破伤风毒素C片段基因的克隆及其在大肠杆菌中的高效表达

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

应用PCR技术直接从破伤风梭菌64008菌株基因组中扩增出1356bp的破伤风毒素C片段基因,该片段是与靶细胞起结合作用的重链C端基因,经DNA序列测定分析,扩增出的基因与GenBank上登陆的序列AF154828的同源性达到99.2%。将此基因克隆入大肠杆菌融合表达载体pET-30a(+),构建成重组表达质粒pET-TetC,并在大肠杆菌Rosetta(DE3)中表达,经SDS-PAGE蛋白电泳鉴定,表达产物约为50000的重组蛋白,其表达量占菌体总蛋白的33.5%,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。动物试验证明,此重组抗原具有良好的免疫原性,能保护动物免受破伤风毒素的攻击。

The gene of tetanus toxin fragment C was amplified from the genomie DNA of Clostridium tetani strain 64008 by PCR. The PCR product was inserted into the high-expression vector peT-30a（＋） for sequencing and expressed in E. coli Rosetta（DE3）. It was shown that the sequence had 99.2% homogeneity to that in Gen- Bank. The expressed protein was purified by His . Bind Resin Kit. The result of SDS-PAGE verified that the desired recombinant protein with molecular weight of 50 000 was expressed and accounted for 33.5% of the total bacterial protein. Western-blot analysis further indicated this expressed product had the immunogenieity of tetanus toxin fragment C. The expressed protein initiated enough antibodies in immunized mice to protect mice against the lethal challenge with tetanus toxin.