多组分重组减毒沙门疫苗菌对幽门螺杆菌感染的免疫保护作用

目的　利用人类幽门螺杆菌 (Helicobacterpylori,Hp)感染的小鼠模型研究多组分重组减毒沙门疫苗菌对幽门螺杆菌感染的免疫保护作用。方法　将二级C5 7BL/ 6小鼠随机分成 6组 ,通过灌胃方法分别给予生理盐水 (A组 )、PBS(B组 )、减毒鼠伤寒沙门菌SL32 6 1(C组 )、重组减毒鼠伤寒沙门菌pTrc99A -ureB +pGSTag -katA(D组 )、重组减毒鼠伤寒沙门菌pTrc99A -ureB +pTrc99A -HPaA(F组 )及重组减毒鼠伤寒沙门菌 pTrc99A -ureB +pGSTag -katA +pTrc99A -HPaA(F组 )。免疫后 4周 ,予Hp进行攻击 (A组不攻击 )。攻击菌量 10 7CFU/只 ,灌胃 2次 ,每日 1。再 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染色观察胃粘膜组织炎症情况。取脾组织行淋巴细胞增殖试验。结果　A组小鼠Hp定植密度为 0 ,B组为 9 4 9× 10 6CFU/ g胃组织 ,C组为 1 4 2× 10 6CFU/ g胃组织 ,D组、E组和F组小鼠分别为 2 92× 10 5CFU/g、5 5 1× 10 5CFU/g和 2 16× 10 5CFU/g胃组织 ,C组、D组、E组和F组与A组和B组相比定植密度均明显降低 (P <0 0 5 )。免疫组与对照组胃粘膜均无明显炎症反应。免疫组脾淋巴细胞增殖试验阳性。结论　口服多组分重组减毒沙门疫苗菌对小鼠Hp感染具有一定免     

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     

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。     

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。     

口服疫苗预防幽门螺杆菌感染的实验研究

目的：利用感染人类幽门螺杆菌（Ｈ．ｐｙｌｏｒｉ）的小鼠模型,研究Ｈ．ｐｙｌｏｒｉ粗制抗原与霍乱毒素（ＣＴ）或重组霍乱毒素Ｂ亚单位（ｒＣＴＢ）组成的口服疫苗预防ｈ．ｐｙｌｏｒｉ感染的作用。方法：无特殊病原菌级Ｃ５７ＢＬ／６小鼠分为４组,通过灌胃方法分别予各组小鼠以Ｈ．ｐｙｌｏｒｉ超声粉碎抗原１ｍｇ加ＣＴ１０μｇ、Ｈ．ｐｙｌｏｒｉ超声粉碎抗原１ｍｇ加ｒＣＴＢ１０μｇ、单纯Ｈ．ｐｙｌｏｒｉ超声粉碎抗原１     

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。     

幽门螺杆菌核酸疫苗的研究

幽门螺杆菌(H.pylori)与多种胃肠道疾病密切相关,在人群中的感染率较高,目前的"三联"或"四联"抗H.pylori治疗由于治疗费用、抗生素耐药等受到一定限制,为了有效控制H.pylori感染,疫苗尤其是核酸疫苗的研制逐渐成为该研究领域的热点,被认为是防治H.pylori最有前景的方法.     

幽门螺杆菌粘膜疫苗的研究进展

胃窦部幽门螺杆菌(Helicobacter pylori,Hp)感染可引起许多胃十二指肠疾病,包括B型(胃窦)胃炎、消化性溃疡、胃粘膜相关淋巴组织淋巴瘤和胃癌.临床上对Hp感染的治疗主要采用以抗生素为主的治疗方案,虽可在一定程度治愈Hp感染,但不能预防Hp再感染,同时抗生素的使用可导致Hp耐药菌株的产生和带来许多严重的副作用.而Hp疫苗可以解决这一难题,使得Hp疫苗接种成为了根治Hp感染和预防再感染的唯一方法.目前,Hp疫苗的研究进展迅速,粘膜疫苗以其有效的保护性免疫效果成为这一研究的热点.我们对Hp粘膜疫苗的组成、接种途径、免疫效果及保护性免疫机制等方面的研究进展加一综述.     

幽门螺杆菌感染的免疫防治

回顾和总结了幽门螺杆蓖疫苗研制中的一些主要问题如免疫原及佐剂的选择,动物模型的建立,免疫途径及疫苗转送系统的确定以及免疫保护机制的探讨,并对今后的研究方向作了进一步展望。     

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。     

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.     

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.     

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.     

Construction of recombinant attenuated Salmonella typhimurium DNA vaccine expressing H pylori ureB and IL-2

AIM: To construct a recombinant live attenuated Salm-onella typhimurium DNA vaccine encoding H pylori ureB gene and mouse IL-2 gene and to detect its immunogenicity in vitro and in vivo. METHODS: H pylori ureB and mouse IL-2 gene fragments were amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified ureB and IL-2 genes was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions resulting in pIRES-ureB and pIRES-ureB-IL-2. The recombinant plasmids were used to transform competent E. coli DH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-ureB and pIRES-ureB-IL-2 were used to transform LB5000 and the recombinant plasmids extracted from LB5000 were finally introduced into the final host SL7207. After that, recombinant strains were grown in vitro repeatedly. In order to detect the immunogenicity of the vaccine in vitro, pIRES-ureB and pIRES-ureB-IL-2 were transfected to COS-7 cells using LipofectamineTM2000, the immunogenicity of expressed UreB and IL-2 proteins was assayed with SDS-PAGE and Western blot. C57BL/6 mice were orally immunized with 1 × 108 recombinant attenuated Salmonella typhimurium DNA vaccine. Four weeks after vaccination, mice were challenged with 1 × 107 CFU of live H pylori SS1. Mice were sacrificed and the stomach was isolated for examination of H pylori 4 wk post-challenge. RESULTS: The 1700 base pair ureB gene fragment amplified from the genomic DNA was consistent with the sequence of H pylori ureB by sequence analysis. The amplified 510 base pair fragment was consistentwith the sequence of mouse IL-2 in gene bank. It was confirmed by PCR and restriction enzyme digestion that H pylori ureB and mouse IL-2 genes were inserted into the eukaryotic expression vector pIRES. The experiments in vitro showed that stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying ureB and IL-2 genes was successfully constructed and the specific strips of UreB and IL-2 expressed by recombinant plasmids were detected through Western blot. Study in vivo showed that the positive rate of rapid urease test of the immunized group including ureB and ureB-IL-2 was 37.5% and 12.5% respectively, and was significantly lower than that (100%) in the control group (P < 0.01). CONCLUSION: Recombinant attenuated Salmonella typhimurium DNA vaccine expressing UreB protein and IL-2 protein with immunogenicity can be constructed. It can protect mice against H pylori infection, which may help the development of a human-use H pylori DNA vaccine.