结核杆菌ESAT-6基因的克隆扩增及其在耻垢分枝杆菌和毕赤酵母中表达

发布时间：2018-04-01 20:26

本文选题：结核分枝杆菌　切入点：ESAT-6　出处：《华中科技大学》2007年硕士论文

【摘要】： 目的构建结核杆菌ESAT-6基因穿梭质粒,在耻垢分枝杆菌中表达蛋白并对重组蛋白进行鉴定,同时利用电转化方法,将重组质粒转化到卡介苗中,为疫苗研究奠定实验基础;构建能在毕赤酵母表达系统中表达结核杆菌ESAT-6蛋白的重组质粒,并对其表达产物进行基本生物学特性鉴定,为新型结核病疫苗的研究开发提供研究依据。方法[1]从GenBank获得结核分枝杆菌ESAT-6基因DNA序列,根据该序列及ps3000表达载体的要求,设计和合成一对引物,利用PCR技术扩增ESAT-6基因,并插入pGEMT载体,进行PCR,双酶切及测序鉴定。[2]亚克隆法构建ps3000—ESAT-6大肠杆菌--分枝杆菌穿梭质粒,经电转化法导入到耻垢分枝杆菌和卡介苗中,Western blot鉴定其生物学活性。[3]设计和合成毕赤酵母表达引物并引入酶切位点,扩增ESAT-6基因,并插入pGEMT载体,进行PCR,双酶切及测序鉴定。[4]亚克隆法构建pPICZaA-- ESAT-6穿梭质粒,经化学转化法整合毕赤酵母GS115染色体中,PCR鉴定目的基因的整合,Zeocin抗性筛选多拷贝重组子并诱导表达,表达产物用Western-blot鉴定其表达。结果[1]成功扩增了结核分枝杆菌ESAT-6基因;正确构建穿梭质粒ps3000-ESAT-6,用pET表达系统ESAT-6纯化蛋白免疫小鼠的多抗血清通过Western blot证实了该重组耻垢杆菌中有表达并具有生物学活性。[2]从结核分枝杆菌H37Rv基因组中扩增出ESAT-6基因片段,成功构建重组表达质粒pPICZaA-ESAT-6,SDS-PAGE显示胞外并无表达,酵母细胞经裂解后ESAT-6基因翻译的蛋白加上载体的信号肽序列和c-myc表位一共18kDa左右在蛋白胶上相应位置有表达,并且能被结核病人血清抗体所识别。结论[1] ESAT-6基因重组耻垢分枝杆菌构建成功,为下一步表达ESAT-6蛋白的重组BCG( Bacilli Calmette-Guérin)疫苗的研究奠定了基础。[2]利用毕赤酵母表达系统获得胞内表达的结核杆菌ESAT-6重组蛋白,为结核病诊断抗原和疫苗研究奠定了基础。
[Abstract]:Objective to construct the shuttle plasmid of ESAT-6 gene of Mycobacterium tuberculosis, express the protein in Mycobacterium smeareum and identify the recombinant protein. At the same time, the recombinant plasmid was transformed into BCG vaccine by the method of electrical transformation, which laid the experimental foundation for vaccine research. The recombinant plasmid which can express the ESAT-6 protein of Mycobacterium tuberculosis in Pichia pastoris expression system was constructed, and its expression product was identified by biological characteristics, which provides the basis for the research and development of new TB vaccine. Methods [1] the DNA sequence of ESAT-6 gene of Mycobacterium tuberculosis was obtained from GenBank. According to this sequence and the requirement of ps3000 expression vector, a pair of primers were designed and synthesized. ESAT-6 gene was amplified by PCR and inserted into pGEMT vector. Ps3000-ESAT-6 Escherichia coli-Mycobacterium shuttle plasmid was constructed by [2] subclone method. The biological activity of Pichia pastoris was identified by Western blot. [3] expression primers of Pichia pastoris were designed and synthesized, and restriction endonuclease sites were introduced to amplify the ESAT-6 gene and insert it into pGEMT vector. The pPICZaA- ESAT-6 shuttle plasmid was constructed by subcloning method. The recombinant plasmid was identified by chemical transformation method and identified by chemical transformation method. The integration of the target gene, Zeocin resistance, was screened for multiple copies of recombinant plasmids and induced expression, and the expression of pPICZaA- ESAT-6 shuttle plasmid was induced by chemical transformation method, which was used to identify the integration of the target gene in the GS115 chromosome of Pichia pastoris. The expressed product was identified by Western-blot. Results [1] Mycobacterium tuberculosis ESAT-6 gene was amplified successfully. The shuttle plasmid ps3000-ESAT-6 was constructed correctly. The polyclonal antibody of mouse immunized with ESAT-6 purified by pET expression system was confirmed to be expressed and bioactive by Western blot. [2] the ESAT-6 gene fragment was amplified from the H37Rv genome of Mycobacterium tuberculosis. The recombinant expression plasmid pPICZaA-ESAT-6 SDS-PAGE showed no extracellular expression. The protein translated by ESAT-6 gene and the signal peptide sequence and c-myc epitope of yeast cells were expressed in the corresponding position on the protein gel. And can be recognized by the tuberculosis patient serum antibody. Conclusion [1] the recombinant Mycobacterium ESAT-6 gene was successfully constructed, which laid a foundation for the further study of recombinant Bacilli Calmette-Gu 茅 rin vaccine expressing ESAT-6 protein. [2] Recombinant protein of Mycobacterium tuberculosis ESAT-6 was obtained by using Pichia pastoris expression system. It lays a foundation for the study of tuberculosis diagnosis antigen and vaccine.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

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