破伤风毒素C片段(TTc)在大肠杆菌中的表达、优化与鉴定

HTSS以一株破伤风生产菌株基因组DNA为模板,通过上游引物中几个碱基的修改,PCR扩增出破伤风毒素C片段(TTc)基因,构建了原核表达质粒pET-42(b)/TTc,在大肠杆菌BL21(DE3)中表达。重组蛋白分子量约50kD,表达量为22%,超声波破碎显示为可溶性重组蛋白。通过对培养基、诱导时间、诱导温度的优化,重组蛋白的表达量和可溶性均有提高。Western blotting检测表达产物可与破伤风C片段单克隆抗体产生特异的免疫反应。该工作为亚单位疫苗或载体蛋白的开发奠定了基础。

Expression,optimization and identification of tetanus toxin fragment C in E. Coli BL21(DE3)

TTc was obtained by PCR through base mutant upstream primer.After a series of DNA recombination manipulation,expression plasmid pET-42(b)/TTc was finally constructed and transformed into E.coli BL21(DE3) for IPTG-induced expression.Expressed protein is soluble,the rate of target protein is about 22%,and MW is about 50kD.After optimization of medium,inducing time,inducing temperature,solubility and expression level of target protein are all improved.Western blotting assay showed that expressed can react with TTc McAb.This study result provide the basis for developing this protein as a protein carrier and recombinant vaccine.