重组破伤风毒素C片段的原核表达、纯化及其活性

Prokaryotic Expression,Purification and Activity of Recombinant Tetanus Toxin Fragment C

目的原核表达、纯化重组破伤风毒素C片段(rTTC),并进行活性鉴定。方法采用PCR法从破伤风梭菌基因组DNA中扩增TTC基因片段,将其插入载体pThioHisA中,构建重组表达质粒rTTC-pThioHisA,转化大肠杆菌BL21,IPTG诱导表达。表达的rTTC蛋白经离子交换、凝胶层析两步纯化后,采用Western blot、免疫双扩散及动物免疫试验进行活性及免疫原性鉴定。结果重组表达质粒经双酶切鉴定证明构建正确;表达的重组蛋白以包涵体和可溶性形式存在;纯化的重组蛋白纯度大于95%,能与全段破伤风毒素(TT)抗体反应,rTTC抗体能与全段TT反应。rTTC能较好地诱导家兔产生免疫反应,但诱导小鼠产生的免疫反应较弱。结论已原核表达并纯化了rTTC,其有望开发为一种新型抗破伤风芽孢梭菌疫苗及细菌多糖类结合疫苗的蛋白载体。

Objective To express recombinant tetanus toxin fragment C（rTTC）in prokaryotic cells,purify the expressed product and determine its activity.Methods TTC gene fragment was amplified by PCR from the genomic DNA of Clostridium tetani and inserted into vector pThioHisA,and the constructed recombinant plasmid rTTC-pThioHisA was transformed to E.coli BL21 for expression under induction of IPTG.The expressed rTTC protein was purified by ion exchange and gel filtration chromatography,and determined for activity and immunogenicity by Western blot,double immunodiffusion test and immunization of animals.Results Restriction analysis showed that recombinant plasmid rTTC-pThioHisA was constructed correctly.The expressed rTTC protein existed in both soluble and inclusion body forms and reached a purity of more than 95%after purification.The purified rTTC protein reacted with the antibody against complete tetanus toxin（TT）,while the antibody against rTTC reacted with complete TT.The rTTC induced high immune response in rabbit but low immune response in mice.Conclusion Recombinant TTC was expressed in prokaryotic cells and purified,which might be used as a novel vaccine against Clostridium tetani and a protein vector of bacterial polysaccharide vaccine.