IBDV血清Ⅱ型VP5蛋白原核表达及型特异性单克隆抗体的制备

Preparation of monoclonal antibody against VP5 protein of serotype Ⅱ infectious bursal disease virus expressed in E.coli

本实验构建表达了传染性法氏囊病病毒(IBDV)血清Ⅱ型23/82株VP5蛋白,并制备出能够区分不同血清型IBDV的单克隆抗体(mAb)。利用EcoRⅠ和XhoⅠ双酶切pUC57-23/82VP5质粒,获得23/82株IBDVVP5基因片段,连接到同样处理的原核表达质粒pET-28a,经酶切鉴定获得重组质粒pET-23/82VP5,转化到宿主菌BL21(DE3),在IPTG诱导下表达融合蛋白,SDS-PAGE分析表明该融合蛋白分子量大小约为23ku。Ni-NTA柱纯化重组蛋白,复性后免疫8周龄BALB/c小鼠,3次免疫后,常规方法进行细胞融合,获得1株阳性杂交瘤细胞株(5D3),IFA和westernblot分析表明该细胞株分泌的mAb仅与血清Ⅱ型IBDV23/82株VP5反应,不与血清Ⅰ型IBDVGt株VP5反应。腹水抗体间接ELISA效价为1×104。该mAb的制备为研究血清Ⅱ型IBDV的VP5的功能和标记疫苗的研制奠定了基础。

The vp5 gene of serotype Ⅱ infectious bursal disease virus 23/82 strain was sub-cloned into a prokaryotic expression vector pET-28a, and the recombinant plasmid was transformed into BL21 （DE3）. Protein expressed was induced by IPTG and confirmed by SDS-PAGE. The recombinant protein was purified by Ni-NTA, renatured, and inoculated to BALB/c mice. A monoclonal hybridoma cell line （5D3） was generated by standard protocols. Tests by IFA and western blot indicated that the mAb reacted only with 23/82 strain, but not Gt strain. The mAb has an acetic titre of 1 × 10^4 measured by indirect ELISA. The preparation of mAb against VP5 of 23/82 strain provided a foundation for studying pathogenesis of serotype Ⅱ IBDV and VP5 based vaccine development.