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目的原核表达A型产气荚膜梭菌α毒素疫苗抗原rCPA-HSP65,并进行纯化。方法以CPA全基因为模板,PCR扩增rCPA基因,插入表达质粒p ET-28a(+)-HSP65,构建重组表达质粒p ET-28a-rCPA-HSP65,转化E.coli BL21(DE3),IPTG诱导表达,表达产物进行12%SDS-PAGE分析;优化表达工艺进行高密度发酵,发酵产物经DEAE阴离子柱上复性后,再经金属螯合层析纯化。结果重组表达质粒p ET-28a-rCPA-HSP65经双酶切及测序鉴定证明构建正确,表达的重组蛋白相对分子质量约90 000,主要以可溶性形式为主,表达量占菌体总蛋白的30.17%。利用TB培养基,IPTG 34℃诱导5 h的表达产物经纯化后,纯度约达95%,蛋白收率为4.5 mg/g湿菌。结论已成功表达并纯化了A型产气荚膜梭菌α毒素疫苗抗原rCPA-HSP65,为后期疫苗生物学活性的研究奠定了基础。
Objective To prokaryotically express rCPA-HSP65 antigen of Clostridium perfringens type A vaccine and purify it. Methods The rCPA gene was amplified by PCR using the CPA gene as a template. The recombinant plasmid p ET-28a (+) - HSP65 was inserted into expression vector p ET-28a-rCPA-HSP65 and transformed into E.coli BL21 (DE3) The expression product was analyzed by 12% SDS-PAGE, and the high density fermentation was performed by optimizing the expression process. The product was purified by DEAE anion column and purified by metal chelate chromatography. Results The recombinant plasmid p ET-28a-rCPA-HSP65 was confirmed by double enzyme digestion and sequencing. The recombinant protein was constructed correctly and the molecular weight of recombinant protein was about 90 000, mainly in soluble form. The expression level of the recombinant protein was 30.17 %. Using TB medium, the expressed product of IPTG induced at 34 ℃ for 5 h was purified to about 95% purity with a protein yield of 4.5 mg / g wet bacteria. Conclusion The rCPA-HSP65 antigen of Clostridium perfringens type A toxin A vaccine has been successfully expressed and purified, which lays a foundation for the study of the biological activity of the vaccine.