IBDV H1株VP2基因的原核表达及其产物的复性

应用RT-PCR技术从传染性腔上囊病病鸡总RNA中克隆出1 461 bp的VP2基因,将其克隆于pET-28a载体,筛选并构建了pVP2表达载体。经IPTG诱导,在其宿主菌BL21(DE3) 中成功表达了53．7 ku的蛋白,SDS-PAGE和Western-blotting分析结果显示,VP2表达产物以包涵体形式存在,可与鸡IBDV抗血清及1株IBDV单抗发生特异性反应。将VP2表达产物进行纯化和复性,用复性前后的蛋白分别与特异性多抗进行Dot-ELISA检测,结果,复性后蛋白的反应活性比复性前增强了10倍;用复性后的蛋白与IBDV单抗进行Dot-ELISA,结果显示,VP2蛋白与单抗1H4、1E12、583、EA6、3C7反应强( );与4E4、1H11反应中度( );与4E5、3C4、 1E11、3H9反应较弱;而与EC6、3D12、1A1无反应。

Prokaryotic expression of VP2 gene of infectious bursal disease virus H1 strain from chickens and renaturation of the expressed protein

A VP2 gene of approximately 1 461 bp in length, which was obtained from the total RNA of chicken IBDV by RT-PCR, was cloned into the vector pET-28a to construct an expression vector pVP2. An expected protein of 53. 7 ku in size was expressed in E. coli BL21(DE3) induced by IPTG. SDS-PAGE and Western-blotting analyses showed that VP2 protein existed mainly in inclusion bodies and could react to anti-IBDV polyclonal and monoclonal antibodies. Then the protein was purified and refolded by lyso-zyme, Triton-100 and carbamide. Analysis using Dot-ELISA showed that activity of the renatured protein was increased by 10 times. The renatured protein was analysed with 16 monoclonal antibodies by Dot-ELISA. Result showed that the renatured protein could react strongly with 1H4,1E12,5B3 ,EA6 and 3C7, moderately with 4E4 and 1H11, and weakly with 4E5,3C4,1E11 and 3H9, but did not react with EC6, 3D12 and 1A1.