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。     

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。     

胃癌MG7-Ag模拟表位减毒鼠伤寒沙门菌活菌疫苗研制及免疫效果

目的以减毒鼠伤寒沙门菌为疫苗载体,构建基于胃癌MG7-Ag模拟表位的口服活菌疫苗,观察Balb/c小鼠口服免疫后诱导的体液和细胞免疫反应。方法构建MG7-Ag模拟表位和HBcAg融合基因的原核表达载体,将其转入减毒鼠伤寒沙门菌得到模拟表位的口服活菌疫苗。用构建的口服鼠伤寒沙门菌疫苗免疫Balb/c小鼠,以PBS和携带pYA3341空载体的减毒鼠伤寒沙门菌作为对照。初次口服接种6周后应用ELISA法检测小鼠血清中抗MG7-Ag抗体的滴度。初次免疫后8周时,取免疫鼠脾细胞,以51Cr释放法检测小鼠脾淋巴细胞对靶细胞的杀伤效果,用表达MG7-Ag的小鼠艾氏腹水瘤细胞进行肿瘤攻击实验,观察疫苗对小鼠的保护作用。结果口服疫苗免疫小鼠后疫苗免疫组小鼠血清抗MG7-Ag抗体较PBS和空载体对照组显著增高,三组分别为0.954±0.040,0.653±0.018和0.692±0.012(P<0.01)。各组小鼠脾淋巴细胞体外杀伤实验未见显著差异,三组分别为234.7±27.7,183.4±26.0和195.7±8.0(P>0.05)。小鼠艾氏腹水瘤的攻击实验显示:疫苗免疫组中5只小鼠有1只未见肿瘤形成,而对照组小鼠则全部成瘤,且免疫组的成瘤重量为(0.05±0.01)g,明显小于两对照组的(0.10±0.04)和(0.09±0.04)g(P<0.01)。结论以胃癌MG7-Ag模拟表位为基础的口服减毒鼠伤寒沙门菌疫苗具有免疫原性,可诱导小鼠产生抗肿瘤免疫,并具有一定的保护作用。     

幽门螺杆菌中性粒细胞激活蛋白核酸疫苗实验研究

目的 构建幽门螺杆菌(Hp)中性粒细胞激活蛋白(Hp-NAP)口服DNA疫苗，初步评价其免疫治疗作用，为Hp疫苗研制奠定基础。方法 PCR扩增全长Hp-NAP基因(napA)，测序并同源性分析后，亚克隆入真核表达载体pIRES，双酶切并PCR鉴定。将重组质粒plRES-napA转化减毒鼠伤寒沙门菌SL7207，口服接种长期感染Hp之BALB／c小鼠，观察疗效。结果PCR扩增出-435bp产物，序列分析表明，所克隆序列与CenBank中SSl-napA核苷酸及蛋白质同源性均＞98％。PCR及双酶切证实，成功构建了携带napA的重组减毒鼠伤寒沙门菌DNA疫苗。疫苗接种后4周，治疗组3／4小鼠快速尿素酶检测阴性，对照组均阳性(P=0．0476)；治疗组血清抗Hp-NAP抗体效价明显升高。结论 成功构建了具有较好免疫治疗作用的Hp—NAP口服重组DNA疫苗，为进一步研制多价抗HpDNA疫苗奠定了基础。     

大肠杆菌不耐热肠毒素无毒突变体mLT63毒性检测及佐剂效果研究

目的对已构建的大肠杆菌不耐热肠毒素无毒突变体mLT63进行表达、纯化、毒性检测,并对其佐剂活性进行研究。方法采用已知的最佳诱导表达条件进行诱导表达,诱导表达产物经亲和层析、纯化浓缩后,进行毒性检测。将纯化后的mLT63联合幽门螺旋杆菌(Hp)亚单位疫苗UreB、Omp11经口途径免疫BALB/c小鼠,免疫后取血清、胃组织提取液、粪便提取液进行ELISA试验,检测其中的特异性抗体水平,将结果进行统计分析。结果经家兔回肠袢毒性试验验证本室构建大肠杆菌不耐热肠毒素无毒突变体mLT63没有毒性,mLT63联合Hp亚单位疫苗UreB、Omp11免疫小鼠实验证实mLT63具有佐剂活性。结论本室构建、表达的大肠杆菌不耐热肠毒素无毒突变体mLT63无毒,并具有免疫佐剂的活性。     

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。     

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。     

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。     

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     

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。     

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.     

Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immunogenicity

INTRODUCTION The discovery of H pylori has brought about a revolution in the research of etiological factors of gastrointestinal diseases[1]. It has been confirmed that H pylori is the main cause of chronic superficial gastritis, chronic active gastritis …