黄热病毒NS1蛋白的制备及免疫原性鉴定

目的 原核系统克隆表达黄热病毒(yellow fever virus, YFV)非结构蛋白1(nonstructural protein 1,NS1),鉴定其免疫原性。方法 以YFV-17D疫苗株为模板,常规分子生物学方法构建pQE30-YFV NS1表达载体,原核表达纯化YFVNS1重组蛋白;免疫印记、免疫荧光和酶联免疫吸附实验验证其免疫原性。结果 成功构建重组质粒pQE30-YFV NS1,制备纯化可溶性YFV NS1蛋白;该重组YFV NS1蛋白与登革病毒、黄热病毒、乙脑病毒、西尼罗河病毒免疫小鼠腹水及寨卡病毒NS1、YFV NS1蛋白免疫小鼠血清均发生反应,重组YFV NS1蛋白免疫小鼠血清与YFV感染细胞超声上清也发生反应。结论 获得高纯度YFV NS1重组蛋白,且具有较好的免疫原性,含天然NS1蛋白表位,为基于NS1黄热病毒早期抗原诊断方法和蛋白功能研究奠定基础。

Preparation and immunogenity identification of nonstructural protein 1 of yellow fever virus

Nonstructural protein 1(NS1) has been a biomarker for different Flavivirus diseases. A recombinant nonstructural protein 1 of yellow fever virus by using the Escherichia coli system was cloned and expressed, and its immunogenicity was evaluated. A new PQE30-YFVNS1 expression vector was constructed by routine molecular biological method using RNA from an YFV-17D vaccine strain as template. A recombinant YFVNS1 protein was expressed in E.coli M15 and purified. The immunogenity of this protein was identified by using Western blot (WB), immunofluorescence(IFA) and enzyme linked immunosorbent assay(ELISA). The sera from the recombinant YFV NS1-immunized mice showed strong reactivity with both recombinant NS1 proteins, YFV-infected C6/36 cell lysates and YFV-infected C6/36 cell. This recombinant protein also could react with ascites of mice immuned by dengue virus, yellow fever virus, Japanese encephalitis virus, West Nile virus and sera immuned by Zika virus NS1 protein, YFV NS1 protein. The high-purity recombinant protein NS1 which had good immunogenicity and contained some natural NS1 protein epitopes, was produced, which may contribute to the development of new YFV diagnostic assays based on NS1 and the functional properties of YFV NS1 protein.