重组猪细小病毒VP2基因的猪伪狂犬病毒rPRV-VP2株的构建及纯化  

The development of recombinant pseudorabies virus expressing porcine parvovirus VP2 gene

作　　者：付朋飞[1] 乔涵[1] 杨兴武[1] 张宇[1] 王林青[2] 郭晓庆[1] 潘鑫龙[1] 陈红英[1] 

机构地区：[1]河南农业大学郑州市猪重大疾病防控重点实验室,河南郑州450002 [2]郑州师范学院,河南郑州450044

出　　处：《中国兽医学报》2016年第9期1476-1483,共8页Chinese Journal of Veterinary Science

基　　金：河南省重大科技专项资助项目(111100110300)

摘　　要：参考GenBank中登录的猪细小病毒(PPV)(M38367.1)基因序列,设计1对可以扩增出约1.6kb PPV VP2基因片段的特异性引物,上、下游引物的5′端均引入BamHⅠ酶切位点,PCR扩增得到VP2目的片段后,连接到pMD18-T载体中得到重组质粒pMD-VP2。取本实验室构建的含绿色荧光蛋白标记基因的伪狂犬病毒(PRV)通用转移质粒pG和构建好的pMD-VP2质粒,用BamHⅠ对质粒pG和pMD-VP2进行酶切,然后用CIAP对酶切产物进行去磷酸化处理,纯化回收后将VP2基因插入pG质粒中获得重组伪狂犬病毒转移质粒pGVP2。用脂质体转染法将PRV HB-98株全基因组与质粒pGVP2共转染ST细胞,得到携带有PPV VP2外源抗原基因和绿色荧光蛋白标记基因的重组伪狂犬病毒rPRV-VP2株。结合VP2基因PCR扩增和绿色荧光观察,经5轮病毒空斑纯化得到了纯化的重组病毒rPRV-VP2株。According to PPV complete gene sequence avaliable in GenBank（GenBank accession NO. M38367.1）,a pair of specific primers was designed in order to amplify VP2 of PPV with a fragment of about 1.6 kb,and BamH I site was introduced on the both upper and lower stream of the primers. The VP2 gene was obtained by polymerase chain reaction（PCR） and subsequently cloned into pMD18-T vector,producing the recombinant plasmid pMD-VP2. The transfer plasmid pG cotaining EGFP gene and pMD-VP2 were digested with BamH I and then gel purified. After dephosphorylation with CIAP,VP2 gene was linked into the transfer plasmid pG,to generate the recombinant plasmid pGVP2. The genome of PRV HB98 attenuated vaccine and the plasmid pGVP2 were transfered into ST cells by transfection with lipofectamine, to generate the recombinant rPRV-VP2 strain that carry EGFP gene and foreign antigenic gene VP2. The recombinant virus rPRV PGVP2 was selected by five cycles of plaque purification and was further identified by fluorescence assay and PCR analysis.

关 键 词：PRV 猪细小病毒 VP2基因 重组 

分 类 号：S852.65[农业科学—基础兽医学]