猪轮状病毒OSU强毒株vp7基因的克隆及其抗原表位原核表达和免疫学活性分析

Cloning and expression of the vp7 gene antigenic region of porcine rotavirus OSU virulent strain

参照GenBank中所收录的猪轮状病毒OSU株序列设计了一对特异性引物,RT-PCR扩增出1062bp的vp7全长基因,与参考OSU序列的核苷酸同源性达99.8%,克隆到pMD18-T载体中,获得pMD18-T-vp7阳性质粒。以阳性质粒为模板,利用引入BamHⅠ和XhoⅠ酶切位点的引物进行PCR扩增得到含有VP7中和抗原表位区域639bp的基因片段,定向克隆到表达载体pGEX-6P-1中,经酶切PCR鉴定的阳性质粒转化大肠杆菌BL21(DE3),经IPTG(终浓度为1.0mmol/mL)37℃诱导4h后,SDS-PAGE电泳显示表达了大小约50ku带GST标签的融合蛋白,薄层扫描显示蛋白表达融合量占菌体总蛋白的33.2%。Western blot分析表明该VP7重组蛋白可与PRV阳性血清特异性反应。

The vp7 gene of porcine rotavirus （PRV） OSU strain was amplified by RT-PCR using a pair of specific primers designed according to the published sequence in GenBank, The PCR products were cloned to pMD18-T vector and sequenced, The vp7 of OSU strain consisted of 1 062 bp and shared 99,8 % sequence homology with PRV vp7 gene from NCBI. A 639 bp fragment containing the neutron-antigenic epitope was amplified from the vp7 gene and subcloned into expression vector pGEX-6P-1. The construct was transformed E.coli BL21 （DE3） and induced by 1PTG. The expressed GST-fusion protein had a molecular weight of 50 ku that accounted for up to 33.2 % of total protein, Western blot showed that the recombinant VP7 protein reacted with polyclonal antibodies against PRV