结核分枝杆菌热休克蛋白Hsp60编码基因的克隆与原核表达
发布时间：2019-06-26 08:36
【摘要】： 一、目的 热休克蛋白(heat shock protein,Hsp)属于广泛存在于自然界中高度保守的蛋白质家族,在热应激下它们大量表达,因此称为热休克蛋白。热休克蛋白表达量的提高与许多应激条件有关,如低氧、营养缺乏、病毒感染、吞噬和转化作用。结核分枝杆菌是结核病病原菌。结核分枝杆菌基因组携带有两组GroEL基因,GroEL1和GroEL2。它们分别编码热休克蛋白60(cpn60.1)和热休克蛋白65(cpn60.2)。当机体感染结核分枝杆菌时,结核杆菌热休克蛋白60显示较强的疫原性,可诱导较强的B细胞和T细胞免疫应答。因此我们克隆结核分枝杆菌热休克蛋白60(Hsp60)编码基因,并在大肠杆菌中表达,为进一步纯化和研究该蛋白的免疫学特性奠定基础。 二、方法 利用PCR技术从结核杆菌H37Rv株中扩增hsp60基因,并将其与pZero-T载体连接,进行DNA测序。用EcoRI+XbaI双酶切,从TBhsp60-pZero-T质粒上切下hsp60基因,然后将该片段插入同样经过双酶切的E.coli表达载体pProEXHTb中。构建重组原核表达质粒pProEXHTb-TBhsp60。并将pProEXHTb-TBhsp60重组原核表达质粒转化入不同的E.coli菌株(BL21, DH5α, JM109),用不同摩尔浓度的IPTG诱导表达。 三、结果 成功地克隆了结核分枝杆菌hsp60基因。DNA测序证实,与GenBank公布的序列一致。含pProEXHTb-TBhsp60基因表达质粒的大肠杆菌经IPTG诱导后,能够高效表达相对分子质量(KD)约为60KD的融合蛋白。 四、结论 获得了结核分枝杆菌hsp60基因,成功地构建了原核表达质粒pProEXHTb-TBhsp60,并在大肠杆菌得到表达,结果显示重组质粒在E.coliBL21中表达时表达量最大,并随时间的增加表达量也增加,但蛋白的表达不受IPTG浓度的影响。为大量获得HSP60蛋白和研究其免疫学特性奠定了基础。
[Abstract]:Objective heat shock protein (heat shock protein,Hsp) belongs to a highly conserved protein family which widely exists in nature and is highly expressed under heat stress, so it is called heat shock protein. The increase of heat shock protein expression is related to many stress conditions, such as hypoxia, nutritional deficiency, virus infection, phagocytosis and transformation. Mycobacterium tuberculosis is the pathogen of tuberculosis. There are two groups of GroEL genes, GroEL1 and GroEL2., carried in the genome of Mycobacterium tuberculosis. They encode heat shock protein 60 (cpn60.1) and heat shock protein 65 (cpn60.2), respectively. When the body was infected with Mycobacterium tuberculosis, Mycobacterium tuberculosis heat shock protein 60 showed strong epidemic genicity and could induce strong B cell and T cell immune response. Therefore, we cloned the heat shock protein 60 (Hsp60) coding gene of Mycobacterium tuberculosis and expressed it in E. coli, which laid a foundation for further purification and study of the immunological characteristics of the protein. Methods hsp60 gene was amplified from Mycobacterium tuberculosis H37Rv strain by PCR and ligated with pZero-T vector for DNA sequencing. The hsp60 gene was cut off from TBhsp60-pZero-T plasmid by EcoRI XbaI double enzyme digestion, and then the fragment was inserted into the E.coli expression vector pProEXHTb, which was also digested by double enzyme digestion. Construction of Recombinant prokaryotic expression plasmid pProEXHTb-TBhsp60. The recombinant prokaryotic expression plasmid of pProEXHTb-TBhsp60 was transformed into different E.coli strains (BL21, DH5 伪, JM109) and induced by IPTG with different molar concentrations. Results the hsp60 gene of Mycobacterium tuberculosis was successfully cloned and confirmed by DNA sequencing, which was consistent with the sequence published by GenBank. E. coli containing pProEXHTb-TBhsp60 gene expression plasmid was induced by IPTG to express fusion protein whose relative molecular weight (KD) was about 60KD. Conclusion the hsp60 gene of Mycobacterium tuberculosis was obtained, and the prokaryotic expression plasmid pProEXHTb-TBhsp60, was successfully constructed and expressed in E. coli. The results showed that the expression of recombinant plasmid in E.coliBL21 was the highest, and also increased with the increase of time, but the expression of protein was not affected by the concentration of IPTG. It laid a foundation for obtaining a large number of HSP60 proteins and studying their immunological characteristics.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R378
本文编号：2506064
[Abstract]:Objective heat shock protein (heat shock protein,Hsp) belongs to a highly conserved protein family which widely exists in nature and is highly expressed under heat stress, so it is called heat shock protein. The increase of heat shock protein expression is related to many stress conditions, such as hypoxia, nutritional deficiency, virus infection, phagocytosis and transformation. Mycobacterium tuberculosis is the pathogen of tuberculosis. There are two groups of GroEL genes, GroEL1 and GroEL2., carried in the genome of Mycobacterium tuberculosis. They encode heat shock protein 60 (cpn60.1) and heat shock protein 65 (cpn60.2), respectively. When the body was infected with Mycobacterium tuberculosis, Mycobacterium tuberculosis heat shock protein 60 showed strong epidemic genicity and could induce strong B cell and T cell immune response. Therefore, we cloned the heat shock protein 60 (Hsp60) coding gene of Mycobacterium tuberculosis and expressed it in E. coli, which laid a foundation for further purification and study of the immunological characteristics of the protein. Methods hsp60 gene was amplified from Mycobacterium tuberculosis H37Rv strain by PCR and ligated with pZero-T vector for DNA sequencing. The hsp60 gene was cut off from TBhsp60-pZero-T plasmid by EcoRI XbaI double enzyme digestion, and then the fragment was inserted into the E.coli expression vector pProEXHTb, which was also digested by double enzyme digestion. Construction of Recombinant prokaryotic expression plasmid pProEXHTb-TBhsp60. The recombinant prokaryotic expression plasmid of pProEXHTb-TBhsp60 was transformed into different E.coli strains (BL21, DH5 伪, JM109) and induced by IPTG with different molar concentrations. Results the hsp60 gene of Mycobacterium tuberculosis was successfully cloned and confirmed by DNA sequencing, which was consistent with the sequence published by GenBank. E. coli containing pProEXHTb-TBhsp60 gene expression plasmid was induced by IPTG to express fusion protein whose relative molecular weight (KD) was about 60KD. Conclusion the hsp60 gene of Mycobacterium tuberculosis was obtained, and the prokaryotic expression plasmid pProEXHTb-TBhsp60, was successfully constructed and expressed in E. coli. The results showed that the expression of recombinant plasmid in E.coliBL21 was the highest, and also increased with the increase of time, but the expression of protein was not affected by the concentration of IPTG. It laid a foundation for obtaining a large number of HSP60 proteins and studying their immunological characteristics.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R378
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