破伤风毒素C片段基因的克隆及在大肠杆菌中的高效表达

本研究对破伤风毒素C片段进行了基因克隆、重组表达、蛋白纯化和免疫原性分析。应用PCR技术直接从破伤风梭菌64008菌株基因组中扩增出大小为1356 bp的破伤风毒素C片段(TetC)基因,经DNA序列测定分析,扩增出的基因与GenBank上登录的序列AF154828的同源性达到99.2%。将此基因克隆至大肠杆菌融合表达载体pGEX-6P-1,构建成重组表达质粒pGEX-6P-1-TetC,并在大肠杆菌BL21中表达,重组蛋白的表达量占菌体总蛋白的21%。经SDS-PAGE蛋白电泳鉴定,表达产物为76 Ku左右的重组蛋白,经免疫印迹试验证实该重组蛋白是破伤风毒素C片段抗原。

Cloning and high-level expression of tetanus toxin fragment C in E.coli

Gene expression and immunogenicity of the freagment C of tetanus toxin was investigated.The gene of tetanus toxin fragment C was amplified by PCR from the genomic DNA of Clostridium tetani strain 64008.The PCR product was inserted into the pMD19-T vector for sequencing and subcloned into the high-expression vector pGEX-6P-1 and expressed in E.coli BL21. The results showed that the fragment C shared 99.2% sequence homology with AF154828 deposited in GenBank.The expressed protein was purified by Redipack GST purification kit.SDS-PAGE revealed that the recombinant protein has a molecular weight of 76 Ku and accounted for 21% of the total bacterial protein.Western blot analysis further indicated that this protein possessed full immunogenicity of tetanus toxin fragment C.