结核分支杆菌Rv0901基因置换型打靶载体的构建

Construction of the targeting vector of Rv0901 gene in Mycobacterium Tuberculosis

目的 :对结核杆菌H3 7Rv的Rv0 90 1基因进行体外扩增 ,构建基因打靶载体 ,利用基因打靶技术进行结核分支杆菌基因Rv0 90 1功能的研究。方法 :结核分支杆菌标准毒力株H3 7Rv体外培养 ,扩增目的基因 ,连接载体及目的片段 ,切除目的基因 ,筛选阳性克隆 ,酶切鉴定。结果 :经酶切鉴定PCR产物及插入片段大小与预期值相符 ,鉴定证实PCR产物及插入片段为所需目的基因片段 ,成功切除靶片段。证实标记基因插入片段插入方向正确。结论 :成功构建了用于结核分支杆菌基因打靶的置换型载体 ,为随后将进行的Rv0 90 1基因敲除株的建立 ,Rv0 90 1基因功能的研究奠定了基础。

Object: In order to investigate the function of Rv0901 gene in Mycobacterium tuberculosis,a fragment of Mycobacterium tuberculosis was amplified and targeting vector was constructed. Methods: Culturing the Mycobacterium tuberculosis in vitro, extracting the genome DNA,amplifying the targeted gene with PCR, constructing the targeting vector and identifying with restricted enzymes. Results: The fragment was amplified successfully.The replacement vector with deleted Rv0901 gene was constructed. Conclusion: Constructing successfully the replacement vector which is used to the gene knockout in Mycobacterium Tuberculosis.