A型产气荚膜梭菌Fba重组干酪乳杆菌的构建与表达

Construction and Expression the Recombinant Lactobacillus casei Anchoring the Gene Fba of Clostridium Perfringens Type A

为了构建重组乳酸菌,以A型产气荚膜梭菌全基因组DNA为模板,利用PCR技术扩增出Fba基因并将其连接到大肠杆菌表达载体pET-28a上,鉴定正确后,用1.0 mmol·L-1 IPTG诱导表达,最佳诱导表达时间为5 h。表达的目的蛋白纯化后与弗氏佐剂乳化制备抗血清,Western blot分析表明,目的蛋白能与制备的抗血清特异结合,具有很好的反应原性。将正确的Fba基因与pLA乳酸菌载体构建成重组质粒并电转至干酪乳杆菌中,Western blot结果显示,重组的干酪乳杆菌表达的蛋白与抗血清具有良好的反应原性。间接免疫荧光、流式细胞仪检测结果表明,目的蛋白能够展示表达到菌体表面。

In order to construct recombinant lactic acid bacteria,Fba gene was amplified by PCR using the whole genome DNA of Clostridium percapsulatus type A as the template and connected to the expression vector pET-28a of Escherichia coli.After correct identification,FBA gene was induced by 1.0 mmol·L-1 IPTG,and the optimal induction expression time was 5 h.The antiserum was prepared by emulsification with Freund adjuvant after purification of the expressed target protein.Western blot analysis showed that the target protein could specifically bind to the prepared antiserum and had good reactivity.The recombinant plasmid was constructed with the correct Fba gene and pLA Lactobacillus carrier and transferred to Lactobacillus casei by electric transfer.Western blot results showed that the protein expressed by the recombinant Lactobacillus casei had good reactivity with antiserum.The results of indirect immunofluorescence and flow cytometry showed that the target protein could be expressed on the surface of the bacteria.