中国致病性小肠结肠炎耶尔森菌生物分型研究

目的初步了解我国致病性小肠结肠炎耶尔森菌O∶3、O∶9血清型菌株的生物型分布,掌握我国致病性小肠结肠炎耶尔森菌的生物学特征。方法用文献报道方法对1986-2008年从我国不同地区的腹泻病人、家禽家畜、鼠类、食品、环境等各类宿主中分离到的427株致病性小肠结肠炎耶尔森菌进行生物分型。结果 427株致病性小肠结肠炎耶尔森中213株属于生物2型(49.9%),全部为O∶9血清型;208株属于生物3型(48.7%),其中191株为O∶3血清型,17株为O∶9血清型;6株属于生物4型(1.4%),全部为O∶3血清型。没有发现生物1A型、1B型与5型的致病性菌株。结论我国O∶3血清型致病性小肠结肠炎耶尔森菌以生物3型为主、O∶9血清型致病性菌株以生物2型为主,没有生物1B型菌株,与国外致病性菌株的生物血清型分布有一定差异。     

胶体金标记免疫层析法检测O∶9型小肠结肠炎耶尔森菌

目的建立一种简便快速的胶体金标记免疫层析(GICA)条用于检测标O∶9血清型小肠结肠炎耶尔森氏菌。方法采用柠檬酸三钠还原法制备胶体金颗粒,标记抗小肠结肠炎耶尔森菌单克隆抗体,以硝酸纤维膜作为抗小肠结肠炎耶尔森氏菌单克隆抗体的包被载体,制成GICA检测条。被检小肠结肠炎耶尔森菌与检测卡上金标记抗体(Au-Ab)结合后,利用硝酸纤维膜的层析作用,与膜上的固相抗体结合形成可见的红色条带。结果GICA小肠结肠炎耶尔森菌检测条灵敏度可达104~105CFU/ml,与其他所选择肠杆菌科细菌均未发现交叉反应,仅与布鲁菌出现交叉。检测了50株O∶9血清型小肠结肠炎耶尔森氏菌,符合率为100%。结论GICA小肠结肠炎耶尔森菌检测条对小肠结肠炎耶尔森菌特异性强;简便快速,不需任何仪器设备;结果易于判断,可望用于标本快速筛查。     

胶体金标记免疫层析法检测O:9型小肠结肠炎耶尔森菌

目的建立一种简便快速的胶体金标记免疫层析（GICA）条用于检测标O：9血清型小肠结肠炎耶尔森氏菌。方法采用柠檬酸三钠还原法制备肢体金颗粒，标记抗小肠结肠炎耶尔森菌单克隆抗体，以硝酸纤维膜作为抗小肠结肠炎耶尔森氏菌单克隆抗体的包被载体，制成GICA检测条。被检小肠结肠炎耶尔森菌与检测卡上金标记抗体（Au-Ab）结合后，利用硝酸纤维膜的层析作用，与膜上的固相抗体结合形成可见的红色条带。结果GICA小肠结肠炎耶尔森菌检测条灵敏度可达10^4～10^5CFU／ml，与其他所选择肠杆菌科细菌均未发现交叉反应，仅与布鲁菌出现交叉。检测了50株O：9血清型小肠结肠炎耶尔森氏菌，符合率为100％。结论GICA小肠结肠炎耶尔森菌检测条对小肠结肠炎耶尔森菌特异性强；简便快速，不需任何仪器设备；结果易于判断，可望用于标本快速筛查。     

小肠结肠炎耶尔森氏菌研究近况

小肠结肠炎耶尔森氏菌是一个很重要的人类和动物肠道致病菌，病人最常见的临床症状有腹泻，胃肠炎，肠系膜淋巴结炎，更为严重者可引起败血症，伴随肝脓肿。其它器官组织也会产生病变，如活动性关节炎和结节性红斑[1，2]。根据O抗原因子将小肠结肠炎耶尔森氏菌分成50个以上血清     

鼠疫耶尔森氏菌外膜蛋白研究进展

耶尔森氏菌属中致病性细菌有3种，即鼠疫耶尔森氏菌、小肠结肠炎耶尔森氏菌和假结核耶尔森氏菌。其中鼠疫耶尔森氏菌是致病性最强的一种。随着分子生物学的研究进展，人们对鼠疫菌有了深入的了解。鼠疫菌外膜蛋白（Yops）是近些年对鼠疫菌的基础研究内容焦点之一。国内在世界上首先发现鼠疫菌锡林郭勒高原型病原体缺少32Kd外膜蛋白和40Kd蛋白位于膜内，     

鼠疫耶尔森菌外膜蛋白及其功能研究进展

鼠疫耶尔森菌和其他革兰阴性杆菌一样,其细胞结构由双层膜结构包绕,内层为内膜(亦称胞浆膜),外层是细胞与外环境的分界,称为外膜.     

耶尔森氏菌外膜蛋白1体外表达条件的观察

使用常规培基孵育耶尔森氏菌一般均能获得满意的增菌效果。有文献报导,不同培基对耶尔森氏菌外膜蛋白(Yops)的表达无显著的影响。然而,我们的实验结果提示,Yop1的体外表达具有严格的培基依赖性,该蛋白仅在只含氨基酸成份的最小培基中才能充分表达,其表达量远高于其他几种常规培基。此外,我们还观测了细菌菌龄对这种蛋白体外表达的影响,结果表明,菌龄也影响着该蛋白的体外表达,但其作用强度不如培基那样显著。转换温度后12小时,膜制剂中Yop1的含量达到高峰,增加孵育时间不能增大含量,反而使其呈明显的下降趋势,温度转换后24小时,表达量仅略高于2.5小时的水平。本文还报导了两种孵育温度(22℃、37℃)以及钙离子对Yop1蛋白表达的作用,所得结论与国外同类研究的结果一致,即与其他Yops不同,yopA基因的表达只受生长温度的控制而与钙离子的存在与否无关。本实验的结果将有助于Yop1蛋白的分析研究,特别是此种蛋白的提取制备。     

小肠结肠炎耶尔森氏菌主要毒力基因分析

目的了解南京地区从不同来源分离的小肠结肠炎耶尔森氏菌毒力基因的分布情况。方法聚合酶链反应(PCR)技术进行不同血清生物型菌株的毒力基因检测。结果从血清型来看,分离到的9株血清型O:3菌株有7株的基因型分布为ail+,ystA+,ystB-,virF+,yadA+。从生物型来看,共分离到致病生物型(3,4)菌株12株,其中9株毒力基因分布特征为ail+,ystA+,ystB-,virF+,yadA+。结论本市小肠结肠炎耶尔森氏菌血清型O:3菌均包含毒力基因,毒力基因主要分布特征为ail+,ystA+,ystB-,virF+,yadA+。所有的致病生物型(3,4)菌株,均包含染色体上的毒力基因ail,毒力基因主要分布特征亦为ail+,ystA+,ystB-,virF+,yadA+。ail与致病生物型之间存在明显关联。     

幽门螺杆菌黏附素HpaA蛋白在大肠杆菌中表达及其免疫保护作用

目的表达和纯化幽门螺杆菌(Helicobacter pylori,H.pylori)黏附素HpaA蛋白,研究其免疫活性和对小鼠的免疫保护作用。方法诱导TB1(pMAL-c2X-hpaA)表达H.pylori黏附素HpaA蛋白,Amyloss树脂预装柱进行分离、纯化,Western blot鉴定其免疫学活性;纯化蛋白免疫小鼠后,H.pylori国际标准菌株NCTC11637攻击感染,观察H.pylori定植情况及免疫保护效果。结果获得了高纯度且免疫活性良好的目的蛋白;HpaA蛋白免疫小鼠的保护率为53.33%(8/15),与对照组统计学差异显著(P=0.002)。结论纯化HpaA蛋白对小鼠有免疫保护作用,可作为H.pylori基因工程疫苗的候选组分。     

Surveillance of human Yersinia enterocolitica infections in Belgium: 1963–1978

Details are given on the epidemiology of human Yersinia enterocolitica infections in Belgium, based on 3167 isolations from 1963 to 1978. A continuing increase of the number of isolations is noted, with 1386 isolations in the last three years covered, excluding repeated isolations from the same patient.Serotype 3 remains predominant accounting for 84 per cent of all isolations. However, a changing pattern of serotypes distribution has been observed in recent years, with an increasing proportion of serotype 9 and serotypes other than 3 or 9.Most isolations are from faeces. From blood and deep abscesses only serotypes 3 and 9 were recovered. In Belgium only the latter serotypes are clearly associated with human disease, such as enteritis, pseudo-appendicular syndrome and septicaemia. The pseudo-appendicular syndrome is seen at an older age than is enteritis, and is relatively more frequent in serotype 9 infections. In contrast, serotypes other than 3 or 9 are relatively more common in persons without any illness or with atypical symptoms. There is evidence that they are non-pathogenic.     

Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

Advances in computational design methods have made possible extensive engineering of soluble proteins, but designed β-barrel membrane proteins await improvements in our understanding of the sequence determinants of folding and stability. A subset of the amino acid residues of membrane proteins interact with the cell membrane, and the design rules that govern this lipid-facing surface are poorly understood. We applied a residue-level depth potential for β-barrel membrane proteins to the complete redesign of the lipid-facing surface of Escherichia coli OmpA. Initial designs failed to fold correctly, but reversion of a small number of mutations indicated by backcross experiments yielded designs with substitutions to up to 60% of the surface that did support folding and membrane insertion.The β-barrel membrane proteins comprise one of the two structural classes of integral membrane proteins. They are found within the outer membranes of bacteria, mitochondria, and chloroplasts, where they perform a range of structural, transport, and catalytic functions (1). In addition to their biological interest they are increasingly relevant to biotechnology, serving as scaffolds for bacterial surface display (2, 3) and atomically precise pores for nanopore-based DNA sequencing. Although the suitability of natural β-barrel membrane proteins for biotechnology has been improved by protein engineering (3–10), the ability to design membrane proteins de novo would deliver tools customized to meet the demands of each application.De novo design provides a stringent test of our understanding of the determinants of protein folding and stability. Protein design software [e.g., Rosetta (11, 12)] has made tremendous strides in addressing the design problem for small water-soluble proteins (13–15), and design of simplified model α-helical membrane proteins including single transmembrane helices and small bundles (16–20) has also been accomplished. In contrast, a designed β-barrel membrane protein has yet to be reported, perhaps as a consequence of the unique design challenges presented by the folding pathway and architecture of these proteins. Unlike the α-helical membrane proteins, nascent β-barrel membrane proteins must transit the periplasm to the outer membrane, where folding and membrane insertion are thought to occur in concert (21, 22). An extensive network of chaperones maintains the solubility of the unfolded barrel and guides membrane insertion. The C-terminal β-strand is known to interact with the BAM chaperone complex (23–25), which assists the folding of all β-barrel membrane proteins. However, despite recent progress (26–30), we do not fully understand how interactions between chaperones and transiting membrane proteins are directed by sequence-encoded information.Further complicating design is the inside-out architecture of β-barrel membrane proteins. In place of a hydrophobic core is either a central water-filled pore or a solid core composed of polar side chains. The lipid bilayer becomes increasingly hydrophobic at greater depths within the membrane (31), and this environmental anisotropy is reflected in the amino acid composition of the barrel surface. Aliphatic side chains are prevalent toward the center of the membrane, and aromatic side chains are common in the lipid head group regions, where they encircle the barrel in external- and periplasmic-side girdles (32).Recently we developed E_zβ, a membrane depth-dependent, residue-level potential calculated from an ensemble of experimentally determined outer membrane protein structures (33, 34). E_zβ can be used to estimate energetics of membrane insertion to predict transmembrane protein orientation within the bilayer, and to detect oligomerization sites on β-barrel surfaces (34). E_zβ and related statistical functions (35, 36) can recapitulate properties of natural outer membrane proteins (37, 38) and predict the effects of mutations on protein stability and oligomerization (39). Similar potentials have driven computational approaches that have fully redesigned α-helical membrane protein surfaces to convert membrane proteins into water-soluble ones (40–42).Here, we considered whether the complete redesign of the lipid-facing surface of an outer membrane protein using a statistical potential such as E_zβ preserves its structure and function. This approach allowed us to investigate whether membrane insertion requires only a lipid-facing surface composed of depth-appropriate hydrophobic residues, or whether folding requires sequence-specific interstrand interactions, chaperone-recruiting sequences, evolutionarily optimized aromatic girdles, folding nucleation sites, or other design features lost during the population averaging inherent in parameter fitting of statistical potentials.Previous studies have explored the sensitivity of the β-barrel fold and its chaperone recognition mechanisms to mutations. The canonical eight-stranded β-barrel membrane protein OmpA tolerates a limited number of mutations to the lipid-facing surface, provided hydrophobicity is maintained (43, 44). More radically, the eight-stranded barrel OmpX has been duplicated to form a 16-stranded barrel capable of membrane insertion (45). However, the lipid-facing residues of transmembrane β-strands are conserved across homologous β-barrel membrane proteins beyond the extent expected from hydrophobicity alone (46, 47), implying a functional role that has yet to be elucidated.To explore the sequence constraints on β-barrel membrane proteins, we extensively redesigned the lipid-facing surface of E. coli OmpA. We created a series of OmpA variants with entirely or partially redesigned lipid-facing surfaces and tested their ability to insert into the outer membrane of E. coli. Our results indicate that the surfaces of β-barrel membrane proteins are amenable to large-scale redesign, provided that energetically destabilizing substitutions are avoided.     

2009-2012年登封市小肠结肠炎耶尔森菌分布调查

目的 调查登封地区家畜家禽小肠结肠炎耶尔森菌感染状况。方法 采集家畜家禽新鲜粪便分离小肠结肠炎耶尔森菌,对分离菌株进行生化鉴定、血清分型、生物分型及毒力基因检测。结果 从1 285份粪便标本中共检出105株,检出率8.17%。其中狗17株(17.35%);猪35株(13.62%);检出O∶3血清型12株(13.48%), O∶5血清型12株(13.48%),O∶8血清型14株(15.73%);Ail+、ystA+、yadA+、virF+的菌株占12.36%,ystB+ 的菌株占42.70%。结论 登封市家禽家畜普遍携带小肠结肠炎耶尔森菌,主要动物宿主为猪和狗,是小肠结肠炎耶尔森菌人兽共患感染性腹泻病的重要传染源。     

不同毒力钩端螺旋体属特异性外膜蛋白抗原的分析

目的　了解不同毒力钩端螺旋体株有无属特异性外膜蛋白抗原。方法　TR/ patocⅠ钩体属特异性抗原免疫家兔获得抗血清 ,以显微镜凝集试验检测TR/patocⅠ属特异性抗原抗血清对我国 15群 17型问号钩体和 2群 2型双曲钩体的凝集情况。采用Auran介绍的方法制备问号钩体黄疸出血群赖型 5 6 6 0 1株、波摩那群波摩那型 5 6 6 0 8株和双曲钩体三宝垄群Patoc型patocⅠ株的外膜蛋白 ,用SDS -PAGE及Westernblot分析不同毒力钩体外膜蛋白的电泳特征及与TR/ patocⅠ属特异性抗血清的反应性。结果　钩体TR/ patocI属特异性抗血清能与上述各株钩体发生凝集反应 ,其效价为 1∶2 5 6～ 1∶5 12。三种不同毒力钩体外膜蛋白有相似的SDS -PAGE图谱 ,其主要蛋白组分的分子量约为 6 0kDa ,Westernblot结果证实在约31kDa处有一共同的能与TR/patocI属特异性抗血清发生反应的阳性条带。结论　钩体细胞表面具有属特异性抗原 ,分子量31KDa外膜蛋白可能是属特异性抗原之一。     

汉赛巴通体重组外膜蛋白Pap31的研制和抗原性分析

目的克隆并表达汉赛巴通体Pap31外膜蛋白基因,并对其抗原性进行初步分析。方法采用PCR从汉赛巴通体基因组DNA扩增外膜蛋白基因pap31,将目的基因片段插入原核表达质粒pQE30,构建重组质粒pQE30/pap31;将构建的重组质粒转化大肠杆菌M15并诱导目的基因表达,以SDS-PAGE电泳以及免疫印迹法分析表达目的蛋白。结果在SDS-PAGE电泳分析发现pQE30/pap31转化菌高效表达一重组蛋白,经免疫印迹分析发现该蛋白与汉赛巴通体免疫血清发生强烈反应;经间接免疫荧光分析发现该重组蛋白免疫血清能特异识别汉赛巴通体。结论汉赛巴通体外膜蛋白基因pap31在大肠杆菌高效表达,表达的重组Pap31外膜蛋白具有良好的抗原性。     

Human monoclonal antibodies against an epitope on the class 5c outer membrane protein common to many pathogenic strains of Neisseria meningitidis.

Neisseria meningitidis is a causative agent of meningitis. Despite vaccination programs, it still causes a large number of deaths in young children. Early diagnosis followed by passive immunization with human monoclonal antibodies could be an approach to effective therapy. Peripheral blood lymphocytes from normal, healthy blood donors and from vaccinated individuals were immunized in vitro, using outer membrane proteins purified from N. meningitidis B:4:P1.15. The immunized human B cells were Epstein-Barr virus transformed and fused to a heteromyeloma. Several stable human hybridoma cell lines were established and two, secreting antibodies against the 31-kDa class 5c outer membrane protein, were characterized further. The human antibodies were of IgG1 and IgG3 isotypes, with kappa light chains. The recognized epitope was commonly found among pathogenic strains of N. meningitidis; thus, these human monoclonal antibodies may be important in the evaluation of N. meningitidis infections.     

多重耐药奇异变形杆菌外膜通透性改变的初步研究

目的 研究奇异变形杆菌诱导耐性后外膜通透性改变与其耐药性的关系。方法 用含头孢噻肟的梯度平板多步诱导２株奇异变形杆菌达稳定耐药;以十二烷基硫酸钠－聚丙烯酰胺凝胶电泳法（ＳＤＳ－ＰＡＧＥ）电泳耐药前后细菌外膜蛋白;以高效液相色谱法（ＨＰＬＣ）测定细菌对环丙沙星的摄取量;以扫描、透射电镜观察形态学变化。结果 所诱导的细菌对氟喹诺酮类、头孢类、青霉素类抗生素多重耐药;耐药株外膜蛋白相对分子质量４００００     

不同方式重组表达鼠疫caf1M蛋白的研究

目的对比不同方式克隆表达鼠疫耶尔森氏菌caf1M蛋白的存在状态及免疫学活性。方法分别选择3种不同载体pET32a(+)、pGEX4t-1、pGBTNH,克隆表达鼠疫caf1M蛋白;并以鼠疫菌免疫血清分别检测其免疫学活性。结果成功构建了4个重组质粒,分别是含去除信号肽编码序列caf1M基因的3个质粒载体,及1个含有完整caf1M基因的pGEX4t-1表达质粒;4个质粒经诱导均在大肠杆菌中得到高效表达;存在状态分析表明,含有去除信号肽编码序列caf1M基因的pGEX4t-1表达的重组蛋白以可溶方式存在,其余3种以包涵体形式存在;重组蛋白与鼠疫菌免疫血清的Western blot分析表明,除pGBTNH表达的蛋白存在非特异反应外,其余3种重组蛋白都发生特异性反应。结论成功克隆表达了4种鼠疫菌caf1M蛋白,其中3种具有良好的特异性免疫反应性;信号肽序列及载体都对重组caf1M蛋白的表达有影响。     

钩端螺旋体OmpL1抗原表位预测、克隆和表达

目的　应用生物信息学方法预测钩体外膜蛋白OmpL1的表位 ,结合基因工程手段进行表位重组、表达和分离纯化。方法　用预测程序ProPred和ANTIGENIC预测OmpL1的表位 ,PCR合成重组表位基因片段 ,克隆PCR产物构建表达质粒 pGEX/Omp Omp ,测序验证。对含有该质粒的大肠杆菌BL2 1(DE3)进行诱导表达 ,表达产物westernblot分析并纯化融合蛋白。结果　预测到 2个既具有MHC结合肽特性又具有B细胞表位特征的肽段。重组表位基因序列与理论设计完全一致。IPTG诱导BL2 1(DE3)中高效表达Mr约 30 0 0 0的融合蛋白 ,纯化后蛋白纯度 >90 %。结论　成功构建原核表达质粒pGEX/Omp Omp ,并进行含重组表位的GST融合蛋白的分离纯化 ,为OmpL1的表位研究和应用于亚单位疫苗奠定了基础。