应用噬菌体随机肽库技术筛选幽门螺杆菌中性粒细胞激活蛋白的抗原表位

目的 筛选和鉴定幽门螺杆菌(Helicobacter pylori,Hp)中性粒细胞激活蛋白(neutrophil-activating protein,NAP)的有效抗原表位,为Hp疫苗的研制提供基础.方法 以抗NAP的单克隆抗体作为固相筛选分子,经3轮吸附-洗脱-扩增免疫,筛选噬菌体随机7肽库,随机挑选噬菌体克隆,经噬菌体酶联免疫吸附试验(ELISA)、交叉反应试验及竞争抑制试验鉴定阳性克隆,测定阳性克隆所携带DNA序列并进行计算机辅助分析.以制备的阳性噬菌体克隆短肽液免疫小鼠,免疫血清与NAP经Western blot分析,以验证NAP的模拟表位.结果 经3轮免疫筛选后挑选到45个阳性克隆,经ELISA鉴定有12个阳性克隆,测序结果显示5种表位,其中P17噬菌体展示肽FAHLATQ与NAP氨基酸序列(137～143)高度同源,位于NAP高抗原区域(118～140),免疫血清可识别NAP.结论 用噬菌体随机7肽库成功筛选到了NAP的模拟表位,为基于NAP的诊断和疫苗的研制提供了基础.     

幽门螺杆菌中性白细胞激活蛋白的研究进展

幽门螺杆菌中性白细胞激活蛋白是150000u蛋白质,具有多种生物学功能①作为抗原能激发宿主产生特异性免疫反应;②对白细胞具有趋化、激活作用;③介导免疫粘附及介导幽门螺杆菌粘附、定植胃粘膜;④激活人白细胞NADPH氧化酶,产生活性氧中间产物.结论幽门螺杆菌中性白细胞激活蛋白是参与幽门螺杆菌致病机制的重要因子;可作为幽门螺杆菌疫苗的重要候选者.     

Hp中性粒细胞激活蛋白研究进展

幽门螺杆菌(Hp)是一种参与多种胃、十二指肠疾病的重要人类病原体,可引起慢性胃炎和消化性溃疡,并与胃癌密切相关.人类幽门螺杆菌感染的特征是胃黏膜炎症部位中性粒细胞持续浸润.幽门螺杆菌中性粒细胞激活蛋白(H.pylori neutrophil-activating protein,HP-NAP)正是由于其能活化中性粒细胞而被如此命名.HP-NAP不仅是重要的毒力因子,而且是主要的候选疫苗抗原.本文综述了HP-NAP的结构、生物学活性和致病机制等的研究进展,并对其应用前景进行了展望.     

幽门螺杆菌napA-ctxB融合蛋白的基因克隆与表达

目的 克隆、表达幽门螺杆菌中性粒细胞激活蛋白napA与霍乱毒素B亚单位ctxB融合基因napA-ctxB(nctB),为制备预防H.pylori感染的疫苗奠定基础.方法 用PCR方法扩增ctxB目的基因片段,克隆至pQE30-napA质粒的napA基因上游,构建含双基因的表达质粒pQE30-napA-ctxB(pQE30-nctB),经测序分析确认后转化E.coli DH5α,经IPTG诱导表达融合蛋白NCTB,融合蛋白NCTB经镍离子柱纯化.结果 PCR扩增出807 bp的目的基因片段nctB.工程菌pQE30-nctB-DH5α经IPTG诱导后,SDS-PAGE显示有新生的蛋白表达条带,Mr为30 000,与预期的一致,约占菌体总蛋白的27%,重组蛋白用Ni2 -NTA 树脂提纯,纯化后的蛋白质经SDS-PAGE分析可见单一条带,图象软件分析表明纯度可达94%以上.Western blot 显示重组蛋白质有良好的抗原性.结论 构建含双基因的表达质粒pQE30-nctB成功,并在大肠杆菌DH5α中高效的表达.     

枯草芽孢杆菌介导的基因工程菌研制现状

枯草芽孢杆菌是一种新型表达载体,它可以表达酶类和生长因子等多种蛋白,本文拟就这方面的研制现状进行综述。     

应用枯草芽孢杆菌表达系统制备肠道病毒71型VP1蛋白

目的利用枯草芽孢杆菌系统表达肠道病毒71型（EV71）病毒VPl蛋白。方法使用基因特异性引物扩增VPl开放读码框（ORF）,构建包含VP1完整开放读码框的pSac—VPl穿梭载体。根据枯草芽孢杆菌表达系统双交叉同源重组的特点,将EV71的结构抗原基因VPl整合在枯草芽孢杆菌sacA染色体,并经测序鉴定。使用VPl特异性抗体,通过蛋白印记（Western—Blot）鉴定枯草芽孢杆菌中VPl蛋白的表达情况。结果成功克隆EV71的VPl基因,长度1361bp,并将其克隆入穿梭载体pSae—Kan。测序结果显示VPl基因成功整合如sacA染色体。Western．Blot证实VPl抗原在枯草芽孢杆菌的成功表达,相对分子质量约35×10^3。结论利用枯草芽孢杆菌表达系统成功制备EV71病毒VPl抗原,为后续EV71诊断试剂、疫苗研发奠定基础。     

中性红摄入法检测幽门螺杆菌空泡毒素活性

空泡变性细胞毒素 (ＶａｃＡ)是幽门螺杆菌 (Ｈ .ｐｙｌｏｒｉ)分泌的一种重要毒素 ,其相对分子质量 (Ｍｒ)约为 (87～ 95 )×10 3 ,最初发现该毒素与哺乳动物细胞共同孵育后可导致细胞内产生大量的空泡。既往多采用光学显微镜下直接计数空泡形成细胞的比例来评价ＶａｃＡ活性 ,我们采用中性红摄入法 (Ｎｅｕｔｒａｌｒｅｄｕｐ ｔａｋｅａｓｓａｙ ,ＮＲＵ)对Ｈｐ菌株ＶａｃＡ进行定量检测 ,评价该方法检测ＶａｃＡ活性的应用价值。标准Ｈｐ菌株ＣＣＵＧ17874经固体平皿培养后 ,混悬于布氏肉汤液体培养基中 ,置 37℃微需氧环境振荡培养 72…     

幽门螺杆菌分子内佐剂三价疫苗的构建、纯化及活性研究

目的构建幽门螺杆菌（helicobacter pylori,Hp）中性粒细胞激活蛋白基因napA、黏附素hpaA、尿素酶B亚单位活性功能片段ureB414与大肠杆菌不耐热肠毒素B亚单位（heat-labile enterotoxin Bsubunit,LTB）的分子内佐剂三价融合蛋白疫苗,并通过13服免疫小鼠研究其抗原性,为疫苗的开发奠定基础。方法利用分子克隆技术构建携带napA-hpaA．ureB414-LTB融合基因的重组原核表达质粒pET-22b-NHUL,转化E．coliB121（DE3）,进行原核表达,重组表达蛋白采用HK26／60Superdex-75分子筛和亲和层析柱Chelating Sepharose进行纯化,Tris-Tricine电泳和Westemblot鉴定,GM1-ELISA检测rNHUL与神经节苷脂的结合。口服免疫Balb／c小鼠,检测重组融合蛋白的抗原性。结果重叠延伸PCR扩增出了约1590bp的NHUL融合基因;其核苷酸序列与GenBank公布的相关序列一致。重组融合蛋白以可溶和包涵体两种形式表达,表达量约占细菌总蛋白的25％,Tris-Tficine初步测定目的蛋白的肘,约59000,纯化后纯度大于90％。Westemblot检测其具有良好的抗原性。该重组蛋白保持了与神经节苷脂GM1结合的生物学活性。动物实验表明,多亚单位疫苗口服免疫小鼠后血清特异性IgG、IsA和胃黏液、肠黏液的抗原特异性sigA抗体显著高于单一亚单位疫苗。结论所获得重组融合蛋白具有良好的抗原性和黏膜佐剂活性,为进一步的疫苗研究奠定了基础。     

幽门螺杆菌alpA基因在乳酸菌中的表达及免疫原性分析

目的:在乳酸菌中表达幽门螺杆菌(Hp)黏附素al-pA基因,口服免疫小鼠后检测其免疫原性。方法:PCR方法从基因组中扩增alpA基因,将其克隆至表达载体pNICE:sec,将重组质粒转化乳酸菌NZ9000株,筛选鉴定阳性菌落,诱导表达的alpA蛋白用SDS-PAGE和Western blot进行鉴定。将雌性ICR(CV级)小鼠随机分为4组,分别用PBS、携带空质粒的乳酸菌、携带pNICE:sec-alpA的乳酸菌、灭活的Hp灌胃。免疫7次后检测其特异性IgG和IgA的产生。结果:扩增alpA基因并构建了重组原核表达质粒pNICE:sec-alpA,转化乳酸菌NZ9000后经nisin诱导可表达Mr约56 000的重组蛋白,表达量约占菌体总蛋白量的9.6%,Western blot分析其能与幽门螺杆菌抗血清特异性反应,具有良好的免疫原性。携带pNICE:sec-alpA质粒的乳酸菌刺激产生的IgG水平明显高于携带空质粒组,与灭活Hp组没有明显的差异,但其刺激产生的IgA水平明显高于其他组(P0.05)。结论:表达al-pA蛋白的乳酸菌口服免疫小鼠后,能够刺激小鼠产生特异的IgG和IgA,可作为幽门螺杆菌口服疫苗候选抗原。为乳酸菌作为抗原递送载体的研究和Hp口服疫苗的开发提供一定的实验基础。     

基于枯草芽孢杆菌系统制备的EV71VP1重组抗原诱导小鼠免疫应答的研究

目的观察基于枯草芽孢杆菌系统制备的EV7lVPl重组抗原诱导BALB／c小鼠产生的免疫应答反应。方法通过建立BALB／c小鼠动物模型,分别将前期构建的基于枯草芽孢杆菌系统制备的EV71重组抗原（rVPl）、灭活EV71病毒免疫组（EV71）和空白对照组（PBS）,经鼻腔接种6～8周龄BALB／c小鼠,ELISA方法检测免疫后不同时间产生的特异性IgG、IgA以及IgGl和IgG2a水平。结果EV71重组抗原可以有效诱导小鼠产生高水平的体液免疫和明显的黏膜免疫应答,且能够诱发均衡的Th1、Th2免疫调节。结论肠道病毒71型重组VPl抗原可诱发小鼠产生明显的特异性体液免疫和黏膜免疫应答。     

The neutrophil-activating protein of Helicobacter pylori (HP-NAP) activates the MAPK pathway in human neutrophils

Infection by Helicobacter pylori causes an acute inflammatory response followed by a chronic infection of the human gastric mucosa characterized by the infiltration of neutrophils andmononuclear inflammatory cells. The neutrophil-activating protein of Helicobacter pylori (HP-NAP) is a virulence factor that activates neutrophils, monocytes, and mast cells. However, the mechanism by which HP-NAP activates these cells is not fully understood. Here, we show that HP-NAP induces extracellular regulated kinase (ERK) and p38-mitogen-activated protein kinase (MAPK) activation in human neutrophils; c-Jun N-terminal kinase is not activated by HP-NAP. A MAPK/ERK kinase inhibitor and a p38-MAPK inhibitor suppress HP-NAP-mediated neutrophil oxidative burst, adhesion, andchemotaxis, but not actin polymerization. Pertussis toxin (PTX) inhibits all these neutrophil functions and the MAPK activation caused by HP-NAP. These results demonstrate that HP-NAP activates neutrophils through a PTX-sensitive pathway and that ERK and p38-MAPK are involved in many neutrophil functions stimulated by HP-NAP.     

人幽门螺杆菌热休克蛋白A亚单位的基因克隆及序列分析

目的 获取人幽门螺杆菌（Ｈｐ）热休克蛋白Ａ亚单位（ＨｓｐＡ）的ＤＮＡ（ｈｓｐＡ）,并将它克隆到质粒ＰｉｎＰｏｉｎｔ＾ＴＭ Ｘａ－３中进行核苷酸序列分析。方法 利用ＰＣＲ技术扩增ＨｓｐＡ,并将其它向插入ＰｉｎＰｏｉｎｔ＾ＴＭ Ｘａ－３载体中通过４种荧光染料标记,激光检测的方法进行核苷酸序列分析。结果 ＤＮＡ序列分析表明,所克隆的ＨａｐＡ ＤＮＡ序列与ＧｅｎＢａｎｋ公布的一致。结论 本研究获得了序列正     

幽门螺杆菌HspA和UreB双价侯选疫苗株的构建

目的 构建表达幽门螺杆菌的组成成分热休克蛋白Ａ亚单位（ＨｓｐＡ）和尿素酶Ｂ亚单位（ＵｒｅＢ）的重组蛋的白质的侯选菌株。方法 用ＰＣＲ方法从幽门螺杆菌的染色休ＤＮＡ上分别扩增出ＨｓｐＡ和ＵｒｅＢ基因片段,将它们融合插入原核表达载体ｐＥＴ－２３ｂ（＋）中,并在ＢＬ２１（ＤＥ３）大肠杆菌表达。结果 经测序,ＨｓｐＡ－ＵｒｅＢ（ＨＵ）事例基因片段由２０６１ｂｐ组成,为编码６８７个氨基酸残基的多肽。ＳＤＳ－     

Significant association between Helicobacter pylori infection and serum C-reactive protein

Background: Helicobacter pylori (H. pylori) infection in gastric mucosa may cause systemic inflammatory reaction. This study aimed to examine the association between the infection and serum high sensitivity C-reactive protein (hsCRP).Methods: Subjects were comprised of three groups; 453 health checkup examinees from Yakumo town inhabitants in Hokkaido, Japan (YTI, 153 males and 300 females), 449 health checkup examinees (ENUH, 273 males and 176 females), and 255 female patients of an infertility clinic (PIC), Nagoya University Hospital. Twenty participants with hsCRP more than 1 mg/dl were excluded from the analysis. Those with hsCRP more than 0.1mg/dl were defined as high hsCRP individuals. H. pylori infection status was examined with a serum IgG antibody test.Results: When the three groups were combined, the geometric mean of hsCRP concentration was significantly higher among the seropositives (0.047mg/dl) than among the seronegatives (0.035mg/dl); p<0.0001 by a t-test. The percentage of high hsCRP individuals was also higher in the seropositives than in the seronegatives among any group; 23.3% and 20.1% in YTI, 22.0% and 16.0% in ENUH, and 32.7% and 18.7% in PIC, respectively, although the difference was significant only in ENUH. The summary odds ratio of the high hsCRP for the seropositives relative to the seronegatives was 1.38 (95% confidence interval, 1.01-1.89), when age, sex, body mass index, smoking, and subject group were adjusted by a logistic model.Conclusions: In three groups, hsCRP was higher among the infected individuals. The summary odd ratio indicated that H. pylori infection could influence the serum hsCRP level.     

The effect of Helicobacter pylori eradication on C-reactive protein: results from a meta-analysis

IntroductionHelicobacter pylori is a bacterium that causes chronic gastroduodenal infection and affects various systemic diseases. An increase in the blood level of C-reactive protein (CRP; a systemic inflammatory marker), at a low-grade chronic inflammation level, is observed in cases of infection. However, the effect of H. pylori eradication on CRP remains undetermined. Therefore, we aimed to evaluate the circulating CRP levels in eradicated patients through a meta-analysis.Material and methodsThe PubMed database was searched from its inception to June 2020. Studies that described the CRP levels following H. pylori eradication were collected. A random-effects meta-analysis was then performed using inverse variance with standardized mean difference.ResultsA total of 10 eligible studies (642 subjects in total) were available. The median age in the studies was 49.9 years. The CRP level was 6.0 (median) mg/l before H. pylori eradication and 5.8 (median) mg/l after eradication. From the results of the overall meta-analysis, there was found to be a significant reduction in the CRP levels with H. pylori eradication (standardized mean difference: –0.64; 95% confidence interval: –1.02 to –0.27). The result was not similarly confirmed in a subanalysis of the available randomized controlled trials.ConclusionsWeak evidence exists regarding the effects of H. pylori eradication on CRP levels. Further research is called for.     

幽门螺杆菌GroEL蛋白重组表达及表达产物小鼠免疫保护作用

目的 检测幽门螺杆菌临床菌株groEL基因序列差异,确定重组表达的GroEL蛋白(r-GroEL)抗原性、免疫反应性以及对幽门螺杆菌感染小鼠的免疫保护性.方法 采用PCR及测序法了解幽门螺杆菌临床菌株groEL基因序列.构建幽门螺杆菌groEL基因pET42a-E.coli BL21DE3原核表达系统,采用SDS-PAGE及凝胶图像分析系统确定rGroEL表达量,Ni-NTA亲和层析法提纯rGroEL.采用ELISA和Western blot检测rGroEL的抗原性和免疫反应性,采用幽门螺杆菌全菌为包被抗原的ELISA确定GroEL膜定位.采用幽门螺杆菌小鼠感染模型了解rGroEL免疫保护作用.结果 35株幽门螺杆菌临床菌株groEL基因序列相似性高达99.4％～ 100％.rGroEL表达量可达细菌总蛋白的56％,SDS-PAGE后提纯的rGroEL在凝胶中显示为单一的条带.rGroEL可诱导家兔和小鼠产生高效价IgG抗体,同时也能被幽门螺杆菌全菌抗体(Hp-IgG)或rGroEL-IgG识别并与之结合.GroEL是幽门螺杆菌表面蛋白抗原.50、100或200 μg rGroEL免疫可分别使50.0％ (6/12)、75.0％ (9/12)和91.7％ (11/12)小鼠免于幽门 螺杆菌SS1株的感染,200 μg rGroEL免疫保护率(91.7％)显著高于50μg rGroEL(50.0％)( P＜0.05).结论 幽门螺杆菌GroEL蛋白是序列保守、具有良好免疫原性和免疫保护性的表面抗原,可作为基因工程疫苗候选抗原.