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目的克隆并原核表达分枝杆菌噬菌体D29裂解酶gp10基因,观察重组蛋白对脓肿分枝杆菌的抑菌作用。方法以噬菌体D29基因组DNA为模板,PCR扩增gp10基因片段,克隆至pET-32a(+)载体上,构建重组原核表达质粒pET-32a-gp10,转化E.Coli BL21(DE3),IPTG诱导表达。表达的重组蛋白经SDS-PAGE分析,亲和层析纯化后,采用杯碟法检测其抑菌活性。结果 PCR扩增获得长1 454 bp的gp10基因片段,重组表达质粒pET-32a-gp10经PCR、双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约为54 800,表达量占菌体总蛋白的97.4%,破菌上清及破菌沉淀中均可见目的蛋白的表达,纯化后纯度可达90%,浓度为0.5 mg/ml;重组蛋白对脓肿分枝杆菌有抑菌作用。结论已成功表达重组分枝杆菌噬菌体D29裂解酶gp10,其对脓肿分枝杆菌具有抑菌作用,为抗脓肿分枝杆菌及其他非结核分枝杆菌感染新药物的研制提供了依据。
Objective To clone and express mycobacterium phage D29 lyase gp10 gene in E. coli and observe the antibacterial activity of the recombinant protein on Mycobacterium abscess. Methods The phage D29 genomic DNA was used as a template to amplify gp10 gene fragment and cloned into pET-32a (+) vector. The prokaryotic expression plasmid pET-32a-gp10 was constructed and transformed into E. coli BL21 (DE3) . The expressed recombinant protein was analyzed by SDS-PAGE and purified by affinity chromatography. The antibacterial activity of the expressed recombinant protein was tested by cup plate method. Results The gp10 gene fragment of 1 454 bp was amplified by PCR. The recombinant plasmid pET-32a-gp10 was confirmed by PCR, double enzyme digestion and sequencing. The relative molecular mass of the expressed recombinant protein was about 54 800, The total protein of 97.4%, broken bacteria supernatant and broken bacteria precipitate can be seen in the expression of the target protein purity of up to 90% after purification, a concentration of 0.5 mg / ml; recombinant protein antibacterial effect on Mycobacterium abscess. Conclusion Recombinant mycobacterium bacteriophage D29 lyase gp10 has been successfully expressed, which has antibacterial activity against Mycobacterium abscess and provides a basis for the development of new drugs against Mycobacterium abscess and other non-mycobacterial infections.