人博卡病毒VP2蛋白的原核表达及血清学检测方法的建立

目的获得高表达量的人博卡病毒（HBoV1、HBoV2）VP2蛋白,建立血清学检测方法。方法按照大肠埃希菌密码子使用偏性,对HBoV1、HBoV2VP2衣壳蛋白基因编码区序列进行优化合成。将合成的目的基因克隆至表达载体pET30a,构建重组表达质粒pET30a-VP2,转化大肠埃希菌BL21（DE3）,使用IPTG诱导表达并摸索最佳表达条件;粗提取蛋白进行Ni亲和层析纯化后建立间接酶联免疫吸附试验（ELISA）,进行临床血清标本筛查,分析人群HBoV1、HBoV2感染情况。结果优化合成的HBoV1、HBoV2VP2基因正确连接至pET30a载体,开放阅读框正确,用1mmol／LIPTG过夜诱导表达（25℃）得到高表达量的VP2蛋白,目的蛋白主要以包涵体的形式存在。粗提取蛋白经Ni—NTA亲和层析,在pH4．5时获得较理想的纯化蛋白,以其为抗原建立ELISA检查方法,筛查临床血清标本IgG抗体水平,结果显示健康儿童HBoV1阳性率为62．2％,HBoV2阳性率为55．5％,混合感染率为37％。结论本研究成功将H-BoV1、HBoV2VP2衣壳蛋白基因编码区序列进行优化,得到了较高的蛋白表达量,所建立的ELISA法可以应用于HBoV1、HBoV2人群血清流行病学调查。

Optimizing expression of the capsid protein VP2 from human Bocavirus and establish it's seroepidemiology assying methord

Objective To obtain sufficient recombinant VP2 protein of human Bocavirus and establish it＇s seroepidemiology assying methord. Methord The capsid protein VP2 DNA genes of HBoV1 and 2 were optimized in accordance with the usage of the favorite codons in E. coil so as to enhance its protein expression in prokaryotic expressing system. The protein was purified by Ni-NTA column, and its antigenieity was determined by Western Blot. Then establish ELISA to detect the specific anti-VP2 IgG antibodies against HBoV1 and 2 in healthy children aged 3 - 6 years in Nanjing, China. Results The recombinant protein 6 ~ His-VP2 was produced in a larger quantity at 25~C induced by IPTG （lmmol/L） over night and purified by Ni-NTA column. Seropositive rates of HBoVland 2 were 62.2% and 55.5% and their mixed seropositivity was 37%. Conclusion The optimizing expression of the capsid protein VP2 from human Boeavirus constructed successfully and get a high yield under certain conditions. The established ELISA could be used to further analyze seroepidemiology of HBoV in china.