大肠杆菌耐热肠毒素基因多拷贝串联表达及其单克隆抗体的制备

Expression of tandem repeat gene of estp in E.coli and preparation of monoclonal antibodies against STp

为制备具有免疫原性的重组大肠杆菌耐热肠毒素蛋白(STp)及抗STp特异性单克隆抗体(MAb),本研究将5拷贝estp串联,克隆于p GEX-6p-1中,构建重组表达质粒p GEX-estp5-his,并转化于大肠杆菌中进行重组蛋白STp5-His(r STp5-His)的表达。以r STp5-His为免疫原,重组蛋白MBP-5STp为检测抗原,制备和筛选阳性杂交瘤细胞株,并采用western blot、阻断ELISA方法对MAb进行鉴定。结果显示,r STp5-His主要以包涵体形式表达,大小约为38 ku,具有良好的免疫原性,以其作为免疫原制备了4株能够识别天然STp的MAb,特异性良好。研究表明,多拷贝基因串联表达可以制备具有免疫原性的重组STp蛋白,所制备的MAb为天然STp和重组STp蛋白检测方法的建立奠定了基础。

To prepare the immunological recombinant heat-stable enterotoxin （STp） and the monoclonal antibodies （MAb） against STp, a gene concatemer of estp5-his was constructed by five estp copies of tandem repeat, and inserted into pGEX-6p-1. The recombinant protein STp5-His （rSTp5-His） was expressed. The rSTp5-His was used as immunogen, the MBP-5STp as detection antigen of ELISA, and the positive hybridoma cells were prepared and screened. The specificity of the MAb obtained was examined by western blot assay and blocking ELISA. The results showed that the rSTp5-His was expressed mainly in the form of inclusion bodies （about 38 ku） and it has immunogenicity. Using it as immunogen, four MAbs （C3, G1, F3 and G9） were prepared and all of them were able to react with natural STp, and with high specificity. This research conformed that the immunological recombinant protein of STp can be prepared by multicopy tandem repeat of estp, and the MAb prepared in this study there is the application value for detection of natural STp and recombinant protein of STp.