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。     

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。     

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。     

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     

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     

重组原核表达质粒PTrc99A-ureB/hlyE 的构建和表达

目的 构建表达幽门螺杆菌（Helicobacter pylori,Hp）尿素酶B亚单位（ureB）基因与大肠杆菌K-12株hlyE基因融合表达的原核表达质粒pTcr99A-hlyE.并表达。方法 用PCR扩增hlyE基因，经本科切连接反应将其克隆入重组原核表达质粒并测序，与GenBank中的核酸和蛋白序列进行BLAST分析，重组的阳性克隆经IPTG诱导培养，SDS-PAGE电泳进行表达分析，薄层扫描分析融合蛋白的含量。结果 对重组质粒进行酶切和PCR检测，证明构建了携带hlyE及ureB基因的重组原核表达质粒pTrc99A-ureB/hlyE,核酸序列测定及同源性分析证实所构建的原核表达质粒pTrc99A-ureB/hlyE中所含的Hp-ureB及hlyE与GeneBank中的Hp-ureB和hlyE序列的同源性分别为97.42%（1663/1707）、99%（910/918）。重组细菌JM109可表达约100kD的融合蛋白含量的15.5%。结论 构建并鉴定了原核表达质粒pTrc99A-hlyE并高效表达，为研究Hp口服疫苗奠定了基础。     

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     

Helicobacter pylori

PURPOSE OF REVIEW: Helicobacter pylori is an important human pathogen, responsible for most peptic ulcer disease, gastritis and gastric malignancies. H. pylori has several unique features: it is highly adapted for gastric colonization, yet it produces clinical consequences in a small minority, its genome is known, and it is the only bacterium strongly associated with cancer. H. pylori is therefore of great interest to clinicians and researchers of many, often disparate, disciplines. We highlight recent advances in this fast changing field from many different areas. RECENT FINDINGS: The major contentious clinical issues relate to the synergistic gastrotoxic interactions of H. pylori with non-steroidal anti-inflammatory drugs, and a possible association of H. pylori with atherosclerotic events. Accumulating evidence implicates genetic variation in the inflammatory response to H. pylori in the etiology of the increased risk of gastric cancer after H. pylori infection. Studies of pathogenesis have been aided by increasingly sophisticated murine models. The effects in gastric epithelial cells of two of the major virulence factors (genes within the cag pathogenicity island and the vacuolating cytotoxin, VacA) of H. pylori illustrate the complex network of cellular reactions activated by H. pylori. The metabolism of H. pylori is dependent on the availability of hydrogen. SUMMARY: Basic science research into H. pylori continues to elucidate the mechanisms by which H. pylori infection causes disease. These findings have implications for the design of novel therapies and for improving clinical strategies to identify at-risk individuals. Many are also worthy of consideration for other epithelial-microbial interactions.     

Helicobacter pylori

The diagnostics and treatment of Helicobacter pylori infections have substantially changed in recent years. Instead of a general test-and-treat strategy, differentiated treatment methods are increasingly being used. Practical problems in many cases were that a useful combination was often not employed after the failure of an initial antibiotic treatment. In 2009 new guidelines on the diagnostics and treatment of Helicobacter pylori infections were published. Various expert groups from gastro-enterology, microbiology and rheumatology provided new general frameworks and concrete treatment suggestions for Helicobacter pylori infections of the stomach. The statements are grouped according to ?should“ and ?can“ recommendations and the consensus opinion is divided into various subgroups. The new S3 guidelines specify the therapy indications with respect to first and second line procedures and now give different durations of therapy (7 days for first line, 10 days for second line after treatment failure) as well as concrete algorithms. Before treatment two positive diagnostic procedures are required because the prevalence in Germany is decreasing. In addition to the rapid test and histological investigations, the 13-C breath test and stool tests with excellent sensitivity and specificity are also now available. Probiotics can improve therapy success especially for long-term antibiotic regimes and in the future bismuth could again play an increasingly more important role because antibiotic resistance to metronidazol and clarithromycin is increasing.     

Helicobacter pylori

Helicobacter pylori is associated with various gastroduodenal diseases such as peptic ulcer, functional dyspepsia, MALT lymphoma and distal gastric cancer. Diagnosis of H. pylori can be established by non-invasive (^13Curea breath test, stool antigen test, serology) and invasive (histology, rapid urease test, culture) tests. In adults, culture and susceptibility testing should or must be performed after failing of first-line therapy in case of a control endoscopy and before third-line therapy, respectively. Peptic ulcer and gastric MALT lymphoma represent obligatory indications for eradication therapy. Other potential indications are functional dyspepsia, prevention of gastric cancer in individuals being at risk, and before starting treatment with traditional non-steroid antiphlogistics. First-line therapy is performed with a 7-days combination of proton pump inhibitor with clarithromycin and amoxicillin or metronidazole. In second-line therapy levofloxacin and rifabutin are good rescue antibiotics.     

Diagnosis of Helicobacter pylori infection and diseases associated with Helicobacter pylori by Helicobacter pylori outer membrane proteins

sAIM: To examine the serological response of patients with upper gastrointestinal diseases and Helicobocter pylon (Hpy/on)infection to two Hpyloriouter membrane proteins (OMPs) (Mr18 000 and Mr26 000) acquired by gene recombinant technique, and to determine the diagnostic significance of serological tests derived from these OMPs.METHODS: Recombinant vectors encoding the two Hpylori OMPs were used to transform and express in BL21 (DE3) E.coli After purification with NP-NTA agarose resin, colloid gold kits were prepared with purified recombinant proteins to detect H pyloH infection and H pylori-associated diseases by the immunity-marker technology. We selected 150 patients with Hpy/on‘infection and digestive symptoms wibhout previous treabnent, including chronic gastritis (n = 60), duodenal ulcer (n = 30), gastric ulcer (n = 30), and gastric cancer (n=30).As controls, 33 Hpylori-negative healthy volunteers were also recruited. Serum samples were collected from all subjects, and the antibodies to specific proteins of Hpylori were tested with the colloid gold test kits. The sensitivity,specificity and accuracy of the colloid gold tests were evaluated, by using the combination of standard diagnostic methods (^13C urea breath test and bacteria culture) and classic enzyme-linked immunosorbent assay (ELISA) as reference.RESULTS: After purification with Ni^2+-NTA agarose resin,the purity of recombinant fusion proteins was about 95%.The recombinant fusion proteins were recognized by the specific monodonal antibodies against bhe two Hpy/oriOMPs,as demonstrated by the ELISA. Of the 150 serum samples from patients infected with Hpy/oH 141 (94.0%) responded positively to the recombinant protein with Mr26 000, while the seropositive rates were 95.0%, 96.7%, 96.7% and 90.0% for patients with H pylori-associated chronic gastritis,duodenal ulcer, gastric ulcer, and gastric cancer respectively.The sensitivity, specificity, and accuracy of the colloid gold kit with Mr26 000 protein were 94.0%, 97.0%, and 94.5%,respe.ctively. Compared with the classic ELISA, bacteria culture and ^13C urea breath test results in detecting Hpyloriinfection, there was no significant difference (P&gt;O.O5). For the colloid gold kit with Mr18 000, the seropositive rates were 52.0%, 40.0%, 40.0%, 53.3% and 86.7%, respectively,in Hpylori-infected palJents, and bhose wibh Hpylori-associated chronic gastritis, duodenal ulcer, gastric ulcer, and gastric cancer. There was a significant difference (P&lt;0.05) in seropositivity between patient with gastric cancer (86.7%) and those with other diseases (43.3%).CONCLUSION: The two colloid gold kits derived from the recombinant OMPs are useful tools either for detecting Hpyloriinfection, or for, predicting Hpylori-associated gastric malignancy.