幽门螺杆菌外膜蛋白的基因克隆及表达

目的：克隆及表达幽门螺杆菌（Hp)相对分子质量为18000的外膜蛋白基因,为Hp的疫苗开发,快速诊断试剂盒的研究奠定基础。方法：用PCR从Hp染色体DNA扩增该外膜蛋白的基因片段,将目的基因,pQE30质粒同时分别经限制性内切酶BamHⅠ,HindⅢ酶切,纯化,连接,转染,筛选以及表达含插入片段的重组载体。结果;经酶切,测序分析插入的基因片段为表达Hp相对分子质量为18000外膜蛋白基因,与文献相比：有2％碱基发生变异,以1．68％氨基酸残基改变,同源性可达到98％以上,SDS－PAGE显示;表达产物相对分子质量为18000,可溶性表达占全菌的18％以上;ELISA法显示：该蛋白可被Hp全菌抗血清识别。结论：Hp外膜蛋白基因克隆表达产物具有良好的抗原性,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。

Gene cloning and expression of outer membrane protein of Helicobacter pylori

OBJECTIVE: To construct a recombinant vector which expresses the 18 kDa outer membrane protein (OMP) from Helicobacter pylori (Hp), and exploit the possibility of obtaining Hp vaccine and diagnostic reagent kit for rapid diagnosis of Hp infection. METHODS: The gene encoding the structural 18 kDa outer membrane protein of Hp was amplified from Hp chromosomal DNA by PCR. The purified PCR product and identified plasmid pQE30 underwent restriction enzyme (Hind III, BamHI, BgLI) digestion and purified by PCR purification reagent kit, and then linked in the proportion of 4:1 (molar weight). The recombinant vector was sequenced with T7 as seqiencing primer. Homology of the determined DNA sequence was analyzed by DNA analysis software. The recombinant vector was selected and identified by restriction enzyme digestion, and then transformed into DH5 a (PREP4) Escherichia coli strain which was cultured and induced by isopropylthio-beta-D-galactosideso as to determine its expressed products. RESULTS: The gene segment inserted into the recombinant vector was identified as the gene experssing the OMP of HP with a molecular mass of 18 fDa. As compared with previously reports, 2% of the gene was mutated, 1.68% of the amino acid residues was changed, and the homogeneity was about 98%. The level of soluble expression products was about 18% of total cellular protein. ELISA results showed that this objective protein could be recognized by anti-serum against Hp. CONCLUSION: The product expressed by Hp OMP gene clone has good antigenicity. The recombinant vector expressing 18 kDa OMP may be a potential source for effective protein vaccine against Hp infection and reagent kit of Hp infection diagnosis.