人博卡病毒VP2蛋白的原核表达及血清学检测方法的建立

摘要

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、HBoV2 VP2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、HBoV2 VP2基因正确连接至pET30a载体,开放阅读框正确,用1 mmol/L IPTG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NiNTA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HBoV1、HBoV2 VP2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.%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 antigenicity 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℃ induced by IPTG (lmmol/L) over night and purified by Ni-NTA column.Seropositive rates of HBoV1and 2 were 62.2％ and 55.5％ and their mixed seropositivity was 37％.Conclusion The optimizing expression of the capsid protein VP2 from human Bocavirus constructed successfully and get a high yield under certain conditions.The established ELISA could be used to further analyze seroepidemiology of HBoV in china.