猪圆环病毒pSCA-Cap-T2A-Rep重组载体的构建及小鼠免疫试验
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摘要
克隆猪圆环病毒1型(PCV1)的Rep基因和猪圆环病毒2型(PCV2)Cap基因,利用T2A片段进行连接,并插入到载体pSCA,构建pSCA-Cap-T2A-Rep重组质粒。转染PK-15细胞后通过Western blot技术检测目的蛋白的表达。大量提取构建的重组质粒后进行小鼠接种试验。56只小鼠随机分为8组,每组7只。第1,2,3组为质粒注射组,注射质粒质量分别为50,100,200μg/只;第4～6组分别为与1～3组注射相同剂量质粒+免疫佐剂对应组;第7组为注射生理盐水对照组;第8组为注射生理盐水+佐剂对照组。首次接种后14 d进行第2次接种,处理方法同第1次。在首次接种后0,14,21,28 d从试验小鼠眼静脉丛采血,ELISA方法检测血清中PCV2 Cap蛋白抗体。结果显示,pSCA-Cap-T2A-Rep重组质粒接种小鼠血清中Cap蛋白特异性抗体在接种后14～28 d均为阳性;质粒+免疫佐剂组比质粒注射组小鼠特异性抗体水平显著升高。结果表明,pSCA-Cap-T2A-Rep重组质粒在试验小鼠中产生有效的特异性抗体,为进一步优化免疫方法、在试验猪上的应用及猪圆环病毒核酸疫苗的研制提供了依据。
In this study,a recombinant vector of porcine circovirus was constructed,which provided basic materials for the development of novel porcine circovirus nucleic acid vaccine vector.The pig porcine circovirus type 1(PCV1) Rep gene and the pathogenic porcine circovirus type 2(PCV2) Cap gene that are not pathogenic to pigs were cloned and then ligated by the T2 A fragment and then inserted into suicide vector pSCA to constructe A pSCA-Cap-T2 A-Rep recombination vector.After transfection of PK-15 cells,the expression of the target protein was detected by Western blot.After a large number of recombinant plasmids were constructed,a mouse inoculation test was conducted.56 mice were randomly divided into 8 groups of 7 each.The 1 st,2 nd and 3 rd groups were plasmid injection groups.Group 1-3 of test mice were injected with plasmid concentration of 50,100,and 200 μg/group;Group 4-6 were the same plasmids concentration as Group 1-3 with adjuvant as counterparts;The 7~(th) group was injected with saline as control group;The 8 th group was injected with saline+adjuvant as control group.The second inoculation was performed at 14 days after the first vaccination,and the treatment method was the same as the first.At the 0,14,21 and 28 dafter the first vaccination,blood was collected from the eye venous plexus of the test mouse.The serum PCV2 Cap protein antibody was detected by ELISA.The results indicated that the specific antibodies of Cap protein in serum of mice inoculated with pSCA-Cap-T2 A-Rep recombinant plasmid were positive from 14 to 28 days after inoculation;the specific antibody level of mice in plasmid+immunized adjuvant group was significantly higher than that in plasmid injection group.The results indicated that pSCA-Cap-T2 A-Rep recombinant plasmid can induce in experimental mice producing effective antibodies.It provided basic data for the optimization of further immunization methods and application in experimental pigs,and also provided basic materials for the development of porcine circovirus nucleic acid vaccine.
In this study,a recombinant vector of porcine circovirus was constructed,which provided basic materials for the development of novel porcine circovirus nucleic acid vaccine vector.The pig porcine circovirus type 1(PCV1) Rep gene and the pathogenic porcine circovirus type 2(PCV2) Cap gene that are not pathogenic to pigs were cloned and then ligated by the T2 A fragment and then inserted into suicide vector pSCA to constructe A pSCA-Cap-T2 A-Rep recombination vector.After transfection of PK-15 cells,the expression of the target protein was detected by Western blot.After a large number of recombinant plasmids were constructed,a mouse inoculation test was conducted.56 mice were randomly divided into 8 groups of 7 each.The 1 st,2 nd and 3 rd groups were plasmid injection groups.Group 1-3 of test mice were injected with plasmid concentration of 50,100,and 200 μg/group;Group 4-6 were the same plasmids concentration as Group 1-3 with adjuvant as counterparts;The 7~(th) group was injected with saline as control group;The 8 th group was injected with saline+adjuvant as control group.The second inoculation was performed at 14 days after the first vaccination,and the treatment method was the same as the first.At the 0,14,21 and 28 dafter the first vaccination,blood was collected from the eye venous plexus of the test mouse.The serum PCV2 Cap protein antibody was detected by ELISA.The results indicated that the specific antibodies of Cap protein in serum of mice inoculated with pSCA-Cap-T2 A-Rep recombinant plasmid were positive from 14 to 28 days after inoculation;the specific antibody level of mice in plasmid+immunized adjuvant group was significantly higher than that in plasmid injection group.The results indicated that pSCA-Cap-T2 A-Rep recombinant plasmid can induce in experimental mice producing effective antibodies.It provided basic data for the optimization of further immunization methods and application in experimental pigs,and also provided basic materials for the development of porcine circovirus nucleic acid vaccine.
引文
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[10] ARAVINDARAM K,KUO T Y,LAN C W,et al.Protective immunity against porcine circovirus 2 in mice induced by a gene-based combination vaccination[J].J Gene Med,2009,11(4):288-301.
[11] SYLLA S,CONG Y L,SUN Y X,et al.Protective immunity conferred by porcine circovirus 2 ORF2-based DNA vaccine in mice[J].Microbiol Immunol,2014,58(7):307-316.
[12] 代振江,王伟丞,曾智勇,等.猪圆环病毒基因组结构与功能研究进展[J].畜牧与兽医,2016,48(3):151-154.
[13] 刘金凤,吴健敏,覃绍敏,等.VP22短肽融合猪圆环病毒2型重组病毒样颗粒的构建及其鉴定[J].中国兽医学报,2017,37(2):218-223.
[14] 赵燕,喻正军.猪圆环病毒2型基因组DNA的结构与功能研究进展[J].中国畜牧兽医,2016,43(5):1176-1181.
[2] 张玲楷,李永锋,谢利豹,等.表达猪圆环病毒2型Cap蛋白的重组猪瘟兔化弱毒疫苗株的构建与鉴定[J].生物工程学报,2018,34(2):216-223.
[3] 杨利勇,李春凤.猪圆环病毒2型在我国的流行及疫苗研究现状[J].畜牧与兽医,2015,47(4):132-134.
[4] 崔美秋,高进涛.猪圆环病毒疫苗应用技术研究[J].山东畜牧兽医,2016,37(7):6-7.
[5] 余建国,周立.姜黄素对猪圆环病毒灭活苗细胞免疫效果的影响[J].中国动物传染病学报,2017,25(5):60-63.
[6] 刘长明,陆月华,张超范,等.猪圆环病毒2型重组Cap蛋白在昆虫杆状病毒中的表达[J].中国预防兽医学报,2005,27(6):41-44.
[7] 李玲,李国新,徐彦召,等.猪圆环病毒衣壳蛋白在昆虫细胞中表达形成病毒样颗粒的研究[J].中国动物传染病学报,2011,19(5):15-20.
[8] BEACH N M,MENG X J.Efficacy and future prospects of commercially available and experimental vaccines against porcine circovirus type 2 (PCV2)[J].Virus Res,2012,164(1/2):33.
[9] FENAUX M,OPRIESSNIG T,HALBUR P G,et al.A chimeric porcine circovirus (PCV) with the immunogenic capsid gene of the pathogenic PCV type 2 (PCV2) cloned into the genomic backbone of the nonpathogenic PCV1 induces protective immunity against PCV2 infection in pigs[J].J Virol,2004,78(12) :6297-6303.
[10] ARAVINDARAM K,KUO T Y,LAN C W,et al.Protective immunity against porcine circovirus 2 in mice induced by a gene-based combination vaccination[J].J Gene Med,2009,11(4):288-301.
[11] SYLLA S,CONG Y L,SUN Y X,et al.Protective immunity conferred by porcine circovirus 2 ORF2-based DNA vaccine in mice[J].Microbiol Immunol,2014,58(7):307-316.
[12] 代振江,王伟丞,曾智勇,等.猪圆环病毒基因组结构与功能研究进展[J].畜牧与兽医,2016,48(3):151-154.
[13] 刘金凤,吴健敏,覃绍敏,等.VP22短肽融合猪圆环病毒2型重组病毒样颗粒的构建及其鉴定[J].中国兽医学报,2017,37(2):218-223.
[14] 赵燕,喻正军.猪圆环病毒2型基因组DNA的结构与功能研究进展[J].中国畜牧兽医,2016,43(5):1176-1181.