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
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国家高技术“863”计划资助项目(102-07-03-06)、国家自然科学基金(30270078)和军队“十五”医药卫生科研课题(OIMA-132)资助项目.


Cloning,Expression,Purification and Identification of Conservative Region of Four Helicobacter pylori Adhesin Genes in AlpA Gene
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This work was supported by grants from The State 863 High Technology R & D Project of China (102-07-03-06), The National Natural Sciences Foundation of China (30270078) and Military ‘ten-five’ Commanding Project.

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    摘要:

    幽门螺杆菌(Helicobacter pylori,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-22b(+)载体,在BL21(DE3)大肠杆菌中表达.测序显示AB基因长588 bp,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22.5 ku,其重组蛋白质表达量占菌体总蛋白质的29%,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.AB蛋白的获得为进一步研究Hp黏附素保守区的分子黏附机制和免疫防治作用提供了基础.

    Abstract:

    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.

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白杨,但汉雷,王继德,张兆山,S. ODENBREIT,周殿元,张亚历.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[J].生物化学与生物物理进展,2002,29(6):922-926

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  • 收稿日期:2002-04-24
  • 最后修改日期:2002-05-28
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