幽门螺杆菌HspA融合蛋白口服疫苗的构建

构建表达幽门螺杆菌的保护性抗原分热休克蛋白A亚单位(HspA）和霍乱毒素B亚单位（CtxB）的重组融合蛋白的生物工程菌株,以此制备幽门螺杆菌的口服疫苗。用PCR方法扩增hspA和ctxB两个目的的基因片段,将它们分别克隆至pSK（＋）质粒上,然后插入含T7启动子ET－22b（＋）的表达载体中,构建嗓基因的表达质量pET－hct,转化E.coliBL21(DE3）,经IPTG诱导表达融合蛋白HCT。经测序,hspA-ctxB(hct)融合基因片段由726bp组成,可以编码242个氨基酸残基的多肽。经SDS－PAGE和免疫印迹分析检测发现,融合基因表达的蛋白质相对分子质量约为30kD。融合蛋白经镍离子柱纯化、复性后,和HspA共同标记同位素^125I,然后给小鼠灌胃,结果观察到HCT组小鼠血清中的^125I的放射量要明显高于HspA组（P＜0．001）,且吸收峰值时间明显提前。融合蛋白中的CtxB可明显促进小鼠对HspA的吸收,HCT融合蛋白可以作为预防和治疗幽门螺杆菌感染的侯选口服疫苗。     

幽门螺杆菌重组腺病毒双价疫苗的构建及免疫学评价

幽门螺杆菌(Hp)是导致胃炎、消化道溃疡和胃癌的的主要病原菌.利用基因重组技术,成功将Hp的ure B和hsp A基因融合.将ure B-hsp A融合基因成功构建到腺病毒载体上.检测结果表明,该重组腺病毒具有侵染真核细胞能力,且能在真核细胞中表达目标抗原.以血清中IgG和新鲜粪便中sIgA,评价其免疫效果,结果显示,该重组腺病毒载体双价疫苗能在小鼠体内激发体液免疫和黏膜免疫反应.     

高效表达幽门螺杆菌UreB重组蛋白工程菌的构建

克隆表达幽门螺杆菌(Hp)的尿素酶B亚单位(UreB)重组蛋白,可为Hp疫苗开发和快速诊断试剂盒的研究奠定基础。用PCR方法由幽门螺杆菌染色体DNA扩增UreB基因片段,将其融合插入原核表达载体pQE30中,并在M15大肠杆菌表达。经酶切、测序分析,包括部分融合载体基因在内的重组UreB基因片段由1773bp组成。为编码591个氨基酸残基的多肽。SDS-PAGE分析显示重组表达的目的蛋白相对分子量约为66kD,表达量点菌体总蛋白的23．5％,并经免疫印迹分析证实被幽门螺杆菌感染的阳性血清可与纯化UreB重组蛋白发生特异性的结合反应。UreB重组蛋白具有良好的抗原性,将有可能成为一种有效蛋白质疫苗以及快速诊断试剂盒用于Hp感染的防治和检测。     

幽门螺杆菌重组蛋白脂质体疫苗免疫保护作用的动物实验研究

探讨幽门螺杆菌重组蛋白脂质体疫苗的免疫预防作用及可能的免疫机制.用逆向蒸发法制备以卵磷脂和胆固醇为膜组分包裹的重组蛋白和/无免疫佐剂的口服疫苗,并用透射电镜测定其粒径.BALB/c小鼠分为6组,分别通过灌胃方法给予PBS、空白脂质体、UreB重组蛋白 CT、脂质体包裹UreB重组蛋白、脂质体包裹UreB重组蛋白和CT、脂质体包裹(UreB Kat CT),每周1次共4次,末次攻击2周再用活幽门螺杆菌(Hp)攻击3次,5周后处死小鼠,行胃组织快速尿素酶试验、Hp的定植半定量、炎症程度及其炎症活动度的评分.RT-PCR检测小鼠脾淋巴细胞中γ-干扰素(INF-γ)和白细胞介素-4(IL-4)表达.脂质体疫苗电镜下负染,显示粒径为(0.7±0.2)μm.PBS组、空白脂质体组、UreB重组蛋白 CT组、脂质体包裹UreB重组蛋白组、脂质体包裹UreB重组蛋白和CT组、脂质体包裹(UreB Kat CT)组的免疫保护率分别为0(0/11)、0(0/11)、58.3%(7/12)、54.5%(6/11)、63.6%(7/11)、75.0%(9/12),UreB重组蛋白 CT组免疫保护率与脂质体包裹UreB重组蛋白组比较无显著性差异(P>0.05),脂质体包裹UreB重组蛋白 CT组免疫保护率与脂质体包裹(UreB Kat CT)组比较有显著性差异(P<0.05).各疫苗组Hp定植评分均明显低于PBS和脂质体对照组(P<0.01),且UreB Kat双价疫苗组较三个单价疫苗组比较也有显著性差异(P<0.05).疫苗组不仅能降低Hp的定植,而且能减轻Hp造成的小鼠胃黏膜的局部慢性炎症反应(P<0.05),但各疫苗组间比较未见显著性差异(P>0.05).Hp攻击后,与对照组比较,各疫苗组脾淋巴细胞INF-γmRNA水平较低(P<0.05),而IL-4mRNA水平则较高(P<0.05),各疫苗组间比较未见显著性差异(P>0.05).研究表明,在小鼠Hp感染模型中脂质体能部分代替霍乱毒素的免疫佐剂作用,在Hp攻击后,未经免疫小鼠体内诱导以Th1为主应答,免疫小鼠体内则诱导以Th2为主的免疫应答.     

幽门螺杆菌疫苗常用抗原的研究进展

幽门螺杆菌(Helicobacter pylori,H.pylori)属于Ⅰ类致癌因子,是慢性胃炎、消化性溃疡、胃癌等的首要病原菌。目前临床疗法存在诸多不足,而疫苗接种被认为是对抗大多数细菌感染的有效手段,也是当前防治H.pylori感染的研究热点,可作为抗生素疗法的补充。目前尚未研发出高效、安全的H.pylori疫苗,选择合适的抗原是研制疫苗成功的重要因素。本文总结了H.pylori疫苗研发领域经典抗原的研究进展,从分泌型毒力蛋白抗原、菌体表面抗原和胞内蛋白抗原三方面作一综述,为H.pylori感染预防的新策略提供更多依据。     

幽门螺杆菌疫苗诱导的免疫保护反应及临床试验研究进展

幽门螺杆菌(Helicobacter pylori,Hp)是一种微需氧、寄生于胃黏膜表面的革兰阴性致病菌,可导致各种不同的疾病,如慢性活动性胃炎、胃和十二指肠溃疡,甚至包括胃黏膜相关淋巴样组织(mucosa-associated lymphoid tissue,MALT)淋巴瘤和胃腺癌等恶性疾病。目前根除Hp主要依靠包括2种抗生素、铋剂和质子泵抑制剂(proton pumpinhibitors,PPI)在内的四联疗法。随着Hp耐药性的增强,研发Hp疫苗成为防治Hp感染的新途径。Hp疫苗能够诱导机体产生抗原特异性血清IgG和黏膜sIgA体液免疫反应以及抗原特异性CD4+T细胞免疫反应等的有效免疫保护反应。     

幽门螺杆菌热休克蛋白A基因的克隆,表达及免疫原性 …

幽门螺杆菌的感染可以诱发人体产生胃炎和消化性溃疡,其组成成分热休克蛋白Ａ可刺激机体产生保护性的免疫反应,用ＰＣＲ方法从幽门螺杆菌的染色体ＤＮＡ上扩增出ＨｓｐＡ基因片段,将其插入原核表达载体ｐＥＴ－２２ｂ（＋）中,并在ＢＬ２１（ＤＥ３）大肠杆菌表达。经测序Ｈｓｐ基因片段有３５４ｂｐ组成,可编码１１８个氨基酸残基的多肽。     

幽门螺杆菌热休克蛋白B亚单位的克隆和序列分析

幽门螺杆菌是消化道疾病的主要致病菌。其有效抗原成份热休克蛋白B亚单位（HspB)可刺激机体产生保护性的免疫反应。用高保真PCR扩增系统扩增出HspB基因片段,将其克隆至质粒PUC19中,对其全序列进行了测定。克隆的HSpB基因序列与报道的序列完全一致。这一研究获得了序列正确的HspB基因,为将来以HspB分子为抗原的疫苗研制工作打下了良好的基础。     

幽门螺杆菌感染引起的免疫应答及其对疫苗研制的启示

幽门螺杆菌是一种全世界范围的人类感染病原菌,人感染该菌后可以被诱导产生较强的体液免疫应答和一定程度的细胞免疫应答反应;但是,自然感染往往不能使机体产生有效的免疫保护,相反可引起对机体的免疫病理损伤,有效的疫苗应该被设计为能够诱导机体产生非病理损伤的特异的免疫保护反应,以此来预防和治疗幽门螺杆菌相关性疾病。本综述了这些方面的研究进展。     

幽门螺杆菌尿素酶B单克隆抗体的制备与鉴定

目的:制备稳定分泌抗幽门螺杆菌尿素酶B单克隆抗体(mAb)的杂交瘤细胞系,并对其分泌的mAb进行鉴定。方法:用初步纯化的重组幽门螺杆菌尿素酶B免疫BALB/c小鼠,利用杂交瘤技术制备抗尿素酶B的mAb,用间接ELISA检测mAb的特异性和亲和力,检测mAb腹水效价,鉴定Ig亚类并测定其抗原决定簇。结果:获得8株能稳定分泌抗尿素酶B的mAb杂交瘤细胞系,这8株单抗与能产生尿素酶的小肠结肠耶尔森氏菌、肺炎克雷伯氏菌和普通变形杆菌均无交叉反应,相对亲和力为1.13×10-8～4.66×10-10,腹水mAb效价可达2×104～3.2×105。其中2株单抗属IgG1亚类,3株单抗属IgG2a亚类。8株单抗分属于3种不同的抗原决定簇。结论:获得了IgG1和IgG2a类型的针对3种不同抗原决定簇的特异性幽门螺杆菌尿素酶B的mAb,为进一步用于幽门螺杆菌的临床诊断和实验研究创造了条件。     

不同幽门螺杆菌菌株ureB和hspA基因同源性分析

目的研究不同来源幽门螺杆菌菌株的ureB基因和hspA基因的同源性。方法在GenBank中调取全球来源于幽门螺杆菌不同菌株的ureB基因序列23个和中国境内来源于幽门螺杆菌不同菌株的hspA基因序列20个,利用ClustalW2生物软件,分别对ureB基因和hspA基因序列进行同源性比较分析,并建立基因进化树,分析其特点。结果不同国家之间ureB基因序列并不一致,同一国家ureB基因序列相似性较高;中国境内hspA基因序列相似性程度很高。结论中国境内不同幽门螺杆菌菌株ureB基因序列和hspA基因序列的相似性程度都很高,都具有很高的同源性。     

Oral vaccination of mice against Helicobacter pylori with recombinant Lactococcus lactis expressing urease subunit B

To determine whether a protective immune response could be elicited by oral delivery of a recombinant live bacterial vaccine, Helicobacter pylori urease subunit B (UreB) was expressed for extracellular expression in food-grade bacterium Lactococcus lactis . The UreB-producing strains were then administered orally to mice, and the immune response to UreB was examined. Orally vaccinated mice produced a significant UreB-specific serum immunoglobulin G (IgG) response. Specific anti-UreB IgA responses could be detected in the feces of mice immunized with the secreting lactococcal strain. Mice vaccinated orally were significantly protected against gastric Helicobacter infection following a challenge with H. pylori strain SS1. In conclusion, mucosal vaccination with L. lactis expressing UreB produced serum IgG and UreB-specific fecal IgA, and prevented gastric infection with H. pylori .     

幽门螺杆菌Lpp20-IL2核酸疫苗免疫活性

【目的】观察pcDNA3.1(+)/Lpp20-IL2免疫C57BL/6小鼠后所产生的体液免疫和细胞免疫应答水平,为研制高效、新型的幽门螺杆菌核酸疫苗提供实验依据。【方法】构建pcDNA3.1(+)/Lpp20-IL2重组载体,并转染HeLa细胞,用Western-blot观察鉴定其在真核细胞得到表达后免疫C57BL/6小鼠,ELISA间接法测定小鼠血清中抗Lpp20IgG抗体水平,ELISA双抗体夹心法检测脾淋巴细胞培养上清中IFN-γ、IL4水平,MTT比色法检测脾淋巴细胞增殖反应,免疫荧光组化法检测Lpp20蛋白在小鼠肌肉组织中的表达情况。【结果】成功构建了pcDNA3.1(+)/Lpp20-IL2真核表达载体,且重组质粒能在HeLa细胞内有效表达目的蛋白;小鼠接种pcDNA3.1(+)/Lpp20-IL2核酸疫苗后能产生特异性IgG抗体,8w后ELISA测定血清抗体A450值明显升高。核酸疫苗pcDNA3.1(+)/Lpp20-IL2免疫组小鼠脾淋巴细胞经特异性抗原刺激后,培养上清中IFN-γ、IL4含量明显升高。pcDNA3.1(+)/Lpp20-IL2和pcDNA3.1(+)/Lpp20核酸疫苗组小鼠脾淋巴细胞经特异性抗原刺激后,刺激指数明显高于空质粒组和PBS组。Lpp20蛋白在小鼠肌肉组织中能够有效表达。【结论】幽门螺杆菌Lpp20-IL2融合基因核酸疫苗和Lpp20单基因核酸疫苗均能刺激机体产生较强细胞免疫应答和体液免疫应答,且前者能诱导更强的细胞免疫应答。     

幽门螺杆菌热休克蛋白A在乳酸乳球菌中的表达及免疫学活性分析

目的构建含幽门螺杆菌(H.pylori)热休克蛋白A编码基因的重组载体,并电转入乳酸乳球菌MG1363,表达目的蛋白并分析其免疫原性,为H.pylori基因工程口服疫苗的研究和开发奠定基础。方法以H.py-loriNCTC 11637株基因组DNA为模板,PCR扩增hspA基因,并克隆至乳酸乳球菌表达载体pMG36e中。将重组质粒转化E.coliDH5α,经鉴定的阳性重组质粒命名为pMG36e/hspA。以电穿孔法将pMG36e/hspA转化乳酸乳球菌MG1363并用Western blot检测HspA蛋白的表达。结果克隆重组后得到pMG36e/hspA。将pMG36e/hspA电转化MG1363后,收集菌体蛋白进行Western blot分析,在HspA的相对分子质量(Mr≈13 kDa)处出现特异性条带。结论首次成功构建了表达H.pyloriHspA的重组乳酸乳球菌MG1363,为进一步口服疫苗的相关研究奠定了基础。     

Development and evaluation of a DNA vaccine based on Helicobacter pylori urease B: failure to prevent experimental infection in the mouse model

BACKGROUND: The development of a vaccine against Helicobacter pylori has become a priority to prevent major morbidity and mortality associated with this infection. Our goal was to prepare and evaluate a DNA vaccine based on the urease B gene (ureB). METHODS: The ureB gene of H. pylori was amplified and cloned into the eukaryotic expression vector pcDNA3.1/TOPO. Plasmid DNA was purified from transformed Escherichia coli cells and used to immunize mice by the intragastric, intramuscular, intrarectal (40 micro g each) and intranasal (16 micro g) route, three doses every 2 weeks, with CpG oligodeoxynucleotide (ODN) as adjuvant. Four weeks after the third dose, animals were orally challenged with Helicobacter felis and were sacrificed 6 weeks later. The stomach was stained to detect the presence of infection. RESULTS: Despite in vitro confirmation of successful cloning and functionality of the ureB gene with expression of a protein morphologically and antigenically identical to urease B, the DNA vaccine did not perform well in vivo. Immunization of mice produced a weak immune response. Overall, intrarectal and intranasal administration seemed more immunogenic than other routes. Protection against challenge was modest and nonsignificant, and slightly better on animals immunized by the intramuscular and intranasal route. CONCLUSION: A DNA vaccine based on H. pylori urease B was poorly immunogenic and nonprotective at the conditions evaluated. Higher doses, better adjuvants or a prime-boost approach may circumvent these limitations.     

Potential animal models of Helicobacter pylori infection in immunological and vaccine research

Abstract Presence of Helicobacter pylori in the human gastric mucosa is associated with chronic gastritis and promotes the formation of peptic ulceration. Furthermore, long-term gastritis caused by the bacteria represents an increased risk of developing gastric cancer. Much controversy remains about the pathogenic mechanisms by which H. pylori can induce disease because of the limitations of animal models and the relevance of in vitro observations to the in vivo disease process. Studies of putative pathogenic factors such as induction of inflammatory mediators and immune evasion are required to understand how to design a vaccine against the infection. Vaccine adjuvants, delivery systems and therapeutic vaccination are likely to be the areas of major progress in the future. Data related to immunological aspects and vaccine development in potential animal models are reviewed.     

Importance of the host genetic background on immune responses to Helicobacter pylori infection and therapeutic vaccine efficacy

In order to investigate the role of host factors in Helicobacter pylori infection and immunity, two different strains of inbred mice, C57BL/6 and BALB/c, were infected with a standard H. pylori strain, SS1. A month later, infected mice were immunized orally with whole-cell lysates of H. pylori SS1 and cholera toxin on days 1, 3, 6, 30, and 54. Ten days after the last immunization, mice were sacrificed and the stomach was collected to assess H. pylori colonization density by quantitative culture. H. pylori SS1 colonization was significantly greater in C57BL/6 than in BALB/c (P<0.02 and P<0.003 at 2 and 13 weeks post-inoculation, respectively). Colonization in C57BL/6 persisted at equivalent levels for 13 weeks but the colonization density in BALB/c decreased significantly during this period. In contrast to the pattern of bacterial colonization, antibody responses following H. pylori SS1 infection were greater in BALB/c than in C57BL/6, suggesting that host factors may modulate the immune responses to H. pylori infection. Following therapeutic immunization, H. pylori colonization in BALB/c mice was also significantly reduced (P<0.03), while no significant differences in bacterial density were observed in C57BL/6. These observations collectively demonstrate the great importance of host factors in H. pylori infection and the development of effective immune responses.     

Progress in vaccine development against Helicobacter pylori

Based on the very high prevalence of diseases caused by Helicobacter pylori, particularly in the developing world, and the rapid emergence of antibiotic resistance among clinical isolates, there is a strong rationale for an effective vaccine against H. pylori. In this review we describe recent promising candidate vaccines and prophylactic or therapeutic immunization strategies for use against H. pylori, as well as studies to identify immune responses that are related to protection in experimental animals. We also describe identification of different types of immune responses that may be related to protection against symptoms based on comparisons of H. pylori-infected patients with duodenal ulcers or gastric cancer and asymptomatic carriers. We conclude that there is still a strong need to clarify the main protective immune mechanisms against H. pylori as well as to identify a cocktail of strong protective antigens, or recombinant bacterial strains that express such antigens, that could be administered by a regimen that gives rise to effective immune responses in humans.