SDS—PAGE垂直板电泳技术鉴定脆弱类杆菌及相关菌株的探讨

本文报告了采用SDS—PAGE垂直板电泳技术对脆弱类杆菌及相关菌株超声波粉碎的可溶性蛋白抗原进行了电泳谱分析。结果证明这些菌株有三种共同的蛋白抗原组分,但不同菌种间蛋白成分存在着差异。     

对流免疫电泳对El Tor型霍乱弧菌与不凝集弧菌抗原相关性的初步探讨

本文对37株NAG 8株El Tor弧菌的抗原性进行了初步分析,用对流免疫电泳不同程度地出现有交叉沉淀线,这说明它们之间有血清学相关性;通过血清吸收试验,发现“H”抗原吸收后的血清大部分仍出现沉淀线,而用同一菌株的可溶性抗原吸收后的血清与5种NAG可溶性抗原进行对流免疫电泳,除少数仍有沉淀线外,大部分均消失,因而证明它们之间的抗原有相关性,而此相关部分并非弧菌共同的H抗原,以对流电泳与糖发酵反应双盲试验结果也证明二者之间亦有一定联系。本文还介绍了用去氧胆酸钠裂解细菌的方法、原理和注意事项。     

52株链霉菌全细胞蛋白组分的数值分类

全细胞可溶性蛋白的凝胶电泳分析作为细菌分类的一种简便而客观的方法得到了广泛应用,在许多报道中已证明蛋白凝胶电泳分析和DNA-DNA杂交的结果有紧密的相关性.目前在许多属中进行了全细胞蛋白组分的数值分类研究,我们对52株链霉菌进行了SDS-PAGE分析,并以全细胞蛋白组分进行聚类分析,以期在全细胞蛋白组分上探讨链霉菌属内种间及种内不同菌株的亲缘关系.     

福氏志贺氏2a及Y变种保护性抗原的研究

福氏志贺菌Y变种曾经作为一种痢疾疫苗的候选株,其特有的抗原结构在疫苗的有效性抗原研究中起主要作用。以Y变种毒株与无毒株、野生型F2a株与T32株及失去Ⅱ型抗原结构的T32-1株之间分别进行了各种毒力表型的检测、四种外膜侵袭蛋白表达、菌株的外膜蛋白提取物(OMPs)分析、质粒DNA图谱和小鼠主动免疫、被动保护试验的对比分析,了解其抗原特性。结果显示：细菌外膜蛋白抗原和具有完整型特异性抗原结构的福氏菌LPS在动物机体免疫中都发挥着重要的作用。这些抗原物质的共同存在似乎能达到更好的免疫效果。     

破伤风毒素保护性抗原在毕氏酵母中的分泌表达

采用PCR方法．从C.Tetani 64008菌株中扩增大小为1353bp的破伤风毒素与靶细胞起结合作用的重链C端基因(Tetc),直接连接pGEM-T栽体进行测序,并以pPIC9K为表达栽体构建重组表达质粒,经线形化的重组质粒电转化毕氏酵母细胞GS1l5和KM71．甲醇诱导获得了分泌表达,表达产物存在于培养上清中,占分泌蛋白的10％,通过免疫印迹可以检测到重组表达产物,活性测定表明,重组蛋白具特异结合活性,本研究通过实现破伤风毒素保护性抗原在酵母系统中的分泌表达、研究其影响因素,为其它细菌毒素蛋白高效可溶性表达,及进一步抗原片段介导保护性免疫研究及抗毒素制备奠定了基础。     

单链抗体scFv-GCN4在大肠杆菌中的重组表达及活性检测

目的:重组表达融合GST或MBP标签的单链抗体scFv-GCN4,对其进行分离纯化,并检测其生物学活性。方法:构建pBAD-MBP-scFv-GCN4及pBAD-GST-scFv-GCN4表达载体,使用大肠杆菌(Escherichia coli)Top10菌株表达并亲和纯化重组蛋白MBP-scFv-GCN4及GST-scFv-GCN4。构建p ET30a-Nus-GCN4载体,使用E.coliBL21(DE3)菌株表达重组蛋白Nus-GCN4及Nus。以重组的GCN4为特异性抗原,通过Pull-down技术和WesternBlot技术,检测MBP-scFv-GCN4及GST-scFv-GCN4的抗体活性及特异性。结果:重组菌株E.coli Top10可高效表达可溶性的MBP-scFv-GCN4和GST-scFv-GCN4蛋白,通过亲和纯化,均得到了高纯度的重组蛋白。重组菌株E.coliBL21(DE3)可高效表达可溶性的Nus与Nus-GCN4蛋白。Pull-down及WesternBlot结果显示,重组蛋白MBP-scFv-GCN4及GST-scFv-GCN4均可以高效、特异地识别重组的GCN4抗原。结论:重组抗体GST-scFv-GCN4及MBP-scFv-GCN4均可在E.coli中高效表达,并且具有良好的抗体活性及特异性。     

生物防治

建立了可以在休外或体内检测的苏云金芽袍杆菌(Baciilus thuringiensis)P2蛋白的可溶性制剂的原毒素(Protoxin)活化方法.对来自5个不同Bt菌株的δ一内毒素品体蛋白的多从分析表明,尽管它们的分一子虽、肤谱、序列不同,但有二个相似的抗原形式,在休内这两种形式的纯化制剂     

裂解系统在细菌载体疫苗中的应用

重组细菌载体疫苗因其能够诱导机体产生粘膜免疫、体液免疫和细胞免疫的特点,已经被广泛用作递送保护性抗原和核酸疫苗的载体来预防某些传染病。但是重组到细菌载体疫苗中的保护性抗原和核酸难以穿越细菌细胞壁释放到宿主细胞内发挥作用,残留在动物或畜禽产品中的疫苗菌株还可能造成环境的污染和疫苗菌株的传播。而有效解决这些问题的方法是构建一种细菌自动裂解系统,使疫苗菌株能够在体外培养时正常生长而在体内环境中自动裂解死亡。目前主要应用的细菌裂解系统包括:基于调控延迟肽聚糖合成的裂解系统、基于噬菌体裂解蛋白调控的裂解系统、基于毒素-抗毒素系统(Toxin-antitoxin system)的裂解系统。此外,一种潜在的基于细菌Ⅵ型分泌系统(Type Ⅵ secretion system,T6SS)的裂解系统也有望成为构建自动裂解菌株的新方法。文中将着重对这几种裂解系统的调控机制进行阐述。     

耐盐碱细菌DQSA1的分离鉴定及盐碱胁迫下对绿豆的促生作用

【背景】近年来,大庆地区土壤盐碱化程度逐渐加剧,而微生物改良盐碱土是当今的研究热点。【目的】从大庆盐碱土中筛选出耐盐碱菌株,验证其促生作用,为改良大庆盐碱土壤提供微生物资源。【方法】采用筛选培养基从大庆盐碱土中获得耐盐碱促生菌,对其进行形态学观察、生理生化和16S rRNA基因鉴定,并测试菌株在盐碱胁迫下对绿豆植株和土壤细菌群落结构的影响。【结果】筛选得到一株耐盐碱促生菌DQSA1,具有固氮、产ACC脱氨酶、产铁载体、产吲哚乙酸(Indole-3-Acetic Acid,IAA)功能;通过生理生化鉴定和系统发育分析,判定该菌株为卓贝尔氏菌属(Zobellella)。在盐碱土中种植绿豆后接种菌株DQSA1,处理后的绿豆较对照相比根系鲜重、根系干重及叶绿素含量分别增加了33%、32%和79%;植株叶部可溶性糖、脯氨酸和可溶性蛋白含量分别升高了10%、80%和73%;根系的脯氨酸及可溶性蛋白含量分别增加了78%和44%。对种植绿豆的土壤细菌进行高通量测序,发现菌株DQSA1可以在盐碱环境下定殖并促进根瘤菌和鞘氨醇杆菌等有益菌的生长。【结论】菌株DQSA1可以在盐碱条件下调节土壤细菌群落结构并促进植物生长,为改良盐碱土地提供了有效的微生物资源。     

炭疽毒素保护性抗原第四结构域在大肠杆菌中的可溶性表达

人工优化设计并合成炭疽毒素保护性抗原第四结构域基因,并与噬菌体gⅢ蛋白N端结构域基因融合,在大肠杆菌中可溶性表达融合蛋白。结果表明合成了炭疽毒素保护性抗原第四结构域基因,并在大肠杆菌中获得了高效可溶性融合表达,可溶性表达产物占细菌总蛋白量的36％左右;经亲和层析纯化获得了重组蛋白;Western印迹分析表明,表达产物能与His单抗（重组蛋白羧基端带有6xHis）发生特异性结合反应。以上结果表明获得了炭疽毒素保护性抗原第四结构域,为利用人抗体库进行筛选抗炭疽毒素的人源性中和抗体奠定了基础。     

Screening for Protein-producing Bacteria

Screening of protein-producing bacteria was conducted to systematically study the ubiquitous nature of the extracellular production of proteins and their excretion mechanisms. A very simple and efficient test revealed that about 15% of bacteria tested (total 1200 strains) accumulated some protein under the cultural conditions employed. Among protein-excretors, five strains produced a large amount of protein in the liquid shake culture.

Many good protein producers including five excellent ones were found to be gram-positive rod and probably belong to Bacillus species. An acid-insoluble product by one of the hyper-protein producers was identified as a protein mixture. Good producer was not found among the known 15 species of bacteria. The implication of these findings is discussed.     

Interaction of rabbit lymph node cells with bacteria (Salmonella paratyphi B)

In five rabbits immunized withSalmonella paratyphi B antigen in all four paws, incubation of the regional lymph node cells with the same bacteria was followed by a significant shift of some of the bacteria from the supernatant of the culture medium to the sediment (as compared with the controls). In six rabbits, bactericidal activity was demonstrated in extracts prepared from the regional lymph nodes only 30 hours after immunization. No antibodies were present in extracts from control rabbits up to the fifth day after immunization.     

Immunological characterization of Rhizobium leguminosarum outer membrane antigens by use of polyclonal and monoclonal antibodies.

Surface antigens of Rhizobium leguminosarum biovar viciae strain 248 were characterized by using polyclonal and monoclonal antibodies. With Western immunoblotting as the criterion, an antiserum raised against living whole cells recognized mainly flagellar antigens and the O-antigen-containing part of the lipopolysaccharide (LPS). Immunization of mice with a peptidoglycan-outer membrane complex yielded eight monoclonal antibodies, of which three reacted with LPS and five reacted with various sets of outer membrane protein antigens. The observation that individual monoclonal antibodies react with sets of related proteins is discussed. Studies of the influence of calcium deficiency and LPS alterations on surface antigenicity showed that in normally grown wild-type cells, the O-antigenic side chain of LPS blocks binding of an antibody to a deeper-lying antigen. This antigen is accessible to antibodies in cells grown under calcium limitation as well as in O-antigen-lacking mutant cells. Two of the antigen groups which can be distinguished in cell envelopes of free-living bacteria were depleted in cell envelopes of isolated bacteroids, indicating that the monoclonal antibodies could be useful tools for studying the differentiation process from free-living bacteria to bacteroids.     

Identification of the p53 protein domain involved in formation of the simian virus 40 large T-antigen-p53 protein complex.

An expression vector utilizing the enhancer and promoter region of the simian virus 40 (SV40) DNA regulating a murine p53 cDNA clone was constructed. The vector produced murine p53 protein in monkey cells identified by five different monoclonal antibodies, three of which were specific for the murine form of p53. The murine p53 produced in monkey cells formed an oligomeric protein complex with the SV40 large tumor antigen. A large number of deletion mutations, in-frame linker insertion mutations, and linker insertion mutations resulting in a frameshift mutation were constructed in the cDNA coding portion of the p53 protein expression vector. The wild-type and mutant p53 cDNA vectors were expressed in monkey cells producing the SV40 large T antigen. The conformation and levels of p53 protein and its ability to form protein complexes with the SV40 T antigen were determined by using five different monoclonal antibodies with quite distinct epitope recognition sites. Insertion mutations between amino acid residues 123 and 215 (of a total of 390 amino acids) eliminated the ability of murine p53 to bind to the SV40 large T antigen. Deletion (at amino acids 11 through 33) and insertion mutations (amino acids 222 through 344) located on either side of this T-antigen-binding protein domain produced a murine p53 protein that bound to the SV40 large T antigen. The same five insertion mutations that failed to bind with the SV40 large T antigen also failed to react with a specific monoclonal antibody, PAb246. In contrast, six additional deletion and insertion mutations that produced p53 protein that did bind with T antigen were each recognized by PAb246. The proposed epitope for PAb246 has been mapped adjacent (amino acids 88 through 109) to the T-antigen-binding domain (amino acids 123 through 215) localized by the mutations mapped in this study. Finally, some insertion mutations that produced a protein that failed to bind to the SV40 T antigen appeared to have an enhanced ability to complex with a 68-kilodalton cellular protein in monkey cells.     

Evidence that mycoplasmas, gram-negative bacteria, and certain gram-positive bacteria share a similar protein antigen.

It was demonstrated that mycoplasmas, gram-negative bacteria, and certain gram-positive bacteria share a similar protein antigen with a molecular weight ranging from 42,000 to 48,000. Western blotting (immunoblotting) with an antibody specific to a 43-kDa membrane protein of Mycoplasma fermentans showed the existence of this protein antigen in all Mycoplasma spp. tested (14 species), Acholeplasma laidlawii (1 strain), and gram-negative bacteria (8 species) but only in Staphylococcus aureus of four gram-positive species tested. Neither Ureaplasma urealyticum nor mammalian cell cultures showed any cross-reactions with this antibody. These proteins were found in both cytoplasmic and membrane fractions of mycoplasma cells but were not exposed on the surface of mycoplasmal or bacterial cells.     

Prevalence of a Vibrio cholerae 18-kDa antigen in vibrios

Abstract An 18-kDa protein that occurs in Vibrio cholerae has been described as an in vivo and low-iron regulated outer membrane antigen. Monoclonal antibodies which recognized this antigen were protective as passive vaccines in the infant rabbit model of cholera disease. In this study, those monoclonal antibodies were used in three immunological assays for surveillance of various bacteria for the 18-kDa antigen. ELISA, and Western blot assays gave variable results with bacteria or outer membrane preparations. The biodot assay was the most sensitive test, detecting the 18-kDa antigen in 29 of 29 V. cholerae strains, independent of biotype or serotype. A few other Gram-negative bacteria and V. parahaemolyicus reacted weakly with our antibodies and antiserum.     

Escherichia coli Antibody: A Screening Test for Immunodeficiency

Six patients suffering from recurrent chest infections were found to lack antibodies to a pooled antigen obtained from six different serotypes of commensal Escherichia coli bacteria. All had normal serum IgG concentrations, but five subsequently benefited from regular gammaglobulin injections. We suggest that the absence of such E. coli antibodies usually indicates a clinically significant defect in antibody production. This simple screening test is of use in the diagnosis of primary and secondary immunodeficiency disorders.     

Molecular characterization of novel red green nonsulfur bacteria from five distinct hot spring communities in Yellowstone National Park.

We characterized and compared five geographically isolated hot springs with distinct red-layer communities in Yellowstone National Park. Individual red-layer communities were observed to thrive in temperatures ranging from 35 to 60 degrees C and at pH 7 to 9. All communities were dominated by red filamentous bacteria and contained bacteriochlorophyll a (Bchl a), suggesting that they represented novel green nonsulfur (GNS) bacteria. The in vivo absorption spectra of individual sites were different, with two sites showing unusual Bchl a protein absorption bands beyond 900 nm. We prepared and analyzed 16S rRNA libraries from all of these sites by using a combination of general bacterial primers and new GNS-specific primers described here. These studies confirmed the presence of novel GNS-like bacteria in all five communities. All GNS-like clones were most similar to Roseiflexus castenholzii, a red filamentous bacterium from Japan that also contains only Bchl a. Phylogenies constructed by using GNS-like clones from Yellowstone red-layer communities suggest the presence of a moderately diverse new "red" cluster within the GNS lineage. Within this cluster, at least two well-supported subclusters emerged: YRL-A was most similar to Roseiflexus and YRL-B appeared to be novel, containing no known isolates. While these patterns showed some site specificity, they did not correlate with observed Bchl a spectrum differences or obvious features of the habitat.     

Amino acid sequence analysis of fragments generated by partial proteolysis from large simian virus 40 tumor antigen

Large simian virus 40 tumor antigen was bound as immune complex to protein A-Sepharose and then subjected to limited proteolysis which yielded several discrete fragments. Primary structures near the cleavage sites were determined by radiosequencing techniques. Experimental data for five fragments matched an amino acid sequence predicted from a nucleotide sequence at 0.51 map unit of the viral genome. We have thus identified the reading frame of translation beyond the intervening sequence at 0.60 to 0.53 map units. A cleavage map of tumor antigen was established on the basis of the sequence data and of the apparent molecular weights of the fragments. The bond most susceptible to cleavage by trypsin was between arginine-130 and lysine-131 in a cluster of five basis amino acids. Other cleavage sites were located in the COOH-terminal half of tumor antigen. Each fragment was analyzed by complete tryptic proteolysis and peptide mapping on an ion exchange column. Peaks occurring in the peptide map of large tumor antigen could thus be assigned to different segments of the protein. Two specific regions of tumor antigen were shown to be phosphorylated.     

Antigen processing and presentation by macrophages

The classical macrophage is one of the most important cells involved in presenting antigen to helper T cells, because of its ability to regulate its expression of Ia molecules and to encounter and process particulate and soluble antigens. We have summarized in this report studies examining the handling by macrophages of two different antigens, the bacteria Listeria monocytogenes and the protein hen egg white lysozyme (HEL). The purpose was to identify potential sources of immunogenic peptides. Presentation of Listeria required an intracellular processing stage sensitive to lysosomotropic drugs. The Listeria required internalization and processing, after which immunogenic molecules were recognized by T cells on the macrophage surface. Metabolic studies showed that Listeria-derived peptides were released by macrophages that had phagocytosized the bacteria. The release of these peptides was a temperature-dependent process, unaffected by inhibiting lysosomal catabolism by treatment with chloroquine. Listeria-derived peptides were also detected on the surface of the macrophage. These peptides behaved like integral membrane proteins, some of which persisted for at least 24 hr at the macrophage surface. When tested for immunogenicity, the released peptides were very weakly immunogenic. The membrane-associated peptides alone could not stimulate Listeria-specific T cells, but could be reprocessed by additional macrophages and subsequently stimulate the T cells. A defined antigen system using HEL-specific T-cell hybridomas was used to examine the processing of HEL. Presentation of HEL required a chloroquine-sensitive intracellular processing stage. In examining two T-cell hybridomas, a differential requirement for antigen processing was determined.(ABSTRACT TRUNCATED AT 250 WORDS)