幽门螺杆菌基因hpaA克隆、融合表达和鉴定

Cloning,fusion expression and identification of the adhesion gene hpaA from Helicobacter pylori

目的克隆幽门螺杆菌粘附素基因hpaA,构建幽门螺杆菌hpaA基因与麦芽糖结合蛋白基因融合表达载体,并进行诱导表达,鉴定融合蛋白免疫原性,为幽门螺杆菌疫苗研究提供依据。方法利用PCR技术从H.pylori郑州分离株MEL-HP27染色体DNA上扩增出hpaA基因,序列分析后,将其克隆到表达载体pMAL-c2X中,转化大肠杆菌(E.coliTB1),用IPTG诱导目的基因表达,SDS-PAGE方法对表达产物进行分析,Western blot鉴定其免疫原性。结果用PCR方法扩增的hpaA基因长度为783bp,编码260个氨基酸,经酶切鉴定和测序,插入到克隆载体的基因片段与预期目的DNA片段相一致;SDS-PAGE结果显示表达产物相对分子质量约为29kDa,融合蛋白的表达量约占全菌总蛋白的26%。结论本研究成功构建了hpaA基因与麦芽糖结合蛋白基因融合原核表达系统,为幽门螺杆菌基因工程组分疫苗的研究奠定基础。

To clone the adhesion gene hpaA of Helicobacter pylori, and to construct the expression vector expressing the fusion of the hpaA gene of Helicobacter pylori and the gene coding maltose binding protein, the hpaA gene of Helicobacter pylori strain MEL-HP27 was amplified by PCR. After being purified, the target fragment was cloned into plasmid pBlueseriptb Ⅱ and subject to nucleotide sequencing. Then,hpaA gene from recombinant plasmid pBluescriptb Ⅱ-hpaA was digested with restriction enzyme and was inserted into expression vector pMAL-c2X. The positive recombinants were transferred into E. coli TB1 and identified by restriction enzyme digestion and PCR. Finally, the genetically engineered bacteria including pMAL- e2X-hpaA plasmids were induced by IPTG, and the expression products were analyzed by SDS-PAGE and Western blotting. Results show that hpaA gene of 783bp, encoding the polypeptides of 260 amino acids, is obtained from the Helicobacter pylori strains MEL-HP27 genome DNA. The gene segment inserted into the recombinant vector was identified by using restriction enzyme digestion and sequencing, Plasmid pMAL-c2X-hpaA could express a specific approximative 72kDa fusion protein（HpaA 29kDa and MBP 42.5kDa） in E, coli TB1, and the fusion protein accounted for 26% of the total protein of recombinant bacterial. In this study, a prokaryotic high expression system for fusion hpaA and maltose gene was successfully constructed, which will help to develop gene recombinant vaccine against Helicobacter pylori infection.