菜蛾盘绒茧蜂多分DNA病毒EP-1-like基因克隆、原核表达与多克隆抗体制备方法

Cloning,prokaryotic expression and polyclonal antibody preparation of Cotesia vestalis polydnavirus (CvBV) EP-1-like gene

本实验提取被菜蛾盘绒茧蜂寄生后24h的小菜蛾幼虫体内总RNA,反转录合成cDNA,以此为模板PCR扩增出菜蛾盘绒茧蜂多分DNA病毒(Cotesia vestalis polydnavirus,CvBV)EP-1-like基因,将其分别克隆到表达载体pET30a、pET28a中,转化宿主菌BL21(DE3),获得单克隆重组质粒pET-30a-EP1L和pET-28a-EP1L。经IPTG诱导,pET-28a-EP1L成功表达了约34.8kDa的包涵体蛋白。将诱导条件优化以后,采用割胶回收的方法纯化包涵体,将纯化后的表达蛋白免疫新西兰大耳兔制备多克隆抗体。ELISA分析表明制备的抗体效价达1:128000,Western blot检测证明抗体具有良好的免疫反应特异性。

The ORF of the CvBV EP-1-like gene was amplified by PCR with cDNA of Plutella xylostella larvae extracted 24 h after parasitism by Cotesia vestalis as template and sequenced. The gene was cloned into pET30a and pET28a prokaryotic expressive vectors, and the constructed recombinant plasmids pET- 30a-EP1L and pET-28a-EP1L were transformed into the host bacteria E. coli BL21 （DE3）. The expressive product induced by IPTG was identified by SDS-PAGE and Western-blot analysis. The results showed that the recombinant vector pET-28a-EP1L produced a 34.8 kDa inclusion body protein, which was purified and then injected into New Zealand rabbit to induce immune serum （polyclonal antibody）. ELISA analysis showed the titer for the polyclonal antibody was 1 ： 128000. Western blot analysis showed the antibody could react specifically with the EP-1-like protein.