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

【摘要】：目的原核表達(dá),并純化幽門螺桿菌NCTC11637菌株的1501基因,為進(jìn)一步研究Hp1501基因表達(dá)蛋白的功能和篩選預(yù)防H.pylori感染疫苗的候選抗原提供實驗材料。 方法用PCR技術(shù)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(xué)軟件分析后,構(gòu)建表達(dá)載體pQE30-Hp1501a,轉(zhuǎn)化E.coli XL1-blue,IPTG誘導(dǎo)表達(dá)重組蛋白,經(jīng)SDS-PAGE及Western blot鑒定,親和層析法純化重組蛋白。 結(jié)果測序分析表明Hp1501基因全長為1164bp,與GeneBank公布的H.pylori國際標(biāo)準(zhǔn)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(gòu)建的表達(dá)質(zhì)粒經(jīng)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(dá)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(dá)成功。 結(jié)論首次成功原核表達(dá)并純化H.pylori NCTC11637菌株Hp1501基因重組蛋白,為Hp外膜蛋白生物學(xué)功能和疫苗的研究奠定了基礎(chǔ)。
[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(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 鄒全明;;幽門螺桿菌疫苗的研究進(jìn)展[J];胃腸病學(xué);2007年09期

2 高志剛;鄒全明;劉鳳英;曹向光;;重組幽門螺桿菌尿素酶B亞單位鼻腔免疫凝膠劑的研制[J];中國現(xiàn)代應(yīng)用藥學(xué);2007年06期

3 王華;邵世和;;幽門螺桿菌OipA、HopX、HopW基因的檢測及序列分析[J];世界華人消化雜志;2007年35期

4 王凱娟,王潤田;中國幽門螺桿菌感染流行病學(xué)Meta分析[J];中華流行病學(xué)雜志;2003年06期

5 毛亞飛,嚴(yán)杰,徐樅;幽門螺桿菌內(nèi)置佐劑UreB和HpaA雙價重組疫苗對實驗感染小鼠的免疫保護(hù)作用[J];浙江大學(xué)學(xué)報(醫(yī)學(xué)版);2005年05期

6 ;Construction of prokaryotic expression system of ureB gene from a clinical Helicobacter pylori strain and identification of the recombinant protein immunity[J];World Journal of Gastroenterology;2004年07期

7 ;Aspirin increases susceptibility of Helicobacter pylori to metronidazole by augmenting endocellular concentrations of antimicrobials[J];World Journal of Gastroenterology;2009年08期

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本文編號：1518620