幽门螺杆菌omp11基因与麦芽糖结合蛋白基因融合表达载体的构建与表达

目的：构建幽门螺杆菌(H．pylori)外膜蛋白基因(omp11)与麦芽糖结合蛋白基因融合表达载体,并进行诱导表达,为幽门螺杆菌疫苗和诊断抗原的筛选奠定基础。方法：提取H．pylori郑州分离株MEL-Hp27染色体DNA,用自行设计的PCR引物,从染色体DNA上扩增出omp11基因,将其克隆到表达载体pMAL-c2x中,用重组质粒转化大肠杆菌(E．coli TB1)。对重组质粒进行酶切鉴定,对目的基因片段进行测序。用IPTG诱导目的基因表达,用SDS-PAGE方法对表达产物进行分析。结果：用PCR方法扩增的omp11基因长度为561bp;经酶切鉴定和测序,插入到载体的基因片段与文献报道相一致;SDS-PAGE的结果显示,目的基因表达产物的相对分子质量为28000,融合蛋白的表达量占全菌总蛋白的30％。结论：作者构建的pMAL-c2x与omp11基因重组质粒在E．coli TB1中能够高效表达目的基因,该重组质粒的构建为H．pylori omp11基因的研究建立了重要的基础。

Construction of the expression vector for the fusant of the omp11 gene of Helicobacter pylori and the gene coding for maltose binding protein and inducement of its expression

Aim: To construct the expression vector for the fusant of the omp11 gene of Helicobacter pylori and the gene coding for maltose binding protein, and induce the expression of the fusional genes in order to build a basis for the investigation work of vaccine and diagnostic antigens. Methods: H.pylori chromosomal DNAs were prepared with the H.pylori strains MEL-HP27. The omp11 gene of H.pylori was amplified from H.pylori chromosomal DNA by PCR. The PCR product was cloned into the expression vector pMAL-c2X. The recombinant vector was identified by restriction enzyme digestion and sequencing method. The recombinant vector was transformed into E. coli TB1, and the expression of the omp11 gene was induced with IPTG. The expression products were analyzed by using SDS-PAGE method. Results: The PCR product consisted of 561 base pairs. The gene segment inserted into the recombinant vector was identified by using restriction enzyme digestion and sequencing methods and was consistent with those papers reported before. The expression product of omp11 gene was a molecular weight of 28 000. The amount of the recombinant gene expression products was 30% in the total proteins of the host. Conclusion: The recombinant vector constructed in this study with pMAL-c2X and omp11 gene could express the inserted gene at a high level, and act as an important basis for the further research on the H.pylori omp11 gene.