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。     

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。     

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。     

幽门螺杆菌热休克蛋白60核酸疫苗的构建和免疫原性鉴定

背景:核酸疫苗是新兴的第三代疫苗。目前幽门螺杆菌(H.pylori)疫苗的研究主要集中在减毒、灭活和亚单位疫苗方面,有关H.pylori核酸疫苗的研究罕见报道。目的:构建含H.pylori热休克蛋白60(hsp60)基因的核酸疫苗,并鉴定其表达蛋白的免疫原性。方法:抽提H.pylori标准菌株CCUG17874基因组DNA为模板,应用聚合酶链反应(PCR)扩增hsp60基因并插入测序载体pUCmT,测定插入的hsp60基因的核苷酸序列。通过一系列酶切、连接反应将hsp60基因克隆入真核表达载体pIRES,然后转化入感受态大肠杆菌DH5α,筛选阳性克隆,行酶切和PCR鉴定。采用脂质体法将所构建的重组载体pIRES鄄hsp60转染真核细胞COS鄄7,蛋白质印迹法(Western blotting)检测pIRES鄄hsp60表达蛋白的免疫原性。结果:以H.pylori基因组DNA为模板,PCR扩增出约1 640 bp的hsp60基因片断,测序结果表明其与GenBank中H.pylorihsp60原序列的同源性达98%。酶切和PCR鉴定结果证实hsp60基因已克隆入真核表达载体pIRES,成功构建了含hsp60基因的H.pylori核酸疫苗pIRES鄄hsp60。蛋白质印迹法检测显示经pIRES鄄hsp60转染的COS鄄7细胞在约60 kDa(1 Da=0.992 1 u)处出现特异性蛋白条带。结论:成功构建了具有免疫原性的含hsp60基因的H.pylori核酸疫苗,为进一步探索其免疫作用奠定了     

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。     

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。     

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。     

人NKp30基因克隆及其真核表达载体的构建

目的对人NKp30（hNKp30）进行基因克隆并在大肠埃希菌中重组表达,为进一步研究NK细胞抗肿瘤作用奠定基础。方法提取人外周静脉血单个核细胞,分离纯化外周血总RNA。用PCR扩增hNKp30片段,克隆至质粒载体pMD18-T,对克隆的DNA片段行序列分析。用限制酶XhoI、EcoRI消化pMD18-T-hNKp30重组质粒,分离hNKp30片段,插入真核表达载体pIRES2-EGFP相应限制酶位点,酶谱分析鉴定重组表达载体pIRES2-EG-FP-hNKp30,并转染COS-7细胞。结果PCR扩增DNA片段与hNKp30 cDNA大小一致。重组质粒pMD18-T-hNKp30的DNA序列分析显示,克隆DNA序列与文献报道hNKp30的cDNA序列一致。重组表达质粒pIRES2-EGFP-hNKp30转染COS-7细胞后,实现了hNKp30基因在COS-7细胞中的体外转染及瞬时表达。结论采用重组技术成功构建了hNKp30真核表达载体,为探讨NK细胞受体的特性及其信号转导机制奠定了基础。     

Helicobacter pylori and pregnancy-related disorders

Helicobacter pylori(H.pylori)infection is investigated in gastric diseases even during pregnancy.In particular,this Gram-negative bacterium seems to be associated with hyperemesis gravidarum,a severe form of nausea and vomiting during pregnancy.During the last decade,the relationship among H.pylori and several extra-gastric diseases strongly emerged in literature.The correlation among H.pylori infection and pregnancy-related disorders was mainly focused on iron deficiency anemia,thrombocytopenia,fetal malformations,miscarriage,pre-eclampsia and fetal growth restriction.H.pylori infection may have a role in the pathogenesis of various pregnancy-related disorders through different mechanisms:depletion of micronutrients(iron and vitamin B12)in maternal anemia and fetal neural tube defects;local or systemic induction of pro-inflammatory cytokines release and oxidative stress in gastrointestinal disorders and pre-eclampsia;cross-reaction between specific anti-H.pylori antibodies and antigens localized in placental tissue and endothelial cells(preeclampsia,fetal growth restriction,miscarriage).Since H.pylori infection is most likely acquired before pregnancy,it is widely believed that hormonal and immunological changes occurring during pregnancy could activate latent H.pylori with a negative impact not only on maternal health(nutritional deficiency,organ injury,death),but also on the fetus(insufficient growth,malformation,death)and sometime consequences can be observed later in life.Another important issue addressed by investigators was to determine whether it is possible to transmit H.pylori infection from mother to child and whether maternal anti-H.pylori antibodies could prevent infant’s infection.Studies on novel diagnostic and therapeutic methods for H.pylori are no less important,since these are particularly sensitive topics in pregnancy conditions.It could be interesting to study the possible correlation between H.pylori infection and other pregnancy-related diseases of unknown etiology,such as gestational diabetes mellitus,obstetric cholestasis and spontaneous preterm delivery.Since H.pylori infection is treatable,the demonstration of its causative role in pregnancy-related disorders will have important social-economic implications.     

Helicobacter pylori infection and drugs malabsorption

Drug absorption represents an important factor affecting the efficacy of oral drug treatment.Gastric secretion and motility seem to be critical for drug absorption.A causal relationship between impaired absorption of orally administered drugs and Helicobacter pylori(H.pylori)infection has been proposed.Associations have been reported between poor bioavailability of l-thyroxine and l-dopa and H.pylori infection.According to the Maastricht Florence Consensus Report on the management of H.pylori infection,H.pylori treatment improves the bioavailability of both these drugs,whereas the direct clinical benefits to patients still await to be established.Less strong seems the association between H.pylori infection and other drugs malabsorption,such as delavirdine and ketoconazole.The exact mechanisms forming the basis of the relationship between H.pylori infection and impaired drugs absorption and/or bioavailability are not fully elucidated.H.pylori infection may trigger a chronic inflammation of the gastric mucosa,and impaired gastric acid secretion often follows.The reduction of acid secretion closely relates with the wideness and the severity of the damage and may affect drug absorption.This minireview focuses on the evidence of H.pylori infection associated with impaired drug absorption.     

Barrett's oesophagus and Helicobacter pylori

Abstract In order to demonstrate the presence of Helicobacter pylori in the metaplastic epithelium of Barrett's oesophagus and to evaluate its possible association with this entity, we examined 29 cases of Barrett's oesophagus where concomitant antral biopsies were also available. These cases were compared with an equal number of age and sex matched controls of uncomplicated reflux oesophagitis. H. pylori was present in 11 of 29 cases of Barrett's oesophagus (38%). No increase in the frequency of H. pylori antral gastritis was found in patients of Barrett's oesophagus compared to the control group of uncomplicated reflux oesophagitis. The positivity of Barrett's oesophagus for H. pylori correlated with the presence of H. pylori antral gastritis ( P < 0.05), although in two cases of H. pylori -positive Barrett's oesophagus antral biopsies were negative for H. pylori. No difference was found in the severity of inflammatory and dysplastic changes of H. pylori- positive and H. pylori -negative Barrett's oesophagus. Presence of H. pylori does not seem to alter the natural history of Barrett's oesophagus.     

Laryngopharyngeal reflux and Helicobacter pylori

Laryngopharyngeal reflux (LPR) occurs when gastric contents pass the upper esophageal sphincter, causing symptoms such as hoarseness, sore throat, coughing, excess throat mucus, and globus. The pattern of reflux is different in LPR and gastroesophageal reflux. LPR usually occurs during the daytime in the upright position whereas gastroesophageal reflux disease more often occurs in the supine position at night-time or during sleep. Ambulatory 24-h double pH-probe monitoring is the gold standard diagnostic tool for LPR. Acid suppression with proton pump inhibitor on a long-term basis is the mainstay of treatment. Helicobacter pylori (H. pylori) is found in many sites including laryngeal mucosa and interarytenoid region. In this paper, we aim to present the relationship between LPR and H. pylori and review the current literature.     

Helicobacter pylori infection in older people

Since the discovery of Helicobacter pylori(H.pylori)infection as the major cause of gastroduodenal disorders three decades ago,H.pylori has been the focus of active research and debate in the scientific community.Its linkage to several diseases,such as peptic ulcer disease,gastritis and gastric malignancy is incontestable.In particular,it has been noticed that,as the aged population is increasing worldwide,older people are at increased risk of developing several gastroduodenal diseases and related complications.At the same time,gastric cancer is definitely more frequent in elderly than in adult and young people.In addition,it has been showed that peptic ulcer and related complications occur much more commonly in aged individuals than in young people,resulting in a significantly higher mortality.Although this infection plays a crucial role in gastrointestinal disorders affecting all age groups and in particular older people,only a few studies have been published regarding the latter.This article presents an overview of the epidemiology,diagnosis,clinical manifestations and therapy of H.pylori infection in elderly people.     

Helicobacter pylori and skin autoimmune diseases

Autoimmune skin diseases are characterized by dysregulation of the immune system resulting in a loss of tolerance to skin self-antigen(s). The prolonged interaction between the bacterium and host immune mechanisms makes Helicobacter pylori(H. pylori) a plausible infectious agent for triggering autoimmunity. Epidemiological and experimental data now point to a strong relation of H. pylori infection on the development of many extragastric diseases, including several allergic and autoimmune diseases. H. pylori antigens activate cross-reactive T cells and induce autoantibodies production. Microbial heat shock proteins(HSP) play an important role of in the pathogenesis of autoimmune diseases because of the high level of sequence homology with human HSP. Eradication of H. pylori infection has been shown to be effective in some patients with chronic autoimmune urticaria, psoriasis, alopecia areata and Schoenlein-Henoch purpura. There is conflicting and controversial data regarding the association of H. pylori infection with Beh et’s disease, scleroderma and autoimmune bullous diseases. No data are available evaluating the association of H. pylori infection with other skin autoimmune diseases, such as vitiligo, cutaneous lupus erythematosus and dermatomyositis. The epidemiological and experimental evidence for a possible role of H. pylori infection in skin autoimmune diseases are the subject of this review.     

Helicobacter pylori:Management in 2013

Helicobacter pylori(H.pylori)is a prevalent,worldwide,chronic infection.Choice of treatment can be modified according to antibiotic-resistance rates of H.pylori.The ideal therapeutic regimen for H.pylori infection should achieve an eradication rate of≥80%.In some countries,triple therapy with a proton-pump inhibitor(PPI),clarithromycin,and amoxicillin or metronidazole is still the best option.Bismuth-containing quadruple therapy consisting of bismuth salts,tetracycline,metronidazole and PPI,may be the preferred option in countries with clarithromycin resistance>20%.Sequential therapy including a PPI and amoxicillin given for the first 5 d,followed by triple therapy including a PPI,clarithromycin,and nitroimidazole antimicrobial(all twice daily)for the remaining 5 d,can be another option for the first-line treatment of H.pylori.Recent data suggest that treatment with PPI,levofloxacin,and amoxicillin for 10 d is a good choice for second-line therapy.Concomitant therapy consisting of PPI,amoxicillin,clarithromycin and metronidazole is another option for second-line treatment.If second-line treatment also fails,it is recommended to culture H.pylori from biopsy specimens and perform antimicrobial susceptibility testing.Rescue treatment should be based on antimicrobial susceptibility.     

Host-bacterial interactions in Helicobacter pylori infection

Helicobacter pylori are spiral-shaped gram-negative bacteria with polar flagella that live near the surface of the human gastric mucosa. They have evolved intricate mechanisms to avoid the bactericidal acid in the gastric lumen and to survive near, to attach to, and to communicate with the human gastric epithelium and host immune system. This interaction sometimes results in severe gastric pathology. H pylori infection is the strongest known risk factor for the development of gastroduodenal ulcers, with infection being present in 60%-80% of gastric and 95% of duodenal ulcers.(1)H pylori is also the first bacterium to be classified as a definite carcinogen by the World Health Organization's International Agency for Research on Cancer because of its epidemiologic relationship to gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma.(2) In the last 25 years, since H pylori was first described and cultured, a complete paradigm shift has occurred in our clinical approach to these gastric diseases, and more than 20,000 scientific publications have appeared on the subject. From the medical point of view, H pylori is a formidable pathogen responsible for much morbidity and mortality worldwide. However, H pylori infection occurs in approximately half of the world population, with disease being an exception rather than the rule. Understanding how this organism interacts with its host is essential for formulating an intelligent strategy for dealing with its most important clinical consequences. This review offers an insight into H pylori host-bacterial interactions.     

Prevention of Helicobacter pylori infection in childhood

Helicobacter pylori(H.pylori)infection is one of the most common infections worldwide.Although infection rates are falling in the developed and developing countries,H.pylori is still widespread in the world.This article has reviewed the important publications on H.pylori in childhood with a focus on its evolving transmission route and the source of infection and preventive strategies in childhood,PubMed was searched up to identify eligible studies.Relevant publications were searched using the following.