猪瘟病毒E2基因自杀性DNA疫苗的构建及动物免疫试验  

Construction of suicidal DNA vaccine of CSFV E2 gene and immunity induction in animals

作　　者：郭抗抗[1] 温肖会[1] 汤智慧[1] 侯勃[1] 张彦明[1] 谢林红[1] 王晶钰[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《中国兽医学报》2011年第5期625-629,共5页Chinese Journal of Veterinary Science

基　　金：国家高技术研究发展计划("863"计划)资助项目(2006AA10A204)

摘　　要：从含有猪瘟病毒(CSFV)Shimen株E2全长基因的重组质粒pMD18-T-E2上扩增得到带有BamHⅠ酶切位点的E2基因片段,构建重组克隆质粒pMD19-T-E2。用BamHⅠ酶切pMD19-T-E2和"自杀性"DNA疫苗表达载体pSCA1,构建"自杀性"DNA疫苗重组质粒pSCA1-E2。对目的基因E2的大小、插入位置、方向和读码框经PCR、酶切和序列测定进行鉴定。用纯化的阳性质粒pSCA1-E2转染PK-15细胞,转染后48 h荧光抗体法检测E2蛋白的表达;纯化的pSCA1-E2质粒分别免疫小鼠(10μg/只)和试验猪(600μg/头),二免后分别检测小鼠脾淋巴细胞刺激指数和猪血清抗猪瘟特异性抗体水平。结果表明,构建的重组质粒中E2基因的大小、插入位置、方向和读码框均正确,获得了重组表达质粒pSCA1-E2;转染后48 h可检测到转染的PK-15细胞中E2蛋白的表达。免疫小鼠产生较高水平的淋巴细胞刺激指数,免疫猪可检测到抗CSFV特异性血清抗体;表明构建的pSCA1-E2重组质粒能激发实验动物的免疫反应。The complete E2 gene of classical swine fever virus（CSFV） with the BamHⅠ restriction site was cloned from the recombinant plasmid pMD18-T-E2 and inserted into pMD19-T plasmid to construct the recombinant plasmid pMD19-T-E2.E2 gene was obtained from pMD19-T-E2 digested with BamHⅠ,and was inserted into the suicidal DNA vaccine vector pSCA1 which was treated with the same enzyme.The recombinant plasmid pSCA1-E2 was obtained and identified the length,insert sites,orientation and reading frame of E2 gene in pSCA1-E2 by polymerase chain reaction（PCR）,enzyme digesting and sequencing.E2 protein was determined in PK15 cells transfected with purified recombinant plasmid pSCA1-E2 at 48 h post-transfection.The stimulation index（SI） of T lymphoid cell in experimental mice immunized with pSCA1-E2（10 μg/each） were analyzed at 10 d,20 d,30 d after the second vaccination.Experimental pigs were immunized with pSCA1-E2（600 μg/each） and the anti-CSFV serum antibody was screened at 7 d,14 d,21 d and 28 d after the second vaccination.The results showed that the insert site,orientation and reading frame of E2 gene were correct in pSCA1-E2.The E2 protein of CSFV was detected in transfected PK-15 cell.A high SI of T lymphoid cell was detected in vaccinated mice and the anti-CSFV serum antibody was screened in immunized pigs.The experiments demonstrated that the pSCA1-E2 could trigger immune responses in experimental animals.

关 键 词：猪瘟病毒 E2基因 “自杀性”DNA疫苗 动物免疫试验 

分 类 号：S862.65[农业科学—野生动物驯养]