不耐热肠霉素和耐热肠毒素基因的融合

用重组基因技术将产肠毒素大肠杆菌（ＥＴＥＣ）的耐热肠毒素（ＳＴａ）基因与不耐热肠毒素Ｂ亚单位（ＬＴ－Ｂ）基因融合。表达的ＬＴ－Ｂ／ＳＴａ融合蛋白，既有ＬＴ－Ｂ的抗原性又有ＳＴａ的抗原性。由于基因融合的方式不同，对ＳＴａ的抗原性影响很大，但对ＬＴ－Ｂ的抗原性没有影响。ＬＴ－Ｂ／ＳＴａ融合多肽保留了ＥＴＥＣ肠毒素结合ＧＭ－１神经节甙酯的能力，却仍具有ＳＴａ的生物毒性。     

应用非放射性标记探针测定小肠结肠炎耶氏菌新亚型耐热肠毒素

由致病性小肠结肠炎耶氏菌产生的耐热肠毒素（Ｙ－ＳＴ）至少有三个亚型（Ｙ－ＳＴａ，Ｙ－ＳＴｂ，Ｙ－ＳＴｃ），但以Ｙ－ＳＴａ型多见。我们构建了０．２５ｋｂ的Ｙ－ＳＴａＤＮＡ克隆片段，经用辣根过氧化酶标记后，具特异性。１９株我国分离的致病性小肠结肠炎耶氏菌由乳鼠试验结果显示均可产生Ｙ－ＳＴ。但用此酶标记Ｙ－ＳＴａＤＮＡ探针测定，仅１３株呈杂交反应，表明携带Ｙ－ＳＴａ基因；其余６株均为阴性反应，免疫学试验显示１株产生Ｙ－ＳＴｂ，另外５株产Ｙ－ＳＴｃ。产生耐热肠毒素的大肠杆菌及非０１霍乱弧菌等也不与此探针杂交。     

产毒性大肠杆菌肠毒素对及豚鼠回肠上皮细胞的影响

产毒性大肠杆菌 (ｅｎｔｅｒｏｔｏｘｉｇｅｎｉｃＥｓｃｈｅｒｉｃｈｉａｃｏｌｉ,ＥＴＥＣ)是引起人和家畜急性腹泻病的主要病原菌之一[1] 。ＥＴＥＣ的致病主要与肠毒素有关。它能产生两类肠毒素 :不耐热肠毒素(ｈｅａｔ ｌａｂｉｌｅｅｎｔｅｒｏｔｏｘｉｎ ,ＬＴ)和耐热肠毒素 (ｈｅａｔ ｓｔａｂｌｅｅｎｔｅｒｏｔｏｘｉｎ,ＳＴ)。为进一步探讨ＥＴＥＣ肠毒素的作用机制 ,本研究观察了ＬＴ和ＳＴ对豚鼠离体回肠上皮细胞的影响。1　材料与方法1 1　豚鼠 :为非纯种短毛白色豚鼠 ,体重 35 0～ 45 0ｇ ,雌雄不限 ,由本校动物所提供。1 2…     

产毒性大肠杆菌不耐热肠毒素 B 亚单位基因克隆及表达

产毒性大肠杆菌不耐热肠毒素Ｂ亚单位基因克隆及表达王万胜方利君王顺林蒋惠秋李宏年顾健人产毒性大肠杆菌（ＥＴＥＣ）是发展中国家婴幼儿腹泻的重要致病原。其产生的不耐热肠毒素（ＬＴ）是其重要致病因素。ＬＴ由一个活性中心Ａ亚单位和５个毒素受体结合点Ｂ亚单位组成...     

肠毒素大肠杆菌耐热肠毒素与谷胱甘肽巯基转移酶融合蛋白的制备和耐热肠毒素抗体测定

目的：弥补ST的弱抗原性，实现ST抗体测定。方法：采用生物工程技术，融合表达GST和带有前导序列的突变耐热肠毒素proSTm，制备ST融合蛋白，测定ST抗体。结果：测到了抗ST的血清IgG和黏膜IgA。结论：融合蛋白GST／proSTm能有效提高ST的抗原活性，可直接用于ST抗体测定。     

分泌抗金黄色葡萄球菌C1型肠毒素杂交瘤细胞系的建立及初步应用

应用常规方法建立了３株稳定分泌抗金黄色葡萄球菌Ｃ１型肠毒素（ＳＥＣ１）单克隆抗体（ＭｃＡｂ）的小鼠杂交瘤细胞系Ｂ３、Ｃ４和Ｇ８。其中Ｂ３和Ｃ４均为ＩｇＧ１（ｋ），Ｇ８为ＩｇＧ２ａ（ｋ）。Ｂ３和Ｇ８与ＳＥＡ，ＳＥＢ及ＳＥＤ均无交叉反应；Ｃ４虽与ＳＥＡ和ＳＥＤ无交叉反应，但与ＳＥＢ有交叉反应。间接ＥＬＩＳＡ测定小鼠腹水效价为１０＾－５～１０＾－８。应用识别不同表位的ＭｃＡｂ建立了双ＭｃＡｂ夹心ＥＬＩＳ     

应用非放射性标记探针测定小肠结肠炎耶氏菌新亚…

由致病性小肠结肠炎耶氏菌产生的耐热肠毒素（Ｙ－ＳＴ）至少有三个亚型（Ｙ－ＳＴａ，Ｙ－ＳＴｂ，Ｙ－ＳＴｃ），但以Ｙ－ＳＴａ型多见。我们构建了０．２５ｋｂ的Ｙ－ＳＴａＤＮＡ克隆片段，经用辣根过氧化酶标记后，具特异性。１９株我国分离的致病性小肠结肠炎耶氏菌由乳鼠试验结果显示均可产生Ｙ－ＳＴ。但用此酶标记Ｙ－ＳＴａＤＮＡ探针测定，仅１３株呈杂交反应，表明携带Ｙ－ＳＴａ基因；其余６株均为阴性反应，免疫学试验     

人细胞色素P4502B6的GST融合蛋白及其抗体的制备

目的：获得纯化抗原用于制备ＣＹＰ２Ｂ６多克隆抗体方法：ＰＣＲ扩增目的基因片段,亚克隆入融合蛋白表达载体ＰＧＥＸ－３ｂ,构建了重组质粒ＰＧＥＸ／２Ｂ６。然后将该重组质粒转化大肠杆菌ＤＨ５α,ＩＰＴＧ诱导表达,ＳＤＳ＿ＰＡＧＥ分离,获纯化融合蛋白ＧＳＴ－２Ｂ６。用ＧＳＴ－３Ｂ２免疫ＢＡＬＢ／Ｃ小鼠,自腹水中获取ＣＹＰ２Ｂ６多克隆抗体。结果：融合蛋白ＧＳＴ－２Ｂ６（ＣＹＰ２Ｂ６２０２￣３５２ａａ）,并获     

中国株白喉杆菌白喉毒素全基因编码序列的克隆及其…

通过聚合酶链式反应（ＰＣＲ）从一中国株中型白喉产毒杆菌的β噬菌体基因组中克隆出１０６５碱基对的白喉毒素全基因编码序列，将ＰＣＲ产物直接克隆到ｐＧＥＭ－Ｔ／载体系统，经有关限制性内切酶消化，核苷酸序列分析表明，成功的克隆出白喉毒素全基因编码序列。利用上下游引物中导入的Ｎｄｅ Ⅰ和ＢａｍＨ Ⅰ位点，将白喉毒素基因插入原核表达载体ＰＥＴ－３ａ，从而构建出白喉毒素表达载体ＰＥＴ／ＤＴ。以ＢＬ２１（ＤＥ３）     

hIL—13融合表达载体的构建及其在大肠杆菌中的表达

通过ＲＴ－ＰＣＲ技术直接从激活的健康人外周血单个核细胞（ＨＰＢＭＣ）中扩增出的０．３ｋｂ的ｈＩＬ－１３基因片段，经ＤＮＡ重组技术，插入到融合蛋白表达载体ｐＧＥＸ－４Ｔ－２中，转化入感受态的大肠杆菌ＴＧ１中，成功地构建了ｈＩＬ－１３的基因工程表达菌株ｈＩＬ－１３「ｐＧＥＸ－４Ｔ－２／ＴＧ１。经异丙基硫代半乳糖苷（ＩＰＴＧ）诱导后表达的ＧＳＴ－ＩＬ－１３融合蛋白经ＳＤＳ－ＰＡＧＥ证明分子量约３６ｋＤ、     

Development of mucosal protection against the heat-stable enterotoxin (ST) of Escherichia coli by oral immunization with a genetic fusion delivered by a bacterial vector.

An LT-B-ST (LT-B/ST) fusion peptide was constructed by genetically joining the 5' terminus of a synthetic gene coding for the heat-stable enterotoxin (ST) of Escherichia coli to the 3' terminus of the gene coding for the binding subunit of the heat-labile enterotoxin (LT-B) of E. coli. An eight-amino-acid, proline-containing linker was included between the LT-B and ST moieties. An aroA mutant of Salmonella dublin transformed with a plasmid carrying this genetic construct was shown to express a fusion peptide with antigenic determinants of both LT-B and ST. Mice were immunized orally with this strain or with a control strain expressing just LT-B from the same plasmid. Sera and mucosal secretions were obtained and analyzed for the presence of serum immunoglobulin G and mucosal immunoglobulin A that were able to recognize LT-B and ST by enzyme-linked immunosorbent assay (ELISA) and, more importantly, were able to neutralize native ST in the suckling mouse assay. Sera and mucosal secretions from animals immunized with the strain expressing the LT-B/ST fusion exhibited detectable ELISA reactivity against LT-B but not against native ST. However, even in the absence of detectable ELISA reactivity, both sera and mucosal secretions from these animals were able to neutralize the biological activity of native ST in the suckling mouse assay. These findings are important because they demonstrate the development of mucosal protection against ST by oral immunization with a genetic fusion delivered by a bacterial vector.     

Construction of a nontoxic fusion peptide for immunization against Escherichia coli strains that produce heat-labile and heat-stable enterotoxins.

The 5' terminus of the gene that codes for the heat-stable enterotoxin of Escherichia coli (ST) was genetically fused to the 3' terminus of the gene that codes for the binding subunit of the heat-labile enterotoxin of E. coli (LT-B). The ST-encoding gene used for these studies was constructed synthetically with appropriate restriction sites to permit in-frame, downstream insertion of the oligomer. For this construction, maximum expression of ST antigenicity was obtained when a seven-amino-acid, proline-containing linker was included between the LT-B and ST moieties. The LT-B-ST fusion peptide was purified by affinity chromatography and consisted of a single polypeptide chain with an apparent molecular weight of 18,000 when examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. There was no evidence of multimer formation and no change in the mobility of the fusion peptide when it was boiled in SDS or in SDS with dithiothreitol. The LT-B-ST fusion peptide was nontoxic, and immunologic determinants of both LT and ST were recognized by antibodies to the native toxins. More importantly, the LT-B-ST fusion peptide was immunogenic. Animals immunized with crude or purified preparations containing the hybrid molecule produced antibodies that were able to recognize native toxin in vitro. Significantly, these antibodies were able to neutralize the biological activity of native ST.     

Escherichia coli heat-labile toxin subunit B fusions with Streptococcus sobrinus antigens expressed by Salmonella typhimurium oral vaccine strains: importance of the linker for antigenicity and biological activities of the hybrid proteins.

A set of vectors possessing the genes for aspartate semialdehyde dehydrogenase (asd) and the B subunit of the heat-labile enterotoxin of Escherichia coli (LT-B) has been developed. These vectors allow operon or gene fusions of foreign gene epitopes at the C-terminal end of LT-B. Two groups of vectors have been constructed with and without leader sequences to facilitate placing of the foreign antigen in different cell compartments. Two Streptococcus sobrinus genes coding for principal colonization factors, surface protein antigen A (SpaA), and dextranase (Dex), have been fused into the 3' end of the LT-B gene. Resulting protein fusions of approximately 120 to 130 kDa are extremely well recognized by antibodies directed against both SpaA and Dex as well as against LT-B domains and retain the enzymatic activity of dextranase and the biological activity of LT-B in that they bind to GM1 gangliosides. Maximum antigenicity was obtained with the vector possessing an intervening linker of at least six amino acids with two proline residues. Some of the fusion proteins also exhibited another property of LT-B in that they were exported into the periplasm where they oligomerized. LT-B-SpaA and LT-B-Dex hybrid proteins are expressed stably and at a high level in avirulent Salmonella typhimurium vaccine strains which are being used to investigate their immunogenicity and types of induced immune responses. The fusion vectors will also be useful for production and purification of LT-B fusion antigens to be used and evaluated in other vaccine compositions.     

Vaccines against enterotoxigenic bacterial pathogens based on hybrid Salmonella that express heterologous antigens

In this report, we examine two aspects in the development of a vaccine against enterotoxigenic bacterial pathogens based on hybrid Salmonella that express heterologous antigens. First, we describe the construction of a non-toxic fusion peptide for immunization against Escherichia coli that produce heat-labile (LT) and heat-stable (ST) enterotoxins. For that construction, the 5' terminus of the gene coding for ST was fused to the 3' terminus of the gene coding for the binding subunit of LT(LT-B). The ST gene was constructed synthetically with appropriate restriction sites to permit in-frame, downstream insertion. Maximum expression of ST antigenicity was obtained when a seven-amino-acid proline-containing linker was included between the LT-B and ST moieties. The purified LT-B/ST fusion peptide consisted of a single polypeptide chain with an apparent molecular weight of 18,000. The LT-B/ST fusion peptide was non-toxic and immunologic determinants of both LT and ST were recognized by antibodies directed against the native toxins. Animals immunized with either crude or purified preparations containing the hybrid molecule produced antibodies that were able to recognize native toxin in vitro. Significantly, these antibodies were able to neutralize the biological activity of native ST. The second aspect reported here examines a mechanism for stabilizing expression of heterologous antigens in attenuated Salmonella mutants by integration of the heterologous gene (LT-B) into the chromosome of the carrier. A comparative in vitro study of the levels of expression of LT-B between the cointegrate strain and an isogenic strain carrying the LT-B gene on a multicopy plasmid demonstrated that the initial levels of expression of both strains is similar, that the plasmid-carrying strain loses the ability to express the heterologous antigen very quickly and that the cointegrate continues to maintain and express the antigen without the requirement for a stabilizing antibiotic.     

Construction of a plasmid for expression of foreign epitopes as fusion proteins with subunit B of Escherichia coli heat-labile enterotoxin.

A novel vector (pFS2.2) for high-level expression of fusion polypeptides with the nontoxic subunit B (LT-B) of Escherichia coli heat-labile enterotoxin in Escherichia coli and salmonellae is presented. It carries the complete coding sequence of LT-B under lac promoter control and a universal polylinker site for the in-frame insertion of foreign genes at the LT-B gene 3' end. By using this vector, fusion proteins comprising parts of the human or woodchuck hepatitis B virus surface and nucleocapsid antigens are expressed in E. coli and salmonella.     

Development of an immunoassay using recombinant maltose-binding protein-STa fusions for quantitating antibody responses against STa, the heat-stable enterotoxin of Escherichia coli.

A set of fusion proteins containing heat-stable enterotoxin (STa) and maltose-binding protein were engineered. These molecules were readily purified and used as solid-phase antigens in an enzyme-linked immunosorbent assay to monitor anti-STa responses in mice immunized with a recombinant vaccine composed of STa and the B subunit of heat-labile enterotoxin.     

Detection of LT and ST genes in the Escherichia coli isolated from the dogs, sheep and poultry

This study was designed to identify if the heat-labile enterotoxin (LT)-, heat-stable enterotoxin (ST)a- and STb-encoding genes are detectable in faecal samples from different healthy hosts and also on which part of the genome (chromosome or plasmid) they are located. Seventy-five samples of Escherichia coli were isolated from dog, sheep and poultry faecal samples (25 from each host). Plasmid and chromosomal DNA were extracted and polymerase chain reaction performed on all plasmid and chromosomal DNA using LT, STa and STb primers. Additionally, the plasmid profile of all E. coli isolates was defined using gel electrophoresis. The results showed that 36% of the E. coli isolates possessed genes for the production of LT toxin. All samples were negative for STa and STb genes. The plasmid profile of different hosts showed that all samples harboured plasmids. The results of this study indicate that enterotoxigenic E. coli are present in the faeces of different hosts. Since the genes encoding for LT and ST toxins reside on plasmids, or occasionally on transposons, they may transfer among the Gram-negative bacteria especially to the enterobacteriaceae family including E. coli which then may infect other hosts, for example, humans.     

Production of Escherichia coli STa-like heat-stable enterotoxin by Citrobacter freundii isolated from humans.

Citrobacter species are often present in the stools of children and are generally considered a normal component of the intestinal microflora. Previous reports suggested that they might act as enteric pathogens. Aiming at defining the role of Citrobacter species in inducing diarrhea, we looked for their presence in the stools of 328 children with diarrhea and in 108 controls. Citrobacter strains were isolated from 46 patients (14%) and 7 controls (6.5%) (P less than 0.05). All isolates were tested for heat-stable (ST) and heat-labile (LT) enterotoxin. No LT-producing organisms were found. Three C. freundii strains, all isolated from children with diarrhea, elaborated an enterotoxin detected by the suckling mouse assay. A crude extract was prepared by acetone precipitation and a sequential ultrafiltration technique. The enterotoxin was heat stable, and its estimated molecular weight was between 2,000 and 10,000. Citrobacter enterotoxin was soluble in methanol and stable at acid and neutral pHs but not above pH 8, and its activity was destroyed by treatment with 2-mercaptoethanol. Citrobacter enterotoxin was inactive in the 18-h rabbit ileal loop test. All these characteristics closely resemble STa produced by Escherichia coli. The time course of Citrobacter enterotoxin-induced intestinal secretion in suckling mice was similar to that of E. coli STa. The enterotoxin produced by C. freundii cross-reacted with monoclonal antibodies raised against E. coli STa. These results suggest that C. freundii is capable of inducing diarrhea through the production of an E. coli-like STa, and its presence in the stools of patients with diarrhea should be considered as that of a possible etiologic agent.     

Blinded, two-laboratory comparative analysis of Escherichia coli heat-stable enterotoxin production by using monoclonal antibody enzyme-linked immunosorbent assay, radioimmunoassay, suckling mouse assay, and gene probes

Heat-stable enterotoxin (ST)-producing enterotoxigenic Escherichia coli (ETEC) can be identified by a variety of assays, including the suckling mouse assay (SMA), radioimmunoassay (RIA), polyclonal or monoclonal antibody enzyme-linked immunosorbent assay (ELISA), and DNA hybridization with STh and STp gene probes. To compare the sensitivity and reliability of these assays, 100 coded ETEC and non-ETEC isolates were blindly tested in two independent laboratories. SMA, RIA, and monoclonal ELISA were performed in Cincinnati, Ohio, while gene probe analysis was performed in Baltimore, Md. The method of storage of organisms had a profound effect on the stability of plasmids in certain strains. Hybridization experiments to determine the presence or absence of the enterotoxin gene showed that strains stored on Dorset egg medium at room temperature better retained their plasmids than strains stored frozen in skim milk. Forty-four of the 100 organisms obtained from the skim milk stock were found to produce STa in liquid culture by the RIA, SMA, and monoclonal ELISA (100% agreement). However, 50 of 54 of the strains stored on Dorset egg medium which were originally classified as STa+ or ST+ LT+ (positive for both heat-stable and heat-labile [LT] enterotoxins) were found to produce STa and retain the plasmid by each of these assays. Three additional strains were found which harbored the plasmid but did not elaborate STa by any of the assays (3% discrepancy). The monoclonal antibody ELISA appears to be highly reliable for determination of STa production by ETEC and can be easily scored visually even by untrained personnel. Furthermore, when this STa assay is coupled with a polyclonal antibody assay, it is possible to predict the genotype of STh- and STp-producing organisms.     

Mechanism of spontaneous loss of heat-stable toxin (STa) production in enterotoxigenic Escherichia coli

Five strains of enterotoxigenic Escherichia coli (ETEC) showing spontaneous loss of heat-stable enterotoxin (STa) production were studied to elucidate the underlying genetic mechanisms. Southern blot analysis revealed that loss of STa production, and the corresponding lack of hybridization with the STa gene probes, were associated with deletions of DNA fragments harboring the relevant toxin genes rather than with loss of plasmids.