幽门螺杆菌NCTC11637株Hp1501基因重组蛋白的原核表达与纯化

发布时间：2018-02-20 03:09

本文关键词： 幽门螺杆菌 Hp1501基因生物信息学分析原核表达　出处：《重庆医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的原核表达,并纯化幽门螺杆菌NCTC11637菌株的1501基因,为进一步研究Hp1501基因表达蛋白的功能和筛选预防H.pylori感染疫苗的候选抗原提供实验材料。方法用PCR技术从H.pylori NCTC11637菌株基因组扩增1501基因,T-A克隆后鉴定、测序;用生物信息学软件分析后,构建表达载体pQE30-Hp1501a,转化E.coli XL1-blue,IPTG诱导表达重组蛋白,经SDS-PAGE及Western blot鉴定,亲和层析法纯化重组蛋白。结果测序分析表明Hp1501基因全长为1164bp,与GeneBank公布的H.pylori国际标准株26695和J99的基因序列一致性为96%～97%,氨基酸H.pylori一致性为97%～98%;构建的表达质粒经测序鉴定,其插入H.pylori完全正确;SDS-PAGE分析表明,在37kDa处有一新生的蛋白带,表达量约占菌体总蛋白的21%;重组蛋白主要以包涵体形式存在,纯化后纯度约91%,Western blot表明重组蛋白表达成功。结论首次成功原核表达并纯化H.pylori NCTC11637菌株Hp1501基因重组蛋白,为Hp外膜蛋白生物学功能和疫苗的研究奠定了基础。
[Abstract]:Objective to express and purify the 1501 gene of Helicobacter pylori (NCTC11637) strain in prokaryotic expression, and to provide experimental materials for further study on the function of Hp1501 gene expression protein and screening of candidate antigens for the prevention of H. pylori infection vaccine. Methods the 1501 gene was amplified from the genome of H. pylori NCTC11637 by PCR and sequenced. The expression vector pQE30-Hp1501a was constructed by bioinformatics software, and transformed into E. coli XL1-blueIPTG to induce the expression of recombinant protein. The recombinant protein was identified by SDS-PAGE and Western blot. The recombinant protein was purified by affinity chromatography. Results sequencing analysis showed that the length of Hp1501 gene was 1164bp. the gene sequence of H.pylori 26695 and J99 published by GeneBank was 96.97, and that of amino acid H.pylori was 970.The constructed expression plasmid was confirmed by sequencing and inserted into H.pylori correctly by SDS-PAGE. There was a new protein band at 37kDa, which accounted for about 21% of the total bacterial protein, and the recombinant protein mainly existed in the form of inclusion body, and the purity of the recombinant protein was about 91% after purification, which indicated that the recombinant protein was successfully expressed. Conclusion the recombinant protein of Hp1501 gene of H.pylori NCTC11637 strain was successfully expressed and purified for the first time, which laid a foundation for the study of the biological function of HP outer membrane protein and the vaccine.
【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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