一株能表达987P和F41两种粘附毒的猪源性大肠杆菌的研究

通过直接凝集试验,免疫荧光试验．ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ印迹,对一株猪源性大肠杆菌的粘附素进行了研究,结果表明,该菌株是一株同时表达９８７Ｐ和Ｆ４１两种粘附素抗原的猪源性大肠杆菌。     

禽致病性大肠杆菌IMT5155 疑似毒力基因在人源及动物源大肠杆菌中的分布

[目的]检测禽致病性大肠杆菌IMT5155自分泌黏附素基因等具有代表性的疑似毒力基因在不同来源大肠杆菌中的分布,为进一步研究其致病机理提供依据.[方法]采用PCR和Dot blot,检测疑似毒力基因在不同地区(101株大肠杆菌中国分离株和121株大肠杆菌德国分离株)、不同来源(人源、禽源及猪源)大肠杆菌中的分布,并分析其和大肠杆菌系统进化分群的关系.[结果]自分泌黏附素基因B11等11个疑似毒力基因在禽致病性大肠杆菌中分布率较高,阳性率分别为:A1 36.4%(32/88)、A8 53.4%(47/88)、A1063.6%(56/88)、B1137.5%(33/88)、F3 59.1%(52/88)等,且疑似毒力基因主要存在于大肠杆菌B2进化群中.值得注意的是,D1、E9和F11基因片段在新生儿脑膜炎大肠杆菌中有较高的分布率,分别为60%(6/10)、80%(8/10)和90%(9/10),而在新生儿脑膜炎大肠杆菌中未检测到B11基因.[结论]自分泌黏附素B11等疑似毒力基因与禽致病性大肠杆菌关系密切,但疑似毒力基因D1、E9和F11与新生儿脑膜炎大肠杆菌密切相关,提示禽致病性大肠杆菌可能是新生儿脑膜炎大肠杆菌的毒力基因储库.     

猪源乳酸杆菌对鼠伤寒沙门氏菌DT104在猪肠道上皮细胞IPEC-J2上粘附的影响

采用猪肠道上皮细胞株IPEC-J2体外培养模型,考察9株猪源乳酸杆菌对IPEC-J2细胞的粘附特性,以及对鼠伤寒沙门氏菌DT104粘附的竞争、排斥、置换和抗侵袭作用.结果显示,9株乳酸杆菌均能粘附IPEC-J2细胞,粘附率在0.1%～10%之间,具有菌株特异性和浓度效应.乳酸杆菌和沙门氏菌同时加入细胞培养,能竞争性抑制沙门氏菌的粘附,并具有浓度效应,高浓度(109 CFU/mL)添加K30、K67和K16时,抑制率可达80%以上.乳酸杆菌预处理细胞后再加入沙门氏菌,高浓度乳酸杆菌可降低沙门氏菌粘附率40%～70%,而中浓度(108 CFU/mL)乳酸杆菌能抑制23%～33%沙门氏菌对细胞的侵入.但是,只有高浓度添加乳酸杆菌能置换已经粘附的沙门氏菌,置换率在12%～84%之间.该结果为临床上筛选乳酸杆菌,有效防治猪沙门氏菌病提供了一条新的途径.     

铜绿假单胞菌PA16株粘附性、菌毛与质粒关系的研究

为探讨PA的质粒与粘附性及质粒与菌毛的关系,围绕PA16株的耐药性与质粒的关系、质粒与菌毛及粘附性的关系作了一系列的研究,结果表明PA16对所测的7种抗生素全部耐药,其MIC＞400 mg/L;PA16仅含有一种27.3 kb(18 Mu)的质粒.转化后此质粒也使JM109获得了对四环素的耐药性.消除此质粒后,PA16对四环素的耐药性消失.粘附试验证明PA16质粒消除株对尿道上皮细胞的粘附能力较野生株显著性减小(P＜0.05),同时,透射电镜照片显示PA16野生株表面有致密、纤细刚直的菌毛,而PA16质粒消除株表面几乎无菌毛可见.     

应用被动免疫溶血试验快速检测大肠杆菌不耐热肠毒素

本文介绍了被动免疫溶血试验检测埃希氏大肠杆菌不耐热肠毒素的方法;比较了四种不同培养基以及抗CT血清和抗LT血清对测定结果的影响。经对236株人源和24株猪源毒素源性大肠杆菌的测定结果表明,该方法与固相放射免疫分析、LT基因探针等方法的测定结果基本相符。说明被动溶血试验是一种快速、敏感和特异的检测方法,不仅可用于流行病学调查,在临床检验上也是一种可行手段。     

高黏附罗伊氏乳杆菌的筛选及其益生特性评价

通过对28株不同罗伊氏乳杆菌(Lactobacillus reuteri)菌株的粘附力、细胞表面疏水性、与大肠杆菌的共聚合等指标的测定及相关性分析,成功筛选出粘附力最高的菌株R28。研究了该菌株的耐酸性和耐胆盐能力。结果表明,R28与其它菌株相比具有良好的粘附能力。该菌株的自聚集能力和疏水性分别为26.25%和75.53%。与大肠杆菌的共聚合能力可高达36.84%。细胞粘附数可达约43个细菌/每个杆菌细胞。菌株R28具有良好的耐酸性(pH=3)和耐胆盐性(0.1%),其能够很好的粘附于宿主肠道细胞发挥益生作用。罗伊氏乳杆菌R28作为一株优良的益生菌在食品和医药领域具有广阔的应用前景。     

致肾盂肾炎大肠杆菌粘附特性的研究

本文对临床肾盂肾炎病人尿标本中分离的大肠杆菌１３２和１３６的粘附特性进行了系统的研究。受试菌的Ｐ血型阳性红细胞血凝试验阳性,能够与人的尿道上皮细胞粘附。利用致肾盂肾炎大肠杆菌Ｐ菌毛粘附基因群抗血清进行免疫学检测,两株菌的全菌ＥＬＩＳＡ结果阳性,免疫电镜证实该抗血清能与受试菌株的菌毛特异性结合。提取临床分离株的菌毛蛋白进行免疫印迹测定,仅有一条蛋白带显色,其分子量为１６．６ｋｄ。致肾盂肾炎大肠杆菌的粘附特性是区别于其他大肠杆菌的重要特征,上述结果表明本文报告的两株大肠杆菌为致肾盂肾炎大肠杆菌。     

Ⅰ型大肠杆菌粘附素的表达受环境因素影响的初步探讨

本文探讨了葡萄糖、半乳糖、麦芽糖、ＰＨ、温度因子对尿路感染Ⅰ型大肠杆菌粘附素表达的影响。结果发现葡萄糖、半乳糖和低温可使粘附素的表达减少,而且葡萄糖和半乳糖与低温因子累加可使细菌几乎完全不表达粘附素。另外分析了与此有关的一些现象。     

新型促细胞粘附重组人源性胶原的原核表达及功能性研究

选择一段与COL1A1基因相应序列同源、适合于在大肠杆菌E.coli中表达、含有编码促细胞粘附位点GER三肽密码子的基因序列。利用RT-PCR技术自人组织扩增该序列,构建pET28a(+)-COL重组表达质粒并转化到大肠杆菌BL21(DE3)pLysS。IPTG诱导表达获得重组人源性胶原RHDC, 表达量达到菌体总蛋白的40%左右。SDS-PAGE和Western Blot结果推测该胶原具有三螺旋结构。细胞粘附试验证明RHDC具有促细胞粘附能力,具有应用在生物材料领域的潜力。     

大肠杆菌O157:H7致病因子及其保护性免疫研究现状

大肠杆菌O157:H7是肠出血型大肠杆菌(EHEC)的主要病原血清型,它可产生特殊的粘附因子粘附靶细胞,产生Vero毒素和肠溶血素毒力因子.近几年对大肠杆菌O157:H7的毒力因子有了深入的了解,对致病机理作了一些探讨,用实验动物对保护性免疫进行了研究.本文对近几年来0157:H7大肠杆菌的致病因子及其主要的保护性免疫的研究作一简要综述.     

抗大肠杆菌987P粘着素单克隆抗体及其初步应用

共研制出ll株抗大肠杆菌987P粘着素单克隆抗体,对其部分免疫学特性作了测定。这些单抗不仅效价很高,而且特异性强,对不具有987P抗原的大肠杆菌及所试的其他肠道杆菌均无交叉反应。以FlTc或Hrpo标记的987P单抗作实验室诊断,具有简易、快速、敏感和准确的优点。酶标EPN3株单抗检测的灵敏度可达2×l03个／ml 987P+菌,对人工发病仔猪的粪样和小肠内容物的阳性检出比分别为4／4和2／2。 EP22株荧光标记单抗对病猪小肠粘膜触片的阳性检出比为6／6。 11株单抗中7株能不同程度地阻断987P+菌对仔猪小肠上皮细胞的吸附作用。EL1sA和荧光阻抑试验表明,1株单抗是针对987p粘着素上三种不同的抗原决定簇。EPN2株单抗的免疫胶体金定位还表明,987P粘着素似呈螺旋状结构,且含有许多相同的抗体结合位点。这些单抗不仅可用于ETEC菌株的987P柔毛鉴定,而且可用于987P分子结构和生物学特性的研究。     

The production of F41 fimbriae by piglet strains of enterotoxigenic Escherichia coli that lack K88, K99 and 987P fimbriae

The enterotoxigenic Escherichia coli strains 1676, 1706, 1751 and KEC96a, which do not produce fimbrial adhesive antigens of the K88, K99 or 987P antigen type reacted both in vitro and in vivo with antiserum to F41 fimbriae in an indirect immunofluorescent antibody technique. Antiserum used to demonstrate material B, an adhesive antigen thought to mediate the adhesive and mannose-resistant (MR) haemagglutinating properties of E. coli strains 1676, 1706 and 1751, reacted in vitro with an F41+ strain. The antiserum also inhibited the MR haemagglutinating activity of F41 antigen and gave an anionic precipitation line in immunoelectrophoresis experiments with an extract containing F41 antigen. The MR haemagglutinating properties of an antigen extract containing material B from E. coli strain 1706 was neutralized by antiserum to F41 fimbriae and by OK antisera to E. coli strains that produce both F41 and K99 fimbriae. These sera also gave an anionic precipitation line with the MR haemagglutinin from E. coli strain 1706 and the MR haemagglutinin gave a line of identity with F41 in gel diffusion experiments with antiserum to F41 fimbriae. OK antisera to K99+ F41- bacteria and OK antisera to K88+ bacteria and 987P+ bacteria did not react with this haemagglutinin. Transmission electron microscopy on the ileum of newborn gnotobiotic piglets infected with E. coli strain 1706 showed irregular, poorly defined filamentous material surrounding some,though not all, bacteria but regular fimbrial structures were not visible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a new fimbrial antigen (CS1541) from a porcine enteroxigenic Escherichia coli O8: KX105 strain

Abstract A fimbrial antigen (CS1541) was extracted and purified from the porcine enterotoxigenic Escherichia coli strain 1541P (O8:K-:H9). CS1541 fimbriae appeared as long thin filaments 3–5 mm in diameter. CS1541 antigen consisted of two peptide bands of about 18 and 19 kDa as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. It was expressed both at 37°C and 15°C and did not demonstrate hemagglutinating properties. It was antigenically distinct from the fimbrial antigens K88, K99, F41, FY(Att25), F165, type 1, CFA-I, and CFA-II complex but demonstrated serological cross-reactions with the 987P fimbrial antigen.     

Hemagglutinin-neuraminidase-independent fusion activity of simian virus 5 fusion (F) protein: difference in conformation between fusogenic and nonfusogenic F proteins on the cell surface

The fusion (F) protein of simian virus 5 (SV5) strain W3A is known to induce cell fusion in the absence of hemagglutinin-neuraminidase (HN) protein. In contrast, the F protein of SV5 strain WR induces cell fusion only when coexpressed with the HN protein, the same as do other paramyxovirus F proteins. When Leu-22 in the subunit F2 of the WR F protein is replaced with the counterpart (Pro) in the W3A F protein, the resulting mutant L22P induces extensive cell fusion by itself. In the present study, we obtained anti-L22P monoclonal antibodies (MAbs) 21-1 and 6-7, whose epitopes were located in the middle (amino acids [aa] 227 to 320) of subunit F1. The amino-terminal region (aa 20 to 47) of subunit F2 was also involved in the formation of MAb 21-1 epitope. Flow cytometric analysis revealed that both the MAbs reacted very faintly with native WR F protein that was expressed on the cell surface whereas they reacted efficiently with native L22P irrespective of whether it is cleaved into F1 and F2. However, by heating the cells at 47 degrees C after mild formaldehyde fixation, the epitopes for MAb 6-7 and mAb 21-1 in the WR F protein were exposed and the reactivity of the MAbs with the WR F protein became comparable to their reactivity with L22P. Thus, the two MAbs seem to distinguish the difference in native conformation between fusogenic mutant L22P and its parental nonfusogenic WR F protein. The native conformation of L22P may represent an intermediate between native and postfusion conformations of a typical paramyxovirus F protein.     

Hemagglutination by pilus antigen 987P of enterotoxigenic Escherichia coli

The hemagglutination (HA) by pilus antigen 987P of an enterotoxigenic Escherichia coli strain 987 was examined using fresh and glutaraldehyde (GA)-fixed erythrocytes (RBC) of various animals. Only when GA-fixed RBC was employed, a strain 987 exhibited striking HA activities. This was also demonstrated by using latex heads sensitized with the 987P antigen. The 987P-specific antiserum inhibited HA of strain 987 and 987P sensitized latex beads against GA-fixed RBC. We concluded that HA of strain 987 against GA-fixed RBC was specifically associated with the presence of 987P pilus antigen but do not exclude a possibility that adhesin is distinct from pili antigen.     

The 14,000-molecular-weight antigen of Mycobacterium tuberculosis is related to the alpha-crystallin family of low-molecular-weight heat shock proteins.

Eight monoclonal antibodies (MAbs) directed against the 14,000-molecular-weight (14K) antigen of Mycobacterium tuberculosis reacted specifically with mycobacteria of the M. tuberculosis complex. The nucleotide sequence of the gene encoding the 14K antigen was determined by using recombinant DNA clones isolated from lambda gt11 and cosmid libraries of the M. tuberculosis genome. The DNA sequence of the 14K protein gene coded for a polypeptide of 144 amino acids with a calculated molecular mass of 16,277 Da. The 14K antigen has a marked homology with proteins belonging to the alpha-crystallin family of low-molecular-weight heat shock proteins, which includes the 18K antigen of M. leprae. The eight MAbs recognized at least four distinct epitopes localized within the following three regions of the 14K protein: amino acids 10 to 92 (MAbs F67-8 and F67-16), amino acids 41 to 92 (F159-1 and F159-11), and amino acids 41 to 144 (F23-41, F24-2, F23-49, and TB68).     

Recognition by human monoclonal antibodies of free and complexed peptides representing the prefusogenic and fusogenic forms of human immunodeficiency virus type 1 gp41

Human immunodeficiency virus type 1 (HIV-1) entry into target cells appears to be triggered when two heptad repeat regions in the ectodomain of gp41 associate, converting the prefusogenic form of gp41 to a fusogenic form. Peptides from these two heptad repeat regions, designated N51 and C43, form a coiled coil consisting of an alpha-helical trimer of heterodimers which approximates the core of the fusogenic form of gp41. To understand the antigenic structures of gp41 in these two configurations, and to examine the specificity of anti-gp41 antibodies produced by HIV-1-infected individuals, human anti-gp41 monoclonal antibodies (MAbs) were tested for their reactivity against N51, C43, and the complex formed by these peptides. Of 11 MAbs, 7 reacted with the complex but with neither of the parent peptides. These MAbs reacted optimally with the N51-C43 complex prepared at a 1:1 ratio and appeared to recognize the fusogenic form of gp41 in which the two heptad repeat regions are associated to form the coiled coil. The existence of antibodies from HIV-infected humans that exclusively recognize the N51-C43 complex constitutes the first proof that the coiled-coil conformation of gp41 exists in vivo and is immunogenic. Two of the 11 MAbs were specific for the hydrophilic loop region of gp41 and failed to react with either peptide alone or with the peptide complex, while the remaining 2 MAbs reacted with peptide C43. One of these two latter MAbs, 98-6, also reacted well with the equimolar N51-C43 complex, while reactivity with C43 by the other MAb, 2F5, was inhibited by even small amounts of N51, suggesting that the interaction of these peptides occludes or disrupts the epitope recognized by MAb 2F5. MAbs 98-6 and 2F5 are also unusual among the MAbs tested in their ability to neutralize multiple primary HIV isolates, although 2F5 displays more broad and potent activity. The data suggest that anti-gp41 neutralizing activity is associated with specificity for a region in C43 which participates in complex formation with N51.     

Expression of a cloned 987P adhesion-antigen fimbrial determinant in Escherichia coli K-12 strain HB101

The properties of three independent enterotoxigenic Escherichia coli isolates known to express 987P adhesion fimbriae in a manner subject to phase variation were examined. Phase variation could not be correlated with any major changes in the plasmid DNA content of these strains or with readily detectable changes in any other tested phenotypic markers. The 987P genetic determinant from one of these strains, E. coli 987, was cloned into the non-fimbriated E. coli K-12 strains HB101, and expressed, using the cosmid vector system. 987P fimbriae produced by cells harbouring these recombinant plasmids (987P+ phenotype) could not be distinguished from 987P fimbriae produced by strain 987. Expression of 987P fimbriae from some recombinant plasmids was unstable but none of the recombinants exhibited the phase variation phenotype displayed by the parental strain. One recombinant plasmid, pPM200, contained an insert of strain 987 DNA of ca. 33 kb. The HB101[pPM200] displayed a rather stable 987P+ phenotype, but this was not true for several hosts, since pPM200 acquired approx. 20-kb deletions following transformations of E. coli K-12 strains other than HB101. The deletions mapped to the same region of pPM200 irrespective of the host strain transformed. Cells harbouring the deleted plasmids did not express 987P fimbriae (987P- phenotype).     

我国呼吸道合胞病毒抗原亚型的初步探讨

An analysis of subtypes of 9 respiratory syncytial (RS) viruses isolated from Guangzhou and Nanjing areas of china was carried out with eight Sweden RS-subtype specific monoclonal antibodies (MAbs) and 7 internal anti-RS MAbs. All these MAbs directed against respectively the large Glycoprotein (G), fusion protein (F), nucleoprotein (NP), and phosphoprotein (P) components of the prototype Long strain of RS virus. The patterns of the reactions of these MAbs to the nine isolated strains of RS virus were compared with indirect immunofluorescence assay (IFA), alkaline phosphoesterase-anti alkaline phosphoesterase (APAAP) enzyme-linked assay and Western blotting. The antigenic variations were founded among the strains of RS virus, and two subtypes allocated to the subtype A and B of RS virus by using the eight RS-subtype specific MAbs. Seven out of the 9 isolated strains of RS virus belonged to the subtype A, and two were being to the subtype B. The antigenic diversities were also founded within the same subtype, and the main pronounced difference were observed on the G glycoprotein by using the internal anti-RS MAbs. These findings are potentially important both for vaccine development and for the understanding of clinical and epidemiological characteristics of RS virus.     

Neutralization of respiratory syncytial virus by individual and mixtures of F and G protein monoclonal antibodies.

We identified several types of neutralization effected by F and G protein monoclonal antibodies (MAbs) reacted individually or as mixtures against respiratory syncytial virus (RSV). Neutralizing activity was identified by a microneutralization test in which virus replication was determined by enzyme immunoassay. Complete neutralization was seen only with MAbs against the F protein. Strain-specific neutralization, complete neutralization against some strains of RSV, and no neutralization against other strains were seen with an additional MAb against the F protein. Partial neutralization, virus replication significantly reduced but still present, and no neutralization were seen with MAbs against both the F and G proteins. Enhanced neutralization, enhanced efficacy of neutralization, or increased neutralizing titer with a mixture of two MAbs over that for the individual MAbs was seen with all MAbs against the F protein and all but three MAbs against the G protein. Most (10 of 13) of the MAbs that exhibited neutralizing activity reacted with some but not all strains of RSV in an enzyme immunoassay. The epitopes corresponding to these 10 MAbs probably contribute to the strain-specific component of the neutralizing antibody response to RSV. Our results suggest that interpretation of RSV neutralization with MAbs is complex and that studies of such neutralization should include mixtures of MAbs and multiple RSV strains.