传染性法氏囊病病毒非结构蛋白VP5的原核表达及多克隆抗体的制备

利用PCR技术,从传染性法氏囊病病毒（IBDV）Gx，Gt毒株中分别扩增出VP5基因,将其克隆到表达载体pET30a、pET28a中。经PCR、酶切和序列分析鉴定获得重组质粒命名为pET28a-GtVP5、pET30a-GxVP5。将pET30a-GxVP5、pET28a-GtVP5分别转化宿主菌BL21(DE3)，在IPTG诱导下均成功表达约24 kDa的Gx-VP5及23kDa的Gt-VP5融合蛋白，并都以包涵体形式存在。将Gx-VP5纯化后的蛋白免疫8周龄BALB/c雌鼠，ELISA分析表明制备的抗血清效价在1:25600以上，Western blot分析VP5表达产物能与抗6×His mAb及抗IBDV多克隆抗血清发生反应，具有良好的免疫反应特异性。

Prokaryotic expression and preparation of polyclonal antibody for IBDV non- structure protein VP5 gene

With a pair of primers designed according to the sequence of Infectious bursal disease virus(IBDV), the VP5 gene of IBDV was amplified from vvIBDV-Gx, IBDV-Gt, respectively. Then IBDV VP5 was cloned into expressing vector pET30a and pET28a. The recombinant plasmid was identified by restrictive digestion, PCR and sequence analysis, then named pET30a-GxVP5, pET28a-GtVP5, respectively. The recombinant plasmid was transformed into E.coli BL21(DE3) and induced by IPTG. SDS-PAGE results indicated that Gx-VP5, Gt-VP5 were about 24kDa, 23kDa, respectively. These recombinant fusion proteins mainly existed as inclusion bodies. High titer anti-VP5 serum was also prepared in BALB/c mice immunized with purified Gx-VP5 fusion protein inclusion. ELISA results showed that titer was above 1:25600 and Western blot analized that the expressed protein Gx-VP5 reacted with polyclonal antibody and 6×His monoclonal antibody. It explained that the expressed protein Gx-VP5 possess specific satisfactory immunological reaction.