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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 and purify recombinant tetanus toxin C fragment (rTTC) in prokaryotic cells and identify its activity. Methods TTC gene fragment was amplified from genomic DNA of Clostridium tetani by PCR and inserted into pThioHisA vector to construct recombinant plasmid rTTC-pThioHisA. The recombinant plasmid was transformed into E. coli BL21 and induced by IPTG. The expressed rTTC protein was purified by ion exchange and purified by gel chromatography. Western blot, double immunodiffusion and animal immunoassay were used to evaluate the activity and immunogenicity. Results The recombinant plasmid was confirmed by double restriction enzyme digestion. The recombinant protein was expressed in inclusion body and soluble form. The purified recombinant protein was more than 95% purity and could react with tetanus toxoid (TT) antibody. The rTTC antibody And the whole section of TT response. rTTC can induce rabbit immune response better, but induced immune response in mice is weak. CONCLUSION Prokaryotic expression and purification of rTTC is expected to be developed as a novel protein carrier against Clostridium tetocaensis vaccine and bacterial polysaccharide conjugate vaccine.