淋病奈瑟菌孔蛋白PIA真核表达系统的构建及序列分析

目的:构建淋病奈瑟菌孔蛋白PIA(Porin IA)基因的真核表达系统,为淋病DNA疫苗的研究提供材料。方法:PCR技术扩增淋病奈瑟菌捷克标准株的PIA基因片段,将其定向插入真核表达载体pC I-neo多克隆位点中,构建重组表达载体。并用酶切分析、PCR扩增及序列测定方法,对重组载体进行鉴定。结果:淋病奈瑟菌PIA基因被正确克隆到真核表达载体pC I-neo中,测序结果与Genebank中AF090819等菌株序列有99.99%同源性。结论:真核表达系统(pC I-PIA)构建成功。     

基于淋病奈瑟菌外膜蛋白pIA-pIB融合基因重组产物酶联免疫吸附试验的建立和应用

目的 克隆淋病奈瑟菌外膜蛋白pIA、pIB基因并构建pIA-pIB融合基因及其原核表达系统以及建立基于rPIA-PIB的酶联免疫吸附试验(ELISA).方法 采用连接引物PCR构建pIA-pIB融合基因,按常规分子生物学方法构建其原核表达系统.采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和BioRad凝胶图像分析系统检测目的重组蛋白rPIA-PIB表达情况,Ni-NTA亲和层析法提纯rPIA-PIB.以rPIA-PIB为包被抗原建立检测淋病患者血清标本中rPIA和/或rPIB特异性IgG的ELISA,以rPIA-PIB抗血清为一抗建立检测淋病患者脓液标本中rPIA和/或rPIB的ELISA,实验中以rPIA、rPIB及其抗血清相关ELISA为对照.结果 pIA-pIB融合基因与原始核苷酸和氨基酸序列相似性均为100%.rPIA-PIB表达量为细菌总蛋白的29.8%,其提纯物SDS-PAGE后显示单一条带.rPIA-PIB-IgG-ELISA检测119例淋病患者血清标本的阳性率(98.3%)明显高于rPIA-IgG-ELISA(30.3%)或rPIB-IgG-ELISA(66.4%)(P＜0.01).rPIA-PIB-ELISA检测119例淋病患者脓液标本的阳性率(91.6%)也明显高于rPIA-IgG-ELISA(27.7%)或rPIB-IgG-ELISA(62.2%)(P＜0.01).结论 成功构建淋病奈瑟菌pIA-pIB融合基因及其原核表达系统;较之单一的rPIA或rPIB,rPIA-PIB作为淋病相关检测试剂盒抗原具有明显的优越性.     

淋病奈瑟菌外膜蛋白PorB编码基因的克隆及序列分析

目的 获取淋病奈瑟菌外膜蛋白PorB编码基因(porB)并构建其重组表达质粒。方法 根据porB已知序列，设计合成一对引物，用PCR方法从淋病奈瑟菌基因组DNA中扩增出porB基因片段，克隆到原核表达质粒pQE30中，转化大肠埃希菌M15感受态细胞，经酶切和PCR鉴定，然后进行测序。结果 porB基因体外扩增产物大小约为911bp。重组质粒经双酶切和PCR鉴定表明为正确重组子，测序结果与已知序列基本吻合。结论 成功克隆了淋病奈瑟菌外膜蛋白PorB编码基因，为下一步PorB蛋白的功能研究和淋病奈瑟菌蛋白质疫苗的研制提供了基本的物质基础：     

问号钩端螺旋体lipL41基因表达及重组抗原分析

目的构建问号钩端螺旋体（钩体）ltB／ctB-lipL41／1融合基因及其原核表达系统。方法采用连接引物聚合酶链反应（PCR）构建ltB—lipL41／1和ctB—lipL41／1融合基因，常规方法构建其原核表达系统。采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳检测目的重组蛋白rLTB—rLipL41／1和rCTB—rLipL41／1表达情况；用免疫印迹和神经节苷脂-酶联免疫吸附试验（GM1-ELISA）分别检测上述目的重组蛋白的免疫原性和佐剂活性；采用PCR和显微镜凝集试验（MAT）分别检测97株问号钩体野生株lipL41／1基因及其表达情况；用ELISA检测228例钩体患者血清lipL41基因产物的抗体。结果与报道的相关序列比较，ltB—lipL41／1和ctB—lipL41／1融合基因核苷酸和氨基酸序列相似性分别为99．6％～99．9％和99．8％～100％。rLTB—rLipL41／1和rCTB—rLipL41／1表达产量均约为细菌总蛋白的10％，主要以包涵体形式存在。rLTB—rLipL41／1和rCTB—rLipL41／1均分别能与rLipL41／1兔抗血清和牛GM1结合。87．6％（85／97）问号钩体野生株含有lipL41基因，84．5％（82／97）问号钩体野生株分别与rLipL41／1和rLipL41／2免抗血清出现效价范围为1：4～1：128的MAT阳性结果。84．6％（193／228）和78．5％（179／228）的患者血清分别rLipL41／1和rLipL41／2抗体阳性。结论成功地构建了ltB—lipL41／1和ctB-lipL41／1融合基因及其原核表达系统。所表达的rLTB-rLipL41／1和rCTB-rLipL41／1融合蛋白有良好的免疫原性和佐剂活性。lipL41基因存在于不同问号钩体血清群中并高频率表达。rLTB—rLipL41／1和rCTB—rLipL41／1具有成为钩体属特异性疫苗抗原的良好前景。     

浙江地区奈瑟淋球菌临床菌株porinⅠ基因优势型及原核表达系统的构建

目的分析浙江地区奈瑟淋球菌临床菌株porinⅠ基因优势型分布情况,构建porinⅠA与porinⅠB基因的原核表达系统。方法在先前研究的基础上,对确定的porinⅠA和porinⅠB基因进行T-A克隆后测定核苷酸顺序,分析porinⅠA和porinⅠB的地区优势基因型。再构建porinⅠA和porinⅠB基因优势基因型的原核表达系统,0.5 mmo L/L IPTG诱导后,10%SDS-PAGE检测蛋白表达情况。结果测序的5株porinⅠA全为血清型ⅠA-6;测序的11株porinⅠB中,血清型ⅠB-3有5株,血清型ⅠB-3/6有3株,血清型ⅠB-6有1株,其余2株为变异株。构建的原核表达系统PET-42-PⅠA的PⅠA表达量占细菌总蛋白量的30%;PET-42-PⅠB的PⅠB表达量占细菌总蛋白量的20%。结论浙江地区奈瑟淋球菌临床菌株porinⅠA以血清型ⅠA-6为优势基因型,porinⅠB以血清型ⅠB-3为优势基因型,porinⅠA相对于porinⅠB而言更加保守。所构建的原核表达系统能高效表达PⅠA与PⅠB重组蛋白。     

无锡地区淋病奈瑟菌TEM-1基因分子流行病学研究

目的 调查无锡地区淋病奈瑟菌株TEM-1基因流行情况。方法 参照国外的β-内酰胺酶阳性的淋病奈瑟菌菌株内含TEM-1基因的质粒序列,自行设计淋病奈瑟菌TEM-1基因的半套式聚合酶链反应(heminested PCR)检测方法。并对2002年1～10月自无锡地区分离的195株菌进行检测。结果 195株淋病奈瑟菌临床分离株中有138株为TEM-1基因阳性,阳性率为70.8％。1株无锡地区分离株的TEM-1基因半套式PCR产物,经DNA测序与核酸库登录的β-内酰胺酶阳性的淋病奈瑟菌质粒pFA7序列99％同源。结论 含TEM-1基因的淋病奈瑟菌株是无锡地区的流行株。     

淋球菌膜蛋白疫苗的研究

目的设计淋球菌膜蛋白疫苗并构建原核表达系统,测定表达蛋白的免疫活性.方法PCR扩增淋球菌膜蛋白(PIA)基因片段,构建重组质粒,重组质粒宿主菌经IPTG诱导,用SDS-PAGE分析PIA的表达情况.淋球菌标准株免疫BALB/c小鼠,体外凝集检测动物血清的免疫效果及ELISA法检测PIA的免疫活性.结果成功构建表达PIA蛋白的重组质粒,SDS-PAGE电泳图上显示1条相对分子量(Mr)约为40KDa的新生条带,能与免疫血清发生特异性结合反应.结论淋球菌膜蛋白疫苗原核表达系统的设计和构建正确,表达蛋白PIA具有免疫活性,为淋病疫苗的开发奠定了基础.     

淋病奈瑟菌标准株外膜蛋白NspA基因重组质粒构建和生物信息分析

[目的]研究淋病奈瑟菌标准株(NG 29403和NG 29400)外膜蛋白NspA基因和NspA融合蛋白结构特点,为淋病奈瑟菌疫苗靶蛋白的选择提供依据。[方法]PCR扩增淋病奈瑟菌标准株NG 29400和NG 29403 NspA基因,与表达质粒pET-30c(g )构建重组子NspA-pET-30c( ),阳性克隆子经双酶切鉴定后进行基因测序。以生物软件对NspA基因进行生物信息分析,并在线预测其蛋白质结构。[结果]成功构建NspA-pET-30c( )重组质粒,双酶切获得0.5kb和5.4kb预期产物。基因序列分析表明淋病奈瑟菌NspA基因有较高同源性,两标准株与GenBank已有序列均有98%同源性,且与脑膜炎奈瑟菌NspA基因高度近似。蛋白预测显示NspA融合蛋白有40%以上为环状结构,可能为主要功能区。三级结构与脑膜炎奈瑟菌NspA相似。[结论]淋病奈瑟菌NspA基因具有高度保守性,与脑膜炎奈瑟菌NspA基因同源性高,蛋白结构相似,有望成为淋病奈瑟菌疫苗的候选抗原。     

淋球菌膜蛋白疫苗的研究

目的 :设计淋球菌膜蛋白疫苗并构建原核表达系统 ,测定表达蛋白的免疫活性。方法 :PCR扩增淋球菌膜蛋白( PIA)基因片段 ,构建重组质粒 ,重组质粒宿主菌经 IPTG诱导 ,用 SDS- PAGE分析 PIA的表达情况。淋球菌标准株免疫 BAL B/c小鼠 ,体外凝集检测动物血清的免疫效果及 EL ISA法检测 PIA的免疫活性。结果 :成功构建表达 PIA蛋白的重组质粒 ,SDS-PAGE电泳图上显示 1条相对分子量 ( Mr)约为 4 0 KDa的新生条带 ,能与免疫血清发生特异性结合反应。结论 :淋球菌膜蛋白疫苗原核表达系统的设计和构建正确 ,表达蛋白 PIA具有免疫活性 ,为淋病疫苗的开发奠定了基础     

淋球菌临床分离株PI基因序列的测定与分析

目的：分析比较4株临床分离的淋球菌和2株标准菌株外膜蛋白IP基因及蛋白质序列，初步探索我国淋球菌的变异情况，为淋球菌多态性研究、IP基因的表达和疫苗研制提供依据。方法：构建pBS-T-IP克隆重组子并进行测序，用hlastn和CLUSTAL W分析基因和蛋白质序列的相似性，并与已发表的序列进行比较，预测并分析所得IP基因的loop(表面暴露区域)结构。结果：在GenBank中搜索到相似性大于98％的序列，6条序列分别属于PIA和PIB两个亚型，其中IP4的序列与GenBank中的相应序列有18个位点的差异，可能为我国特有的流行株。位于7个loop结构中的变异位点占3条PIA蛋白间总变异的67％，占3条FIB蛋白间总变异的84％，变异程度较高。结论：成功克隆了临床分离的淋球菌株和标准菌株的IP基因并进行序列分析，为深入研究我国流行的淋球菌菌株的多态性、IP基因的表达和疫苗研制奠定了基础。     

孔蛋白对蜱虫及其相关病原体的作用

蜱虫能携带很多病原体,在畜牧生产和公共卫生上具有十分重要的意义.研究蜱虫与病原体的相互关系,有助于开发、研究防蜱控病的药物、疫苗.孔蛋白存在于大量生物体中,也包括蜱虫.它穿梭于线粒体膜,形成膜通道,通过阴阳离子调节电压开闭通道,进而控制细胞代谢过程中的物质和能量流.本文阐述了孔蛋白的结构、进化关系和功能,并重点概述了蜱...     

淋球菌孔蛋白疫苗在动物体内诱导的免疫应答

目的:观察淋球菌孔蛋白疫苗免疫日本大耳白兔后所诱导的免疫应答。方法:兔背皮下注射经原核构建表达并纯化的淋球菌孔蛋白B,ELISA动态检测免疫后大耳兔血清抗体滴度的变化及最高效价;后腿皮内注射孔蛋白B,观察迟发型超敏反应(DTH)、免疫血清体外玻片凝集及抗菌作用。结果:初次免疫后2周,大耳兔体内已产生抗体,但效价较低;第2次免疫后2周血清效价达到1∶2000;第3次免疫后2周血清抗体水平成倍升高,效价为1∶4000;第4次免疫后2周血清最高效价达到1∶12000。大耳兔腿部发生DTH反应,出现明显红肿,于注射后第4天消退。血清体外凝集试验呈阳性,加入补体后发生淋球菌菌体裂解。结论:淋球菌孔蛋白疫苗可诱发动物特异性免疫应答,这为淋病疫苗的进一步研究提供了理论和实验依据。     

多药耐药肺炎克雷伯菌β-内酰胺酶基因及膜孔蛋白突变检测

目的了解多药耐药肺炎克雷伯菌耐药性及耐药基因的表达,为临床治疗肺炎克雷伯菌致呼吸机相关性肺炎(VAP)提供理论依据和指导。方法收集医院2011年7月-2012年6月入住儿童重症监护病房(PICU)20例呼吸机相关性肺炎患儿下呼吸道痰液标本,通过药敏试验检测耐药率,PCR法及基因测序检测β-内酰胺酶和膜孔蛋白的表达。结果共20株肺炎克雷伯菌对头孢类抗菌药物均存在耐药,对头孢唑林、头孢呋辛、头孢他啶、头孢噻肟、头孢吡肟、头孢西丁耐药率分别为100.0%、95.0%、70.0%、70.0%、55.0%、30.0%;共检出6种β-内酰胺酶基因,分别为A类SHV、TEM、CTX-M-1、LAP,C类DHA-1和D类OXA-1,ompK35膜孔蛋白基因检测10株为基因缺失和8株为基因突变,ompK36膜孔蛋白基因7株缺失和2株突变;6株非产ESBLs株均同时存在ompK35/36的突变或缺失,且伴有β-内酰胺酶基因的表达。结论 20株肺炎克雷伯菌均检出1³种β-内酰胺酶基因;ompK35/36膜孔蛋白基因检测结果显示,该蛋白几乎均有缺陷;提示该组耐β-内酰胺类药物为产β-内酰胺酶和ompK膜孔蛋白缺陷的双重作用,特别是非产ESBLs菌株。     

Characterisation and immune responses to meningococcal recombinant porin complexes incorporated into liposomes

We have analysed the structure of meningococcal outer membrane complexes and found that the main complexes are formed by different combinations of PorA and/or PorB molecules, associated to other proteins such as RmpM. In view of the growing knowledge of the importance of conformational epitopes in the immune responses to many pathogens, our aim in this study was to analyse the interactions of PorA and PorB by reconstitution of both recombinant porins into liposomes and determine the relevance of these interactions for the immune response. Recombinant PorA and PorB incorporated into liposomes associate forming complexes that are homomeric when only one of the porins is present, but heteromeric when both neisserial porins are present, mimicking those found previously in native outer membrane vesicles (OMVs). Association of PorA and PorB to form heterocomplexes modifies the immunogenicity of at least PorB, allowing the production of antibodies that recognise conformational epitopes, and produces new epitopes that react with a 50 kDa outer membrane protein not yet identified.     

产碳青霉烯酶肺炎克雷伯菌膜孔蛋白OmpK35与OmpK36基因转录水平

目的通过对产碳青霉烯酶肺炎克雷伯菌携带耐药基因和分子流行特点及膜孔蛋白OmpK35、OmpK36基因转录水平、膜孔蛋白表达情况分析,了解膜孔蛋白OmpK35、OmpK36在耐药机制中的作用。方法对32株产碳青霉烯酶肺炎克雷伯菌,进行MLST基因分型;利用PCR技术对产碳青霉烯酶,头孢菌素酶、超广谱β内酰胺酶相关耐药基因和膜孔蛋白OmpK35、OmpK36基因进行检测,并对OmpK35、OmpK36基因进行序列分析;实时荧光定量RT-PCR检测膜孔蛋白OmpK35、OmpK36基因转录水平,用SDS-PAGE检测膜孔蛋白的表达情况。结果 32株耐碳青霉烯类肺炎克雷伯菌MLST分型,29株为ST11、2株ST15、1株为ST2193;PCR检测出31株KPC-2阳性,1株NDM-1阳性,32株都检出SHV,其中一株同时检出OXA-48基因。OmpK35、OmpK36基因序列存在点突变、单个碱基和小片段缺失;OmpK35、OmpK36基因转录水平与肺炎克雷伯菌ATCC 13883相比,转录水平不一,大部分明显上调;SDS-PAGE未检测有膜孔蛋白OmpK36缺失株。结论产KPC-2、NDM-1型碳青霉烯酶是导致肺炎克雷伯菌对碳青霉烯类药耐药并表现多药耐药的主要原因,而膜孔蛋白OmpK35、OmpK36在其耐药中作用未体现。     

耐药大肠埃希菌β-内酰胺酶基因及膜孔蛋白基因研究

目的 调查大肠埃希菌中β-内酰胺酶基因和膜孔蛋白ompC基因的存在及变异情况.方法 收集医院2009年6月-2010年6月临床分离的耐药大肠埃希菌共20株,采用聚合酶链反应(PCR),分析A、B、C、D等4类β-内酰胺酶24种基因和膜孔蛋白ompC基因.结果 20株耐药大肠埃希菌共检出TEM、CTX-M-1群和OXA-1群等3种β-内酰胺酶基因,阳性率分别为45.0％、40.0％和10.0％,其余21种基因均未检出;20株耐药大肠埃希菌共检测到19株占95.0％,膜孔蛋白编码基因ompC,经测序比对除11号株外,其余18株占90.0％均存在突变.结论 20株耐药大肠埃希菌对β-内酰胺类药物耐药为产β-内酰胺酶和膜孔蛋白变异的共同结果.     

Effect of adjuvant composition on immune response to a multiple antigen peptide (MAP) containing a protective epitope from Neisseria meningitidis class 1 porin.

A variety of adjuvants with the potential for use with experimental human vaccines were used for immunisation of mice, in an attempt to augment the humoral immune response to a multiple antigen peptide (MAP) containing a protective epitope from the sero-subtype specific class 1 porin protein of Neisseria meningitidis, in tandem with a Th-cell epitope. Surface plasmon resonance showed that combinations of the immunomodulators pluronic block co-polymer, muramyl dipeptide and monophosphoryl lipid A (MPL), increased the magnitude and avidity of the immune response in comparison with both Al(OH)3 and Freund-type adjuvants. In addition, the incorporation of MPL was essential for the induction of a broad distribution of antibody isotypes. The antibodies induced recognised the native protein in meningococcal outer membranes in a subtype-specific manner. The formulations containing these multiple immunomodulators which have already been used in human phase I/II trials with experimental vaccines, are candidates for inclusion in future human vaccines based on synthetic peptides containing defined, protective epitopes.     

The PorB porin from commensal Neisseria lactamica induces Th1 and Th2 immune responses to ovalbumin in mice and is a potential immune adjuvant

Porins from pathogenic Neisseriae are among several bacterial products with immune adjuvant activity. Neisseria meningitidis (Nme) PorB, has been shown to induce immune cells activation in a TLR2-dependent manner and acts as a vaccine immune adjuvant. The PorB porin from Neisseria lactamica (Nlac), a common nasopharyngeal commensal, shares significant structural and functional similarities with Nme PorB. In this work we ask whether the immune adjuvant ability of porins from pathogenic Neisserial strains is a characteristic shared with porins from non-pathogenic Neisserial species or whether it is unique for bacterial products derived from microorganisms capable of inducing inflammation and disease. We evaluated the potential immune adjuvant effect of Nlac PorB in mice using ovalbumin (OVA) as a prototype antigen. Immunization with Nlac PorB/OVA induced high OVA-specific IgG and IgM titers compared to OVA alone, similar to other adjuvants such as Nme PorB and alum. High titers of IgG1 and IgG2b were detected as well as production of IL-4, IL-10, IL-12 and INF-gamma in response to Nlac PorB, consistent with induction of both a Th1-type and a Th2-type immune response. OVA-specific proliferation was also determined in splenocytes from Nlac PorB/OVA-immunized mice. In addition, B cell activation in vitro and cytokine production in response to Nlac PorB was found to be mediated by TLR2, in a similar manner to Nme PorB.     

Role of efflux pumps: MexAB-OprM and MexXY(-OprA), AmpC cephalosporinase and OprD porin in non-metallo-β-lactamase producing Pseudomonas aeruginosa isolated from cystic fibrosis and burn patients

Purpose of the researchIn order to gain a better understanding of the role of several mechanisms in antibiotic resistance in Pseudomonas aeruginosa clinical isolates obtained from CF and burn patients, we evaluated gene expression of efflux pumps MexAB-OprM and MexXY(-OprA), the natural β-lactamase AmpC and outer membrane porin protein OprD. Also, the presence of genes encoding Ambler classes A, B β-lactamases and aminoglycoside modifying enzymes (AMEs) was examined.Principal resultsPiperacillin–tazobactam and amikacin retained the highest in vitro activities among 21 CF and 27 burn P. aeruginosa isolates. Based on Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR, 15 distinct patterns were detected. There were 5 CF and 6 burn isolates harbored PER-1 and VEB-1, respectively. Among AMEs, involved in resistance of anti-Pseudomonas aminoglycosides, aac(6′)-Ib was the most prevalent gene. Among CF isolates, mexA overexpression was the most prevalent mechanism (47.6%) followed by mexX (42.8%), ampC (9.5%) and oprD downregulation (4.7%). Among burn isolates, the prevalence of mexX, mexA, and ampC overexpression was 62.9%, 74%, and 11.1%, respectively. Downregulation of oprD was observed in 14.8% of burn isolates.Major conclusionsAmong CF isolates, mexX and mexA overexpression were the major contributing factors to aminoglycoside (gentamicin) and carbapenem (meropenem) resistance, respectively while among burn isolates, AMEs in conjunction with mexX hyperexpression were identified to be responsible for aminoglycoside resistance. Also mexA overexpression was partially associated with carbapenem resistance. Moreover, cephalosporin resistance was linked to overexpression of mexA and/or mexX. The impact of interplay between different resistance mechanisms on resistant phenotypes was more complicated among burn than CF isolates.     

A Community-wide epidemic of hepatitis A in Ohio

Between June 1, 1983 and August 30, 1984, an epidemic involving 313 cases of hepatitis A occurred in Muskingum County, Ohio. One hundred ninety-seven cases occurred in the city of Zanesville, with 34.7% of cases concentrated in two neighborhoods in the eastern part of the city. Case characteristics were similar to those reported in previous community-wide outbreaks, including a maximum attack rate among 5-9-year-olds and a very low attack rate in adults over 30 years. Case households were larger, and their members were less educated than the mean for households in the city. Forty-eight per cent of the cases reported exposures to other cases which temporally could have been the source of infection. A case-control study failed to show differences in several behavioral factors between case and control households, but did confirm that lower socioeconomic status was a risk factor for the disease. Broad use of immunoglobulin was effective in preventing clinical disease among family contacts, but did not stop the outbreak. This outbreak typifies a genre of hepatitis A epidemic transmitted from person to person in which exact routes of spread are poorly understood and control is difficult. Lower socioeconomic status may be a marker for some unidentified behaviors that promote hepatitis A transmission.