高效表达幽门螺杆菌UreB重组蛋白工程菌的构建

Recombination and expression of urease B subunit protein of Helicobacter pylori

克隆表达幽门螺杆菌 (Hp)的尿素酶B亚单位 (UreB)重组蛋白 ,可为Hp疫苗开发和快速诊断试剂盒的研究奠定基础。用PCR方法由幽门螺杆菌染色体DNA扩增UreB基因片段 ,将其融合插入原核表达载体pQE30中 ,并在M15大肠杆菌表达。经酶切、测序分析 ,包括部分融合载体基因在内的重组UreB基因片段由 1773bp组成 ,为编码 5 91个氨基酸残基的多肽。SDS PAGE分析显示重组表达的目的蛋白相对分子量约为 6 6kD ,表达量点菌体总蛋白的 2 3.5 % ,并经免疫印迹分析证实被幽门螺杆菌感染的阳性血清可与纯化UreB重组蛋白发生特异性的结合反应。UreB重组蛋白具有良好的抗原性 ,将有可能成为一种有效蛋白质疫苗以及快速诊断试剂盒用于Hp感染的防治和检测。

To construct a recombinant engineering bacteria which express the Urease B subunit (UreB)of Helicobacter pylori(Hp),and cxploit the possibility of preparing Hp vaccine and diagnostic reagent kit for rapid diagnosis of Hp infection,the gene encoding UreB of Hp was amplified from Hp chromosomal DNA by PCR.The gene UreB and the plasmid pQ30 were Linked following restriction enzyme digestion and sequence analysis,the recombinant plasmid were transformed into E.coli M15. the gene segment inserted to the recombinant vector was identified as the gene expressed UreB protein with a molecular mass of 66kD. The level of UreB expression products was about 23.5% of total celluar protein. Westeyn blot results showed that the objective protein could be reacted specificly with anti-serum against Hp.The product expressed by Hp UreB gene clone has good antigenicity.The recombinant engineering bccteria cxpressing UreB protein may be a potential source for developing effectiv protein vaccine against Hp infection and reagent kit of Hp infection diagnosis.