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。     

表达幽门螺杆菌NAP蛋白的减毒沙门疫苗菌株预防幽门螺杆菌感染

目的建立表达幽门螺杆菌(Helicobacterpylori,Hp)中性粒细胞活化蛋白(Hp-NAP)的减毒沙门疫苗菌,并利用人类幽门螺杆菌感染的小鼠模型研究疫苗菌对Hp感染的免疫保护作用。方法利用分子克隆技术构建携带Hp-NAP基因的重组原核表达质粒pTrc99A-NAP,经PCR及酶切鉴定后测定其基因序列,并与美国国立医学图书馆基因文库中相关序列的同源性进行比较。重组质粒pTrc99A-NAP转化减毒伤寒沙门菌SL3261,培养后筛选阳性菌落,抽提质粒进行PCR及酶切鉴定。表达的Hp-NAP蛋白用SDS-PAGE和West-blotting进行鉴定,用薄层扫描分析蛋白含量。C57BL/6小鼠随机分成4组,分别灌胃给予生理盐水(A组)、减毒鼠伤寒沙门菌SL3261(B组)、携带pTrc99A的SL326组(C组)、携带pTrc99A-NAP的SL326组重(D组)。免疫后4周,予Hp攻击,攻击菌量107CFU/只,灌胃2次,每日1次。攻击4周后处死动物,取胃组织分别行改良Giemsa染色及定量细菌培养,观察Hp定植情况。结果核苷酸序列测定及同源性分析证实,克隆的Hp-NAP基因与GenBank中相关序列的同源性为98%(397/402),氨基酸序列的同源性为98%(131/133)。pTrc99A-NAP转化的减毒沙门菌,可表达Mr约15000的Hp-NAP蛋白,表达量约占菌体蛋白量的37·5%;表达的新生蛋白能与小鼠抗Hp血清特异性反应,具有良好的免疫原性。同空白对照组、SL3261组和SL3261(pTrc99A)组相比,表达Hp-NAP的疫苗株组实验动物的胃组织Hp定植密度明显下降(P<0·05)。结论成功构建表达Hp-NAP的减毒沙门疫苗菌;重组疫苗菌对小鼠Hp感染具有一定免疫预防作用,可作为未来多组分重组减毒沙门疫苗菌的侯选菌株之一。     

表达幽门螺杆菌尿素酶B亚单位基因减毒鼠伤寒沙门疫苗菌的构建和鉴定

目的构建表达幽门螺杆菌(Helicobacter     

携带幽门螺杆菌热休克蛋白B亚单位基因重组活减毒鼠伤寒沙门疫苗菌的构建和鉴定

背景：幽门螺杆菌(h.pylori)是慢性活动性胃炎和消化性溃疡的重要致病菌，以减毒鼠伤寒沙门菌为载体构建活疫苗己成为探索新型H．pylori疫苗的重要途径。目的：构建携带H．pylori热休克蛋白B亚单位(hspB)基因的重组活减毒鼠伤寒沙门疫苗菌。方法：应用基因工程技术将1640bp的hspB基因克隆入原核表达质粒pTrc99A。对重组质粒进行序列测定，并将测序结果与基因文库中H.pylori-hspB的基因和蛋白序列进行BLAST分析，再将重组质粒导入活减毒鼠伤寒沙门菌SL3261。结果：重组质粒经聚合酶链反应(PCR)和双酶切，证实构建了携带hspB基因的重组原核表达质粒pTrc99A—hspB，后者成功转化活减毒鼠伤寒沙门菌SL3261。所构建的重组质粒pTrc99A—hspB中所含的H.pylori-hspB与基因文库中量H.pylori-hspB基因和蛋白的同源性均为97％。结论：成功构建并鉴定了携带量H.pylori-hspB基因的重组活减毒鼠伤寒沙门疫苗菌，为研制H.pylori口服疫苗奠定了基础。     

重组减毒沙门菌尿素酶B亚单位和过氧化氢酶疫苗治疗幽门螺杆菌感染的实验研究

目的　利用人类幽门螺杆菌 (Hp)感染的小鼠模型研究重组减毒沙门菌尿素酶B亚单位和过氧化氢酶疫苗在治疗Hp感染中的作用。 方法　将 30只二级C5 7BL/ 6小鼠随机分成 3组 ,通过灌胃方法每只小鼠均用活力良好的Hp菌株隔日攻击 2次。在第二次Hp攻击后 4周 ,分别给予重组减毒沙门菌尿素酶B亚单位疫苗 (A组 )、过氧化氢酶疫苗 (B组 )和生理盐水 (C组 )灌胃 1次 ,4周后处死动物 ,取胃组织分别行尿素酶试验、改良Giemsa染色及定量细菌培养 ,观察Hp定植情况。行HE染色观察胃黏膜组织炎症情况。取脾组织行淋巴细胞增殖试验。结果　C组小鼠Hp定植密度为 1.92× 10 6CFU/g胃组织 ,A组和B组小鼠分别为 1.5 8× 10 5CFU/g和 4 .88× 10 5CFU/g胃组织 ,两个治疗组的定植密度明显降低 (P <0 .0 5 )。治疗组与对照组胃黏膜均无明显炎症反应。治疗组脾淋巴细胞增殖试验阳性。结论　重组减毒沙门菌尿素酶B亚单位疫苗和过氧化氢酶疫苗对Hp感染有治疗作用。     

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。     

携带幽门螺杆菌hpaA基因减毒鼠伤寒沙门疫苗菌的构建

构建携带幽门螺杆菌（H.pylori)hpaA基因的重组活减毒鼠伤沙门疫苗菌。方法：用分子生物学方法将hpaA基因克隆入原核表达质粒pTrc99A,并进行核苷酸测序,重组质粒经再导入活减毒鼠伤寒沙门菌SL3261,提取重组菌苗质粒,聚合酶链反应（PCR）和酶切鉴定,筛选目的克隆。结果：经PCR和酶切证实,构建了携带hpaA基因（560bp)的重组核表达质粒pTrc99A-hpaA,并将后者成功转化活减毒鼠伤寒沙门菌SL3261。结论：S我建并鉴定了携带H.pylorihpaA基因的重组活减毒鼠伤寒沙门疫苗菌,为探索制备H.ylori口服份活疫苗奠定了基础。     

幽门螺杆菌感染的免疫致病及疫苗的保护作用机制

幽门螺杆菌是定植于人体胃粘膜的重要致病菌。在其多种致病中,由机体对细菌的免疫反应导致的免疫／炎症性组织损伤占有重要地位,包括ＩｇＧ,ＩｇＥ抗体介导的体液免疫,辅助性Ｔ细胞介导的细胞免疫,以及由于Ｈｐ抗原与机体组织抗原的“分子模拟”导致的自身免疫。     

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。     

幽门螺杆菌疫苗免疫保护机制及其研究进展

幽门螺杆菌(H.pylori)是慢性活动性胃炎和消化性溃疡的主要病因,与胃恶性肿瘤的发生密切相关。根除H.py-lori对上述疾病的好转及治愈至关重要。目前常用药物(抗生素加秘剂或质子泵抑制剂)根除H.pylori,但因副作用大。复发率高及耐药菌株出现等使药物根除其的疗效受到影响。许多学者证实了免疫方法的可行性,但关于免疫保护的机制尚无定论。本文就H .pylori疫苗的免疫保护机制及其研究进展作一综述。保护机制一。抗体的作用病原微生物感染肠道后可刺激机体免疫反应,产生抗体分泌细胞,分泌以IgA为主的抗体,从而杀伤病原微生物。这是粘膜免疫…     

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.     

Systemic immune responses to oral administration of recombinant attenuated Salmonella typhimurium expressing Helicobacter pylori urease in mice

AIM: To evaluate whether attenuated Salmonella typhimurium producing Helicobacter pylori(H pylori) urease subunit B (UreB) could induce systemic immune responses against Hpylori infection. METHODS: Attenuated 5. typhimurium SL3261 was used as a live carrier of plasmid pTC01-UreB, which encodes recombinant H pylori UreB protein. Balb/c mice were given oral immunization with two doses of SL3261/pTC01-UreB at a 3-wk interval. Twelve weeks after oral immunization of mice, serum IgG antibodies were evaluated by ELISA assay. Gamma interferon (IFN-γ) and interleukin 10 (IL-10) in the supernatant of spleen cell culture were also assessed by ELISA. RESULTS: After oral immunization of mice, serum specific IgG antibodies against UreB in vaccine group were much higher than that in PBS and native Salmonella SL3261 control groups (A450, 0.373±0.100 vs 0.053±0.022, 0.142±0.039, respectively, P<0.01). Moreover, IFN-γ in vaccine group was on average 167.53±29.93 pg/mL, which showed a significant increase vs that of PBS control group (35.68±3.55 pg/mL, P<0.01). There was also a tremendous increase of IL-10 in vaccine group compared to PBS and SL3261 control groups (275.13±27.65 pg/mL vs 56.00±7.15 pg/mL, 68.02±15.03 pg/mL, respectively, P<0.01). In addition, no obvious side effects in mice and no change in gastric inflammation were observed. CONCLUSION: The multiple oral immunizations with the attenuated 5. typhimurium expressing Hpylori UreB could induce significant systemic immune responses, suggesting it may be used as oral vaccine against H pylori infection.     

Oral immunization with a live recombinant attenuated Salmonella typhimurium protects mice against Toxoplasma gondii

The natural site of infection for T. gondii is the mucosal surface of the intestine, so the protective immunity obtained after natural infection with T. gondii points to the importance of developing a vaccine that stimulates mucosal defences. In this study, an aroA- and aroD- attenuated strain of Salmonella typhimurium (BRD509) has been used to deliver the recombinant eukaryotic plasmid pSAG(1-2)/CTA2/B expressing a multi-antigenic gene encoding SAG1 and SAG2 of T. gondii linked to A2/B subunits of cholera toxin as a candidate oral T. gondii vaccine. Immunoblot analysis showed compound gene expression in HeLa cells in vitro and intragastric immunization of mice with the recombinant salmonella resulted in the induction of humoral and Th1 type cellular immune responses and afforded protection against RH strain T. gondii challenge. Anti-T. gondii IgG values increased markedly in the BRD509/pSAG(1-2)-CTA2/B immunized group; these values were significantly higher than those in the negative controls (P = 0.008). With CTA2/B genetic adjuvant, the T. gondii-specific response was predominantly Th1, indicating that the CTA(2)/B genetic adjuvant was able to overcome the strong Th2-bias of the antigen (IgG2a > IgG1). Antigen-specific T cell proliferative responses and CTL activity were significantly enhanced when cholera toxin CTA2/B genetic adjuvant was used (P = 0.009; P = 0.006). Culture supernatants from antigen-stimulated splenocytes from mice in these groups were also examined by ELISA for Th1- and Th2-type cytokines; mean IFN-gamma levels produced after oral immunization with BRD509/pSAG(1-2)-CTA2/B were about nine-fold higher than after immunization with BRD509/pSAG(1-2) (P = 0.007). On the other hand, the levels of IL-4 were low for all groups and no increase was seen in the presence of CTA2/B genetic adjuvant. When the immunized mice were intraperitoneally challenged with 10(3) tachyzoites of the highly virulent RH strain, the survival time of the mice immunized with BRD509/pSAG(1-2)-CTA2/B was markedly longer than other groups (P = 0.003) and a 40% survival rate was achieved. This is the first report that demonstrates that an oral attenuated salmonella DNA vaccine can induce protective immunity against the acute phase of T. gondii infection.     

Construction of an attenuated Salmonella typhimurium vaccine carrying the Helicobacter pylori hpaA gene

OBJECTIVE : Helicobacter pylori is documented to have infected more than half of the world’s population. It colonizes the human stomach and is associated with the development of chronic active gastritis, peptic ulcer disease, gastric adenocarcinoma and gastric mucosa‐associated lymphoid tissue (MALT) lymphoma. Immunization against this bacterium represents a cost‐effective strategy to reduce global gastric cancer rates and would have a major impact on H. pylori‐related peptic ulcer disease. HpaA, a subunit protein of H. pylori adhesin, is known to be the pathogenic factor and attenuated Salmonella typhimurium is a live vaccine vector. The present study aimed at constructing and identifying a live attenuated S. typhimurium vaccine carrying the hpaA gene of H. pylori. METHODS : The hpaA gene was amplified from H. pylori genomic DNA by polymerase chain reaction (PCR) and cloned into the NcoI‐SalI site of the prokaryotic expression plasmid pTrc99A. After sequence analysis of the hpaA gene, the identified recombinant plasmid was then used to transform a live attenuated S. typhimurium, namely SL3261, and positive clones were screened by PCR and restriction enzyme digestion. RESULTS : Confirmed by PCR, restriction enzyme digestion and sequence analysis, a recombinant prokaryotic expression plasmid, namely pTrc99A‐hpaA, harboring approximately 560 bp hpaA was constructed and the recombinant plasmid was then successfully introduced into a live attenuated S. typhimurium SL3261. CONCLUSION : A recombinant live attenuated S. typhimurium vaccine harboring the H. pylori hpaA gene was constructed and identified. This work will help develop an oral recombinant live vaccine against H. pylori infection.     

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.     

重组幽门螺杆菌疫苗诱导小鼠产生THL型保护性应答及免疫后胃炎

目的 研究以减毒鼠伤寒沙门氏菌为载体构建的重组幽门螺杆素酶A亚单位及过氧化氢酶、／尿素酶A亚单位融合蛋白疫苗对小鼠的免疫保护作用及相关机制。方法 将表达幽门螺杆菌(Helicobacter pylori,HP)尿素酶A亚单位(UreA)、过氧化氢酶(KatA)及尿素酶A亚单位／过氧化氢酶融合蛋白(Urea／KatA)的减毒鼠伤寒沙门氏菌(Salmonella typhimurium)分别灌胃免疫小鼠,另设单纯减毒鼠伤寒沙门氏菌免疫鼠和生理盐水免疫鼠为对照。免疫4周后以幽门螺杆菌活菌攻击,比较各组胃粘膜的幽门螺杆菌定植密度,攻击前后三清中抗幽门螺杆菌抗体IgGI,IgG2a和IgA的变化,脾脏和胃粘膜中γ干扰素(IFN-γ)和白介素4(IL-4)mRNA表达变化及胃粘膜炎症情况。结果(1)KarA及Urea／Kara组的幽门螺杆菌定植密度显下降,Urea组下降不明显。(2)和生理盐水组相比攻击前各鼠伤寒沙门氏菌免疫组IgGI,IgG2a均轻度升高而IgA无变化,攻击后各鼠伤寒沙门氏菌免疫组IgG2a升高显,KatA和Urea／KatA组尤为明显,而IgGI和IgA的升高无统计学差异。(3)胃粘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鼹伤寒沙门氏菌免疫组。IL-4在攻击前后各组均无表达。(4)脾IFN-γ和IL-4在所有组攻击前后均全部表达。(5)攻击前各组鼠胃粘膜仅有散在少量白细胞,攻击后各鼠伤寒沙门氏菌免疫组出现明显以淋巴及单核细胞浸润为特征的炎症反应。结论以减毒鼠伤察沙门氏菌为载体构建的重组幽门螺杆菌疫苗(Katk、urek/kalk)可在小鼠体内诱导出以TH1反应为主并伴随免疫后胃炎均保护性免疫应答。     

Construction and identification of a recombinant live attenuated Salmonella typhimurium vaccine strain carrying Helicobacter pylori heat shock protein subunit B gene

OBJECTIVE: Because Helicobacter pylori is the principal cause of chronic active gastritis and peptic ulcer disease, the present study explored the possibility of constructing a recombinant live attenuated S. typhimurium vaccine strain carrying H. pylori heat shock protein subunit B (HspB) gene. METHODS: A 1640‐bp HspB gene was cloned into a prokaryotic expression plasmid pTrc99A. After sequence analysis, the result was compared with the sequence of H. pylori‐HspB gene and protein provided by the Genebank using BLAST analysis. The identified recombinant plasmid was then introduced into a live attenuated S. typhimurium strain SL3261. RESULTS: Using polymerase chain reaction (PCR) and restriction enzyme digestion, a recombinant pro­karyotic expression plasmid pTrc99A‐HspB harboring the HspB gene was constructed and successfully introduced into a live attenuated S. typhimurium strain SL3261. Most of the H. pylori‐HspB in the recombinant plasmid pTrc99A‐HspB was consistent with the H. pylori‐HspB sequence provided by the Genebank. The exchange of A/T or C/G in the cloned sequence hardly changed the encoded amino acids; the homology for both the genes and proteins of H. pylori SS1 strain was 97%; the homology with other common H. pylori strains J99 and 26695 was 96% for both. CONCLUSION: A recombinant live attenuated S. typhimurium vaccine strain harboring H. pylori‐HspB gene was successfully constructed and verified, which may help in the development of an oral vaccine against H. pylori infection.