禽传染性支气管炎病毒多表位嵌合DNA疫苗构建及免疫研究
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摘要
禽传染性支气管炎(Infectious bronchitis,IB)是危害养鸡生产重要的病毒性传染病之一。由于禽传染性支气管炎病毒(Infectious bronchitis virus,IBV)血清型众多,不同血清型之间交叉保护力弱,而常规疫苗常引起IB的免疫失败。因此,从基因水平上研制DNA疫苗等新型基因工程疫苗,对提高IBV免疫保护具有重要意义。在前期研究基础上,本研究对IBV肾型SAIBk株结构蛋白的抗原表位进行系统分析预测,筛选出IBV S1、S2和N蛋白的抗原表位片段,构建含鸡属特异性CpG基序的IBV多表位嵌合基因,将嵌合基因进行原核表达并建立了检测IBV抗体的间接ELISA方法,构建了多表位嵌合基因的真核表达质粒,并结合禽白细胞介素分子佐剂对其免疫效果进行了比较研究,为研制安全、高效的新型IBV DNA疫苗提供了新的方法。
1、采用生物信息学软件及相关网站分析IBV S1、S2和N结构蛋白的二级结构、B细胞表位(主要是中和抗原表位)和T细胞表位(主要是CTL抗原表位),同时结合文献研究报道,共筛选出IBV抗原表位区域7段F1-F7(S1:24-150AA、240-255AA、290-400AA、532-537AA;S2:1-65AA;N:1-120AA、N:290-410AA)。以柔性肽GP或GA作为Linker将7个抗原表位基因依次串联成一条全新的多表位嵌合基因F。嵌合基因5'端引入Kozak序列,3'端引入鸡属特异性CpG免疫刺激基序,分析表明,构建的IBV多表位嵌合基因F有良好的亲水性、柔性及抗原性等。本研究对IBV S1、S2和N蛋白的抗原表位进行系统预测分析,成功构建了含鸡属特异性CpG基序的IBV结构蛋白多表位嵌合基因,分析表明该嵌合基因编码蛋白有良好的亲水性和抗原性等,国内外未见报道,该研究丰富了IBV结构蛋白生物信息学资料,为IBV多表位嵌合基因的原核表达、ELISA方法建立,以及DNA疫苗的制备提供了基础材料。
2、将IBV结构蛋白多表位嵌合基因F片段用BamHI和XhoI双酶切后亚克隆入原核表达载体pET-32a(+),转化E.coli Rosetta,采用IPTG进行诱导,研究表明嵌合基因F编码的蛋白以包涵体形式融合表达,Western-blots显示,该融合蛋白能与抗IBV阳性血清发生特异性反应。通过优化重组质粒的表达条件,确定了嵌合基因F的重组蛋白表达的最佳IPTG诱导物浓度为1 mmol/L,诱导时间为3 h,诱导温度为30℃。以纯化的融合重组蛋白为抗原包被酶标板,筛选出重组抗原最佳包被浓度为20μg/mL,抗体最佳稀释度1∶40,酶标二抗(HRP标记兔抗鸡IgG)最佳稀释度1∶3000,血清样品阴阳性临界值为0.133,建立了以嵌合基因重组蛋白为诊断抗原的IBV抗体间接ELISA方法。本研究将IBV多表位嵌合基因进行原核表达研究,表达产物有良好免疫反应活性,建立了以IBV多表位嵌合蛋白为诊断抗原的间接ELISA方法,该方法安全、灵敏度高(血清稀释度可达1∶300)、特异性强,优于常规的IBV抗体检测方法,国内外未见报道,为IBV抗体的检测提供了新的方法。
3、将IBV多表位嵌合基因F分别亚克隆进真核表达载体pcDNA3.1、pVAX1、VR1020中,构建了禽白细胞介素IL-1β、IL-2、IL-18基因的pcDNA3.1真核表达质粒。重组质粒经酶切和测序鉴定后,通过脂质体转染COS-7细胞,利用RT-PCR及间接免疫荧光检测重组真核质粒在体外的表达。重组质粒经酶切和测序表明IBV多表位嵌合基因F、禽白细胞介素的真核表达质粒构建正确。RT-PCR检测表明,转染了pcDNA-F、pVAX1-F、VR1020-F、pcDNA-IL-1β、pcDNA-IL-2和pcDNA-IL-18的COS-7细胞中均能扩增出特异性的目的基因片段;间接免疫荧光检测表明,转染了pcDNA-F、pVAX1-F、VR1020-F的COS-7细胞显示出特异性绿色荧光。本研究构建了含鸡属特异性CpG基序的IBV结构蛋白多表位嵌合基因的真核表达质粒,检测表明真核质粒表达的IBV多表位嵌合蛋白有良好的免疫反应性,国内外未见报道,为IBV多表位嵌合基因DNA疫苗的研制提供了试验材料。
4、采用本课题组获得专利(专利授权号为200410081443.4)的质粒提取纯化方法制备IBV结构蛋白多表位嵌合基因及禽白细胞介素真核表达重组质粒,将纯化的重组质粒溶液(1mg/mL)与脂质体溶液(10mg/mL)等体积混合配制成DNA疫苗,通过腿部肌肉多点注射7日龄非免疫雏鸡,21日龄加强免疫一次,二免疫后7、14、21、28采血检测外周血CD4+、CD8+T淋巴细胞亚群的数量和ELISA抗体效价,二免后五周用IBV强毒攻击测定免疫保护率;同时对pVAX1-F疫苗免疫组不同时间的质粒分布及整合可能性进行了检测。外周血CD4+、CD8+T淋巴细胞亚群数量检测结果显示:pVAX1-F、pcDNA-F、VR1020-F疫苗免疫组在二免后7～21d,与PBS组和空质粒免疫组相比差异均极显著(P＜0.01),但组间差异不显著。pcDNA-F+pcDNA-IL-1β、pcDNA-F+pcDNA-IL-2、pcDNA-F+pcDNA-IL-18疫苗免疫组在免疫后7～28d,与pcDNA-F单独免疫组相比差异均显著(P＜0.05)。ELISA抗体效价结果显示:pVAX1-F、pcDNA-F、VR1020-F疫苗免疫组在二免后7～28d,与PBS组和空质粒免疫组相比差异均极显著(P＜0.01),但组间差异不显著。质粒pcDNA-F分别与禽IL-1β、IL-2、IL-18共同免疫组在免疫后14～28d,与pcDNA-F疫苗组相比差异均显著(P＜0.05)。攻毒结果表明,pVAX1-F、pcDNA-F、VR1020-F疫苗组的保护率分别为80%、80%和75%,前两者优于常规灭活疫苗(75%)。pcDNA-F+pcDNA-IL-1β、pcDNA-F+pcDNA-IL-2、pcDNA-F+pcDNA-IL-18疫苗免疫组的保护率分别为85%、90%和85%,表明禽IL-2的免疫增强效果优于IL-1β和IL-18。病理切片观察DNA疫苗免疫鸡群的肾脏正常、无病理变化。疫苗质粒分布及整合安全性检测结果显示,pVAX1-F免疫接种后24h,在血液和所有检测的组织中检测到质粒,免疫后90d可在心、脾、肺等组织检测到质粒;纯化后的组织基因组DNA经PCR扩增均呈阴性,未发现整合现象。本研究研制了含鸡属特异性CpG基序的IBV结构蛋白多表位嵌合基因的DNA疫苗,研究表明该多表位嵌合DNA疫苗能够诱导良好的细胞免疫和体液免疫,免疫鸡攻毒保护率为80%,高于常规IBV灭活疫苗(75%);与禽IL-2分子佐剂共同免疫鸡群的攻毒保护率达到90%;该多表位嵌合基因DNA疫苗安全性好,未检测到与宿主基因组整合的现象。
本论文开展的禽传染性支气管炎病毒多表位嵌合DNA疫苗构建及免疫研究,在国内外未见报道,该研究为研制安全、高效的新型IBV DNA疫苗提供了新的方法。
Infectious bronchitis virus(IBV) is one of the primary causes of respiratory disease in domestic fowl.Multitude serological types of IBV are occurred due to variation,and there is no cross-protection between different serological types.In addition,conventional vaccines against infectious bronchitis applied in the field have respective weakness. Therefore,it is of significance to develop a safety and effective genetically engineering vaccine such as DNA vaccine to control the disease.Based on former achievement of our research group,we analyzed and predicted the structural protein of nephritic IBV SAIBk strain,screened several epitopes from S1,S2 and N protein of IBV,and constructed a chimeric multi-epitope gene by using of the screened epitopes genes.The constructed chimeric gene was used to perform prokaryotic expression and a corresponding ELISA assay was established by using the expressing recombination protein.Moreover,the constructed chimeric multi-epitope gene was subcloned into eukaryotic expression vectors and then prepared DNA vaccines,and the immunoreactivity of the chimeric multi-epitope DNA vaccines were evaluated,as well as coadministration with avian interleukins.The aim of the study was to provide a new candidate target for IBV vaccine development.
1.The dominant epitopes of S1,S2,and N protein of IBV SAIBk strain were analyzed on base of computer bioinformatics softwares and reported references,and then seven T-and B-cell epitopes from S1,S2 and N protein(S1:24-150,240-255,290-400,532-537;S2: 1-65;N:1-120,290-410) were selected.The seven multi-epitope minigenes were paiallelled as a single chimeric gene separated from each other with the GA/GP spacers with an unique open reading frame by using Splicing by Overlap Extension(SOEing) and polymerase chain reaction(PCR),and the constructs included a Kozak sequence at the N-terminus and avian specific CpG motif in the end of the chimeric gene.The analysis for the chimeric gene indicated that the coded chimeric protein showed favourable hydrophilicity,flexibility and antigenicity.The study provided foundations for the prokaryotic expression,ELISA method,eukaryotic expression,and research for DNA vaccine of the multi-epitope chimeric gene.
2.The constructed chimeric multi-epitope gene of IBV was digested by BamHI and XhoI to generate chimeric gene F,which was subcloned into the prokaryotic expression vector pET-32a(+).The recombinant plasmid was transformed into E.coli Rosetta.The recombinant bacterium was induced by IPTG.The chimeric F protein was expressed fusedly in the form of cytorrhyctes.Western-bloting results showed the expressed recombinant protein occurred specific reaction with positive serum of IBV.The expression conditions of chimeric F protein were optimized with proper inducing conditions of 1 mmol/L IPTG for 3 hours at 30℃temperature.Using the puridied chimeric recombinant protein as coating antigen,the optimal concentration of F protein for coating of plate was 20μg/mL;the dilution of antibody was 1:40;the working concentration of HRP rabbit-anti-chicken IgG was 1:3000;the threshold value of ELISA assay was 0.133. According to the determination of condition of enzyme-linked immunosorbent assay (ELISA),an indirect ELISA based on the chimeric F protein was developed.The study provided new approachs for the development of subunit vaccine and detection of ELISA antibodies for IBV.
3.The constructed chimeric multi-epitope gene F of IBV was subcloned into eukaryotic expression vectors pcDNA3.1,pVAX1 and VR1020,respectively.In addition,the avian interleukin-1β,interleukin-2 and interleukin-18 gene were inserted into the plasmid pcDNA3.1,respectively.Identification of the constructed eukaryotic expression plasmids were performed by restriction enzyme digestion and DNA sequencing.The positive recombinant plasmids were transfected into COS-7 cells by lipofectamine,and the expression of heterologous genes were detected by RT-PCR and indirect immunofluorescence assay(IFA).The results of restriction enzyme digestion and DNA sequencing showed that the eukaryotic expression plasmids were constructed successfully, The corresponding mRNA transcripts were detected by RT-PCR in the COS-7 ceils which were transfected with plasmids pcDNA-F,pVAX1-F,VR1020-F,pcDNA-IL-1β, pcDNA-IL-2 and pcDNA-IL-18,respectively.The expression of chimeric multi-epitope recombinant protein were detected by indirect immunofluorescent assay in the COS-7 cells which were transfected with plasmids pcDNA-F,pVAX1-F and VR1020-F,respectively. The study provided bases for the further development of chimeric multi-epitope DNA vaccine against IBV.
4.The eukaryotic recombinant plasmids pcDNA-F,pVAX1-F,VR1020-F,pcDNA-IL-1β, pcDNA-IL-2 and pcDNA-IL-18 used for vaccination were extracted from DH5a,and purified by a Chinese invention patent method which was established previously in our laboratory.The diluted recombinant plasmids(1mg/mL) were encapsulated by liposome(10mg/mL) in equal volume,and administered to the 7-day-old chickens by intramuscularly injection.After two weeks,the chickens were boosted by equivalent dosage of DNA vaccine.The percentage of CD4+ and CD8+ T-lymphocytes from peripheral blood and anti-IBV specific antibodies were measured after the booster, respectively.Five weeks after booster,chickens were challenged by virulent IBV strain.In addition,the distribution in different times and possibility of integration into host genomic DNA of plasmid pVAX1-F were detected.The results of percentage of CD4+ and CD8+ T-lymphocytes showed that,the number of CD4+ and CD8+ T-lymphocytes of chickens immunized with pVAX1-F,pcDNA-F and VR1020-F were extremely significantly higher (P＜0.01) than that of PBS and control empty plasmids groups 7-21d post the booster, respectively,but showed no distinguished differences between interclass of themselves, and the number of CD4+ and CD8+ T-lymphocytes of chickens immunized with pcDNA-F+pcDNA-IL-1β,pcDNA-F+pcDNA-IL-2,pcDNA-F+pcDNA-IL-18 were significantly higher(P＜0.05) than that of the single plasmid pcDNA-F inoculated group 7-28d post the booster,respevtively.The results of specific antibodies detection showed that,the antibody titers of chickens immunized with pVAX1-F,pcDNA-F and VR1020-F were extremely significantly higher(P＜0.01) than that of PBS and control empty plasmids groups 7-28d post the booster,respevtively,but showed no noticeable difference between interclass of themselves,and the antibody titers of chickens co-administrated with pcDNA-F and pcDNA-IL-1β,pcDNA-IL-2,pcDNA-IL-18 were significantly higher (P＜0.05) than that of the pcDNA-F alone group 14-28d post the booster,respevtively.The protection rate of pVAX1-F,pcDNA-F and VR1020-F group were 80%,80%and 75%. The protection rates of groups inoculated with pcDNA-F+pcDNA-IL-1β, pcDNA-F+pcDNA-IL-2 and pcDNA-F+pcDNA-IL-18 were 85%,90%and 85%, respectively,which indicated that IL-2 showed better immune enhancement contribution than IL-1βand IL-18.The tissue section indicated that the kidneys of chickens protected by DNA vaccines were normal and physical fitness,without pathological changes.The distribution and integration detection of plasmid pVAX1-F showed that the plasmid was detected in blood and all the other checked tissues 24h post inoculation and last 90d in some tissues at least,and there was no detective phenomenon of integration during the period of experiment.The study provided a new way to enhance the immunogenicity of IBV DNA vaccine and reduce costs.
1、采用生物信息学软件及相关网站分析IBV S1、S2和N结构蛋白的二级结构、B细胞表位(主要是中和抗原表位)和T细胞表位(主要是CTL抗原表位),同时结合文献研究报道,共筛选出IBV抗原表位区域7段F1-F7(S1:24-150AA、240-255AA、290-400AA、532-537AA;S2:1-65AA;N:1-120AA、N:290-410AA)。以柔性肽GP或GA作为Linker将7个抗原表位基因依次串联成一条全新的多表位嵌合基因F。嵌合基因5'端引入Kozak序列,3'端引入鸡属特异性CpG免疫刺激基序,分析表明,构建的IBV多表位嵌合基因F有良好的亲水性、柔性及抗原性等。本研究对IBV S1、S2和N蛋白的抗原表位进行系统预测分析,成功构建了含鸡属特异性CpG基序的IBV结构蛋白多表位嵌合基因,分析表明该嵌合基因编码蛋白有良好的亲水性和抗原性等,国内外未见报道,该研究丰富了IBV结构蛋白生物信息学资料,为IBV多表位嵌合基因的原核表达、ELISA方法建立,以及DNA疫苗的制备提供了基础材料。
2、将IBV结构蛋白多表位嵌合基因F片段用BamHI和XhoI双酶切后亚克隆入原核表达载体pET-32a(+),转化E.coli Rosetta,采用IPTG进行诱导,研究表明嵌合基因F编码的蛋白以包涵体形式融合表达,Western-blots显示,该融合蛋白能与抗IBV阳性血清发生特异性反应。通过优化重组质粒的表达条件,确定了嵌合基因F的重组蛋白表达的最佳IPTG诱导物浓度为1 mmol/L,诱导时间为3 h,诱导温度为30℃。以纯化的融合重组蛋白为抗原包被酶标板,筛选出重组抗原最佳包被浓度为20μg/mL,抗体最佳稀释度1∶40,酶标二抗(HRP标记兔抗鸡IgG)最佳稀释度1∶3000,血清样品阴阳性临界值为0.133,建立了以嵌合基因重组蛋白为诊断抗原的IBV抗体间接ELISA方法。本研究将IBV多表位嵌合基因进行原核表达研究,表达产物有良好免疫反应活性,建立了以IBV多表位嵌合蛋白为诊断抗原的间接ELISA方法,该方法安全、灵敏度高(血清稀释度可达1∶300)、特异性强,优于常规的IBV抗体检测方法,国内外未见报道,为IBV抗体的检测提供了新的方法。
3、将IBV多表位嵌合基因F分别亚克隆进真核表达载体pcDNA3.1、pVAX1、VR1020中,构建了禽白细胞介素IL-1β、IL-2、IL-18基因的pcDNA3.1真核表达质粒。重组质粒经酶切和测序鉴定后,通过脂质体转染COS-7细胞,利用RT-PCR及间接免疫荧光检测重组真核质粒在体外的表达。重组质粒经酶切和测序表明IBV多表位嵌合基因F、禽白细胞介素的真核表达质粒构建正确。RT-PCR检测表明,转染了pcDNA-F、pVAX1-F、VR1020-F、pcDNA-IL-1β、pcDNA-IL-2和pcDNA-IL-18的COS-7细胞中均能扩增出特异性的目的基因片段;间接免疫荧光检测表明,转染了pcDNA-F、pVAX1-F、VR1020-F的COS-7细胞显示出特异性绿色荧光。本研究构建了含鸡属特异性CpG基序的IBV结构蛋白多表位嵌合基因的真核表达质粒,检测表明真核质粒表达的IBV多表位嵌合蛋白有良好的免疫反应性,国内外未见报道,为IBV多表位嵌合基因DNA疫苗的研制提供了试验材料。
4、采用本课题组获得专利(专利授权号为200410081443.4)的质粒提取纯化方法制备IBV结构蛋白多表位嵌合基因及禽白细胞介素真核表达重组质粒,将纯化的重组质粒溶液(1mg/mL)与脂质体溶液(10mg/mL)等体积混合配制成DNA疫苗,通过腿部肌肉多点注射7日龄非免疫雏鸡,21日龄加强免疫一次,二免疫后7、14、21、28采血检测外周血CD4+、CD8+T淋巴细胞亚群的数量和ELISA抗体效价,二免后五周用IBV强毒攻击测定免疫保护率;同时对pVAX1-F疫苗免疫组不同时间的质粒分布及整合可能性进行了检测。外周血CD4+、CD8+T淋巴细胞亚群数量检测结果显示:pVAX1-F、pcDNA-F、VR1020-F疫苗免疫组在二免后7～21d,与PBS组和空质粒免疫组相比差异均极显著(P＜0.01),但组间差异不显著。pcDNA-F+pcDNA-IL-1β、pcDNA-F+pcDNA-IL-2、pcDNA-F+pcDNA-IL-18疫苗免疫组在免疫后7～28d,与pcDNA-F单独免疫组相比差异均显著(P＜0.05)。ELISA抗体效价结果显示:pVAX1-F、pcDNA-F、VR1020-F疫苗免疫组在二免后7～28d,与PBS组和空质粒免疫组相比差异均极显著(P＜0.01),但组间差异不显著。质粒pcDNA-F分别与禽IL-1β、IL-2、IL-18共同免疫组在免疫后14～28d,与pcDNA-F疫苗组相比差异均显著(P＜0.05)。攻毒结果表明,pVAX1-F、pcDNA-F、VR1020-F疫苗组的保护率分别为80%、80%和75%,前两者优于常规灭活疫苗(75%)。pcDNA-F+pcDNA-IL-1β、pcDNA-F+pcDNA-IL-2、pcDNA-F+pcDNA-IL-18疫苗免疫组的保护率分别为85%、90%和85%,表明禽IL-2的免疫增强效果优于IL-1β和IL-18。病理切片观察DNA疫苗免疫鸡群的肾脏正常、无病理变化。疫苗质粒分布及整合安全性检测结果显示,pVAX1-F免疫接种后24h,在血液和所有检测的组织中检测到质粒,免疫后90d可在心、脾、肺等组织检测到质粒;纯化后的组织基因组DNA经PCR扩增均呈阴性,未发现整合现象。本研究研制了含鸡属特异性CpG基序的IBV结构蛋白多表位嵌合基因的DNA疫苗,研究表明该多表位嵌合DNA疫苗能够诱导良好的细胞免疫和体液免疫,免疫鸡攻毒保护率为80%,高于常规IBV灭活疫苗(75%);与禽IL-2分子佐剂共同免疫鸡群的攻毒保护率达到90%;该多表位嵌合基因DNA疫苗安全性好,未检测到与宿主基因组整合的现象。
本论文开展的禽传染性支气管炎病毒多表位嵌合DNA疫苗构建及免疫研究,在国内外未见报道,该研究为研制安全、高效的新型IBV DNA疫苗提供了新的方法。
Infectious bronchitis virus(IBV) is one of the primary causes of respiratory disease in domestic fowl.Multitude serological types of IBV are occurred due to variation,and there is no cross-protection between different serological types.In addition,conventional vaccines against infectious bronchitis applied in the field have respective weakness. Therefore,it is of significance to develop a safety and effective genetically engineering vaccine such as DNA vaccine to control the disease.Based on former achievement of our research group,we analyzed and predicted the structural protein of nephritic IBV SAIBk strain,screened several epitopes from S1,S2 and N protein of IBV,and constructed a chimeric multi-epitope gene by using of the screened epitopes genes.The constructed chimeric gene was used to perform prokaryotic expression and a corresponding ELISA assay was established by using the expressing recombination protein.Moreover,the constructed chimeric multi-epitope gene was subcloned into eukaryotic expression vectors and then prepared DNA vaccines,and the immunoreactivity of the chimeric multi-epitope DNA vaccines were evaluated,as well as coadministration with avian interleukins.The aim of the study was to provide a new candidate target for IBV vaccine development.
1.The dominant epitopes of S1,S2,and N protein of IBV SAIBk strain were analyzed on base of computer bioinformatics softwares and reported references,and then seven T-and B-cell epitopes from S1,S2 and N protein(S1:24-150,240-255,290-400,532-537;S2: 1-65;N:1-120,290-410) were selected.The seven multi-epitope minigenes were paiallelled as a single chimeric gene separated from each other with the GA/GP spacers with an unique open reading frame by using Splicing by Overlap Extension(SOEing) and polymerase chain reaction(PCR),and the constructs included a Kozak sequence at the N-terminus and avian specific CpG motif in the end of the chimeric gene.The analysis for the chimeric gene indicated that the coded chimeric protein showed favourable hydrophilicity,flexibility and antigenicity.The study provided foundations for the prokaryotic expression,ELISA method,eukaryotic expression,and research for DNA vaccine of the multi-epitope chimeric gene.
2.The constructed chimeric multi-epitope gene of IBV was digested by BamHI and XhoI to generate chimeric gene F,which was subcloned into the prokaryotic expression vector pET-32a(+).The recombinant plasmid was transformed into E.coli Rosetta.The recombinant bacterium was induced by IPTG.The chimeric F protein was expressed fusedly in the form of cytorrhyctes.Western-bloting results showed the expressed recombinant protein occurred specific reaction with positive serum of IBV.The expression conditions of chimeric F protein were optimized with proper inducing conditions of 1 mmol/L IPTG for 3 hours at 30℃temperature.Using the puridied chimeric recombinant protein as coating antigen,the optimal concentration of F protein for coating of plate was 20μg/mL;the dilution of antibody was 1:40;the working concentration of HRP rabbit-anti-chicken IgG was 1:3000;the threshold value of ELISA assay was 0.133. According to the determination of condition of enzyme-linked immunosorbent assay (ELISA),an indirect ELISA based on the chimeric F protein was developed.The study provided new approachs for the development of subunit vaccine and detection of ELISA antibodies for IBV.
3.The constructed chimeric multi-epitope gene F of IBV was subcloned into eukaryotic expression vectors pcDNA3.1,pVAX1 and VR1020,respectively.In addition,the avian interleukin-1β,interleukin-2 and interleukin-18 gene were inserted into the plasmid pcDNA3.1,respectively.Identification of the constructed eukaryotic expression plasmids were performed by restriction enzyme digestion and DNA sequencing.The positive recombinant plasmids were transfected into COS-7 cells by lipofectamine,and the expression of heterologous genes were detected by RT-PCR and indirect immunofluorescence assay(IFA).The results of restriction enzyme digestion and DNA sequencing showed that the eukaryotic expression plasmids were constructed successfully, The corresponding mRNA transcripts were detected by RT-PCR in the COS-7 ceils which were transfected with plasmids pcDNA-F,pVAX1-F,VR1020-F,pcDNA-IL-1β, pcDNA-IL-2 and pcDNA-IL-18,respectively.The expression of chimeric multi-epitope recombinant protein were detected by indirect immunofluorescent assay in the COS-7 cells which were transfected with plasmids pcDNA-F,pVAX1-F and VR1020-F,respectively. The study provided bases for the further development of chimeric multi-epitope DNA vaccine against IBV.
4.The eukaryotic recombinant plasmids pcDNA-F,pVAX1-F,VR1020-F,pcDNA-IL-1β, pcDNA-IL-2 and pcDNA-IL-18 used for vaccination were extracted from DH5a,and purified by a Chinese invention patent method which was established previously in our laboratory.The diluted recombinant plasmids(1mg/mL) were encapsulated by liposome(10mg/mL) in equal volume,and administered to the 7-day-old chickens by intramuscularly injection.After two weeks,the chickens were boosted by equivalent dosage of DNA vaccine.The percentage of CD4+ and CD8+ T-lymphocytes from peripheral blood and anti-IBV specific antibodies were measured after the booster, respectively.Five weeks after booster,chickens were challenged by virulent IBV strain.In addition,the distribution in different times and possibility of integration into host genomic DNA of plasmid pVAX1-F were detected.The results of percentage of CD4+ and CD8+ T-lymphocytes showed that,the number of CD4+ and CD8+ T-lymphocytes of chickens immunized with pVAX1-F,pcDNA-F and VR1020-F were extremely significantly higher (P＜0.01) than that of PBS and control empty plasmids groups 7-21d post the booster, respectively,but showed no distinguished differences between interclass of themselves, and the number of CD4+ and CD8+ T-lymphocytes of chickens immunized with pcDNA-F+pcDNA-IL-1β,pcDNA-F+pcDNA-IL-2,pcDNA-F+pcDNA-IL-18 were significantly higher(P＜0.05) than that of the single plasmid pcDNA-F inoculated group 7-28d post the booster,respevtively.The results of specific antibodies detection showed that,the antibody titers of chickens immunized with pVAX1-F,pcDNA-F and VR1020-F were extremely significantly higher(P＜0.01) than that of PBS and control empty plasmids groups 7-28d post the booster,respevtively,but showed no noticeable difference between interclass of themselves,and the antibody titers of chickens co-administrated with pcDNA-F and pcDNA-IL-1β,pcDNA-IL-2,pcDNA-IL-18 were significantly higher (P＜0.05) than that of the pcDNA-F alone group 14-28d post the booster,respevtively.The protection rate of pVAX1-F,pcDNA-F and VR1020-F group were 80%,80%and 75%. The protection rates of groups inoculated with pcDNA-F+pcDNA-IL-1β, pcDNA-F+pcDNA-IL-2 and pcDNA-F+pcDNA-IL-18 were 85%,90%and 85%, respectively,which indicated that IL-2 showed better immune enhancement contribution than IL-1βand IL-18.The tissue section indicated that the kidneys of chickens protected by DNA vaccines were normal and physical fitness,without pathological changes.The distribution and integration detection of plasmid pVAX1-F showed that the plasmid was detected in blood and all the other checked tissues 24h post inoculation and last 90d in some tissues at least,and there was no detective phenomenon of integration during the period of experiment.The study provided a new way to enhance the immunogenicity of IBV DNA vaccine and reduce costs.
引文
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