空肠弯曲菌共同抗原重组质粒的基因测序及表达产物的特性分析

Gene Sequencing of Campylobacter jejuniCommon Antigen Recombinant Plasmid and Property Analysis of Its Expression Products

将自行构建的表达空弯菌共同抗原的重组质粒用EcoR、I、HindⅢ进行双酶切 ,将获得的外源插入片段克隆到pBluescriptⅡKS(+)质粒 ,并对其插入外源基因片段进行了核苷酸序列分析 ,结果表明该插入子的全长为 1.36 2kb ,含有 34 0个氨基酸的开放阅读框架 ,其A +T含量为 6 7.75 %。将表达重组子的菌体裂解产物进行SDS_PAGE ,并用免疫印迹对表达的抗原用 4A7单抗体探针进行反应 ,结果与 39kD的表达特异蛋白发生特异性免疫反应。用表达菌体的裂解产物免疫小鼠 ,于免疫后出现了对空弯菌CA的特异性免疫应答 ,表明重组表达的CA具有较强的免疫原性。该表达产物可作为空弯菌感染诊断和构建基因工程菌苗的候选抗原。

The recombinant plasmids expressing C.jejuni CA was digested with EcoR I and Hind Ⅲ,the restriction fragments containing the specific gene were cloned into pBluescriptⅡKS(+)for DNA sequencing and analysing.Determination of the nucleotide sequence of the DNA insert of this recombinant plasmid revealed that the full length of the insert is 1.362kb and contains an open reading frame encoding 340 amino acids,and its A+T content is 67.75%.The lysatic bacterial cells were separated by SDS_PAGE,the 4A7 monoclonal antibody probe against C.jejuniCA specifically recognized the 39.0kD protein of recombinant expression bacteria on western blots.The lysate derived from expressing 39.0kD proteins E.coli was immunized into mice,the results showed that the specific immune response against CA 1 were raised in mice inoculated by the recombinant E.coli, and the expressed 39.0kD proteins have highly immunogenic.It is therefore a good candidate as an antigen for serological diagnosis and the genetically engineering vaccine of C.jejuni infections.