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

Prokaryotic Expression and Preparation of Polyclonal Antibody for IBDV Non-structure Protein VP5 Gene

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诱导下成功表达了约24kDa的Gx-VP5及23kDa的Gt-VP5融合蛋白,它们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,Westernblot分析VP5表达产物能与抗6×HismAb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。

With a pair of primers designed according to the sequence of infectious bursal disease virus （IBDV）, the VP5 gene of IBDV was amplified from vvlBDV-Gx, IBDV-Gt, respectively. Then IBDV VP5 was cloned into expressing vector pET30a and pET28a. The recombinant plasmicl 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 IPFG. 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 analysis showed 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.