凡纳滨对虾桃拉综合征病毒主要结构蛋白基因的克隆及原核表达

Cloning and expression of main structure protein genes of Taura Syndrome Virus in Litopenaeus vannamei Shrimp

根据GenBank中桃拉病毒的全基因组序列,分别设计扩增该病毒主要结构蛋白基因vp1、vp2、vp3的相应引物,以发病的凡纳滨对虾中提取的总RNA为模板,利用RT-PCR技术对目的基因进行了扩增,分别获得分子量为1 164、849、507bp的基因片段,与预期一致.再将目的基因与pET-28a载体连接,分别构建重组表达载体pET-VP1、pET-VP2和pET-VP3,重组子经酶切和测序鉴定后导入大肠杆菌BL21(DE3),IPTG诱导表达后进行SDS-PAGE检测.SDS-PAGE电泳的结果表明,在37℃下,0.5mmol/dm3的IPTG诱导表达的重组蛋白均以包涵体的形式存在,重组蛋白VP1、VP2、VP3的大小分别为48、36、24KDa.本研究的结果为制备桃拉病毒主要结构蛋白的特异性抗体,进而研制桃拉病毒快速检测试剂盒奠定了基础.

According to the complete genome sequences of Taura Syndrome Virus （TSV） published on GenBank, three pairs of primers were designed to amplify the main structure protein genes （vp1 vp2,vp3） of TSV respectively. These amplified products were 1 164bp,849bp and 507bp. They were constructed to the expression vector pET-28a and named pET-VP1, pET-VP2 ,pET-VP3. The recombinant plasmids were transformed into E. cloi BL21 （ DE3 ） after the identification of the nuclear restriction enzymes cutting and sequencing. After IPTG induction and SDS-PAGE analysis, the fusion proteins of about 48KDa,36KDa and 24KDa in molecule weight were detected. TSV is a major cause of mortality and morbidity in shrimp, and has a profound economic impact on commercial shrimp farming all over the world. The results should be a basis for obtaining all antibodies against TSV and the rapid detection of TSV in the future.