幽门螺杆菌oipA DNA重组质粒的构建及免疫保护作用  

Construction, expression and immunological protection of DNA vaccine of pVAX1-oipA of Helicobacter pylori

作　　者：吴小茜[1] 佘菲菲[1] 丁惠[1] 张静[1] 陈月秀[1] 

机构地区：[1]福建医科大学分子医学微生物学实验室,福州350004

出　　处：《中国免疫学杂志》2006年第8期696-699,706,共5页Chinese Journal of Immunology

基　　金：福建省自然科学基金重大项目(2001F003)

摘　　要：目的:探讨幽门螺杆菌(Hp)oipADNA重组质粒在防御Hp感染中的作用。方法:将HpoipA基因的开放读码框架定向克隆入pVAX1载体,获得的重组子pVAX1oipA转染SGC7901细胞,用RTPCR及ELISA方法检测细胞oipA转录及翻译水平的表达。抽提纯化重组质粒pVAX1oipA,经肌肉注射免疫BALB/c小鼠(100μg/只)每周1次,共3次,以质粒pVAX1(空载组)和生理盐水(空白组)做对照,分别于末次注射1个月和2个月后ELISA法检测血清中相应抗体。在证实有免疫应答的基础上,以HpSS1攻击小鼠(0.5×1090.5ml/只)3次,于末次攻击4周后,取胃黏膜组织作细菌学(包括胃黏膜快速尿素酶试验、细菌培养鉴定)和组织学检测。结果:pVAX1oipA转染的SGC7901细胞在转录水平和翻译水平均有相应的表达;免疫小鼠产生抗oipA抗体。免疫小鼠经细菌攻击后,实验组、空载组和空白组胃黏膜组织快速尿素酶实验阳性率分别为0(0/10)、90%(9/10)和100%(5/5),细菌培养阳性率分别为20%(2/10)、90%(9/10)和100%(5/5),组织学检测结果:空载组和空白组小鼠胃黏膜均出现轻度或重度糜烂,而实验组小鼠胃黏膜80%(8/10)正常,显示oipADNA重组子具有免疫保护作用。结论:oipADNA重组子能有效诱导抗Hp保护性免疫反应。Objective ：To investigate the effect of DNA vaccine expressed Helicobacter pylori（Hp） oipA on protecting against Hp infection. Methods：The ORFs of Hp oipA had been inserted into the eukaryotic expressing vector pVAX1 and SGC-7901 cells had been transfected with recombinant plasmid pVAXl-oipA. The expression of oipA had been detected by RT-PCR and ELISA. After extracted and puritied, pVAXl-oipA had been injected into BALB/c mice through muscles of right leg one time each week for three weeks （ 100 p.g each mouse）, pVAX1 blank and normal saline had been used as the control groups. Titer of antibodies had been detected by ELISA two months after the last immunization. Based on the confirmation of immunological response in the pVAX1 groups, mice had been given orogastric challenged with live Hp Sydney strain three times（0. 5 ×10^9/0. 5 ml each mouse）. Four weeks after challenge,mice had been sacrificed. Histological change and the colonization of Hp in the gastric mucosa had been detected by urease test, the culture of Hp, and electronic microscopy. Results ：SGC-7901 cells transfected with pVAX1-oipA had expressed corresponding production at the level of transcription and translation. Immunized mice had been induced anti-oipA antibodies. After challenged with bacterium,as contrast to immunized mice groups injected with pVAX1-oipA, pVAX1 blank, normal saline, the positive rote of urease test of gastric mucosa was 0（0/10）, 90% （9/10）, 100% （5/5）respectively,the positive rate of cultures of Hp was 20% （2/10）, 90% （9/10）, 100% （5/5）respectively. Histological findings： the different degree of erosion had been observed in control group, but 80% （8/10）of gastric mucosa were normal in immunized mice. Conclusion：oipA DNA could induce effective immune response in protection against Hp infection.

关 键 词：幽门螺杆菌 OIPA DNA 免疫保护 

分 类 号：R378[医药卫生—病原生物学]