艰难梭菌A毒素的纯化

目的建立一种纯化艰难梭菌A毒素的方法。方法将艰难梭菌VPI 10 4 6 3菌株经透析培养 ,5 0 %饱和硫酸胺盐析 ,酸沉淀 ,再经DEAE Toyopearl 6 5 0M离子交换色谱。结果精制的艰难梭菌A毒素Native PAGE及对流免疫电泳均为单一带 ,相对分子质量为 5 5 0 0 0 0 ,小鼠毒力为 1× 10 6MLD/ml,Vero细胞毒性为 1× 10 7CU/ml。凝血活性为 1∶5 12 ,肠袢试验为阳性。结论该法简便、实用、可行。     

艰难梭菌A毒素的抗肿瘤活性研究

目的　研究艰难梭菌A毒素的抗肿瘤活性。方法　从艰难梭菌VPI 1 0 4 63菌株脑心浸液培养物 ,经甲状腺球蛋白亲和层析和QSepharose层析提纯得到精制A毒素。通过应用苔盼蓝染色测定细胞脱落率 ,四氮噻唑蓝 (MTT)比色测定细胞存活率 ,[3H] Uridine(尿嘧啶 )测定细胞膜损伤 ,光学、荧光显微镜观察细胞的形态变化等方法测定比较A毒素对TPC 1细胞 (人甲状腺乳头癌细胞 )和Vero细胞 (非洲绿猴肾细胞 )的作用。结果　在A毒素的作用下 ,TPC 1细胞的增长抑制、凋亡指数、细胞脱落率及细胞膜损伤情况均高于Vero细胞 ,且这种作用具有时间和剂量依赖性。结论　A毒素对TPC 1细胞的杀伤作用远大于对Vero细胞的作用。以上结果对细菌毒素的开发利用、抗癌新药的探索研制具有潜在意义     

钝顶螺旋藻异藻蓝蛋白多克隆抗体的制备

异藻蓝蛋白（allophycocyanin,APC）是螺旋藻细胞中的主要蛋白成分之一,具有多种生理功能和广阔应用前景。本研究采用纯化的钝顶螺旋藻（Spirulina platensis）APC为抗原,免疫新西兰兔,制备APC的多克隆抗体。 ELISA法提示制备的抗体效价高达1∶32000, western blot印迹法确定获得的抗体能与APC产生特异性免疫反应。这为今后进一步开展螺旋藻APC的相关研究提供了有力的工具。     

艰难梭菌感染的治疗及预防

目的探讨艰难梭菌毒素测定在医院相关性腹泻患者中的应用。方法艰难梭菌产生毒素A/B。结果艰难梭菌毒素在抗生素相关性腹泻患者检出率较高。结论治疗的重点在于预防。     

大鼠LC3蛋白的原核表达及多克隆抗体的制备

微管相关蛋白l轻链3-β (LC3)在哺乳动物细胞中定位于自噬泡上,可作为一种特定的标记用来检测自噬.从SD大鼠脑组织提取总RNA,经RT-PCR扩增得到LC3全长编码基因,并克隆到pMD18-T载体.以测序验证正确的重组子为模板扩增LC3基因,并构建pUC 18-LC3重组质粒,将其转化到E.coli BL21 (DE3)中.SDS-PAGE结果表明,菌体诱导后形成包涵体形式的表达产物,以电泳回收目的蛋白并免疫新西兰白兔,获得大鼠LC3蛋白多克隆抗体.western blot 结果显示,制备的多克隆抗体可特异性识别LC3蛋白.     

降低艰难梭菌感染复发新药——人源化单克隆抗体bezlotoxumab

bezlotoxumab为一人源化单克隆抗体,能够中和艰难梭菌B型毒素。bezlotoxumab由美国默克制药公司开发,于2016年10月在美国获批用于艰难梭菌感染复发的治疗。本文从药物的作用机制、药动学、临床疗效及不良反应等方面对bezlotoxumab进行了介绍。     

艰难梭菌感染的临床治疗策略及新药物研究进展

艰难梭菌(Clostridium difficile)是一种革兰阳性厌氧芽孢杆菌,因导致医院内腹泻备受关注。由于C. difficile的分离培养需要在厌氧状态下实现,这给国内外研究者带来非常大的限制和挑战。目前临床治疗手段主要采取抗生素和粪便微生物群移植(fecal microbiota transplantation, FMT)。据统计艰难梭菌感染(C. difficile infection, CDI)经万古霉素治疗后复发率高达30%,死亡率达15%,是临床上亟待解决的难题。目前,许多国家在大力开发有效的新型药物来治疗CDI。本文根据目前的临床治疗现状和正在开发中的新药物进行了简要总结,更新了目前5种已有临床实验的新型抗菌药物和11种新开发的不同抗菌剂,前者包括ridinilazole、surotomycin、cadazolid、CRS3123和DS-2969b;后者包括黄芩苷、rhodomyrtone、OPS-2071、rakicidin B、依拉环素、双环胍、CM-A、月桂酸、NCK-10、Raja 42和AJ-024。针对现有临床前和临床抗菌数据,将这些潜在药物与传统抗生素...     

艰难类梭菌耐药机制及其适应性代价的研究进展

摘要：艰难类梭菌是一种革兰阳性、产芽胞的专性厌氧菌,是抗菌药物相关性腹泻最常见的病原菌。近年来,随着抗菌药 物的广泛应用,艰难类梭菌的耐药率不断攀升。获得抗性基因、药物靶点突变、生物膜生成以及代谢途径发生改变是艰难类梭 菌产生耐药性的主要原因。此外,耐药艰难类梭菌的适应性代价较低,即使在缺乏抗菌药物选择压力的环境中仍然可维持耐药 性状,促使多重耐药菌株的出现及流行,为抗菌药物治疗埋下了巨大隐患。本文对艰难类梭菌的耐药机制及其适应性代价进行 综述,旨在为减少耐药性、开发新疗法提供理论参考。     

Clostridium difficile toxin A and its effects on cells

Clostridium difficile toxin A in its native form is a high molecular weight (520-540 K) aggregate with five major biological activities. It is lethal, enterotoxic, cytotoxic and cytotonic, and induces hemagglutination of rabbit red blood cells. Possibly these activities are contained in separate components. A major subunit of c. 230-310 K has been defined but lower molecular weight components cannot be excluded. The major component has been cloned, and sequence analysis indicated a complicated pattern of repeating sequences in the C-terminal third of the molecule. This review deals mainly with the effects of toxin A on cultured cells. Most mammalian cells are sensitive to toxin A whose major effect is to stop cell division irreversibly. The toxin binds via its repeat sequences to a trisaccharide receptor expressed on rabbit red cells and on brush border membranes from hamster intestine. This receptor seems to be functional in the hemagglutination reaction and the enterotoxicity. Its role in the cytotoxic effect of the toxin is not clear, but no other receptor structure has as yet been identified. In order to exert its cytotoxic (antiproliferative) effect toxin A must first be internalized by endocytosis. Thus a latency period of at least 30 min after toxin binding to cells is consistently observed, and all cytotoxic effects can be prevented by blocking the endocytosis pathway. The first microscopically visible signs of cytotoxicity consist in retraction and rounding of intoxicated cells. In addition the nucleus becomes polarized to one side of the cell while other cell organelles are not significantly affected. These morphological changes seem to be the consequence of a cytoskeletal rearrangement, mainly involving some components of the microfilament system. Inhibition of macromolecular syntheses as well as permeabilization of the plasma membrane may follow the early cytoskeletal effects and finally lead to cell death. Attempts to identify metabolic pathways of significance in the cytotoxicity suggest that the cytosolic level of Ca2+ is not important, thus excluding certain mechanisms for cell killing. In this respect the cytotoxic mode of action of toxin A clearly differs from that of toxin B. However, the biochemical basis for the antiproliferative effect of toxin A remains unknown.     

Fas胞外区基因的构建、表达、纯化及多克隆抗体的制备

目的构建Fas胞外区(eFas)基因的表达载体,表达纯化重组蛋白,进行多克隆抗体的制备,为进一步功能研究奠定基础。方法通过重叠PCR获得eFas基因的编码序列,构建pET-22b(+)/eFas表达载体,转化大肠杆菌Rosetta-gami,IPTG诱导表达,Ni-NTA柱亲和纯化,SDS-PAGE鉴定重组蛋白的纯度。将纯化的eFas融合蛋白免疫新西兰白兔制备多克隆抗体,通过ELISA方法检测多克隆抗体的效价。结果获得了eFas的编码序列与表达载体,目的蛋白主要在包涵体中表达,表达量占菌体总蛋白的30%以上,纯化的重组蛋白纯度达95%以上。结论eFas融合蛋白基因的构建、表达、纯化以及多克隆抗体的制备,为进一步研究Fas提供了材料。     

Clostridium difficile toxin in chronic idiopathic colitis

Clostridium difficile toxin was isolated from the stools of three patients with chronic idiopathic colitis. Two patients were known to have chronic idiopathic colitis before Cl difficile toxin was isolated. The third patient was subsequently found to have ulcerative colitis after presentation with Cl difficile toxin in the stool. Two patients were on sulphasalazine at the time of diagnosis of Cl difficile infection and one had taken sulphasalazine two months previously. Only one patients had antibiotic exposure and that was at least three months before presentation. In each patient, treatment with vancomycin was accompanied by symptomatic improvement and disappearance of the toxin. The underlying colitis remained unaffected. In patients with inflammatory bowel disease in relapse, the presence of Cl difficile toxin should be sought as this may be a factor in the relapse. In any patient presenting with diarrhoea, the presence of Cl difficile toxin may obscure the presence of underlying inflammatory bowel disease.     

Inhibition of insulin-stimulated glucose transport in 3T3-L1 cells by Clostridium difficile toxin B, Clostridium sordellii lethal toxin, and Clostridium botulinum C2 toxin

The role of the actin cytoskeleton and/or GTPases of the Rho/Rac-family in glucose transport regulation was investigated in 3T3-L1 cells with clostridial toxins which depolymerize actin by inactivation of Rho/Rac (Clostridium difficile toxin B and Clostridium sordellii lethal toxin (LT)) or by direct ADP-ribosylation (Clostridium botulinum C2 toxin). Toxin B and C2 reduced insulin-stimulated, but not basal, 2-deoxyglucose (2-DOG) uptake rates in 3T3-L1 fibroblasts. In parallel, the toxins produced morphological alterations of the cells reflecting disruption of the actin cytoskeleton. Both toxins reduced the maximum response to insulin but failed to alter the half-maximally stimulating concentrations of insulin. In 3T3-L1 adipocytes, the lethal toxin reduced the effect of insulin on 2-DOG uptake, whereas toxin B and C2 failed to affect glucose transport or cell morphology. When cells were exposed to the toxins after treatment with insulin, both toxin B and the lethal toxin, in contrast to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin, failed to reduce the 2-DOG uptake rates. Thus, both translocation to the plasma membrane and internalization of glucose transporters were inhibited by the toxins, whereas the PI 3-kinase inhibitor selectively affects translocation. The data suggest that the effects of the clostridial toxins on trafficking of glucose transporters are mediated by the depolymerization of the actin cytoskeleton and are an indirect consequence of Rho or Rac inactivation. It is suggested that pathways signalling through Rac or Rho may play a modulatory role in glucose transport regulation through their effects on the actin network. Received: 28 October 1997 / Accepted: 19 January 1998     

柯萨奇病毒A组5型VP1蛋白原核表达及多克隆抗体的制备

目的 原核表达柯萨奇病毒A组5型(coxsackievirus A5,CV-A5)VP1蛋白,并制备抗VP1多克隆抗体(多抗),为CV-A5相关定性定量研究制备试剂。方法 逆转录PCR扩增N端截短的CV-A5VP1-ΔN56后,克隆至原核表达载体pGEX-6P-1,获得pGEX-6P-1-VP1-ΔN56。将其转化大肠埃希菌BL21(DE3),表达重组蛋白并纯化。用纯化的谷胱甘肽巯基转移酶-VP1-ΔN56融合蛋白经背部皮下免疫日本大耳白兔,制备多抗。结果 重组表达载体构建成功,融合蛋白以不可溶包涵体存在。ELISA、蛋白质印迹法检测表明,获得的兔多抗效价为107,可特异性识别重组和天然CV-A5 VP1蛋白。结论 成功制备重组CV-A5 VP1蛋白及特异性多抗。为中和抗原表征研究及VP1定性定量分析奠定了基础。     

肝癌相关基因HTA的重组表达及多克隆抗体的制备

目的将肝癌相关基因HTA进行克隆、表达并制备多克隆抗体,为进一步研究其作为肝癌导向治疗靶点的可行性及临床意义奠定基础。方法应用RT-PCR技术,从人肝癌细胞系HepG2细胞中扩增得到HTA3＋cDNA,再将其克隆到原核表达载体pET21a（＋）-MBP内,用IPTG诱导其在大肠杆菌BL21（DE3）中表达。His-tag磁珠纯化试剂盒纯化重组蛋白MBP-HTA,通过Westernblot和ELISA方法检测重组蛋白的抗原性。将纯化的重组蛋白免疫BALB／c小鼠制备多克隆抗体,并采用ELISA、Westernblot和免疫组化的方法检测抗体的灵敏度和特异性。结果成功地构建了表达MBP-HTA融合蛋白的原核表达质粒pET21a（＋）-MBP-HTA。重组MBP-HTA融合蛋白在大肠杆菌BL21（DE3）内得以高效表达,且以包涵体的形式存在。His-tag磁珠纯化试剂盒纯化和Westernblot分析,得到了分子量约为52kDa的目的蛋白。获得了高效价的特异性多克隆抗体,经ELISA检测抗体的效价为1：3200。用Westernblot检测的效价为1：400。免疫组织化学检测表明：肝癌组织中HTA蛋白的阳性表达率明显高于正常肝组织俨〈0．01）。结论成功地制备出抗MBP-HTA多克隆抗体,该抗体有较高的效价和特异性;能用于免疫组织化学的检测,且HTA在肝癌组织中的阳性表达率明显高于正常肝组织。HTA蛋白有望成为肝癌导向治疗的潜在靶点。     

Comparative study of immunological properties and cytotoxic effects of Clostridium difficile toxin B and Clostridium sordellii toxin L.

We compared the immunological properties and cytotoxic effects of Clostridium difficile toxin B and Clostridium sordellii toxin L. These two cytotoxins are immunologically related in that the cytotoxic effect of either toxin can be neutralized by the polyclonal antiserum prepared against either cytotoxin. On the other hand, polyclonal antiserum prepared against Clostridium difficile enterotoxin A did not cross-react with the cytotoxins B and L when tested by cytotoxic neutralization test nor by double immunodiffusion assay. However, despite this immunological relationship between toxins B and L, the morphological modifications observed in MacCoy cells induced by treatment with these cytotoxins are clearly distinct. We describe the first quantitative analysis of specific cellular parameters which illustrates the morphological differences induced by these cytotoxins. Moreover, immunocytochemical experiments show that, whereas disruption of microfilaments is observed with toxin B- and L-treatments, alterations of F-actin network are different in the cells treated with toxin B or L. The observation that the cellular modifications induced by toxin B- and toxin L-treatment differ suggests that the molecular mechanisms involved in the respective cytotoxicities are also likely to be different.     

A comparative biochemical, pharmacological and immunological study of Clostridium novyi alpha-toxin, C. difficile toxin B and C. sordellii lethal toxin

The three clostridial cytotoxins, i.e. alpha-toxin of C. novyi (Tox alpha-nov), toxin B of C. difficile (ToxB-dif) and lethal toxin of C. sordellii (LT-sor) consist of single peptide chains of about 200,000 (Tox alpha-nov), 250,000 (LT-sor) and 275,000 (ToxB-dif) mol. wts. ToxB-dif and LT-sor but not Tox alpha-nov cross-reacted with rabbit polyclonal antibodies. Toxicity upon i.v. injection in mice was similar (LD50, 100 hr, 50-200 ng/kg) and was characterized by a slowly developing fluid loss into the interstitial space. When injected into the rat paw the toxins caused a delayed local edema lasting for days. In vitro the three toxins provoked a persistent retraction of endothelial cells cultured from pig pulmonary artery. ToxB-dif and Tox alpha-nov triggered the accumulation of F-actin in the perinuclear region at the expense of the tight peripheral bands whereas LT-sor led to a random loss of microfilament structure. The toxins inhibited uridine incorporation into endothelial or chicken embryonic cells whereas T 84 cells responded by an about 10-fold increase of uridine incorporation. Neither toxin ADP-ribosylated actin. The similarities between the three cytotoxins warrant their arrangement into a common group which perturbs the microfilament system.     

谷胱甘肽-S转移酶-中期因子融合蛋白原核表达及多克隆抗体的制备

目的 构建谷胱甘肽-S-转移酶(GST)和中期因子(MK)融合蛋白的原核表达质粒,并表达和纯化蛋白,制备多克隆抗体.方法 通过RT-PCR技术从人胃癌组织中扩增入MK编码序列,克隆入表达载体pGEX-1λT中,获得表达质粒pGEX-MK,并在大肠杆菌BL21 (DE3)中经IPTG诱导表达,通过亲和层析纯化表达的GST-MK融合蛋白,并以重组蛋白免疫兔子.结果 成功构建了GST-MK融合蛋白的原核表达载体,经诱导表达纯化得到GST-MK融合蛋白.免疫兔子后取多抗血清以间接ELISA检测效价达1∶64 000,Western blotting分析显示多克隆抗血清对MK蛋白特异结合.结论 MK在大肠杆菌中成功表达及其多克隆抗体的获得,为研究MK生物功能奠定了基础.