构建携带幽门螺杆菌oipA核酸的减毒沙门重组菌株及其免疫保护作用的初步研究  

Construction and immunological protection analysis of attenuated Salmonella typhimurium harbouring Helicobacter pylori oipA DNA vaccine

作　　者：林妙端[1] 陈建森[1] 佘菲菲[1] 陈豪[1] 

机构地区：[1]福建医科大学病原生物学系福建省感染与肿瘤重点实验室,福州350004

出　　处：《中国免疫学杂志》2010年第4期335-340,共6页Chinese Journal of Immunology

基　　金：2009年福建省科技计划重点项目(2009Y0024)

摘　　要：目的:优化幽门螺杆菌(Helicobacter pylori,H.pylori)oipA核酸疫苗,构建含oipA核酸的SL7207(减毒伤寒沙门氏菌)重组菌株,并研究其在防御H.pylori感染中的作用。方法:对oipA基因进行密码子优化,Kozak前导序列添加、CpG序列优化,将oipA优化前后的基因分别克隆入pVAX1真核表达载体,获得pVAX1-oipA和pVAX1-optioipA重组子,将上述重组子与空载体pVAX1瞬时转染AGS细胞,应用Western blot检测转染细胞中OipA蛋白的表达。将重组子转化LB5000进行甲基化修饰,利用修饰后重组子转化终宿主菌SL7207,提取SL7207/pVAX1-oipA,SL7207/pVAX1-optioipA菌株质粒进行PCR,酶切鉴定。用SL7207/pVAX1-oipA和SL7207/pVAX1-optioipA免疫C57BL/6小鼠,末次免疫2周后检测抗OipA抗体,并在末次免疫4周后予H.pyloriSS1(5×108CFU/只)攻击,攻击3次,隔天一次,攻击4周后处死动物,取胃组织做快速尿素酶实验和H.pylori定量培养以检测H.pylori感染状况。结果:pVAX1-oipA、pVAX1-optioipA均可在AGS细胞中表达,且pVAX1-optioipA的OipA蛋白表达量高于pVAX1-oipA;从SL7207/pVAX1-oipA和SL7207/pVAX1-optioipA抽提的质粒,经PCR,酶切鉴定正确;免疫小鼠血清中产生抗OipA蛋白的特异性抗体IgG,且pVAX1-optioipA疫苗组诱导IgG水平明显高于pVAX1-oipA疫苗组(P<0.01);pVAX1-oipA、pVAX1-optioipA疫苗组H.pylori感染率分别为60%(9/15)、26.67%(4/15),明显低于对照组100%(10/10)(P<0.01)。结论:成功构建携带pVAX1-oipA,pVAX1-optioipA的SL7207重组菌株,并验证其对C57BL/6小鼠H.pylori感染具有免疫预防作用,且优化oipA基因有助于提高免疫保护效果。Objective：To optimize Helicobacter pylori（H.pylori）oipA gene and develop strain of vaccine in Attenuated Salmonella typhimurium SL7207 harboring recombinant plasmid pVAX1-oipA and pVAX1-optioipA,then to study its protection against H.pylori in C57BL/6 mice.Methods：oipA gene was modified by codon optimization,inserting the Kozak sequence and engineering CpG motifs.The optioipA gene and oipA gene was inserted into the eukaryotic expressing vector pVAX1 respectively to develop recombinant plasmid pVAX1-optioipA and pVAX1-oipA.The AGS cells were transfected by pVAX1-oipA,pVAX1optioipA and pVAX1 respectively.Then the OipA protein expression was confirmed by Western blot.pVAX1-oipA,and pVAX1-optioipA were converted to LB5000 for methylation and then the modified eukaryoticvector was electrotransfered to final host SL7207 respectively,SL7207/pVAX1-oipA,SL7207/pVAX1-optioipA was identified with PCR and enzyme digestion.Specific serum IgG antibody was detected by indirect ELISA after oral inoculation with vaccine strains.Mice were challenged with live Sydney strain（SS1）three times at 0,2,4 d（5×108CFU/each mouse）.Four weeks after challenge,the mice were sacrificed.The colonization of H.pylori in gastric mucosa were detected by rapid urease test and quantitative culture of H.pylori.Results：The AGS cells transfected with pVAX1-oipA and pVAX1-optioipA had expressed corresponding production,moreover,the expression level of pVAX1-optioipA was higher than those of pVAX1-oipA,SL7207/pVAX1-oipA and SL7207/pVAX1-optioipA confirmed with PCR,enzyme digestion;Two weeks after last immunization,immunized mice.were induced to produce anti-OipA IgG antibodies,and the levels of anti-OipA IgG antibody in pVAX1-optioipA group was obviously higher than in pVAX1-oipA group（P〈0.01）;After challenged with SS1,the infection rate of pVAX1-oipA and pVAX1-optioipA group was 60%（9/15） and 26.67%（4/15）,which was significantly lower than 100%（10/10） in the control group（P〈0.01）.Conclusion：Attenuated Salmonell

关 键 词：幽门螺杆菌 oipA基因 核酸疫苗 优化 减毒沙门菌 

分 类 号：R378[医药卫生—病原生物学]