大肠杆菌外膜蛋白研究概述

大肠杆菌具有一种独特的外膜蛋白结构,此结构在大肠杆菌生长的任何一个阶段都起着非常重要的作用。本文主要对大肠杆菌外膜蛋白的组成和功能、致病作用以及免疫原性等方面的研究进展作一概述。     

鸡大肠杆菌的致病因素及其研究进展

本文就鸡大肠杆菌的致病因素及其研究进展做了分析与讨论,阐述了目前研究的现状与进展。通过分析得出:大肠杆菌表面的菌毛物质可以作为抗原,能有效地被识别,对于预防鸡大肠杆菌病具有很大的作用;通过对类脂A成分的疫苗研发,分析怎样有效减少毒素对于鸡身体的破坏作用,也逐渐的成为毒素免疫研究的重点;对大肠杆菌自身的外膜蛋白进行提纯所得到的物质,与人工合成的外膜蛋白物质都拥有非常明显的免疫原性。     

新技术新产品

能分离出鸡大肠杆菌的新菌株 我国科研人员历经2年多的研制,“鸡大肠杆菌异型外膜蛋白细胞工程菌株的构建”,日前通过专家技术鉴定,并被确认为达到国内领先水平。鸡大肠杆菌病是鸡腹膜炎、输卵管炎、眼球炎、大肠杆菌败血病的总称,是危害养鸡业的严重疫病之一。科技工作者研制成的新型融合菌株,其稳定性     

牛源大肠杆菌不同生长阶段的比较蛋白质组学研究

为了分析牛源大肠杆菌生长周期中不同阶段蛋白的表达变化,本研究采用二维凝胶电泳结合质谱技术分离并鉴定了大肠杆菌生长周期中延迟期、对数期和平稳期菌体全蛋白的表达变化。结果显示,大肠杆菌菌体全蛋白组成在延迟期、对数期和平稳期存在差异,大肠杆菌生长延迟期的外膜蛋白W和磷酸丙酮酸羧化酶表达量高于对数期和平稳期,而周质蛋白、外膜蛋白Ⅱ和磷酸转乙酰酶表达量低于对数期和平稳期。本研究结果初步揭示了菌体蛋白表达变化与细菌生长的关系,为进一步用于大肠杆菌奶牛乳房炎的防控提供参考。     

革兰氏阴性菌外膜蛋白研究进展

外膜蛋白对细菌本身和宿主都具有重要的作用。本文综述了革兰氏阴性菌外膜蛋白的组成、结构、功能及遗传调控,外膜蛋白可作为大肠杆菌的克隆标志,在细菌的致病机理及免疫机理中都有重要作用,研制开发外膜蛋白疫苗具有广阔的应用前景,必将为细菌性疾病的防治提供有力的措施。     

大肠杆菌外膜蛋白改变与四环素耐药的关系

大肠杆菌K-12外膜蛋白改变对四环素耐药起着重要作用,但其机制尚不明确。本文采用2-DE、Western-blotting方法研究发现,大肠杆菌对四环素耐药时,其FimD、Tsx、OmpW、OmpC、TolC表达量上调,LamB表达量下调。利用转基因技术构建大肠杆菌突变株(基因敲除或补救),探究6种外膜蛋白的功能,发现tolC、ompC的表达可以降低MIC,而lamB的表达可以升高MIC。通过基因补救的方法可使基因敲除株恢复正常。基因敲除株通过增加ΔlamB或减少tolC、ompC、ompW、tsx使细菌活性增强,且tolC、ompC减少比ompW和tsx减少更为明显,可在基因修补株上检测到相同结果。因此,LamB、OmpC、TolC三种外膜蛋白在大肠杆菌对四环素耐药中起重要作用。此外,细菌外膜蛋白的改变是否影响细菌的血清型有待进一步研究。     

香芪口服液对大肠杆菌耐药抑制作用

通过香芪口服液对耐药大肠杆菌作用前后环丙沙星和庆大霉素最小抑菌浓度测定、质粒图谱分析以及外膜蛋白图谱分析,研究香芪口服液对耐药大肠杆菌的体外耐药性的抑制作用.结果表明:中药作用后,大肠杆菌对环丙沙星和庆大霉素最小抑菌浓度无变化;中药作用后的大肠杆菌的质粒图谱部分质粒消失,1号菌株ompA恢复表达,2号菌株ompF恢复表达;此中药与大肠杆菌质粒拷贝、恢复外膜通道蛋白的正常表达有密切关系,为今后耐药抑制剂的研制奠定理论基础.     

禽致病性大肠杆菌外膜蛋白SDS-PAGE分析

提取4种血清型的禽致病性大肠杆菌外膜蛋白,进行SDS-PAGE分析,证明这些血清型的外膜蛋白主要由2条带组成,位于30～43KD.用血清型O78的免疫血清进行免疫印迹反应,结果显示,其与这4种血清型的外膜蛋白均发生反应,表明4种血清型的主要外膜蛋白不仅具有抗原性,而且具有交叉免疫性.     

犬布氏杆菌外膜蛋白Omp31基因的原核表达

克隆了犬布氏杆菌外膜蛋白Omp31基因并构建原核表达系统,并对表达产物进行了初步的血清学鉴定。利用PCR技术扩增犬布氏杆菌RM6/66参考株Omp31基因,然后将其克隆到pGEMT-easy载体上进行测序。测序正确后,将该基因插入到pET-32a载体中构建原核表达载体,转化大肠杆菌BL21感受态细胞,诱导表达融合蛋白,Western blot分析融合蛋白的免疫反应性。结果构建了犬布氏杆菌Omp31基因的原核表达载体pET-Omp31,并且在大肠杆菌中成功表达融合蛋白,经Western Blot鉴定该蛋白能被犬布氏杆菌阳性血清所识别。犬布氏杆菌外膜蛋白Omp31的表达成功,为犬布氏杆菌病血清学诊断方法的建立提供了基础资料。     

肠道外致病性大肠杆菌外膜蛋白研究进展

肠道外致病性大肠杆菌病(Extraintestinal pathogenic Escherichia coli,ExPEC)是一种常发肠道外疾病,危害人类、动物的健康,也导致禽类产蛋量的下降及肉用动物的上市延迟,严重的则引起死亡。因此,对于肠道外致病性大肠杆菌的致病机制也是许多学者研究的热点,肠道外致病性大肠杆菌致病机理的研究近些年主要集中在脂多糖(LPS)、外膜蛋白(Outer membrane protein,Omp)、菌毛、鞭毛、等毒力因子方面。     

Secretion of virulence factors by Escherichia coli.

In order to interact with their host, pathogenic strains of E. coli need to secrete some virulence factors which can modify the metabolism of host cells, contributing to disease. Since E. coli is a Gram-negative bacteria, this secretion process involves the crossing of both the inner and the outer membranes. E. coli uses mainly four secretion mechanisms called type I, type II, type III and type IV secretion systems. In the type I secretion system, the secretion machinery is composed of three proteins forming a channel through the inner and outer membranes. It is a one-step mechanism. The secretion signal is present in the carboxyterminal region of the secreted protein but without proteolytic cleavage. In E. coli, the best studied type I secreted protein is haemolysin. In type II and type IV secretion systems, the crossing of the inner membrane involves the sec machinery with the cleavage of an aminoterminal signal sequence. The crossing of the outer membrane involves the formation of a pore either by other proteins (type II) or by the carboxyterminal region of the protein (type IV). The A-B toxins, such as heat labile enterotoxin, are secreted by the first mechanism and members of the IgA proteases are secreted by the second. The type III secretion system involves at least 20 proteins including cytoplasmic, inner membrane and outer membrane proteins. The originality of this system is the ability to inject secreted bacteria into the cytosol of the host cells. Such a system is found in attaching and effacing E. coli and in diffusely adhering E. coli.     

Expression of the major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis in Escherichia coli using an arabinose-inducible plasmid vector

The ompA genes encoding the 40 kDa major outer membrane protein (MOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were cloned into the arabinose-inducible plasmid vector pBADMycHis, and recombinant MOMPs (rMOMP) from the three chlamydial species were expressed at high levels in Escherichia (E.) coli. The proteins lacking the 22 aa N-terminal signal peptide were expressed as insoluble cytoplasmic inclusion bodies which were readily purified using immobilized metal-affinity chromatography. The rMOMPs including the N-terminal signal peptide were expressed and translocated as a surface-exposed immunoaccessible protein into the outer membrane of E. coli. Transformants expressing this full-length rMOMP were significantly reduced in viability. Purified native elementary bodies (EB) and rMOMPs of the three chlamydial species purified from the E. coli cytoplasm were used for immunization of rabbits. The resulting sera were analysed for their ability to recognize homologous and heterologous rMOMP and native EB. When testing rMOMP antisera against rMOMP and EB antigens, marked cross-reactivities were detected between the three species. Using EB antisera and rMOMPs as antigens, a significant species-specific reactivity was measured.     

Prokaryotic Expression of the Truncated 43K OMP Genes of Bovine Fusobacterium necrophorum

Four pairs of primers containing BamHⅠ and XhoⅠ sites were designed for amplification of 43 ku outer membrane protein （43K OMP） of bovine Fusobacterium necrophorum strain H05 according to the GenBank. The PCR products of the truncated 43K OMP genes were digested with BamHⅠ and XhoⅠrestriction endonuclease, and then the digested products were ligated to the pET-32a vector with His tag. The positive plasmids of four truncated 43K OMP genes were transformed into E.coli BL21(DE3). Protein expression of four truncated 43K OMP genes were induced using 1.0 mmol/L IPTG. The result indicated that the four truncated 43K OMP genes were successfully expressed in E.coli, and molecular weights of the expressed proteins were all about 30 ku. This study would provide some basis for further research of immunogenicity of the outer membrane protein of Fusobacterium necrophorum.     

鸡大肠杆菌异型外膜蛋白原生质体融合菌株的构建

以鸡大肠杆菌Ww1株(O78,OMP-3,AmpR,KS)和WD2株(O2,OMP-1,AmpS,KR)作为亲本菌株,采用溶菌酶-EDTA法制备原生质体,以PEG作为助溶剂,进行原生质体融合,得到4株双耐药融合菌株,融合菌株的形态、染色特性和生化特性均符合大肠杆菌的特性,且均能同时表达两个亲本菌株的外膜蛋白(OMP)抗原(OMP-3,OMP-1)和O抗原(O78,O2),表明两个亲本菌株发生了融合和染色体重组。经多次传代证明融合菌株是稳定的。     

禽巴氏杆菌C48-1株成熟外膜蛋白H基因的原核融合表达

根据禽巴氏杆菌5：A C48-1株OmpH全长基因序列设计一对特异性引物，经PCR扩增获得成熟外膜蛋白H基因（OmpmH），将OmpmH片段非定向插入原核表达载体pGEX-6p-1中，构建重组表达质粒pGEX—OmpmH。转化大肠杆菌BL21（DE3），在IPTG诱导下表达融合蛋白GST—OmpmH。SDS—PAGE结果显示．GST—OmpmH约为63Ku，与预期的大小一致。Westernblot检测结果表明，GST—OmpmH能与C48-1外膜蛋白免疫血清发生特异性反应，证明C48-1 ompmH原核融合表达成功。其可溶性蛋白经亲和层析纯化，得到了纯度较高的目的蛋白OmpmH。为进一步研究禽巴氏杆菌C州OmpmH的免疫原性奠定了基础。     

布鲁氏菌外膜蛋白OMP15.6表达及免疫反应原性测定

表达羊布鲁氏菌16M预测外膜蛋白OMP15.6,探索其作为诊断抗原的可能性。采用降落PCR方法,从羊布鲁氏菌16M基因组DNA中扩增出423bp的基因片段,将该片段克隆于原核表达载体PGEX-4T-2,构建重组表达载体。经IPTG诱导,SDS-PAGE检测,结果表明,在大肠杆菌中成功表达了外膜蛋白OMP15.6。经West-ern-blotting检测,该蛋白能与豚鼠抗布鲁氏菌阳性血清发生特异性免疫反应,为其之后的抗原性以及免疫原性研究奠定了良好的基础。     

Adhesion of outer membrane proteins containing tandem repeats of Anaplasma and Ehrlichia species (Rickettsiales: Anaplasmataceae) to tick cells

Infection of cells by tick-borne rickettsiae appears to be mediated by outer membrane proteins that allow pathogens to adhere to host cells. Major surface protein (MSP) 1a of Anaplasma marginale, the type species for the genus Anaplasma, was shown previously to be an adhesin for tick cells. The A. marginale MSP1a has a variable number of tandem 28 or 29 amino acid repeats located in the amino terminal region of the protein that contains an adhesion domain that is necessary and sufficient for infection of tick cells. The MSP1a studies demonstrated the importance of combining structural and functional characteristics for identification of adhesive proteins. In the present study other outer membrane proteins containing tandem repeats were selected from organisms of the family Anaplasmataceae and studied for their adhesive properties to tick cells. The adhesive properties and protein characteristics were then analyzed in order to provide a predictor of the adhesion function of proteins identified from genome sequences. Proteins selected included the A. marginale MSP1a, A. phagocytophilum 100 and 130 kDa, Ehrlichia chaffeensis 120 kDa, E. canis 140 kDa and E. ruminantium "mucin", which were all cloned and expressed in Escherichia coli and then tested as adhesins for cultured IDE8 cells. Of the proteins studied, the A. marginale MSP1a and the E. ruminantium "mucin" were found to be adhesins for tick cells. Although all of these recombinant outer membrane proteins were glycosylated, the A. marginale MSP1a and E. ruminantium "mucin" adhesins shared a common feature of having a high Ser/Thr content in the tandem repeats. The results reported herein provide new information on the role of E. ruminantium "mucin" as an adhesin for tick cells and also suggest a role of glycans in adhesin molecules.     

牛布鲁氏菌OMP25的原核表达及表达产物的免疫学特性

目的研究布鲁氏菌外膜蛋白OMP25的克隆、测序和表达,并对其进行免疫特性检测。方法采用PCR技术对牛布鲁氏菌外膜蛋白OMP25基因进行了扩增和克隆测序,并成功构建了原核表达质粒pGEX-OMP25。将其转入E.coliBL21,经IPTG诱导表达,用电洗脱纯化后获得了高纯度的目的蛋白。纯化后的蛋白与弗氏佐剂乳化后免疫家兔得到了较高效价的抗血清。结论通过ELISA、Western-blotting分析及特异性检测试验,证明目的蛋白不仅具有抗原性且有良好的免疫反性。