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     

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     

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。     

幽门螺杆菌疫苗口服免疫小鼠后胃肠免疫应答研究

目的 观测Hp全菌抗原和粘膜佐剂LT口服免疫Balb/c小鼠后的胃肠免疫反应。方法 采用ELISPOT和ELISA法检测了胃粘膜、PP结抗原特异性形成细胞(AFC)和小肠粘液sIgA。结果 抗原免疫组、抗原 佐剂组胃肠粘膜抗原特异性sIgA-AFC、IgG-AFC数量明显增加,尤以sIgA-AFC为甚,并且抗原 佐剂组明显高于单纯抗原组和对照组;小肠粘液特异sIgA两免疫组明显高于对照组。结论 口服免疫可有效诱导胃肠道粘膜免疫,局部sIgA可能在抗Hp感染中具有重要作用。     

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。     

以白细胞介素-2为免疫佐剂的幽门螺杆菌尿素酶B亚单位核酸疫苗的构建及其免疫保护作用

背景：细胞因子具有免疫佐剂效应，但将其用作幽门螺杆菌（H．pylori）核酸疫苗佐剂的研究报道尚少。目的：构建同时含H．pylori尿素酶B亚单位（ureB）基因和小鼠白细胞介素-2（IL-2）基因的重组活减毒鼠伤寒沙门菌核酸疫苗，体外鉴定其表达蛋白的免疫原性，体内检测其对H．priori感染的免疫保护作用。方法：以聚合酶链反应（PCR）扩增H．priori ureB基因和小鼠IL-2基因，分别插入pUCmT载体，测序，通过一系列酶切、连接反应分别克隆人真核表达载体pIRES，酶切、PCR鉴定；重组质粒pIRES-ureB和pIRES-ureB-IL-2分别转化减毒鼠伤寒沙门菌LB5000，抽提质粒，进一步转化终宿主菌SL7207，反复传代培养。以Lipofectamine^TM2000将重组质粒分别体外转染COS-7细胞，蛋白质印迹法检测表达蛋白的免疫原性。以疫苗菌经口接种小鼠，4周后予H．pylori攻击，攻击后4周鉴定H．pylori感染状况。结果：测序结果显示扩增出的ureB和IL-2基因序列与GenBank中的H．pylori ureB和小鼠IL-2序列一致，酶切、PCR鉴定证实ureB和IL-2基因已克隆人pIRES载体，并成功构建了稳定的含H．pylori ureB和小鼠IL-2基因的重组活减毒鼠伤寒沙门菌核酸疫苗。蛋白质印迹法显示，pIRES-ureB-IL-2转染的COS-7细胞表达特异性UreB和IL-2蛋白。体内实验显示，疫苗接种组小鼠的免疫保护率显著高于PBS对照组（P〈0．01），其中ureB-IL-2疫苗组又显著高于ureB疫苗组（87．5％对62．5％，P〈0．05）。结论：成功构建了编码H．pylori ureB和免疫佐剂IL-2基因的重组活减毒鼠伤寒沙门菌核酸疫苗，体外实验证实其可表达具有免疫原性的抗原蛋白和佐剂蛋白，体内实验证实其对小鼠H．pylori感染具有免疫保护性，免疫佐剂IL-2可提高核酸疫苗的免疫保护率。     

幽门螺杆菌疫苗免疫小鼠的Th免疫应答及作用

目的 研究幽门螺杆菌(Helicobacter pylori,Hp)疫苗诱导小鼠保护性免疫应答的机制。方法 以霍乱毒素(CT)加Hp全菌超声粉碎抗原免疫小鼠，并设CT组和生理盐水(NS)组为对照。免疫4周后以Hp攻击；在攻击前后不同时间点分批处死小鼠，比较免疫组和对照组胃黏膜唧定植及各项免疫指标的变化。结果 (1)攻击后5、26周后免疫组和CT组、NS组相比胃黏膜Hp定植量明显降低。(2)各时间点免疫组IgG1、IgG2a、IgA均高于NS组、CT组。(3)攻击前和攻击后5周免疫组和CT组胃黏膜Th1型细胞因子表达较NS组明显升高。攻击后26周免疫组和CT组除白细胞介素(IL)-12外，干扰素(IFN)-γ和IL-2表达均明显降低，而NS组Thl型细胞因子表达明显升高。(4)Th2型细胞因子在攻击前免疫组和CT组胃黏膜均有轻度表达；攻击后5周免疫组和CT组IL-4和IL-10均无表达，IL-6表达低于NS组；攻击后26周免疫组IL-4和IL-10再次表达，CT组仅表达IL-10，IL-6表达各组无明显差异。(5)各细胞因子在所有组脾脏攻击前后各时间点表达无差异。(6)与NS组相比，攻击后5周免疫组鼠胃黏膜出现明显的炎症反应，CT组也有轻度炎症反应；攻击后26周NS组炎症加重而免疫组则有所减轻。结论 (1)以CT为佐剂的Hp疫苗可诱导Thl和Th2混合型初始免疫应答；(2)Hp攻击后早期增强的Thl反应起主要免疫保护作用同时导致免疫后胃炎；晚期随Th2反应的增强Thl反应和免疫后胃炎强度减弱。     

幽门螺杆菌疫苗研究现状

接种疫苗通常被认为是预防和治疗幽门螺杆菌（Hp）感染及其相关疾病最有效的方法。因此，Hp疫苗可以是：（1）预防性的，也就是在感染Hp前给予疫苗。（2）治疗性的，即在感染Hp后，给予疫苗，这样做的目的是减少Hp感染定植的数量甚至根除Hp感染。至今已在免疫途径、剂型（抗原和佐剂等方面）的选择、疫苗筛选评价平台、疫苗免疫机制等方面进行了广泛而深入的研究。本文就Hp疫苗的研究现状从以上方面作一综述。     

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。     

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.     

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。     

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。     

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.     

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.