分支杆菌噬菌体D29裂解酶gp10的克隆表达及抗菌活性鉴定

发布时间：2018-01-19 08:08

本文关键词： 噬菌体裂解酶表达抗菌活性　出处：《重庆医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：研究目的:克隆表达分支杆菌噬菌体D29裂解酶gp10,观察该重组裂解酶对脓肿分支杆菌等多重耐药菌的抑菌作用,为开发治疗多重耐药菌感染的药物打下基础。方法:以噬菌体D29基因组DNA为模板,用PCR扩增gp10基因片段,克隆入PET32a质粒,构建重组原核表达载体PET32a-gp10,转化入大肠杆菌BL21,IPTG诱导表达。SDS—PAGE分析表达蛋白的相对分子质量和表达形式,离子交换层析纯化重组蛋白后,用杯碟法测定其抗菌活性。结果:PCR扩增获得长1454bp的gp10基因片段,经PCR、双酶切及测序证明重组质粒PET32a-gp10构建正确。SDS-PAGE显示表达蛋白相对分子量约为54.8KD,与预期相符;目的蛋白在表达上清,碎菌上清和碎菌沉淀中均可见,纯化后均一性可达90%。杯碟法测定重组蛋白对脓肿分支杆菌出现抑菌环,对金黄色葡萄球菌没有抑菌环。结论:重组分支杆菌噬菌体D29裂解酶gp10对脓肿分支杆菌有明显的抑菌作用,为治疗脓肿分支杆菌感染提供了新路径。
[Abstract]:Objective: to clone and express Mycobacterium bacteriophage D29 lyase gp10 and observe the bacteriostatic effect of the recombinant lyase on multidrug resistant bacteria such as Mycobacterium abscess. To lay a foundation for the development of drugs for the treatment of multidrug resistant bacteria infection. Methods: using the genomic DNA of phage D29 as template, the gp10 gene fragment was amplified by PCR and cloned into PET32a plasmid. The recombinant prokaryotic expression vector PET32a-gp10 was constructed. The expression was induced by IPTG. SDS-PAGE was used to analyze the relative molecular weight and expression form of the expressed protein. The recombinant protein was purified by ion exchange chromatography. The antibacterial activity was determined by cup-disc method. Results the 1454bp gp10 gene fragment was amplified by PCR. Double enzyme digestion and sequencing proved that the recombinant plasmid PET32a-gp10 was constructed correctly. SDS-PAGE showed that the relative molecular weight of the expressed protein was about 54.8 KD, which was consistent with the expectation. Objective protein was expressed in supernatant, supernatant and precipitate of broken bacteria, and the homogeneity of purified protein was up to 90. The inhibitory ring of recombinant protein on mycobacterium abscess was determined by cup-disc method. There is no bacteriostasis ring to Staphylococcus aureus. Conclusion: recombinant Mycobacterium bacteriophage D29 lyase gp10 has obvious inhibitory effect on mycobacterium abscess and provides a new pathway for the treatment of mycobacterium abscess infection.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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