血清细胞因子变化及临床意义[J].中国免疫学杂志,2000,16(7):391.
119.郎洪武,张广川,吴发权.断奶猪多系统衰弱综合征血清抗体检测[J].中国兽医科技,2000, 30(3):3-5.
120.李丽译.猪圆环病毒相关病的并发感染及其有效预防疫苗[J].Feedstuffs,2007,6(10):11
121.刘海鹏,赵玉军.核酸疫苗的研究和应用[J].中国兽医杂志,2001,37(9):34-37.
122.陆彬.药物新剂型与新技术,第-版[M].北京:人民卫生出版社.1998:107-110.
123.罗玉子,仇华吉,刘建华等.猪2型圆环病毒ORF2基因在塞姆利基森林病毒RNA复制子衍生的新型真核表达载体中的表达[J].生物工程学报,2004,20(6):947-951.
124.秦晓冰,金宁一,郑敏,等.猪2型圆环病毒核酸疫苗免疫动物实验的启示[C].:2005第二届全国核酸疫苗与免疫研讨会论文集,北京,2005:19.
125.曲连东,陈溥言.猪圆环病毒2型地方株的分离鉴定和诊断方法的建立及应用[D].南京农业大学博士论文,江苏南京,2006,5.
126.萨姆布鲁克J,拉塞尔DW著,黄培堂等译.《分子克隆实验指南》(第三版)[M].北京:科学出版社,2002.
127.施海晶,胡云章,胡凝珠等.CpG DNA中ISS数量与免疫刺激效果间的关系初探,中国免疫学杂志[J],2001,17(5):531-533.
128.宋云峰,肖少波,金梅林,等.猪2型圆环病毒核酸疫苗免疫效应研究[J].畜牧兽医学报,2005,36(10):1049-1054.
129.隋慧,杨金声.猪圆环病毒2型疫苗研究进[J].动物医学进展,2007,28(12):90-92.
130.孙树汉.核酸疫苗[M].上海:第二军医大学出版社,2000.5.
131.谈国蕾,胡志华,芦银华,等.猪圆环病毒2型ORF2基因真核表达载体的构建及表达[J].中国病毒学,2003,18(4):367-370.
132.田志刚,杨贵贞,等.人白细胞介素6和白细胞介素6mRNA的诱生动力学[J].中华微生物免疫学杂志,1991,11(1):49.
133.王红宁,李进.猪圆环病毒2型感染的PCR测及防治[J].四川畜牧兽医,2004,31(7):25-26.
134.王可,孔健.2型猪圆环病毒ORFZ、ORF3基因的克隆表达与潜在应用研究[D].山东农业大学博士论文,山东济南,2008,4.
135.王忠田,杨汉春,郭鑫.规模化猪场猪圆环病毒2型感染的流行病学调查[J].中国兽医杂志,2002,38(10):3-6.
136.温立斌,杨汉春.猪圆环病毒2型分子流行病学研究[D].中国农业大学博士论文,中国北京,2005,5.
137.吴丹,郭碱军,孙树汉.猪囊尾蝴抗原ccl与7-干扰素融合蛋白在大肠杆菌中的表达,中国 寄生虫学与寄生虫病杂志,1999,17(4):215-221.
138.吴凯源,高荣.新型CpG分子与猪IL6基因的融合及其免疫调节效应研究,四川大学硕士学位论文[M],2005.
139.熊一力,刘惠萍,祖萍,等.大剂量HBsAg疫苗对小鼠细胞免疫应答的调节作用[J].中国病理生理杂志,2003,19(9):1250-1252.
140.许洪林,王四清,王世峰等.两种CpG基序能高度活化人免疫细胞.中华微生物学和免疫学杂志,2001,9(21):471-475.
141.杨汉春.猪免疫抑制性疾病的流行特点与控制对策[J].中国畜牧兽医,2004,31(5):41-43.
142.殷震,刘景华主编.动物病毒学(第二版)[M].北京;科学出版社,1997.
143.殷中琼,贾仁勇,等.细胞因子作为分子佐剂基因免疫中的作用[J].畜禽业,2002,7:47.
144.曾滢,汪恩浩,易学瑞,等.不同剂量重组(酵母)乙肝疫苗对小鼠细胞免疫应答的研究[J].中国药师,2003,6(9):532-534.
145.赵渝,赵冰,陆苹.DNA疫苗的分子佐剂应用研究进展[J]生物技术通报,2002,5(10):10-13.
146.郑连军,王红宁.猪圆环病毒真核表达质粒的构建及其免疫原性的初步研究[J].中国畜牧兽医,2005,32(10):36-38.
147.周绪斌,许秀梅.猪圆环病毒疫苗的研究进展[J].中国兽医杂志,2007,4:26.
148.祝卫国,杨增岐.圆环病毒2型(PCV2)的研究.西北农林科技大学博士学位论文[D].陕西西安杨陵,2008,6.
2. Allan G M. Ellis J A. Porcine circoviruses: a review[J]. J Vet Diagn Invest. 2000a, 12(5):3-14.
3. BallasZK, RasmussenW L, Krieg AM, et al. Induction of NK activity inmurine and human cells by CpG motifys in oligodoxynucleotides and bacterial DNA[J]. J Immunol, 1996, 157(5):1840-1845.
4. Barry MA, Johnston SA. Biological features of genetic immunization[J]. Vaccine, 1997, 15(8):788-791.
5. Blanchard P, Mahe D, Cariolet R, et al. Protection of swine a-gainst post-weaning multisystemic wasting syndrome (PMWS)by porcine circovirus type 2 (PCV2) proteins[J]. Vaccine, 2006, 21(31):4565-4575.
6. Blanchard P, Mahe D, Cariolet R, et al. Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins[J]. Vaccine, 2003, 21(3):4565-4575.
7. Bodmeier R, Chen HG, Paeratakul et al. A novel approach to the delivery of micro or nanoparticles [J]. Pharm Res, 1989, 6(5):413-417.
8. Bodmeier R, Chen HG, Paeratakul et al. A novel approach to the delivery of micro or nanoparticles [J]. PharmRes, 1989, 6(5):413-417.
9. Canals A, Grimm DR, Gasbrre LC, et al. Molecular cloningof cDNA encoding porcine interleukin-15[J]. Gene, 1997, 195:337-339.
10. Cencic A, LeFevre F, Koren S, et al. Tetracycline-controlled expression of glycosylated porcine interferon-gamma in mammlian cells [J]. Anim Biotechnol, 1999, 10(1-2):63-79.
11. CencicA, LeFevreF, Koren S, et al. Tetracycline-controlledexpression of glycosylated porcine interferon-gamma inmammlian cells[J]. Anim Biotechnol. 1999, 10 (1-2): 63-79.
12. Chakrabarti R, Chang Y, Song K. Plasmids encoding membrane-bound IL-4 or IL-12 strongly costimulates DNA vaccination against carcinoembryonic antigen (CEA). Vaccine, 2004, 22(9-10):1199-1205.
13. Choi C, Chae C. Colocalization of porcine reproductive and respiratory syndrome virus and porcine circovirus 2 in porcine dermatitis and nephrology syndrome by double-labeling technique[J]. Vet Pathol, 2001, 38(4): 436.
14. Choi C, Kwon D, Min K, et al. In-situ hybridization for thedetection of inflammatory cytokines (IL-1, TNF-and IL-6) inpigs naturally infected with Actinobacillus pleuropneumoniae[J]. J Comp Pathol, 1999, 121 (4): 349-356.
15. Crawley A, Raymond C, Wilkie BN. Control of immunoglob-ulin isotype by porcine B-cells cultured withcytokines [J]. Vet Immunol Immunopathol, 2003, 91:141-154.
16. Darwich L, Pi S, Rovira A, et al. Cytokine mRNA expression profiles in lymphoid tissues of pigs naturally affected by postweaning multisystemic wasting syndrome[J].Gen Virol, 2003, 84: 2117-2125.
17. Darwich L, Segalds J, Domingo M, et al. Changes in the CD~4+, CD~8+, CD~4/CD~8 double positive cells and postweaning multisystemic wasting syndrome afected pigs and age matched uninfected Wasted and healthy pigs[J]. Clin Diagn Lab Immunol, 2002, 9(4): 236-242.
18. Darwich L, Segales J, Domingo M, et al. Multisystemic wasting syndrome-affected pigs and age-matched unifected wasted and healthy pigs correlate with lesions and Porcine circovirus type 2 load in lympoid tissues american society for Microbiology[J]. Clinical and Diagnostic Laboratory Immunology, 2002, 9(2):236-242.
19. Davis HL, Brazolot Millan CL, Mancini M, et al. DNA-based immunization against hepatitis B surface antigen (HBsAg) in normal and HBsAg-transgenic mice [J]. Vaccine, 1997, 15(8):849-852.
20. Davis HL, Suparto II, Weeratna RR, et al. CpG DNA overcomes hypo-responsiveness to hepatitis B vaccine in orangutans[J].Vaccine, 2000, 18:1920-1924.
21. Davis HL. Use of CpG DNA for enhancing specific immune responses[J]. Cun Top Microbiol Immunol, 2000, 247:171-183.
22. DavisH L, Weeratna R, Waldschmidt TJ , et al. CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen[J]. J Immunol, 1998. 160(2):870-876.
23. Doolan D L, Segales M, Hedstrom R C, et al. Circumventing genetic restriction of protection against malaria with multigene DNA immunization: CD8+ cell-, interferon gamma-, and nitricoxide-dependent immunity [J]. J Exp Med, 1996. 183(4): 1739-1746.
24. Eberl M, Beck E, Coulson C P, et al. IL-18 potentiates the adjuvant properties of IL-12 in the induction of a strong Thl type immune response against a recombinant antigen[J]. Vaccine, 2000. 18:2002-2008.
25. Ellis J. Hassard L. Clark E, et al. Isolation of circovirus from lesions of pigs with postweaning multisyste ic wasting syndrome[J]. Can Vet J, 1998. 39(2):44-51.
26. Ellis J. Krakowka S, Larimore M, et al. Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic pigs[J]. J Vet Diagn Invest. 1999. 11(2):3-14.
27. Fenaux M, Halbur PG. Haqshenas G. et al. Cloned genomic DNA of type 2 porcine circovirus is infectious when injected directly into the liver and lymphnodes of pigs: characterization of clinical disease, virus distribution, and pathologic lesions[J]. J Virol. 2002, 76(2):541-551.
28. Fenaux M, Halbur PG; Haqshenas G et al. Cloned genomic DNA of type 2 porcine circovirus is infectious when injected directly into the liver and lymphnodes of pigs: characterization of clinical disease, virus distribution, and pathologic lesions[J]. J Virol. 2002, 76 (2):541-551.
29. Fenaux M, Opriessnig T, Halbur PG, et al.Two amino acidmutations in the capsid protein of type 2 porcine circovirus(PCV2) enhanced PCV2 replicationin vitroand attenuated thevirusin vivo[J].J Virol, 2004, 78:13440-13446.
30. Fenaux M, Opriessnig T, Halbur P G, et al.A chimeric porcinecircovirus (PCV) with the immunogenic capsid gene of thepathogenic PCV type 2 (PCV-2) cloned into the genomic back-bone of the nonpathogenic PCV1 induces protective immunityagainst PCV2 infection in pigs[J].J Virol, 2004, 78:6297-6303.
31. Foss DL, Zilliox MJ. Murtaugh MP. Bacterially induced actiation of interlerkin-18 in porcine intestinal mucosa[J]. Vet Immunol Immunopathol, 2001, 78: 263-277.
32. Foumout S, Dozois C M, Yerle M, et al. Cloning, chromoso-mal location, and tissue expression of the gene for piginterleukin-l8[J]. Immunogenetics, 2000, 51:358-365.
33. Francisco Ruiz-Fons, Joaqun Vicente, Dolo Vidal, et al. Seroprevalence of six reproductive pathogensin European wild boar(Sus scrofa)from Spain:The effect on wild boar female reproductive performance[J].Theriogenology. 2006, 65:731-743.
34. Gallichan WS, Woolstencroft RN, Guarasci T, et al. In tranasal immunization with CpG oligodeoxynucleotides as an adjuvant dramatically increases IgA and protection against herpess implex virus-2 in the genitaltract[J].J Immunol, 2001, 166:3451-3457.
35. Gregor SD, Kevin She, Luke Terret, et al. CpG stimulation of precursor B-lineage acute lymphoblastic leukemia induces adistinct change incostimulatory molecule expression and shift sallogeneic T cells toward a Th1 response[J]. Blood, 2005, 105:3641-3647.
36. Guo YJ, Wu D, Chen RW, et al. Cloning, high level expression and purification of porcine IFN gamma[J]. Sheng Wu Gong Cheng Xue Bao, 2001, 17(2):183-186.
37. Guo YJ, Wu D, Chen RW, et al. Cloning, high level expression and purification of porcine IFN gamma[J]. Sheng Wu GongCheng Xue Bao, 2001, 17 (2): 183-186.
38. Haddad D , Ramprakash J , Sedegah M , et al. Plasmid vaccine expressing granulocyte-macrophage colony-stimulating factor attracts infiltrates including immature dendritic cdeel intoinjected muscles[J]. J Immunol, 2000, 165: 3772-3781.
39. Hamel AL, Lin LL, Nayar GS. Nucleoitde sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs[J]. J Virol, 1998, 72(6):5262-5267.
40. Hartmann G, Krieg AM, et al. Mechanism and function of a newly identified CpG DNA motify in human primary Bcells[J].J Immunol, 2000, 164:944-952.
41. Hui G, Hashimoto C. 2007. Interleukin26 has differential influence onthe ability of adjuvant formulations to potentiate antibody reponses to aplasmodium falciparum blood2stage vaccine[ J ]. Vaccine, 25:6598-660.
42. Inumaru S, Takamatsu HC. Cloning of porcine granulocy-te-macrophage colony-stimulating factor [J]. Immunol CellBiol, 1995, 73: 474-476.
43. Ishii N, Fukushima J, Kaneko T, et al. Cationic liposomes area strong adjuvant for a DNA vaccine of human immunodeficiency virus type[J]. Aids Research in Human Retroviruses, 1997, 13:1421-1428.
44. Ju C, Fan H, Tan Y, et al. Immunogenicity of a recombinantpseudorabies expressing ORF1-ORF2 fusion protein of porcine circovirus type 2[J].Vet Microbiol , 2005 , 109(3-4):179-190.
45. Kamstrup S, Barfoed A M, Frimann T H, et al. Immunisation against PCV2 structural protein by DNA vaccination of mice[J]. Vaccine, 2004, 22:1358-1361.
46. Kamstrup S, Barfoed AM. Frimann TH, et al. Immunisation against PCV2 structural protein by DNA vaccination of mice[J]. Vaccine, 2004, 22(11-12):1358-1361.
47. Kamstrup S, Barfoed AM. Immunisation against PCV2 struc-tural protein by DNA vaccination of mice[J].Vaceine, 2004, 22:1358-1361.
48. Kiatipattanasakul-Banlunara W, Tantilertcharoen R, Suzuki K, et al. Detection of porcine circovirus 2 (PCV-2) DNA by nested PCR from formalin-fixed tissues of postweaning multi-systemic wasting syndrome (PMWS) pigs in Thailand [J]. J Vet Med Sci, 2002, 64(5): 449-452.
49. Kim JJ, Yang JS, Vancot TC. Modulation of Antigen-specific Humoral Responses in Rhasus Macaques by using cytokine cDNAs as DNA vaccine adjuvant[J]. J Virol, 2000, 74 (7):3427-3429.
50. KojimaY, Xin KQ, Ooki T, et al. Adjuvant effect of multi-CpG motif on an HIV-DNA vaccine[J]. Vaccine, 2002, 20:2857-2865.
51. Kokuho T, Watanabe S, Yokomizo Y, et al. Production ofbiologically active, heterodimeric porcine interleukin-12 using amonocistronic baculoviral expression system[J]. Vet Immunol Immunopathol, 1999, 72:289-302.
52. Krieg AM, Yi AK, Matson S, et al. CpG motifs in bacterial DNAtrigger direct B-cell activation[J]. Nature, 1995, 374(6522):546-549.
53. Ladekjaer-Mikkelsen AS, Nielsen J, Stadejek T, et al. Reproduction of postweaning multisystemic wasting syndrome(PMWS) in immunostimulated and non-immunostimulated 3-week-old piglets experimentally infected with porcine circovirus type2 ( PCV2) [J]. Vet Microbiol, 2002, 89(5):97-114.
54. Lagging LM, Meyer K, Hoft D, et al. Immune responses to plasmid DNA encoding the hepatitis C virus core protein [J]. J Virol, 1995, 69(9):5859-5863.
55. Lee SW, Youn JW, Seong BL, et al. IL-6 induces long-term protective immunity against a lethal challenge of influenza virus[J].Vaccine, 1999. 17:490-496.
56. Lefevre F, Guillomot MD, Andrea S, et al. Interferon-delta:the first member of a noval type I interferon family [J]. Biochimie, 1998, 80 (8-9): 779-788.
57. Levy F, Kristofic C, HeusseC, et al. Role of IL-13 in CD, T cell-dependent IgE productio in atopy[J]. Int Arch Alleray Immunol, 1997, 112(1): 49.
58. Lewis PJ. Babiuk LA. DNA vaccines: a review[J].Adv Res, 1999, 94:129-188.
59. Libin Wen, Xin Guo, Hanchun Yang. Genotyping of porcine circovirus type 2 from a variety of clinicalconditions in China[J].Veterinary Microbiology, 2005, 110:141-146.
60. Lillum. Chitosan and its use as pharmaceutical excipient[J]. Pharm Res. 1998, 15:1326-1331.
61. Mahe D, Blanchard P. Truong C, et al. Differential recognition of ORF2 protein from type I and type 2 porcine circovinises and identification of immunorelevant epitoes[J]. F Gen Virol, 2000, 81(5):1815-1824.
62. Manickan E, Rouse RJ, Yu Z, et al, Genetic immunization against herpes simplex virus. Protection is mediated by CD4+ T lymphocytes [J]. J Immunol, 1995, 155(1):259-265.
63. Mankertz A, Hillenbrand B. Analysis of transcription of porcine circovirus type 1[J]. J Gen Virol, 2002, 83:2743-2751.
64. Maroushek BK, Bosworth BT, Stabel TJ, et al. Cloning and expression of porcine recombinant soluble tumor necrosisfactor receptor 1[J]. Am J Vet Res, 1998. 59 (10): 1317-1322.
65. McNeilly F, McNair I, Mackie DP, et al. Production, characterization and applications of monoclonal antibodies to porcine cercovirus 2[J]. Arc Virol, 2001, 146(6):909-922.
66. Montgomery DL, Shiver JW, Leander KR, et al. Heterologous and homologous protection against influenza A by DNA vaccination: optimization of DNA vectors[J]. DNA Cell Biol, 1993, 12(9):777-783.
67. Muneta Y, Mori Y, Shimoji Y, et al. Porcineinterleukin-18:cloning, characterization of the cDNA and expression with the baculovirus system [J]. Cytokine, 2000, 12:566-572.
68. Muneta Y, Zhao HK, Inumaru S, et al. Large-scale product-ion of porcine mature interleukin-18 (IL-18) in silkworms using a hybrid baculovirus expression system[J]. J Vet Med Sci2003, 65 (2): 219-223.
69. Mwangi SM, Stabel TJ, Kehrli ME. Development of abaculovirus expression system for soluble porcine tumornecrosis factor receptor type I and soluble porcine tumornecrosis factor receptor type Ⅰ IgG fusion protein [J]. VetImmunol Immunopathol. 2002, 86 (3-4): 251-254.
70. Nawagitgul P, Haris P A, Morozov I, et al. Modified indirect porcine circovirus (PCV) type2-based and recombinant capsid protein (ORF2)-based enzyme-linked immunosorbent assays for detection of antibodies to PCV[J]. Clin Diag Lab Immunol, 2002, 9(2):33-40.
71. Nawagitgul P, Morozov I, Bolin SR, et al. Open reading frame 2 of porcine cercovirus type 2 encodes a major capsid protein[J]. J Gen Virol, 2000, 81(8):2281-2287.
72. Nishimura H, Hiromatsu K, Kobayashi N, et al. IL-15 is anovel growth factor for murinedγ T cells induced by Salmonellainfection[J]. J Immunol, 1996, 156: 663-669.
73. P.Blanchard, D.Mahe, R.Cariol et al. Protection of swine against posting- weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type2 (PCV2) proteins[J].Vaccine, 2003, 21:4565-4575.
74. P.Blanchard, D.Mahe, R.Cariolet. Protection of swine against posting- weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type2 (PCV2) pro teins [J].Vaccine, 2003, 21:4565-4575.
75. Pasquini S, Xiang Z, Wang Y, et al. 1997. Cytokine and costimulatorymolecules as genetic adjuvants[ J ]. Immunology and Cell Biology, 75:397-401.
76. Raz E, Watanabe A, Baird SM, et al. Systemic immunological effects of cytokine genes injected into skeletal muscle[J]. PricNalt Acad Sci USA, 1993, 90:45234534.
77. Raz E, Watanse A, Baird S M, et al. Systemic immunological effects of cytokine genes injected into skeletal muscle [J]. Proc Natl Acad Sci USA, 1993, 90(10):4523-4527.
78. Rosell C, Segales J, Ramos-Vara JA, et al. Identification of porcine circovirus in tissues of pigs withporcine dermatitis and nephropathy syndrome[J]. Vet Rec, 2006, 146(2):40-43.
79. Rotinghaus S T, Poland G A, Jacobson R M, et al. Hepatitis BDNA vaccine induces protective antibody responses in human non responders to conventional vaccination[J]. Vaccine, 2003, 21 (31):4604-4608.
80. Sanchez J r RE , Meerts P, Nauwynck HJ, et al. PCV2 replication in lymph nodes of pigs inoculated in lategestation or postnatally: Virus quantification, immunophenotyping of target cells and relation to clinical and pathological outcome of infection [A] . Proceedings of the 4th International Symposium on Emerging and Re-emerging Pig Diseases[C]. Rome: Palazzo dei Congressi, 2003:162-163.
81. Sato Y, Roman M, Tighe H. et al. Immunostimulatory DNA sequences necessary for effective intradermal gene immunization[J]. Science, 1996, 273:352-354.
82. Sebastien Racine, Ali Kheyar, Carl A. Gagnon, et al Eucaryotic Expression of the Nucleocapsid Protein Gene of Porcine Circovirus Type 2 and Use of the Protein in an Indirect Immunofluorescence Assay for Serological Diagnosis of Postweaning Multisystemic Wasting Syndrome in Pigs[J].Clin Diagn Lab Immunol, 2004, 11(4):736-741.
83. Sebastien Racine, Ali Kheyar, Carl A. Gagnon, et al. Eucaryotic Expression of the Nucleocapsid Protein Gene of Porcine Circovirus Type 2 and use of the Protein in an Indirect Immunofluorescence Assay for Serological Diagnosis of Postweaning Multisystemic Wasting Syndrome in Pigs[J]. Clin Diagn Lab Immunol, 2004, 11(4):736-741.
84. Segalds J, Rosell C, Domingo M .Pathological findings as sociated with naturally acquired porcine circovirus type 2 associated diseases[J]. Vet Microbiol, 2004, 98(7): 137-149.
85. Segales J, Alonso F, Rosell C, et al. Changes in peripheral blood leukocyte populations in pigs with natural postweaning multisystemic wasting syndrome[J]. Veterinary Immunology and Immunopathology, 2001, 81:37-44.
86. Sin B, MisirligilZ, AybayC, et al. Effect of allerge specific immunotherapy (IT) on natural killer cell actirity (NK), IgE, IFN-gamma levels and clinical response in patients with allergic rhinitis and asthma[J]. Investig Allgol Clin Immunol, 1999, 6(6):341.
87. Skjolaas KA, Grieger DM, Hill C M, et al. Glucocorticoidregulation type 1 and type 2 cytokines in cultured porcinesplenocytes[J]. Vet Immunol Immunopathol, 2002, 87:79-87.
88. Song Y, Jin M, Zhang S, et al. Generation and immunogenicityof a pseudorabies virus expressing cap protein ofporcine circovirus type 2[J].Vet Microbiol, 2007, 119(2-4):97-104.
89. Sorden SD. Update on porcine circovirus and postweaning multisystemic wasting syndrome (PMWS) [J]. Swine Health Prod, 2000, 8(6):133-136.
90．Spie R E,章建康．用于预防传染病的核酸疫苗及其管理[J]．国外医学：预防、诊断、治疗用生物制品分册, 1997, 20(3)：122—125．
91. Sruadhat S, Intrakamhaeng M, Damrongwatanapokin S. The correlation of virus specific interferon-gamma production and protection against classical swine fever virus infection[J]. Vet Immunol Immunopathol, 2001, 83:177-189.
92. Sruadhat S, Intrakamhaeng M, Damrongwatanapokin S. Thecorrelation of virus-specific interferon-gamma production andprotection against classical swine fever virus infection[J]. Vetlmmunol Immunopathol, 2001, 83:177-189.
93. Stevenson GW, Kiupel M, Mittal SK, et al. Tissue distribution and genetic typing of porcine circoviruses in pigs with naturally occurring congenital tremors [J]. J Vet Diagn Invest, 2001, 13(1): 57-62.
94. Sun JH, Yan YX, Jiang J. DNA immunization against very virulent infectious bursal disease virus with VP2-4-3 gene and chicken IL-6 gene[J]. J Vet Med B Infect Dis Vet Public Health, 2005, 52(1):1-7.
95. Suradhat S. Thanawongnuwech R, Poovorawan Y. Upregula-tion of IL-10 gene expression in porcine peripheral bloodmononuclear cells by porcine reproductive and respiratorysyndrome virus[J]. J Gen Virol, 2003, 84(2): 453-459.
96. T Tokunaga, O Yano, E Kuramoto, et al. Synthetic oligonucleotides with particular base sequences from the cDNA encoding proteins of Mycobacterium bovis BCG induce interferons and activate natural Killer cells[J]. Microbiol Immunol, 1992, 36(1):55-66.
97. Tang D C, DeVit M, Johnston SA. Genetic immunization is a simple method for eliciting an immune response[J]. Nature, 1992, 356(6365):152-154.
98. Tischer J, Gelderblom H, Vetermann W, et al.Avery small porcine virus with circular single -stranded DNA[J]. Nature, 1982, 295(2):64-66.
99. Ueha A, Kitazawa H, Tomioka Y, et al. cDNA cloning andexpression of swine IL-7 from neonatal intestinal epithelium[J]. Biochim Biophys Acta. 2001, 1517(3): 468-471.
100. Uenishi H, Hiraiwa H, Sawazaki T, et al. Genomic organiza-tion and assignment of the interleukin 7 gene (IL7) to porcinechromosome 4q11-q13 by FISH and by analysis of radiationhybrid panels[J]. Cytogenet Cell Genet, 2001, 93 (1-2): 65-72.
101. Wang XW, Jiang WM, Jiang P, et al. Construction and im-munogenicity of recombinant adenovirus expressing the capsidprotein of porcine circovirus 2(PCV2) in mice[J].Vaccine, 2006, 24:3374-338.
102. Wei Zhao, Hidenobu Soejima, Ken Higashimoto, et al. The Essential Role of Histone H3 Lys9 Di-Methylation and McCP2 Binding in MGMT Silencing with Poor DNA Methylation of the Promoter CpG Island[J]. J. Biochem. (Tokyo), 2005, 137:431 -440.
103. Wellenberg GJ, Pesch S, Berndsen FW. Isolation and characterization of porcine circovirus type 2 from pigs showing signs of postweaning multisystemic wasting syndrome in the Netherlands[J].Veterinary Quarterly, 2000, 22(3):167-172.
104. YamamotoS. YamamotoT, TokunagaT. et al. Unique palindromic sequence sinsynthetic oligonucleotides are required to induce IFN (correction of INF) and augment 1FN -mediated (correction of INF)natural killer activity[J].J Immounl,1992,148:4072-4076.
105.Yi A K,Yoon J G,Krieg AM.Convergence of CpG DNA and BCR mediated signals at the c-Jun N-terminal kinase and NF-kappaB activation pathways-regulation by mitigen activated protein kinase[J].Int Immunol,2003,15(5):577-591.
106.Zelphati O,Nguyen C,Ferrari M,et al.Stable and monodisperse lipoplex formulations for gene delivery[J].Gene Ther,1998,5(9):1272-1282.
107.Zhou J Y,Shang SB,Gong H,et al.In vitroexpression,mon-oclonal antibody and bioactivity for capsid protein of porcinecircovirus type Ⅱ without nuclear localization signal[J].J Bio-technol,2005,118(2):201-211.
108.陈创夫,余兴龙,乔军等.IL-2与猪瘟病毒E2基因真核双表达载体的构建及其免疫增强作用的研究[J].石河子大学学报,2001,5(2):90-94.
109.陈庆新,周继勇.猪圆环病毒2型感染的流行病学分析及其突变体致病性[D].浙江农业大学博士学位论文,浙江杭州,2006,7.
110.陈香美,于力方.白细胞介素对系膜细胞增殖的影响及系膜细胞产生白细胞介素的观察[J].中华微生物免疫学杂志.1991,11(2):109.
111.程凯慧,李俊.含CPG基序系列猪圆环病毒核酸疫苗的构建[J].中国兽医科技,2008,38(12):32.
112.樊惠英,陈焕春.猪圆环病毒2型基因工程疫苗的研究[D].华中农业大学博士学位论文,中国武汉,2007,6.
113.冯鹰,符林春,张奉学.AIDS鼠模型最新免疫学研究进展[J].微生物学免疫学进展,2005,33(2):87-88.
114.胡青海,焦新安,徐耀辉,等.鸡CD4和CD8基因cDNA的克隆及其真核表达质粒的构建[J]扬州大学学报(农业与生命科学版),2004,25(1):56-60.
115.胡青海,焦新安.鸡CD4和CD8分子研究进展[J].动物医学进展,2005,26(4):16-20.
116.姜新.核酸疫苗:一种新型疫苗[J].生物技术通报,1998,3:21-24.
117.金宁一,秦晓冰,郑敏,等.猪2型圆环病毒核酸疫苗的接种Balb/c小鼠实验免疫研究[J].中国生物工程杂志,2005,25(7):76-79.
118.金珍蜻,李东复等.慢性肝病患者血清细胞因子变化及临床意义[J].中国免疫学杂志,2000,16(7):391.
119.郎洪武,张广川,吴发权.断奶猪多系统衰弱综合征血清抗体检测[J].中国兽医科技,2000, 30(3):3-5.
120.李丽译.猪圆环病毒相关病的并发感染及其有效预防疫苗[J].Feedstuffs,2007,6(10):11
121.刘海鹏,赵玉军.核酸疫苗的研究和应用[J].中国兽医杂志,2001,37(9):34-37.
122.陆彬.药物新剂型与新技术,第-版[M].北京:人民卫生出版社.1998:107-110.
123.罗玉子,仇华吉,刘建华等.猪2型圆环病毒ORF2基因在塞姆利基森林病毒RNA复制子衍生的新型真核表达载体中的表达[J].生物工程学报,2004,20(6):947-951.
124.秦晓冰,金宁一,郑敏,等.猪2型圆环病毒核酸疫苗免疫动物实验的启示[C].:2005第二届全国核酸疫苗与免疫研讨会论文集,北京,2005:19.
125.曲连东,陈溥言.猪圆环病毒2型地方株的分离鉴定和诊断方法的建立及应用[D].南京农业大学博士论文,江苏南京,2006,5.
126.萨姆布鲁克J,拉塞尔DW著,黄培堂等译.《分子克隆实验指南》(第三版)[M].北京:科学出版社,2002.
127.施海晶,胡云章,胡凝珠等.CpG DNA中ISS数量与免疫刺激效果间的关系初探,中国免疫学杂志[J],2001,17(5):531-533.
128.宋云峰,肖少波,金梅林,等.猪2型圆环病毒核酸疫苗免疫效应研究[J].畜牧兽医学报,2005,36(10):1049-1054.
129.隋慧,杨金声.猪圆环病毒2型疫苗研究进[J].动物医学进展,2007,28(12):90-92.
130.孙树汉.核酸疫苗[M].上海:第二军医大学出版社,2000.5.
131.谈国蕾,胡志华,芦银华,等.猪圆环病毒2型ORF2基因真核表达载体的构建及表达[J].中国病毒学,2003,18(4):367-370.
132.田志刚,杨贵贞,等.人白细胞介素6和白细胞介素6mRNA的诱生动力学[J].中华微生物免疫学杂志,1991,11(1):49.
133.王红宁,李进.猪圆环病毒2型感染的PCR测及防治[J].四川畜牧兽医,2004,31(7):25-26.
134.王可,孔健.2型猪圆环病毒ORFZ、ORF3基因的克隆表达与潜在应用研究[D].山东农业大学博士论文,山东济南,2008,4.
135.王忠田,杨汉春,郭鑫.规模化猪场猪圆环病毒2型感染的流行病学调查[J].中国兽医杂志,2002,38(10):3-6.
136.温立斌,杨汉春.猪圆环病毒2型分子流行病学研究[D].中国农业大学博士论文,中国北京,2005,5.
137.吴丹,郭碱军,孙树汉.猪囊尾蝴抗原ccl与7-干扰素融合蛋白在大肠杆菌中的表达,中国 寄生虫学与寄生虫病杂志,1999,17(4):215-221.
138.吴凯源,高荣.新型CpG分子与猪IL6基因的融合及其免疫调节效应研究,四川大学硕士学位论文[M],2005.
139.熊一力,刘惠萍,祖萍,等.大剂量HBsAg疫苗对小鼠细胞免疫应答的调节作用[J].中国病理生理杂志,2003,19(9):1250-1252.
140.许洪林,王四清,王世峰等.两种CpG基序能高度活化人免疫细胞.中华微生物学和免疫学杂志,2001,9(21):471-475.
141.杨汉春.猪免疫抑制性疾病的流行特点与控制对策[J].中国畜牧兽医,2004,31(5):41-43.
142.殷震,刘景华主编.动物病毒学(第二版)[M].北京;科学出版社,1997.
143.殷中琼,贾仁勇,等.细胞因子作为分子佐剂基因免疫中的作用[J].畜禽业,2002,7:47.
144.曾滢,汪恩浩,易学瑞,等.不同剂量重组(酵母)乙肝疫苗对小鼠细胞免疫应答的研究[J].中国药师,2003,6(9):532-534.
145.赵渝,赵冰,陆苹.DNA疫苗的分子佐剂应用研究进展[J]生物技术通报,2002,5(10):10-13.
146.郑连军,王红宁.猪圆环病毒真核表达质粒的构建及其免疫原性的初步研究[J].中国畜牧兽医,2005,32(10):36-38.
147.周绪斌,许秀梅.猪圆环病毒疫苗的研究进展[J].中国兽医杂志,2007,4:26.
148.祝卫国,杨增岐.圆环病毒2型(PCV2)的研究.西北农林科技大学博士学位论文[D].陕西西安杨陵,2008,6.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |