结核分支杆菌热休克蛋白70原核表达载体的构建、表达和纯化

发布时间：2018-01-25 03:44

本文关键词： 分支杆菌结核热休克蛋白质70 基因表达原核细胞蛋白质类/分离与提纯　出处：《吉林大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:构建结核分支杆菌HSP70原核表达载体pQE30-Mtb HSP70并诱导其表达、纯化及鉴定目的蛋白。方法:利用PCR技术从牛型结核分支杆菌基因组中扩增出Mtb HSP70 DNA序列,构建重组质粒pGEM T-Mtb HSP70,经酶切、PCR和测序鉴定后,将Mtb HSP70基因亚克隆到原核表达质粒pQE30,构建重组表达质粒pQE30-Mtb HSP70,在大肠杆菌M15中诱导表达。用镍凝胶亲和层析的方法纯化目的蛋白,Westren Blot杂交鉴定纯化蛋白。结果:经测序证实,获得的目的基因与GeneBank公布的结核杆菌Mtb HSP70基因序列一致。构建的原核表达载体pQE30-Mtb HSP70在大肠杆菌M15中经1mmol·L-1 IPTG诱导后表达出相对分子量约为70 400的蛋白,镍柱纯化后经抗组氨酸单克隆抗体进行Westren blotting,在相对分子量约70400处可见特异性着色带。结论:成功构建了原核表达载体pQE30-Mtb HSP70,并成功诱导了Mtb HSP70蛋白的表达,通过镍凝胶亲和层析法获得纯度较高的Mtb HSP70蛋白,为Mtb HSP70蛋白作为免疫佐剂和免疫原在抗肿瘤、抗结核等方面的进一步研究提供条件。
[Abstract]:Aim: to construct the prokaryotic expression vector pQE30-Mtb HSP70 of Mycobacterium tuberculosis (HSP70) and induce it to express, purify and identify the target protein. Methods: the Mtb HSP70 DNA sequence was amplified from the genome of Mycobacterium bovis by PCR technique, and the recombinant plasmid pGEM T-Mtb HSP70 was constructed and digested by enzyme. After PCR and sequencing, Mtb HSP70 gene was subcloned into prokaryotic expression plasmid pQE30 and the recombinant expression plasmid pQE30-Mtb HSP70 was constructed. Expression was induced in E. coli M15 and purified by nickel gel affinity chromatography. The purified protein was identified by Westren Blot hybridization. Results: it was confirmed by sequencing. The obtained target gene was identical to the sequence of Mtb HSP70 gene of Mycobacterium tuberculosis published by GeneBank. The prokaryotic expression vector pQE30-Mtb was constructed. HSP70 was induced by 1 mmol 路L -1 IPTG in Escherichia coli M15 and expressed a relative molecular weight of 70,400. After purification by nickel column, Westren blotting was carried out by anti histidine monoclonal antibody. The specific band was observed at the relative molecular weight of about 70400. Conclusion: the prokaryotic expression vector pQE30-Mtb HSP70 was successfully constructed and the expression of Mtb HSP70 protein was induced successfully. High purity Mtb HSP70 protein was obtained by nickel gel affinity chromatography. Mtb HSP70 protein was used as immune adjuvant and immunogen in anti-tumor. Further research on anti-tuberculosis is necessary.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

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