阿龙山病毒VP2蛋白原核表达纯化以及抗原性鉴定

Prokaryotic Expression,Purification and Characterization of VP2 Protein of Alongshan Virus

阿龙山病毒(ALSV)是一种新发现的分节段黄病毒,可通过蜱虫叮咬感染家畜和人。为了获得高纯度ALSV-VP2蛋白,利用密码子优化软件对ALSV H3株VP2蛋白质氨基酸序列进行优化。将优化后的基因克隆到原核表达载体pET-30a中,构建重组质粒pET30a-VP2,重组质粒转化到E.coli BL21(DE3)表达菌株利用IPTG、16℃低温诱导,表达的蛋白再通过亲和层析(Ni-IDA树脂)得到纯度较高ALSV-VP2蛋白。SDS-PAGE电泳结果显示,在27 kDa处出现目的条带,与预期相符合,BCA检测蛋白浓度为0.236 mg/mL,证明ALSV-VP2蛋白在大肠杆菌中高效表达。经Westen blot分析,该重组蛋白具有良好特异性。本研究为ALSV的流行病学监测和致病机制研究奠定基础。

Alongshan virus(ALSV)is a newly discovered segmented flavivirus that can infect livestock and humans through tick bites.In order to obtain highly purified ALSV VP2 protein,the amino acid sequence of VP2 protein of ALSV H3 strain was optimized by codon optimization software.The optimized gene was cloned into prokaryotic expression vector pET-30a.The resulting recombinant plasmid pET30a-VP2 was then transformed into E.coli BL21(DE3).The VP2 protein was expressed at a low temperature(16℃)by induction of IPTG and purified by affinity chromatography(Ni-IDA resin).The protein concentration of the purified VP2 was 0.236 mg/mL,indicating high expression in E.coli.The purified VP2 appeared at the relative molecular weight of 27 kDa as visualized by SDS-PAGE,which was consistent with the expectation.Besides,the recombinant VP2 had good specificity by Western blot.This study laid a solid foundation for research of epidemiological surveillance and pathogenic mechanism of ALSV.