發(fā)布時(shí)間：2018-05-31 15:00

  本文選題：幽門螺桿菌 + 細(xì)胞空泡毒素　； 參考：《重慶醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的:構(gòu)建H.pylori細(xì)胞空泡毒素(VacA)毒性片段與霍亂毒素B亞單位(CtxB)融合基因的原核表達(dá)載體,并誘導(dǎo)表達(dá),以獲得重組蛋白,鑒定其免疫原性,為制備防治H.pylori感染的口服疫苗奠定基礎(chǔ)。 方法: 1)用PCR擴(kuò)增出vacA目的基因片段,構(gòu)建原核表達(dá)質(zhì)粒pQE30-vacA。 2)用PCR擴(kuò)增出ctxB目的基因片段,克隆至pQE30-vacA質(zhì)粒vacA 的基因上游,構(gòu)建含雙基因的表達(dá)質(zhì)粒pQE-vctB。 3) pQE-vctB轉(zhuǎn)化E.coli DH5α,IPTG誘導(dǎo)表達(dá)重組蛋白VCTB,Western blotting分析抗原性,鎳離子柱純化。 4)重組蛋白VCTB口服免疫小鼠,ELISA檢測(cè)小鼠血清特異性IgG、小腸沖洗液IgA,以鑒定其免疫原性。 結(jié)果:經(jīng)測(cè)序vctB融合基因由1092bp組成,為編碼364個(gè)氨基酸殘基的多肽。重組蛋白VCTB經(jīng)SDS-PAGE分析相對(duì)分子量(Mr)約為40KD,表達(dá)量占全菌的20%以上,親和層析后可獲得純度為92%以上的蛋白。Western blotting分析顯示能分別與VacA抗血清和CT抗血清反應(yīng)。ELISA檢測(cè)顯示,免疫小鼠的血清特異性抗體IgG,腸粘液IgA顯著高于VacA對(duì)照組(P0.01 )。 結(jié)論:vacA和ctxB融合基因原核表達(dá)質(zhì)粒構(gòu)建成功,轉(zhuǎn)化E.coli DH5α表達(dá)菌獲得了重組蛋白VCTB,表達(dá)量較高,純度較高,有良好的抗原性和免疫原性,口服免疫小鼠可明顯提高其免疫效果,產(chǎn)生較高水平的IgA,可用于制備防治H.pylori感染的口服疫苗。
[Abstract]:Objective: to construct the prokaryotic expression vector of the fusion gene of H.pylori cell vacuolating toxin (Vaca) and cholera toxin B subunit (CtxB), and to obtain the recombinant protein and identify its immunogenicity. It lays a foundation for the preparation of oral vaccine for the prevention and treatment of H.pylori infection. Methods: 1) the vacA target gene fragment was amplified by PCR, and the prokaryotic expression plasmid pQE30-vacAwas constructed. 2) ctxB target gene fragment was amplified by PCR and cloned into pQE30-vacA plasmid vacA. The expression plasmid pQE-vctB. 3) the expression of recombinant protein was induced by pQE-vctB transformation into E.coli DH5 偽 -IPTG. The antigenicity of the recombinant protein was analyzed by Western blotting and purified by nickel ion column. 4) the immunogenicity of the recombinant protein VCTB was determined by Elisa. Results: the vctB fusion gene was composed of 1092bp and was a polypeptide encoding 364 amino acid residues. The relative molecular weight of the recombinant protein VCTB was about 40 KD by SDS-PAGE analysis, which accounted for more than 20% of the total bacterial expression. After affinity chromatography, the protein with purity of more than 92% was obtained. Western blotting analysis showed that the recombinant protein could react with VacA antiserum and CT antiserum respectively. Elisa analysis showed that the recombinant protein could react with the antiserum of VacA and CT, respectively. The serum specific antibody IgG and intestinal mucus IgA of immunized mice were significantly higher than those of VacA control group (P0.01). Conclusion the prokaryotic expression plasmid of the fusion gene of Vaca and ctxB was successfully constructed, and the recombinant protein VCTB was obtained by transforming E.coli DH5 偽 expression strain. The recombinant protein VCTB was obtained with high expression quantity, high purity, good antigenicity and immunogenicity. High level of IgA can be used to prepare oral vaccine to prevent and treat H.pylori infection.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R378

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 于拽拽;腸產(chǎn)毒性大腸埃希菌F4ac菌毛蛋白FaeG亞單位的原核表達(dá)及免疫學(xué)鑒定[D];重慶醫(yī)科大學(xué);2011年

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本文編號(hào)：1960177