A型产气荚膜梭菌α毒素疫苗抗原rCPA-HSP65的原核表达及其纯化

Prokaryotic expression and purification of Clostridium perfringens αtoxin vaccine antigen rCPA-HSP65

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 express Clostridium perfringens α toxin vaccine antigen r CPA-HSP65 in prokaryotic cells and purify the expressed product. Methods Recombinant CPA gene was amplified by PCR using whole CPA gene as a template and inserted into expression vector p ET-28a（＋）-HSP65. The constructed recombinant plasmid p ET- 28a-r CPAHSP65 was transformed to E. coli BL21（DE3） for expression under induction of IPTG. The expressed product was identified by 12% SDS-PAGE. The expression procedure was optimized to realize the high density fermentation, and the fermentation product was purified by DEAE anion exchange chromatography and metal chelating chromatography. Results Restriction analysis and sequencing proved that recombinant plasmid p ET-28a-r CPA-HSP65 was constructed correctly.The expressed recombinant protein, with a relative molecular mass of about 90 000, mainly existed in a soluble form and contained 30. 17% of total somatic protein. The expressed product in E. coli in TB medium after induction with IPTG at34 ℃ for 5 h reached a purity of 95%, of which the yield was 4. 5 mg / g wet bacteria. Conclusion Clostridium perfringens α toxin vaccine antigen r CPA-HSP65 was expressed successfully, which laid a foundation of further study on biological activity of the vaccine.