重组幽门螺杆菌疫苗免疫保护机制的研究

目的 研究以减毒鼠伤寒沙门菌为载体构建的重组幽门螺杆菌（Hp)疫苗诱导小鼠产生保护性免疫应答的机制。方法 将表达Hp尿素酶B亚单位（UreB)，黏附素（HpaA)及尿素酶B亚单位/黏附素融合蛋白（UreB/HpaA)的减毒鼠伤寒沙门菌（Salmonella typhimurium)给小鼠分别灌胃，另设单纯减毒鼠伤寒沙门菌和生理盐水免疫鼠为对照，免疫4周后以Hp活菌攻击，观察各组小鼠的免疫保护率，攻击前后血清中抗Hp抗体IgC1,IgG2a和IgA的变化。小鼠脾脏和胃黏膜中γ干扰素（IFN-γ）和白介素-4（IL-4）mRNA表达变化。结果 UreB,HpaA及UreB/HpaA组的免疫保护率分别为50%，41%和77%，和生理盐水组相比，攻击前各鼠伤寒沙门菌免疫组IgG1,IgG2a均轻度升高而IgA无变化，攻击后各鼠伤寒沙门菌免疫组IgG2a升高显著并以UreB/HpaA组为最，而IgG1和IgA的升高无统计学差异。胃黏膜攻击前生理盐水组无IFN-γ表达，其余各组均100%表达；攻击后生理盐水组IFN-γ轻度表达，但仍明显低于各鼠伤寒沙门菌免疫组，IL-4在攻击前后各组均无表达，脾IFN-γ和IL-4在所有组攻击前后均全部表达。结论 以减毒鼠伤寒沙门菌为载体构建的Hp疫苗在小鼠体内诱导出以TH1反应为主的保护性免疫应答。     

以白细胞介素-2为免疫佐剂的幽门螺杆菌粘附素核酸疫苗制备及其免疫预防作用

目的构建含幽门螺杆菌（Hp）粘附素（HpaA）基因和白细胞介素（IL）-2的核酸疫苗，体外转染COS-7细胞，鉴定其表达蛋白的免疫原性和免疫保护作用。方法应用聚合酶链反应（PCR）技术从Hp标准菌株CCUG17874基因组DNA扩增HpaA基因；从重组质粒pCIneo—IL-2扩增小鼠IL-2基因，并通过TA克隆分别克隆人pUCmT载体。检测HpaA及IL-2的核苷酸序列，酶切、连接反应将HpaA和IL-2同时克隆人真核表达载体pIRES，再经PCR法和酶切反应进行鉴定；通过脂质体法将重组载体pIRES-HpaA—IL-2转染COS-7细胞，SDS-PAGE及Western印迹法检测表达蛋白的免疫原性。重组载体转化减毒鼠伤寒沙门菌LB5000，抽提质粒，转化人SL7207，反复传代，鉴定重组核酸疫苗菌的稳定性。以该疫苗菌经口接种小鼠，4周后再用Hp攻击，鉴定感染状况。结果测序结果证实扩增的HpaA基因与HpHpaA序列一致，IL-2序列和小鼠IL-2序列一致。PCR和酶切鉴定结果证实，HpaA和IL-2基因克隆人载体pIRES，成功构建含HpaA和IL-2基因的核酸疫苗质粒pIRES-HpaA—IL-2，Western印迹法检测到相对分子质量分别为30000和14000的HpaA和IL-2蛋白条带。小鼠体内实验显示HpaA—IL-2及HpaA组分别有75．0％、58．4％获免疫保护，与PBS组差异有统计学意义（P〈0．01）。结论成功构建了HpaA和IL-2的Hp减毒沙门核酸疫苗菌，其免疫原性和保护性均得到证实，免疫佐剂IL-2可提高免疫保护率。     

重组抗原LTB-UreB-HpaA对幽门螺杆菌感染小鼠的免疫保护性及内置佐剂LTB的免疫增强作用(英文)

目的构建大肠杆菌不耐热肠毒素B亚单位(LTB)和幽门螺杆菌(Hp)尿素酶B亚单位(UreB)、幽门螺杆菌粘附素A(HpaA)的融合基因ltBureBhpaA及其原核表达系统,鉴定重组表达产物rLTBUreBHpaA的免疫原性、佐剂活性及对Hp感染小鼠的保护作用。方法采用连接引物PCR构建ltBureBhpaA融合基因,TA克隆后测序。采用pET42a质粒及其宿主菌E.coliBL21DE3亚克隆构建原核表达系统pET42altBureBhpaAE.coliBL21DE3,并用不同浓度IPTG诱导表达。SDSPAGE用于检测rLTBUreBHpaA的表达及其产量,免疫双扩散试验及Western印迹法检测rLTBUreBHpaA抗原性和免疫反应性。建立牛GM1的ELISA(GM1ELISA)检测重组蛋白中rLTB的佐剂活性。采用HpSS1株BaLb/C小鼠感染模型,检测rLTBUreBHpaA的免疫保护作用。结果ltBureBhpaA核苷酸序列与各原始基因序列完全相同。不同浓度的IPTG均可诱导pET42altBureBhpaAE.coliBL21DE3表达rLTBUreBHpaA,其产量约为细菌总蛋白的15%。rLTBUreBHpaA家兔抗血清的双扩效价为1∶8。商品化兔抗Hp全菌抗体、兔抗UreB或HpaA均能识别rLTBUreBHpaA并与之结合。GM1ELISA结果证实rLTBUreBHpaA仍具有结合牛GM1的活性。rLTBUreBHpaA(200μg/每只小鼠)免疫后,可使小鼠100%免于HpSS1株的感染。10μgrLTB与rUreB或rHpa共同免疫小鼠,可使保护率分别从66.7%提高至81.8%和83.3%。结论本研究成功地构建了ltBureBhpaA融合基因及其原核表达系统。目的表达产物rLTBUreBHpaA有良好的免疫原性、佐剂活性及免疫保护效果,具有作为Hp基因工程疫苗的应用前景。     

金属螯合亲和层析法对重组幽门螺杆菌尿素酶B亚单位的纯化

背景：幽门螺杆菌（H.pylori)疫苗是近年研究的热点。H.pylori尿素酶B亚单位（UreB)是理想的候选抗原。目的：获得纯化的重组H.pylori UreB(rUreB)，为进一步的H.pylori疫苗制备打下基础。方法：采用金属螯合亲和层析法（MCAC）在变性条件下纯化rUreB,SDS-聚丙烯酰胺凝胶电泳（PAGE）和免疫印迹法鉴定纯化产物的分子量、纯度和抗原性。结果：纯化产物的分子量与预计分子量相符，具有良好的特异性UreB免疫原性，且一步即可达到90％以上的纯度。结论：MCAC是一种简单、高效的rUreB纯化方法。     

优化构建UreB/HpaA双价幽门螺杆菌减毒活菌疫苗的免疫保护作用

目的 以减毒鼠伤寒沙门菌为载体，通过在UreB和HpaA间引入由3个甘氨酸残基组成的三肽柔韧接头，构建成UreB／HpaA双价抗幽门螺杆菌(Hp)活疫苗，并对照相应单价疫苗和空白载体研究其对C57BL／6小鼠的免疫保护效果。方法 用序列重叠延伸聚合酶链反应扩增带3个甘氨酸残基柔韧接头的融合基因UreB／HpaA，进一步以减毒鼠伤寒沙门菌SL3261为载体构建UreB／HpaA双价活疫苗，观察其在小鼠体内的稳定性。用双价活疫苗株免疫Ⅱ级C57BL／6小鼠1次，对照单价活疫苗和空白载体观察其在体内诱导的特异抗体反应和对小鼠的免疫保护作用。结果 测序结果显示，3个甘氨酸残基的编码序列GGTGGAGGC已成功地插入UreB／HpaA融合基因中。双价疫苗灌喂小鼠后，至少能在脾脏和回肠末段存留10d。双价疫苗在小鼠体内诱导血清特异性IgGl和IgG2a水平明显升高。UreB／HpaA双价疫苗的免疫保护率为77．3％(17／22)，而UreB疫苗和HpaA疫苗的免疫保护率分别为50．0％(12／24)和43．5％(10／23)。结论 引入柔韧接头，优化构建表达UreB和HpaA的双价抗Hp活疫苗。UreB／HpaA双价活疫苗对Ⅱ级C57BL／6小鼠有更好的免疫保护作用。     

表达幽门螺杆菌尿素酶B亚单位的减毒鼠伤寒杆菌口服疫苗的制备

目的制备抗幽门螺杆菌(Hp)尿素酶B亚单位(UreB)减毒鼠伤寒杆菌活菌疫苗,观察其免疫效果.方法构建表达UreB的原核表达载体PTc01-UreB并转化减毒鼠伤寒杆菌SL3261,得到重组菌SL3261/PTc01-UreB.应用抗Hp菌体蛋白兔血清行Western-blot检测UreB在SL3261中的表达.将SL3261/PTc01-UreB口服免疫Balb/c小鼠,12周后应用ELISA检测肠液和血清中的特异性抗体反应.SL3261/PTc01-UreB在Luria-Bertani培养液中连续传代60代以确定其稳定性.结果成功构建PTc01-UreB原核表达载体.Western-blot显示,其转化减毒鼠伤寒杆菌SL3261后能表达相对分子质量约61×103的蛋白,与HpUreB亚单位相符,具有抗原性.口服免疫小鼠后,在肠液和血清中可分别检测到针对UreB的特异性IgA和IgG抗体.体外连续培养60代未见PTc01-UreB质粒丢失及对宿主细胞毒性.结论表达HpUreB的减毒鼠伤寒杆菌SL3261/PTc01-UreB可用作抗Hp感染口服疫苗.     

幽门螺杆菌临床株粘附素HapA基因的克隆表达及在诊断中的价值

目的　构建表达幽门螺杆菌临床株粘附素 (HpaA)的重组质粒 ,在大肠杆菌中表达获得重组蛋白 ,探讨重组蛋白作为Hp抗原在感染诊断中的价值。 方法　用PCR方法从幽门螺杆菌临床株DNA中扩增HpaA基因片段。克隆及序列分析后在大肠杆菌中进行高效表达 ,表达产物经纯化和Westernblot鉴定后 ,作为Hp抗原 ,ELISA法对 10 0例临床标本进行相应抗体检测 ,并与细菌培养、组织学染色和快速尿素酶试验比较来评价其应用的可行性。结果　经酶切、测序分析插入的基因片段全长 783bp ,与基因文库中的粘附素基因同源性达 98 87%。表达蛋白经SDS -PAGE分析 ,相对分子量 (Mr)约为 300 0 0 ,可溶性表达占全菌的 4 1 6 7%以上 ,经亲和层析后可获得纯度为 90 %以上的重组蛋白。Westernblot证实了其免疫反应性。通过对临床病例的检测结果显示细菌培养、组织学染色、快速尿素酶试验和HpaA抗体检测的敏感性、特异性和准确性分别为 10 0 0 %和 80 9% ;10 0 0 % ;和 90 5 % ;90 5 %和 85 8% ;93 3%和 85 9% ,相差不多。结论　HpaA能在大肠杆菌中高效表达 ,具有较强的免疫反应性 ,作为检测试剂敏感性和特异性均较好 ,有望成为Hp新的非侵入性的检测方法。     

幽门螺杆菌金属蛋白酶原核表达载体的构建与重组表达及纯化北大核心CSCD

目的构建幽门螺杆菌(Helicobacter pylori, Hp)金属蛋白酶(metalloprotease, Mtp)基因mtp与麦芽糖结合蛋白基因mbp融合表达载体,诱导Mtp重组表达并纯化表达产物,为Hp致病机制和疫苗研究奠定基础。方法应用PCR从Hp郑州分离株MEL-Hp27的DNA扩增mtp基因,经纯化回收后与克隆载体pMD19-T连接,并进行测序验证。对重组质粒pMD19-T-mtp双酶切,酶切目的片段克隆至表达载体pMAL-c2X,然后用重组质粒pMAL-mtp转化大肠埃希菌(E.coli TB1)。从大肠埃希菌重组子中提取重组质粒,进行酶切和测序鉴定。用分光光度计测定重组菌的生长曲线。用IPTG诱导mtp表达,用SDS-PAGE法分析表达产物,并用Amylose树脂预装柱纯化Mtp蛋白。结果 PCR扩增的mtp基因片段长度为621 bp,重组菌株的酶切和测序鉴定正确;重组菌生长曲线显示重组质粒的转入对受体菌的生长无显著影响;Mtp诱导表达和纯化产物的SDS-PAGE显示,Mtp表达产物为相对分子质量为66.4×10^3的水溶性融合蛋白(rMtp),纯化产物的纯度约为85.6%。结论成功构建mtp基因与麦芽糖结合蛋白mbp基合表达载体,并纯化制备了重组蛋白,为探索Mtp在Hp感染发病机制和抗Hp感染疫苗研究奠定了基础。     

幽门螺杆菌抗原基因ureB在乳酸菌中的克隆表达与免疫反应性研究

目的为了开发幽门螺杆菌（Helicobacter pylori,Hp）口服疫苗,将Hp尿素酶B（UreB）在食品级乳酸乳球菌NZ3900菌株中进行表达,并研究其免疫反应性。方法 PCR扩增Hp MEL-HP27菌株ureB基因（基因号：FJ436980）,将其克隆入大肠杆菌-乳酸乳球菌穿梭质粒pNZ8110中并转化乳酸乳球菌NZ3900;采用正交试验确定目的蛋白表达的适宜条件;应用western-blot鉴定其免疫反应性。结果成功扩增了Hp MEL-HP27菌株ureB基因,构建了ureB基因的乳酸菌NICE（Nisin-controlled expression）原核表达系统;UreB蛋白适宜的表达条件为：在ureB重组菌生长至对数生长前期（OD600≈0.3～0.4）加入终浓度为40ng/mL的nisin,诱导表达5h,可溶性UreB蛋白表达量最高,可达27.26μg/mL培养基。可溶性UreB蛋白的表达占上清蛋白的比例最高可达20.19%;Western-blot结果显示乳酸乳球菌表达的UreB抗原蛋白具有良好的免疫反应性。结论结果提示应用乳酸乳球菌构建幽门螺杆菌食品级疫苗可能具有较好前景。     

表达幽门螺杆菌hpaA基因的减毒鼠伤寒沙门氏菌对小鼠的免疫保护作用

目的　克隆幽门螺杆菌 (H pylori)全长hpaA基因 ,构建表达HpaA蛋白的重组减毒鼠伤寒沙门氏菌 ,并研究其对小鼠的免疫保护作用。方法　用PCR扩增全长hpaA基因 ,经适当的酶切 -连接反应将其连入原核表达质粒 pTrc99A ,并进行了基因测序。重组质粒经鉴定后再导入减毒鼠伤寒沙门氏菌SL32 6 1,提取重组菌质粒 ,PCR和酶切鉴定 ,筛选阳性克隆。用SDS -PAGE电泳和Westernblot进行HpaA表达分析和鉴定 ,用薄层扫描分析HpaA含量。重组菌 3× 10 8CFU/ 0 4ml/只免疫C5 7BL/ 6小鼠 ,4周后H pyloriSS110 5CFU/只攻击小鼠 ,再 5周后处死小鼠 ,取腺胃做快速尿素酶试验和Giemsa染色 ,以明确H pylori定植情况 ,对照观察免疫保护效果。 结果　经PCR和酶切证实 ,构建了含 783bphpaA基因的重组原核表达质粒 pTrc99A -hpaA ,并将后者成功转化了减毒鼠伤寒沙门氏菌。重组菌能表达约 30kDaHpaA蛋白 ,重组HpaA量约占全菌体蛋白量的 38 9% ,Westernblot证实其有免疫反应性。重组菌对小鼠免疫保护率为 4 3 4 8% (10 / 2 3) ,与空白对照组比统计差异显著 (P =0 0 1)。结论　构建了表达H pyloriHpaA的重组减毒鼠伤寒沙门氏菌 ,该菌株对C5 7BL/ 6小鼠有免疫保护作用。     

Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA

AIM: To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity. METHODS: Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed, then doned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coliDH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207. After that, the recombinant strain was grown in vitro repeatedly. In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using Lipofectamine~(TM)2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot. RESULTS: The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot. CONCLUSION: The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo.     

幽门螺杆菌尿素酶B基因的克隆、表达及免疫原性研究

目的应用基因工程方法构建表达幽门螺杆菌尿素酶Ｂ亚单位（ＵｒｅＢ）的菌株．方法用ＰＣＲ方法从幽门螺杆菌染色体ＤＮＡ上扩增出ＵｒｅＢ基因片段,将其克隆至ｐＳＫ（＋）质粒上,然后插入原核表达载体ｐＥＴ２２ｂ（＋）中,在大肠杆菌ＢＬ２１（ＤＥ３）中表达．结果经测序ＵｒｅＢ片段有１７０７ｂｐ组成,为编码５６９个氨基酸残基的多肽．ＳＤＳＰＡＧＥ和免疫印迹分析检测发现,ＵｒｅＢ基因表达的蛋白质分子质量为６５０００Ｕ,并证实该重组蛋白质可以被幽门螺杆菌感染阳性患者的血清所识别,同时将其免疫小鼠可刺激机体产生抗该重组蛋白质的抗体．结论重组的ＵｒｅＢ可以作为一种有效的蛋白质疫苗用于幽门螺杆菌感染的预防和治疗．     

Diversity of Helicobacter pylori isolates in expression of antigens and induction of antibodies

AIM： To obtain evidence for selection of antigens used in genetically engineered vaccine against Helicobacter pylori （H pylori）. METHODS： Enzyme linked immunoabsorbent assay （ELISA） was established on the basis of recombinant protein antigens rUreB, rHpaA, rVacA, rCagA1, rNapA, rFlaA and rFlaB of Hpylori to detect expression rates of the antigens in bacterial isolates as well as positive rates of the antibodies in sera from H pylori-infected patients. PCR was applied to the detection of carrying rates of the genes encoding antigens in the isolates. RESULTS： The outputs of rUreB, rHpaA, rVacA, rCagA1, rNapA, rFlaA and rFlaB were approximately 35%, 32%, 15%, 23%, 56%, 25% and 20% of the total bacterial proteins, respectively. One hundred and fifty-one strains of H pylori were isolated from 347 biopsy specimens of chronic gastritis, peptic ulcer or gastric adenocarcinoma, with a positive rate of 43.5%. All of the isolates expressed UreB, HpaA, FlaA and FlaB while 52.3%, 92.1% and 93.4% of the isolates expressed VacA, CagA and NapA, respectively. In the sera of 151 Hpylori-infected patients, the positive rates of IgG antibodies against UreB, HpaA, VacA, CagA, NapA, FlaA and FlaB were 100%, 87.4%, 43%, 71.5%, 89.4%, 84.8% and 79.5%, respectively. Furthermore, the expression frequencies of VacA and NapA were found to be relative to the severity of gastric diseases （P = 0.016 and P 〈 0.0001, respectively）. CONCLUSION： UreB antigen is the top option of developing genetically engineered vaccine against H pylori followed by NapA or HpaA.     

幽门螺杆菌融合蛋白HspA-UreB在大肠杆菌中的表达与鉴定

目的在大肠杆菌中表达幽门螺杆菌(简称Hp)HspA-UreB融合蛋白,并探索其免疫反应性,为Hp基因工程疫苗的研制奠定基础。方法用PCR方法扩增郑州分离Hp菌株MEL-HP27的hspA和ureB基因,分别克隆入pNEB193中。测序后,回收两种基因片段,并以hspA-ureB的顺序连接插入原核表达载体pMAL-C2x进行融合表达。采用蛋白印迹法对表达产物进行鉴定。结果特异PCR法和酶切鉴定证实融合基因hspA-ureB克隆入表达载体中;重组质粒转化大肠杆菌TB1后,经IPTG诱导3h,SDS-PAGE电泳显示在119kDa处出现一条特异蛋白带,即麦芽糖结合蛋白(MBP)与HspA-UreB的融合表达形式,约占细菌总体蛋白含量的31%;该融合蛋白与Hp免疫小鼠血清和Hp阳性病人血清的Westernblot分析结果显示,在119kDa处出现特异杂交带。结论成功地在大肠杆菌中实现了Hp融合蛋白HspA-UreB的高效表达,并证实其具有良好的免疫反应性。     

Construction of prokaryotic expression system of ureB gene from a clinical Helicobacter pylori strain and identification of the recombinant protein immunity

AIM: To clone ureB gene from a clinical isolate of Helicobacter pyloriand construct a prokaryotic expression system of the gene and identify immunity of the expressed recombinant protein. METHODS: ureBgene from a clinical Hpyloristrain Y06 was amplified by the high fidelity polymerase chain reaction technique. The target DNA fragment amplified from ureB gene was sequenced after T-A cloning. Prokaryotic recombinant expression vector pET32a inserted with ureB gene (pET32a-ureB) was constructed. The expression of recombinant UreB protein (rUreB) in E. coli BL21DE3 induced by isopropylthio-β-D-galactoside (IPTG) at different concentrations was examined by SDS-PAGE. Western blot using commercial antibodies against whole cell of Hpylori and an immunodiffusion assay using a self-prepared rabbit anti-rUreB antibody were applied to determine immunity of the target recombinant protein. ELISA was used to detect the antibody against rUreB in sera of 125 Hpyloriinfected patients and to examine rUreB expression in 109 Hpylori isolates. RESULTS: In comparison with the reported corresponding sequences, the nucleotide sequence homology of the cloned ureB gene was from 96.88-97.82% while the homology of its putative amino acid sequence was as high as 99.65-99.82%. The rUreB output expressed by pET32a-ureB-BL21DE3 was approximate 30% of the total bacterial proteins, rUreB specifically combined with the commercial antibodies against whole cell of Hpylori and strongly induced rabbits to produce antibody with a 1:8 immunodiffusion titer after the animals were immunized with the recombinant protein. Serum samples from all Hpyloriinfected patients were positive for UreB antibody and UreB expression were detectable in all tested Hpyloriisolates. CONCLUSION: A prokaryotic expression system with high expression efficiency of Hpylori ureBgene was successfully established. The expressed rUreB showed qualified immunoreactivity and antigenicity. High frequencies of UreB expression in different Hpyloriisolates and specific antibody against UreBin sera of Hpyloriinfected patients indicatet hat UreB is an excellent antigen candidate for developing H pylori vaccine.     

Construction of hpaA gene from a clinical isolate of Helicobacter pylori and identification of fusion protein

AIM: To clone hpaA gene from a clinical strain of Helicobacter pylori and to construct the expression vector of the gene and to identify immunity of the fusion protein. METHODS: The hpaA gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The recombinant expression vector inserted with hpaA gene was constructed. The expression of HpaA fusion protein in E.coli BL21DE3 induced by IPTG at different dosages was examined by SDS-PAGE. Western blot with commercial antibody against whole cell of H.pylori as well as immunodiffusion assay with self-prepared rabbit antiserum against HpaA fusion protein were applied to determine immunity of the fusion protein. ELISA was used to detect the antibody against HpaA in sera of 125 patients infected with H.pylori and to examine HpaA expression of 109 clinical isolates of H.pylori. RESULTS: In comparison with the reported corresponding sequences, the homologies of nucleotide and putative amino acid sequences of the cloned hpaA gene were from 94.25-97.32 % and 95.38-98.46 %, respectively. The output of HpaA fusion protein in its expression system of pET32a-hpaA-BL21DE3 was approximately 40 % of the total bacterial proteins. HpaA fusion protein was able to combine with the commercial antibody against whole cell of H.pylori and to induce rabbit producing specific antiserum with 1:4 immunodiffusion titer after the animal was immunized with the fusion protein. 81.6 % of the serum samples from 125 patients infected with H.pylori (102/125) were positive for HpaA antibody and all of the tested isolates of H.pylori (109/109) were detectable for HpaA. CONCLUSION: A prokaryotic expression system with high efficiency of H.pylori hpaA gene was successfully established. The HpaA expressing fusion protein showed satisfactory immunoreactivity and antigenicity. High frequencies of HpaA expression in different H.pylori clinical strains and specific antibody production in H.pylori infected patients indicate that HpaA is an excellent and ideal antigen for developing H.pylori vaccine.     

幽门螺杆菌粘附素与大肠杆菌LtB融合疫苗的口服免疫与预防试验

目的　观察幽门螺杆菌粘附素HpaA与大肠杆菌LtB融合蛋白质经口服免疫BALB/c小鼠后机体产生的免疫应答和蒙古沙鼠免疫预防作用。方法　用PCR方法扩增HpaA和LtB目的基因片段 ,将LtB与 pPIM质粒连接后 ,再与HpaA基因连接 ,转化大肠杆菌DH5α ,经酶切获得含有ltB -hpaA的pPIM -ltB/hpaA融合质粒。将重组质粒转化E coliBL2 1(DE3) ,筛选获得重组工程菌 ,经发酵、包涵体提取 ,复性 ,纯化获得LtB -HpaA。BABL/c小鼠设正常对照组、单独rHpaA对照组、CT +HpaA实验组、融合蛋白LtB -HpaA组和rLtB +rHpaA实验组 ,免疫 4周后检测血清抗原特异性IgG、IgA和胃冲洗液、肠冲洗液中sIgA。蒙古沙鼠分 3个实验组 ,正常对照 (PBS)组 ,单独HpaA免疫组和LtB -HpaA组 ,免疫 4周 ,于末次免疫后 14天灌活力良好的H pyloriSS1活菌 ,1个月处死动物 ,采集标本 ,用细菌培养和病理切片检查评价蒙古沙鼠的免疫保护作用。结果　融合蛋白LtB -HpaA组及将CT和LtB作为外粘膜佐剂的CT +HpaA组和LtB +HpaA组中血清IgA和IgG、胃冲洗液和肠冲洗液中sIgA含量明显高于单独HpaA组和对照组 ,差异非常显著 (P <0 0 0 1)。蒙古沙鼠融合蛋白rLtB-HpaA组比单纯rHpaA组效果明显 (rLtB -HpaA组保护率为 80 % ,单独rHpaA为 2 0 % )。 结论　融合蛋白质LtB     

Expression of Helicobacter pylori AlpA protein and its immunogenicity

AIM: To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori (H pylori) and to study the immunogenicity of adhesin AlpA. METHODS: Gene Ab, which was amplified from H pylori chromosomal DNA by PCR technique, was sequenced and the biological information was analyzed, and inserted into the Nco I and Not I restriction fragments of the expression vector pET-22b(+) using T4 DNA ligase. The resulting plasmid pET-AlpA was transformed into competent E.coli BL21(DE3) cells using ampicillin resistance for selection. Recombinant strains were incubated in 5 mL LB with 100 μg/mL ampicillin overnight at 37 ℃. Sonication of BL21(DE3)pET-22b(+)/AlpA was analyzed by Western blot to detect AlpA immunogenicity. RESULTS: The gene encoding AlpA protein was amplified by PCR with chromosomal DNA of H pylori Sydney strain (SS1) as templates. It revealed that AlpA DNA fragment amplified by PCR had approximately 1 500 nucleotides, compatible with the previous reports. The recombinant plasmid pET-22b(+)/AB was successfully constructed. DNA sequencing showed one open reading frame with the length of 588 bp. It encoded seven conservative regions that showed good antigenicity and hydrophobicity by Parker and Welling method. Furthermore, INTERNET EXPASY, NNPREDICT and ISREC predicted that it was a porin-like structure consisting of β-pleated sheets that were embedded in the outer membrane. BLAST analyzed 836 767 protein sequences and found that the similar sequences were all belonging to H pylori OMP sequences. SDS-PAGE and scan analysis showed that the molecular weight of AB was 22.5 ku and recombinant protein amounted to 29% of the total bacterial protein, among which dissolved expression amounted to 21.9% of sonicated supernatant. The rAB purity amounted to 96% through affinity chromatography. Western blot analysis of rAB confirmed that it could be specially recognized by serum form rabbit immunized with AlpA and H pylori infected. CONCLUSION: Adhesin AlpA recombinant protein may be a potential vaccine for control and treatment of H pylori infection.