以SecA为靶点的新型抗绿脓杆菌药物细胞水平筛选模型的建立和应用

Sec途径(即分泌途径secretion pathway)是蛋白质转运的主要途径.其中,最为关键的组分之一是SecAATP酶,是蛋白质转运途径中的"动力泵",通过ATP的水解循环驱使蛋白质前体穿过细菌内膜,在细菌中是不可缺少的.我们推测抑制SecAATP酶活性的化合物.必然会在一定程度上抑制蛋白质的转运和分泌.通过绿脓杆菌与大肠杆菌SecA蛋白的互补作用,利用本实验室构建的高效表达SecA蛋白的基因工程菌,建立了SecA蛋白ATP酶活性抑制剂的细胞水平筛选模型.利用所纯化的绿脓杆菌SecA蛋白的ATP酶活测定体系,验证了所建立的细胞水平筛选模型具有一定的特异性.研究结果表明其中两个酯相组分在细胞水平和蛋白水平均具有活性,值得进行深入的研究.     

蛋白酪氨酸磷酸酶1B抑制剂高通量筛选模型建立和应用

本文以蛋白酪氨酸磷酸酶1B（protein tyrosine phosphatase,PTP1B）作为靶点,采用分子克隆GST融合蛋白的方法,重组表达获得PTP1B,结合自动化操作技术和比色分析,建立了一种PTP1B抑制剂的高通量筛选模型.经在96孔板优化各种反应条件并对17940个样品的筛选结果表明,靶向PTP1B建立的高通量筛选模型具有微量、快速、特异、灵敏等特点,平均日筛选量可达15000样次以上,为寻找新的抗糖尿病药物和先导化合物提供了一种先进的手段.     

BST-2和Vpu相互作用抑制剂筛选模型的建立

骨髓基质细胞抗原2(Bone marrow stromal cell antigen 2,BST-2,又称Tetherin、CD317、HM1.24)是宿主先天性免疫应答的组成部分,可抑制人类免疫缺陷病毒1(Human immunodeficiency virus 1,HIV-1)的释放,HIV-1的辅助蛋白Vpu可通过跨膜区与BST-2的跨膜区产生相互作用,进而将其降解,下调其在细胞表面的数量,拮抗BST-2的抗病毒功能。本研究将海肾荧光素酶(Renilla luciferase,Rluc)与BST-2的N端连接,增强型黄色荧光蛋白(Enhanced yellow fluorescent protein,EYFP)与Vpu的C端连接,分别构建质粒RB和VE,使两种融合蛋白在细胞内共表达,产生生物发光共振能量转移(Bioluninescence resonance energy transfer,BRET)信号,进而建立稳定双表达细胞系,以BST-2和Vpu的跨膜区相互作用为靶点,应用BRET技术,建立两种蛋白相互作用抑制剂的筛选模型,以期通过BRET信号变化筛选出相互作用抑制剂,发展新型的艾滋病治疗手段。     

以蛋白激酶G为靶点的抗结核药物筛选模型的建立和初步应用

结核分枝杆菌可以产生11种丝氨酸/苏氨酸蛋白激酶,其中蛋白激酶G(PknG)对于结核分枝杆菌在巨噬细胞内以"持留"状态长期存活有着重要作用。本研究以结核分枝杆菌基因组DNA为模板,在大肠杆菌中克隆表达了MTBPknG蛋白,并分离纯化得到PknG纯酶。本研究还采用三步级联反应方法测定了PknG酶活性,建立和优化了PknG抑制剂高通量筛选模型。利用此模型共筛选发酵液样品2120个,化合物样品2300个,筛选得到阳性化合物1个,阳性发酵液13个,阳性率0.32%。     

以蛋白激酶B为靶点的新型抗结核药物高通量筛选模型的建立和应用

【目的】建立以结核分枝杆菌蛋白激酶B为靶点的高通量筛选模型,并运用此模型进行化合物的筛选。【方法】克隆和表达结核分枝杆菌蛋白激酶B,并以其为靶酶建立并优化PknB抑制剂高通量筛选模型,利用该模型对化合物样品进行筛选,并对筛选到的阳性化合物进行抗菌和抑酶活性评价。【结果】利用该模型筛选了化合物样品18 000个,得到具有抑酶活性的阳性化合物8个,其中3个化合物具有较好的对结核分枝杆菌、海分枝杆菌、耻垢分枝杆菌的抑菌活性。【结论】建立的以PknB为靶点的抗结核药物高通量筛选模型具有灵敏度高、稳定性强等优点,可成功用于化合物的高效筛选。筛选得到3个在抑酶水平和抗菌方面均具有良好活性的阳性化合物样品,值得进一步研究。     

一种基于绿色荧光蛋白的蛋白酶体抑制剂细胞筛选模型

为建立基于绿色荧光蛋白(GFP)的药物筛选模型,并用此模型从包括中药提取物在内的化合物中筛选新型蛋白酶体抑制剂,本研究构建了pGC-E1-ZU1-GFP融合蛋白慢病毒表达载体并感染A549细胞,筛选稳定表达细胞株,用已知蛋白酶体抑制剂PS-341处理细胞,荧光显微镜检测处理前后细胞GFP水平变化。结果获得了稳定表达pGC-E1-ZU1-GFP的A549细胞,这些细胞用PS-341处理24h后用荧光显微镜检测,发现细胞绿色荧光强度相对于对照组明显增强。利用这一模型对一些化合物进行筛查,发现了一些新的蛋白酶体抑制剂。     

斑马鱼模型在药物筛选中的应用

斑马鱼具有子代数量多、体外受精、胚胎透明、可以做大规模遗传突变筛选等生物学特性, 因此成为一种良好的脊椎动物模式生物。随着研究的深入, 斑马鱼不仅应用于遗传学和发育生物学研究, 而且拓展和延伸到疾病模型和药物筛选领域。作为一种整体动物模型, 斑马鱼能够全面地检测评估化合物的活性和副作用, 实现高内涵筛选。近年来, 科学家们不断地发展出新的斑马鱼疾病模型和新的筛选技术, 并找到了一批活性化合物。这些化合物大多数在哺乳动物模型中也有相似的效果, 其中前列腺素E2(dmPGE2)和来氟米特(Leflunomide)已经进入临床实验, 分别用来促进脐带血细胞移植后的增殖和治疗黑素瘤。这些成果显示了斑马鱼模型很适合用于药物筛选。文章概括介绍了斑马鱼模型的特点和近年来在疾病模型和药物筛选方面的进展, 希望能够帮助人们了解斑马鱼在新药研发中的应用, 并开展基于斑马鱼模型的药物筛选。     

人源多巴脱羧酶的表达、纯化以及高通量抑制剂筛选模型的建立

帕金森病是一种常见的神经系统退行性疾病。多巴脱羧酶（DDC）是帕金森病研究的靶点蛋白之一,但是目前没有高通量的测活模型。因此,需要构建一种高通量多巴脱羧酶抑制剂的筛选模型,用于发现新型抑制剂。采用克隆表达纯化得到多巴脱羧酶和用于酶偶联反应的磷酸烯醇式丙酮酸羧化酶（PEPC）。基于一系列酶联反应将CO2固定,检测其含量,从而测定多巴脱羧酶的活性。结果得到人源多巴脱羧酶和磷酸烯醇式丙酮酸羧化酶的体外纯酶,建立了一种高通量筛选模型,并且从70个天然化合物中,筛选得到2个多巴脱羧酶的抑制剂。成功构建了一种基于体外纯酶高通量多巴脱羧酶抑制剂的筛选模型。     

An Efficient and Novel Screening Model for Assessing the Bioactivity of Extracts against Multidrug-Resistant Pseudomonas aeruginosa Using Caenorhabditis elegans

As a large number of multidrug-resistant bacteria have emerged, and there is an urgent need for the development of new antibacterial agents. In this study, we developed a liquid-based slow killing assay to be carried out in standard 96-well microtiter plates. This screening method was designed to facilitate high-throughput screening of small molecules and extracts. In antibiotic rescue assays, the Caenorhabditis elegans multidrug-resistant Pseudomonas aeruginosa infection model displayed a high degree of drug resistance in vivo and in vitro. We used the method to screen 1,300 extracts, and found 36 extracts (2.7%) which prolonged the survival of infected nematodes, and four (0.3%) of these extracts showed in vitro and in vivo anti-multidrug resistant P. aeruginosa activity. These results indicate that the whole-animal C. elegans multidrug-resistant bacterial model can be used to screen antibacterial compounds, and can also be useful for bioactive compounds which most likely cannot be identified in vitro.     

以蛋白激酶G为靶点的抗结核药物筛选模型

<正>结核分枝杆菌在被巨噬细胞吞噬、形成吞噬体后,可以通过阻止吞噬体的成熟及其与溶酶体的融合,而使其自身不被溶酶体酶降解,从而在巨噬细胞内长期存留下来。此时的细菌代谢活动降至最低,生长繁殖几乎停止,不易被抗菌药物杀灭,这种类似于休眠的长期存活状态被称为"持留"状态。当机体免疫力下降时,     

Expression, purification, and characterization of Pseudomonas aeruginosa SecA

A secA gene from Pseudomonas aeruginosa PAO1 was amplified and expressed in Escherichia coli BL21.19 (secA13) under conditions where E. coli SecA was depleted. The binding of P. aeruginosa SecA (PaSecA) to the SP-Sepharose column was facilitated by ammonium sulfate fractionation but was not necessary for E. coli SecA (EcSecA) as the later bound more efficiently. PaSecA and EcSecA were purified by the single chromatographic step to greater than 98% purity and had a recovery of more than 20 and 40%, respectively, from the soluble fraction. This simple step purification obtained a higher homogeneity than previously reported. Cross-reactivity by immunoblotting showed that the purified PaSecA contained little EcSecA if any. The purified PaSecA is a dimer in solution, as judged by size exclusion chromatography, and is slightly larger than its counterpart EcSecA with an estimated molecular weight of 240 kDa. Further studies by the sedimentation velocity method indicate that PaSecA tends to remain as a monomer in solution. The purified PaSecA possessed ATPase activity; the intrinsic and liposome-stimulated ATPase specific activities of PaSecA were approximately 50% of EcSecA.     

Expression and purification of Pseudomonas aeruginosa SecA N-terminal domain: stimulation of ATPase activity of the SecAL43P mutant protein

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium which secretes a wide range of hydrolytic enzymes, toxins, and virulence factors into the extracellular medium. Although P. aeruginosa possesses numerous specific systems for the export of proteins across its double-membrane envelopes, the Sec system is still the major and essential mechanism. However, very little is known about its molecular basis. We constructed, cloned, and expressed the N-terminal 236 amino acids of PaSecA domain (PaSecAN236), and SecAL43P mutants of P. aeruginosa in Escherichia coli BL21.19 (secA(ts)). Here, we describe the purification of PaSecAN236 by using osmotic shock as the first step to efficiently release targeted protein from cells, followed by cation-exchange and size exclusion columns to obtain homogeneous PaSecAN236. The purified PaSecA N-terminal domain was functional in stimulating the ATPase activity of mutant SecAL43P protein of P. aeruginosa.     

铜绿假单胞菌的药敏试验中D-现象的探讨

了解铜绿假单胞菌的药敏试验（K-B法）中,亚胺培南（IPM）诱导哌拉西林（PIP）、哌拉西林/他唑巴坦（TZP）出现D-现象的概率,并对这种现象发生的机制及临床意义进行探讨。采用美国临床实验室标准化研究所（CLSI）推荐的K-B法。其中,将TZP、PIP分别贴在IPM的两侧,纸片中心距离为25 mm,看IPM能否诱导TZP、PIP出现D-现象。结果可见,150株铜绿假单胞菌的药敏试验中,出现D-现象的有120株,占80%;未出现D-现象的有30株,占20%,其中5株耐IPM。铜绿假单胞菌的药敏试验中,IPM诱导TZP、PIP出现D-现象非常普遍,这可能与IPM可诱导AmpC酶的大量产生有关。     

铜绿假单胞菌的分布特点耐药性分析

目的分析3年来我院患者铜绿假单胞菌的耐药特点,为临床合理用药提供依据,并有助于医院感染预防与控制。方法对2010年至2012年我院各类感染患者标本中分离获得的铜绿假单胞菌,采用纸片扩散法（K-B法）检测抗菌药物的敏感性,并用WHONET5．3软件对药敏结果进行统计分析。结果3年分离的铜绿假单胞菌共计369株,其对多黏菌素B无耐药,对亚胺培南、美罗培南、头孢哌酮／舒巴坦、哌拉西彬他唑巴坦和阿米卡星的耐药率较低（6．81％～22．73％）,对其他抗菌药物的耐药率较高（32．47％～73．38％）。结论铜绿假单胞菌对多种抗菌药物均具有较高的耐药性,临床上治疗该菌感染时应根据药敏检测结果选择抗菌药物。     

哈尔滨市112株绿脓杆菌的血清学分型和耐药性测定

对从临床分离的112株绿脓杆菌进行系统鉴定后,血清学分型表明:6、2和3型分别占32.14％、15.18％、15.18％,为主要流行型,共占总分离株的62.50％。耐药性测定结果为:对10种抗生素5耐以上者占69.6％。其中对多粘菌素、妥布霉素、丁胺卡那霉素三种抗生素最为敏感,敏感率分别为100％、70.6％、86.5％。     

Adherence of Pseudomonas aeruginosa to inanimate polymers including biomaterials

Cells of Pseudomonas aeruginosa were adhered to polymethyl methacrylate, polyvinyl acetate, polyvinyl chloride, polyhydroxyethyl methacrylate, mixed-acrylic, silicone, and natural latex materials. Planktonic bacteria and bacteria that adhered to the test materials were compared for their uptake of either L-[3,4,5-³H] leucine or [methyl-³H] thymidine during growth in a minimal medium. Leucine incorporation was reduced and thymidine uptake was negligible in adherent bacteria for up to 8 h following primary attachment by which time cells in the planktonic state showed active uptake of both substrates. These reduced uptake periods correlated with lag phases of growth of adherent cells as determined with a sonication-release plate count procedure and analyses of adenosine triphosphate (ATP). The extent of the lag phase of the adherent populations was dependent on initial densities of adhered cells and the nature of the substratum. Received 02 December 1998/ Accepted in revised form 25 April 1999