破伤风毒素C片段基因克隆、重组表达及免疫原性研究

STUDY ON GENE CLONING, EXPRESSION AND IMMUNOGENICITY OF TETANUS TOXIN FRAGMENT C

应用PCR技术从破伤风梭状芽孢杆菌DNA中扩增出1．4kbDNA基因，将其克隆到pUC18质粒中，经测序证明该DNA片段为破伤风片段C的基因，并将此基因片段亚克隆到pGEX-4T-2,构建成表达质粒pGEX－TC在E．coli中进行表达。经聚丙烯酰胺凝胶电泳和免疫印迹鉴定，表达产物为76KD的特异性重组蛋白。动物实验显示：1μg重组蛋白免疫动物，可使动物产生1IU／ml的破伤风抗毒素单位，动物血清的抗体滴度可达1：800。

1. 4kb DNA fragment was obtained by amplification from DNA of Clostridium tetani (TC) by polymerase chain reaction method. The DNA fragment was confirmed to be TC gene after cloned into PUC 18and sequencing. Then, the TC DNA fragment was subcloned into expression vector PGEX - 4T - 2,termed as PGEX- TC. Moreover, pGEX-TC was expressed in E. coli JM107, and the expressed fusion protein was visualized by SDS - PAG. The apparent recombinant fusion protein was shown at 76KD and reached with antitetanus toxoid antibody in Western blot analysis. Immunogenicity of the fusion protein was further evaluated in guinea pigs. The animals injected with 1μg recominant fusion protein could produce 1UI. /ml anti - toxin unit of tetanus. The antibody titer of animals was 1:800 by ELISA analysis.