鸡传染性法氏囊病病毒非结构蛋白基因的克隆、表达及多克隆抗体制备

利用RT-PCR技术从传染性法氏囊病病毒(IBDV)TL2004株感染鸡胚尿囊液中扩增到VP5基因,进而构建了T7启动子控制下的N端GST-Tag融合表达质粒pGEX-VP5。序列测定表明VP5基因全长438bp,编码一个由145个氨基酸组成的VP5蛋白。将pGEX-VP5转化大肠杆菌BL21,在IPTG的诱导下高效表达了GST-VP5融合蛋白(44kD)。通过包涵体纯化的方法,获得的较高纯度的融合蛋白,免疫新西兰兔,Western blot和ELISA分析表明,制备的融合蛋白抗血清效价在1∶12800以上,并具有良好的免疫反应特异性,为进一步研究VP5在IBDV复制与致病中的作用,以及研制IBDVVP5基因缺失疫苗打下了良好的基础。

Cloning, expression and preparation of polyclonal antibody for IBDV non-structure protein gene

Infectious bursal disease virus (IBDV) VP5 gene was amplified and cloned into an N-terminal GST-Tag fusion expression vector, pGEX-4T-2, which was controlled by T7 promoter. The sequencing result showed that the VP5 gene was composed of 438 base pairs, and coded 145 amino acids. High VP5 product was expressed in E.coli BL21 induced by IPTG, and the GST-VP5 fusion protein existed in inclusion. High titer anti-VP5 serum was also prepared in New Zealand rabbit immunized with purified fusion protein inclusion. These results gave a basis for further research for VP5 function in IBDV replication and pathogenicity, which also paved the way for developing VP5 gene deleted IBDV live vaccine.