幽门螺杆菌AlpA基因中四种黏附素基因保守区的克隆、表达、纯化及鉴定

Cloning,Expression,Purification and Identification of Conservative Region of Four Helicobacter pylori Adhesin Genes in AlpA Gene

幽门螺杆菌 (Helicobacterpylori,Hp)感染是慢性活动性胃炎和消化性溃疡的主要病因 ,与胃腺癌、胃黏膜相关淋巴样组织 (MALT)淋巴瘤的发生亦密切相关 .鉴于Hp已证实的四种粘附素保守区 (AB)是外膜蛋白 (OMP)和膜孔素 (porin)样成分 ,而外膜蛋白和膜孔素样成分是优秀的疫苗候选抗原 .用PCR技术扩增AB基因 ,将其定向插入 pET 2 2b (+)载体 ,在BL2 1(DE3)大肠杆菌中表达 .测序显示AB基因长 5 88bp ,编码 195个氨基酸 .SDS 聚丙烯酰胺凝胶电泳和凝胶扫描分析 ,AB基因表达的蛋白质分子质量约为 2 2 5ku ,其重组蛋白质表达量占菌体总蛋白质的 2 9% ,表达产物经亲和层析纯化后蛋白质纯度达 96 % .经免疫印迹证实该重组蛋白可以被AlpA免疫兔血清所识别 .

Helicobacter pylori infection is the major etiological factor of chronic active gastritis and most peptic ulcer disease,and is closely associated with gastric cancers such as adenocarcinoma and MALT lymphoma. Since the Hp adhesin conservative region(AB) is outer membrane protein and porin type component, while these two kinds of protein are the excellent immunogen candidates of vaccination. The gene ab was amplified by PCR and inserted into the prokaryotie expression vector pET 22b(+)and expressed in the BL21(DE3) E.coli strain. DNA sequenced showed one open reading frame of 588 bp,which encoded polypeptides of 195 amino acids. SDS PAGE and scan analysis show the AB molecular mass is 22 5 ku and recombinant protein amounted to 29% of the total bacterial protein. The AB purity amounted to 96% through affinity chromatography. Western blot analysis of AB confirmed it could be specially recognized by serum from rabbit immunized with AlpA.Acquire of AB established foundation for further studying the molecular adhesin, prevention and cure immunity mechanism of the adhesin.