乙型脑炎病毒非结构蛋白NS1基因的重组表达及其与黄病毒属病毒抗原抗体反应研究

目的阐明重组表达乙脑病毒非结构蛋白1(Non-structural protein 1,NS1)与包括乙脑病毒在内的多种蚊传黄病毒的抗原抗体反应,以及对乙脑病毒感染患者急性期血清标本的抗原抗体反应。方法本研究使用大肠杆菌原核表达载体(prokaryotic expression vector,pET)系统,重组表达乙脑病毒NS1基因。使用Western Blot实验方法检测重组表达蛋白质与多种,包括乙脑病毒在内的蚊传黄病毒抗体的反应,以及乙脑病毒感染患者急性期血清的抗原抗体反应。结果乙脑病毒(P3株)NS1基因表达产物以包涵体形式存在,经过变性和复性的表达产物在聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,PAGE,简称变性胶)显示为单一条带,分子量约为45000。进一步的抗原抗体分析结果表明,表达产物与乙脑病毒(蚊虫分离株,脑炎患者分离株)多克隆或者单克隆抗体以及乙脑病毒感染病人急性期血清标本可以获得完全一致的抗原/抗体杂交反应。重组表达产物与蚊传黄病毒,如登革病毒和黄热病毒多克隆抗体之间抗原/抗体杂交反应为阴性,但与西尼罗病毒和墨累谷脑炎病毒多克隆抗体呈现抗原/抗体阳性杂交反应。结论本文成功获得乙脑病毒NS1蛋白的重组表达并分析了重组蛋白对多种蚊传黄病毒及乙脑病毒感染患者标本之间的抗原/抗体反应,研究结果对于阐明乙脑病毒NS1蛋白与蚊传黄病毒之间抗原抗体反应提供了重要的基础数据。本研究所获得重组表达蛋白为进一步研究乙脑病毒NS1蛋白质功能等提供了重要的物质基础。

Recombinant expression of Japanese encephalitis virus non-structural protein NS1 gene and its reaction with Flavivirus antigen and antibody

Objective To elucidate the antigenic antibody reaction of recombinant expression of non-structural protein 1 (NS1) of Japanese encephalitis (JE) virus with various mosquito-borne flaviviruses, including JE virus, and the antigenic antibody reaction of serum samples of patients infected with JE virus in acute stage. Methods In this study, Escherichia coli prokaryotic expression vector (pET) system was used to recombinant express Japanese encephalitis virus NS1 gene. Western Blot assay was performed to detect the antibody responses of the recombinantly expressed protein against a variety of mosquito-transmitted flaviviruses, including JE virus, as well as antigen-antibody reactions of serum from patients with acute JE virus infection. Results The NS1 gene expression product of JE virus (P3 strain) was in the form of an inclusion body, and the denatured and renatured expression product was displayed as a single band in the denatured gel (polyacrylamide gel electrophoresis, PAGE), with a molecular weight of about 45 000. The results of further antigen-antibody analysis showed that the antigen/antibody hybridization reaction of the expression product with polyclonal or monoclonal antibody of JE virus (mosquito isolates, encephalitis isolates) and serum samples of patients with acute JE virus infection could be completely consistent. The recombinant product showed negative antigen/antibody hybridization reactions with mosquito-transmitted flaviviruses, such as dengue virus and yellow fever virus polyclonal antibodies, but positive reactions with polyclonal antibodies to West Nile virus and Murray Valley encephalitis virus. Conclusions In this study, the recombinant expression of the NS1 protein of JE virus was successfully obtained, and the antigen/antibody reaction between the recombinant protein and samples of patients infected with mosquito-borne flavivirus and JE virus was analyzed. The study results provide important basic data for elucidating the antigen-antibody reaction between the NS1 protein of JE virus and mosquito-borne flavivirus. The recombinant expression protein obtained in this study provides an important material basis for further research on the function of JE virus NS1 protein. © 2023 China National Research Institute of Food and Fermentation Industries Co. Ltd. All rights reserved.