本文關(guān)鍵詞： 球形幽門螺桿菌 cagA基因 vacA基因 克隆 表達(dá)　出處：《安徽理工大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 克隆球形H.pylori vacA、cagA基因,并進(jìn)行序列測定及表達(dá),研究球形H.pylori的致病性,并為抗H.pylori疫苗的研制奠定實驗基礎(chǔ)。 方法 采用亞抑菌濃度的氨芐青霉素誘導(dǎo)幽門螺桿菌標(biāo)準(zhǔn)株NCTC11637產(chǎn)生球形變異,收集球形H.pylori。從球形幽門螺桿菌基因組DNA中特異擴(kuò)增出vacA、cagA基因片段,擴(kuò)增的目的片段經(jīng)純化回收后克隆到pMD-18T中,轉(zhuǎn)化入大腸桿菌JM109。陽性重組質(zhì)粒用PCR及雙酶切鑒定,并進(jìn)行序列測定。采用亞克隆技術(shù),用BamH Ⅰ和Sac Ⅰ從重組質(zhì)粒pMD-18T-vacA、pMD-18T-cagA上切下vacA、cagA基因,插入表達(dá)載體pET32a(+)質(zhì)粒,轉(zhuǎn)化大腸桿菌BL21,陽性重組子經(jīng)雙酶切及PCR擴(kuò)增鑒定。重組質(zhì)粒pET32a(+)-vacA、pET32a(+)-cagA轉(zhuǎn)化大腸桿菌,IPTG誘導(dǎo)表達(dá)后進(jìn)行SDS-PAGE電泳和凝膠掃描定量分析。 結(jié)果 從球形幽門螺桿菌基因組DNA中分別擴(kuò)增出3888bp、3444bp的vacA、cagA基因,構(gòu)建重組質(zhì)粒pMD-18T-vacA、pMD-18T-cagA及pET32a(+)-vacA、pET32a(+)-cagA,雙酶切及PCR擴(kuò)增均篩選出含目的片段的陽性克隆。測序結(jié)果顯示,球形H.pylori與已公布的H.pylori vacA、cagA基因序列的同源性分別達(dá)99.8%、99.7%。推測氨基酸同源性分別達(dá)99.3%、99.2%。vacA、cagA基因在大腸桿菌中經(jīng)誘導(dǎo)表達(dá)后分別獲得約156kD、148kD的蛋白,VacA蛋白含量占全菌體蛋白含量的15.5%。 結(jié)論 成功地對球形幽門螺桿菌vacA、cagA基因進(jìn)行體外擴(kuò)增及構(gòu)建原核重組質(zhì)粒pMD-18T-vacA、pMD-18T-cagA、pET32a(+)-vacA、pET32a(+)-cagA,并經(jīng)酶切及序列分析所驗證,證明球形幽門螺桿菌含有完整的vacA、cagA基因,并且能在大腸桿菌中獲得高效表達(dá)。球形H.pylori含有vacA、cagA基因并能體外克隆和表達(dá),可能與其潛在的致病性有關(guān)。
[Abstract]:Objective to clone, sequence and express Vaca cagA gene of spherical H.pylori, to study the pathogenicity of H.pylori and to lay an experimental foundation for the development of anti-H.pylori vaccine. Methods the subinhibitory concentration of ampicillin was used to induce the spherical variation of Helicobacter pylori standard strain NCTC11637. The Vaca cagA gene fragment was specifically amplified from the genomic DNA of Helicobacter pylori. The amplified fragment was purified and recovered and cloned into pMD-18T and transformed into E. coli JM109.The positive recombinant plasmid was identified by PCR and double enzyme digestion and sequenced. Vaca cagA gene was cut off by BamH 鈪，

本文編號：1547440