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UreA和KatA双价抗原的鼠伤寒菌疫苗株的初步构建

目的　构建表达幽门螺杆菌 (Hp)尿素酶A亚单位 (UreA)和过氧化氢酶 (KatA)的减毒鼠伤寒沙门氏菌疫苗株 ,对研制抗Hp感染重组疫苗的可行性进行探讨。方法　PCR方法从Hp基因组中扩增出ureA和katA片段 ,将其插入pGSTag表达载体中 ,重组质粒再转入减毒鼠伤寒沙门氏菌。结果　对重组质粒进行限制酶切分析和PCR检测 ,证实两种基因已被克隆入pGSTag ,并转入减毒鼠伤寒沙门氏菌。结论　本研究成功地将表达HpureA和katA融合基因的重组质粒转入减毒鼠伤寒沙门氏菌中 ,构建了UreA/KatA双价口服活疫苗 ,为进一步研究其在预防Hp感染中的作用奠定了基础     

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。     

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。     

幽门螺杆菌hpaA核酸疫苗的构建及免疫原性检测

目的 构建含幽门螺杆菌(Hp)hpaA基因的核酸疫苗。方法 抽提Hp标准菌株CCUG17874基因组DNA，应用聚合酶链式反应(PCR)技术从基因组DNA扩增hpaA基因，克隆入pUCmT载体，检测hpaA基因序列，经过一系列酶切、连接反应将其克隆入真核表达载体plRES，转入大肠杆菌，筛选阳性克隆，通过PCR和酶切反应鉴定。通过脂质体法将构建好的重组载体pIRES-hpaA转染COS-7细胞，SDS-PAGE及Western印迹法检测pIRES-hpaA表达HpaA蛋白的免疫原性。结果 成功扩增出长约750bp的hpaA基因．测序结果表明扩增出的hpaA基因与Hp hpaA序列一致，PCR和酶切鉴定结果证实hpaA基因克隆入真核表达载体pIRES，成功构建了含hpaA基因的Hp核酸疫苗pIRES-hpaA，并经Western印迹法检测到特异性蛋白条带。结论 构建了hpaA基因的Hp核酸疫苗，为进一步探索其免疫作用奠定了基础。     

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。     

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。     

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UreA和KatA双价抗原的鼠伤寒菌疫苗株的初 …

目的 构建表达幽门螺杆菌（Ｈｐ）尿素酶Ａ亚单位（ＵｒｅＡ）和过氧化氢酶（ＫａｔＡ）的减毒鼠伤寒沙门氏菌疫苗株,对研制抗Ｈｐ感染重组疫苗的可行性进行探讨。方法 ＰＣＲ方法从Ｈｐ基因组中扩增出ｕｒｅＡ和ｋａｔＡ片段,将其插入ｐＧＳＴａｇ表达载体中,重组质粒再转入减毒鼠伤寒沙门氏菌,结果 对重组质粒进行限制酶切分析和ＰＣＲ检测,证实两种基因已被克隆入ｐＧＳＴａｇ,并转入减毒鼠伤寒沙门氏菌。结论 本研究成     

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 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 …     

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 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.     

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     

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。