大肠杆菌耐热性肠毒素STI三重串联突变体与黏附素K99融合基因的表达研究

产肠毒素大肠杆菌（ETEC）是一种导致仔畜和婴儿腹泻的主要病原之一,它的毒力因子主要有两类：黏附素（CFAs）和耐热性肠毒素（ST）或不耐热性肠毒素（LT）。通过PCR技术及双酶切连接技术,成功构建了含有3个STI突变体和1个黏附素K99基因的重组表达质粒pE3S(S)LK和pE3S(G)LK。重组菌株BL21(DE3) (pE3S(S)LK)和BL21(DE3)(pE3S(G)LK)的表达产物经SDS-PAGE和免疫印迹分析,表明以上两种重组菌株均能高效表达3STI(S)-K99和3STI(G)-K99融合蛋白,且融合蛋白能够被产肠毒素性大肠杆菌强毒株C83922 抗血清特异性识别。其次,利用乳鼠灌胃实验检测重组蛋白的生物学毒性,结果均为阴性(G/C值≤0.083),这表明该菌株已无STI生物学毒性。这些为研发预防大肠杆菌性腹泻的新型高效多价基因工程疫苗提供了基本素材和理论指导。     

人源产肠毒素大肠杆菌疫苗的研发进展

腹泻是全球范围内引起5岁以下幼童死亡的第二大病因,而产肠毒素大肠杆菌(ETEC)是引起腹泻的最常见病原菌,其产生的细菌定植因子(CFs)和肠毒素是关键的毒力因子。CFs介导细菌黏附宿主小肠上皮细胞并完成定植,产生热敏肠毒素(LT)和热稳定肠毒素(ST)破坏宿主上皮细胞内的体液平衡,使体液和电介质过量分泌从而导致腹泻。预防ETEC腹泻的首选方法是使用能激发宿主产生抗黏附素免疫力和抗肠毒素免疫力的疫苗,阻断ETEC黏附和定植并中和肠毒素。目前一种名为Dukoral~的霍乱疫苗因能刺激机体产生抗热敏毒素免疫,已经被一些国家批准用于短期保护和预防旅行者腹泻。新型试验性ETEC候选疫苗正在研发中,旨在提供保护期长、反应谱广的抗ETEC感染免疫保护力。本文针对疫苗研发的关键问题和研究现状作一综述,并对未来的研究作出展望。     

肠产毒性大肠杆菌分子生物学及基因工程疫苗的研究进展

综述了肠产毒性大肠杆菌(ETEC)的分子生物学和基因工程疫苗的最新进展。ETEC的肠毒素基因和菌毛蛋白基因对于ETEC的致病性起主要作用,肠毒素基因工程疫苗为预防ETEC引起的腹泻开辟了新的途径。     

肠毒素大肠杆菌定居因子CS3和肠毒素融合抗原基因在减毒鼠伤寒沙门氏菌中的共表达

不耐热肠毒素（LT）和耐热肠毒素（ST）是产肠毒素大肠杆菌的主要致病因素,CS3为该菌的优势定居因子,是定居因子CFA/Ⅱ菌毛抗原的共有抗原组分。采用基因操作技术将编码CS3和融合肠毒素蛋白基因转化到减毒鼠伤寒沙门氏菌疫苗侯选株X4072中进行表达。用重组菌株口服免疫小鼠后,免疫动物能产生抗CS3、LT和ST的血清抗体。特别有意义的是,所产生的抗ST抗体能中和天然ST的生物活性。这一结果为研究载体疫苗防治肠毒素大肠杆菌腹泻疾病奠定了基础。     

动物源产肠毒素大肠杆菌(ETEC)黏附素研究进展

动物源产肠毒素大肠杆菌(enterotoxigenic Escherichia coli,ETEC)是引起动物(尤其是幼龄动物)腹泻的主要病原菌。已知黏附素和肠毒素是ETEC中两种重要的毒力因子,在致病性中两者缺一不可。其中黏附素结合到宿主易感肠上皮细胞是ETEC感染的第一步,也是最重要的关键步骤。动物源ETEC的菌毛黏附素主要包括K88、K99、987P、F18、F17和F41等。人们从20世纪60年代就开始了ETEC菌毛黏附素的相关研究,包括菌毛的基因、结构组成、生物合成、菌毛表达的调控机制以及黏附素和宿主受体相互作用等,这些研究基础有助于我们深入了解ETEC病原菌的感染机理;并且在疾病诊断和新疫苗的开发中具有重大意义。     

CTB/CS3融合蛋白与GM1结合能力和免疫原性的分析

免疫霍乱毒素B亚单位（CTB）或肠毒素大肠杆菌（ETEC）定居因子CS3可使人体对ETEC的侵染有保护作用.为探索研制ETEC双组分亚单位疫苗的可行性,利用大肠杆菌诱导表达系统表达了CTB与CS3的融合蛋白（CTB/CS3）.蛋白质印迹结果表明,诱导表达的29 ku蛋白具有CTB和CS3蛋白双重抗原性.经Ni-NTA亲和层析纯化获得重组蛋白CTB/CS3,复性的重组蛋白可以部分形成五聚体并保留了与神经节苷脂GM1的结合能力.动物实验表明,融合蛋白CTB/CS3具有CTB和CS3蛋白的双重免疫原性,同时,CTB的免疫载体作用提高了CS3的免疫强度.     

产肠毒素大肠杆菌快速检测方法的建立和评价

目的利用环介导等温扩增(LAMP)技术,建立产肠毒素大肠杆菌(ETEC)的快速、便捷、敏感、特异的检测方法,并对该方法的特异性和敏感性进行评价,为实验动物检测和细菌性腹泻的诊断提供技术支持。方法根据GenBank公布的产肠毒素大肠杆菌的LT毒素基因序列(S60731.1)设计外引物和内引物进行LAMP扩增,对LAMP特异性和敏感性与PCR方法做比较。结果建立的LAMP方法检测最低浓度为100 pg/μL,灵敏度是PCR的10倍以上并具有较高的特异性,利用该方法对27份猴腹泻样品进行LAMP和PCR方法检测,发现PCR检出率为33.3%,LAMP(60 min内)结果与PCR相同,而LAMP(90 min内)检出率为92.6%,约是PCR检出率的3倍。结论建立了一种用于检测肠毒性大肠杆菌(ETEC)的LAMP检测方法,该方法特异性强,灵敏度高,方便快捷,适合于ETEC临床快速检测。     

产肠毒素大肠杆菌的热敏肠毒素B亚基(LT-B)和耐热肠毒素(ST)基因融合及其表达

采用基因重组技术构建了表达产肠毒素大肠杆菌(ETEC)的耐热肠毒素(ST)基因和热敏肠毒素B亚基(LT-B)基因融合抗原的疫苗候选株。将ST基因的5’端与LT-B基因的3’端连接,并置于同一阅读框。编码ST的基因是通过PCR从pSLM004质粒中扩增得到的,含有ST的pro序列(其编码ST前体的pro区域),并应用寡核苷酸定点突变技术将编码ST的第14位氨基酸残基发生突变,使ST的第14位氨基酸残基Ala突变为Leu。在所构建的结构中,于LT-B和proST之间分别插入了不同长度的氨基酸Linkers。表达的融合多肽同时具有ST和LT-B的抗原性,并保留结合GM-1神经节苷脂的能力,且无LT和ST的生物毒性。表达的融合蛋白免疫动物,能诱导产生相应的特异性抗体。     

用于预防牛肠炎的商品菌苗中K_(99)(F_5)纤毛粘附素的鉴定

<正>产肠毒素大肠杆菌(ETEC)是犊牛、羔羊和仔猪新生期腹泻的重要病因。从犊牛分离的ETEC大多数菌株表面具有K_(99)纤毛粘附素,它促进对小肠微绒毛的粘附与定居。腹泻犊牛中ETEC的分离率一般在4—30%范围内,看来有地区性差异。 用含有纤毛抗原的菌苗免疫母畜,通过其初乳的免疫力,可对ETEC引起的新生期腹泻提供保护。如今一些厂家已把有纤毛抗原的ETEC菌株纳入他     

肠毒素大肠杆菌CS3定居因子抗原和融合肠毒素基因在减毒痢疾杆菌中的共表达

肠毒素和定居因子抗原 (CFAs)是肠毒素大肠杆菌 (ETEC)两种主要的致病因素。有效的ETEC疫苗应包括这两种成分。采用基因重组技术 ,将定居因子CFA/II的共有抗原成分CS3菌毛抗原和LT B/ST融合肠毒素基因克隆至以asd基因为选择标记的重组质粒上 ,与asd基因剔除的弗氏志贺氏菌Fwl0 1构成了宿主 载体平衡致死系统。结果表明 ,在无抗生素条件选择的情况下 ,该重组菌可稳定表达CS3菌毛抗原和LT B/ST融合肠毒素抗原。通过口服和鼻饲方式免疫小鼠 ,可诱生相应的血清IgG抗体 ,同时能够检测到分泌型IgA的产生 ,表明该重组菌可以有效的诱导产生粘膜免疫。     

人SUMO3基因突变体(SUMO3-K11R)真核表达质粒的构建及其鉴定

构建人SUMO3基因K11R突变体的真核表达质粒并对其进行功能鉴定。方法:PCR扩增人SUMO3基因,将其克隆入真核表达载体pEGFP-C1内,构建含人SUMO基因K11R突变体的真核表达质粒pEGFP-SUMO3-K11R。使用真核转染,Western blot和免疫荧光实验的方法,鉴定SUMO-K11R在真核细胞中的表达和功能。结果:克隆的人SUMO-K11R基因,测序结果显示完全正确。瞬时转染真核细胞后,Western blot在预期的位置检测出目的条带,免疫荧光实验显示SUMO3-K11R突变体蛋白其功能与SUMO1蛋白相似,可作为研究SUMO1蛋白和SUMO3蛋白功能差异的有力工具。结论:成功构建了含人SUMO-K11R基因的真核表达质粒。     

Synthesis and biological properties of the lipophilic technetium-99m complex 99mTc(acac)3

In the development of technetium-99m radiopharmaceuticals for the evaluation of regional cerebral perfusion, one series of complexes that has remained unexplored is the neutral lipophilic tris complexes formed with β-diketonato ligands. The prototype complex of this series, tris(2,4-pentanedionato) technetium(III), has been prepared via a new synthetic route and chemically characterized using ⁹⁹Tc and the biodistribution of the no-carrier-added ^99mTc complex has been determined. The ^99mTc complex was found to be distributed throughout the body with persistant high blood levels indicative of a high degree of protein binding. The primary route of excretion was the hepatobiliary system as indicated by the appearance of ^99mTc in the gut and feces at longer sample times post-injection. Although this complex was not retained by the brain, it does provide a starting point from which a more effective agent might be developed.     

Immunobiology of Lipopolysaccharide (LPS) and LPS-Derived Immunoconjugates Vaccinate Mice against Salmonella typhimurium

The immunobiology of lipopolysaccharide (LPS) of Salmonella typhimurium LT2–71 was studied in its native, modified and conjugated states using mice as the experimental model. An alkali-treated detoxified fraction of LPS (D-LPS) was found to be not only non-toxic but also equally immunogenic, like LPS. In addition D-LPS alone or conjugated with enterotoxin or hemolysin was also non-pyrogenic and non-indurogenic. The immunoprophylactic activity of D-LPS conjugates to a 100 ID₅₀ challenge dose of S. typhimurium was also higher than that of detoxified LPS or native LPS.     

Pig Intestinal Membrane-Bound Receptor (Guanylyl Cyclase) for Heat-Stable Enterotoxin: cDNA Cloning,Functional Expression,and Characterization

A cDNA encoding the receptor protein for a heat-stable enterotoxin (STa) produced by enterotoxigenic Escherichia coli was cloned from intestinal epithelial cells of a 10-week-old pig. The cDNA had an open reading frame of 3,219 base pairs and coded for a protein with 1,073 amino acid residues. The mature protein consisted of 1,050 amino acid residues with a molecular mass of ca. 121 kDa and was 87% and 82% identical with the human and rat protein, respectively. The CHO cell line overexpressing the pig recombinant STa receptor specifically bound to a photoaffinity-labeled analog of STa and showed marked elevation of the cellular content of cGMP in response to STa.     

Detection of heat-stable and heat-labile enterotoxin genes of Escherichia coli in diarrhoeic faecal samples of mithun (Bos frontalis) calves by polymerase chain reaction

Aims:  To find out the prevalence of different serogroups of Escherichia coli ( E. coli ) and to detect heat-stable (ST) and heat-labile (LT) enterotoxin genes of enterotoxigenic E. coli (ETEC) from the faeces of mithun calves with diarrhoea.
Methods and Results:  Faecal samples obtained from 65 diarrhoeic mithun calves of under 2 months of age were examined for E. coli using polymerase chain reaction (PCR). Fifty-four E. coli isolates were obtained from those samples, which belonged to 38 different serogroups. Out of 54 isolates tested by PCR, two isolates (3·70%) belonging to serogroups O26 and O55 were found to possess gene that code for ST enterotoxin and one isolate (1·85%) belonging to serogroup O125 was found to carry LT enterotoxin gene.
Conclusions:  Escherichia coli isolates from diarrhoeic mithun calves were found to possess ST and LT enterotoxin genes, which are designated as ETEC, and these isolates can be detected through PCR using specific primers.
Significance and Impact of the Study:  This study reports the isolation of ETEC possessing ST and LT enterotoxin genes for the first time and ETEC could be a cause of diarrhoea in mithun calves leading to calf mortality.     

Detection of genes encoding cholera toxin (CT), zonula occludens toxin (ZOT), accessory cholera enterotoxin (ACE) and heat-stable enterotoxin (ST) in Vibrio mimicus clinical strains

A total of 51 clinical strains of Vibrio mimicus were searched for the presence of virulence-associated genes, like ctx, zot or ace genes which locate in "cholera virulence cassette," and the st gene by polymerase chain reaction. Moreover, the pathological potential of each clinical strain was also examined by rabbit ileal loop (RIL). Three strains showed to have the ctx gene, of which only one strain was zot gene-positive. Meanwhile, one other strain was zot+ but ctx-. All of these four strains were found to have the ace gene and to belong to serogroup O115. Nine strains showed to carry the st gene. However, none of these ST-gene-positive strains was indicated to contain the genes located in the "cholera virulence cassette." It is of interest to note that all of the RIL-positive and/or virulence gene-positive strains were restricted to three serogroups, O20, O41 and O115. These results suggest a significant association between O antigens and enterotoxic activities in V. mimicus clinical strains, and clearly demonstrate multifactorial virulence potentials of this human pathogen.     

99mTc-labeling of colchicine using [99mTc(CO)3(H2O)3]+ and [99mTc triple bond N]2+ core for the preparation of potential tumor-targeting agents

Multidrug resistance (MDR) mediated by over-expression of P-glycoprotein (Pgp) is one of the major causes of failure of chemotherapy in cancer treatment. Colchicine, a naturally occurring alkaloid, is a Pgp substrate and acts as an antimitotic agent by binding to microtubules. Hence, Colchicine and its analogues radiolabeled with 99mTc may have potential for visualization of MDR in tumors. Here we report 99mTc-labeling of colchicine derivatives using [99mTc(CO)3(H2O)3]+ and [99mTc triple bond N]2+ cores. Trimethylcolchicinic acid synthesized from colchicine was used as the precursor to prepare iminodiacetic acid and dithiocarbamate derivatives which were then radiolabeled with [99mTc(CO)3(H2O)3]+ and [99mTc triple bond N]2+ cores, respectively. Radiolabeling yield for both the complexes was > 98% as observed by HPLC and TLC patterns. In vitro studies in tumor cell lines showed significant uptake for 99mTc-carbonyl as well as for 99mTc-nitrido colchicine complexes. Biodistribution studies in Swiss mice bearing fibrosarcoma tumor showed 4.1 +/- 1.2% ID/g of uptake at 30 min pi for 99mTc(CO)3-complex as against 0.42 +/- 0.24% ID/g for the 99mTcN-complex. 99mTc(CO)3-colchicine complex exhibited better pharmacokinetics with lower liver accumulation as compared to the 99mTcN-complex. Thus, colchicine radiolabeled with [99mTc(CO)3(H2O)3]+ core is more promising with respect to in vivo distribution characteristics in tumor model.