肺炎链球菌 TCSs 中 WalR 的克隆表达、保守性及抗原表位分析

摘要

This work aims to express WalR protein in Escherichia coli BL21 through constructing recombinant expression vector PET28a-WalR and to analyze the immunogenicity in the experimental animal C57 mice,as well as to test the conservation in different Streptococcus pneumonia serotypes and B cell antigenic epitopes. Using the WalR gene of S. pneumonia D39 as the template,the recombinant expression vector PET28a-WalR was constructed and then transferred into Escherichia coli BL21 for induced expressing target protein. The conservation of WalR protein in varied S. pneumonia serotypes was analyzed by ClustalX 2.1 software,and the B cell antigenic epitopes was analyzed by DNASTAR Lasergene v7.1. As result,recombinant expression vector PET28a-WalR was constructed successfully and much target protein was obtained by inducement of IPTG,however mainly in inclusion body. The conservation of WalR protein was up to 99.4% among different S. pneumonia serotypes,and the antigenic epitopes of WalR protein were likely located in 9-16,35-42,95-111,113-135,150-161, and 200-218 of amino acid sequence. In conclusion,recombinant S. pneumonia WalR protein in the form of inclusion body was successfully acquired,and the conservation and antigenic epitope were analyzed.%通过构建 PET28a-WalR 表达载体并在大肠杆菌 BL21中表达 WalR 重组蛋白，以评价其对 C57小鼠的免疫原性；并分析其在不同血清型肺炎链球菌中的保守性和 B 细胞的抗原表位。以肺炎链球菌 D39血清型 WalR 基因为模板，构建重组质粒PET28a-WalR 并转入 BL21诱导表达目的蛋白质。采用 ClustalX 2.1软件对 WalR 蛋白在不同血清型 S.pn 中的保守性进行分析；利用 DNASTAR 软件对其 B 细胞抗原表位进行分析。结果显示，成功构建 PET28a-WalR 重组表达载体，经 IPTG 诱导后有大量高纯度的目的蛋白质，但其主要以包涵体形式存在；WalR 蛋白在不同血清型肺炎链球菌中高达99.4%，其 B 细胞抗原表位可能位于9-16、35-42、95-111、113-135、150-161、200-218等氨基酸序列位点。成功获得大量以包涵体形式表达的肺炎链球菌 WalR 重组蛋白，并对其保守性和抗原表位进行了系统分析。