本文關鍵詞： 幽門螺桿菌 Hp1501基因 生物信息學分析 原核表達　出處：《重慶醫(yī)科大學》2011年碩士論文　論文類型：學位論文

【摘要】：目的原核表達,并純化幽門螺桿菌NCTC11637菌株的1501基因,為進一步研究Hp1501基因表達蛋白的功能和篩選預防H.pylori感染疫苗的候選抗原提供實驗材料。 方法用PCR技術從H.pylori NCTC11637菌株基因組擴增1501基因,T-A克隆后鑒定、測序;用生物信息學軟件分析后,構建表達載體pQE30-Hp1501a,轉化E.coli XL1-blue,IPTG誘導表達重組蛋白,經SDS-PAGE及Western blot鑒定,親和層析法純化重組蛋白。 結果測序分析表明Hp1501基因全長為1164bp,與GeneBank公布的H.pylori國際標準株26695和J99的基因序列一致性為96%～97%,氨基酸H.pylori一致性為97%～98%;構建的表達質粒經測序鑒定,其插入H.pylori完全正確;SDS-PAGE分析表明,在37kDa處有一新生的蛋白帶,表達量約占菌體總蛋白的21%;重組蛋白主要以包涵體形式存在,純化后純度約91%,Western blot表明重組蛋白表達成功。 結論首次成功原核表達并純化H.pylori NCTC11637菌株Hp1501基因重組蛋白,為Hp外膜蛋白生物學功能和疫苗的研究奠定了基礎。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R346

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