香坊肠球菌和罗伊氏乳杆菌在无菌猪肠道中的定殖

【背景】已有研究表明,微生物在宿主肠道中的定殖受宿主、肠道环境、微生物物种特性和菌株来源等多个因素的影响。一般认为,来源于同类宿主的微生物菌株,在该类宿主肠道中具有定殖优势,但缺乏在物种和菌株水平上研究微生物自身特性在宿主肠道中定殖的研究报道。【目的】将不同来源(同类宿主肠道、非同类宿主肠道和非肠道环境)、具有不同生物学特性的3株香坊肠球菌(Enterococcus xiangfangensis)和4株罗伊氏乳杆菌(Lactobacillus reuteri)对无菌猪肠道进行定殖,在物种和菌株2个水平上探究物种特性和菌株来源对宿主肠道定殖的偏好性,揭示影响微生物定殖效率的关键因素。【方法】在本项研究中,将从藏猪(Tibetan pigs)、小鼠(ob/ob mice)、食蟹猴(Macaca fascicularis)和发酵食品中分离得到的多株香坊肠球菌和罗伊氏乳杆菌,制成混合菌剂对无菌巴马香猪(Bama miniature pig)进行为期4周的饲喂,并通过实时荧光定量PCR方法检测这7株菌在无菌猪肠道中的定殖情况。【结果】在物种水平上,香坊肠球菌和罗伊氏乳杆菌在无菌猪体内具有相近的定殖细胞数目。但是在菌株水平上,小鼠来源的香坊肠球菌(EX-MS)和罗伊氏乳杆菌(LR-MS)定殖的细胞数目显著高于其他5株菌,表明小鼠来源的菌株具有定殖优势。在体外混合培养中,将3株香坊肠球菌和4株罗伊氏乳杆菌分别等比例混合培养72 h后,发现香坊肠球菌EX-MS菌株的细胞数目高于香坊肠球菌EX-MK和EX-PG菌株;在罗伊氏乳杆菌中,4株菌的细胞数目相近。7株菌相互作用表明,EX-MS菌株对EX-MK和EX-PG菌株均有明显的抑制作用,但对4株罗伊氏乳杆菌无抑制作用。【结论】本次定殖实验的结果表明,在物种水平上,香坊肠球菌和罗伊氏乳杆菌均具有在无菌猪肠道中良好的定殖能力;但在菌株水平上,小鼠来源的香坊肠球菌EX-MS菌株和罗伊氏乳杆菌LR-MS菌株具有定殖优势;同时发现,小鼠来源的香坊肠球菌EX-MS菌株通过抑制同物种的EX-PG和EX-MK菌株,获得了在宿主肠道中的定殖优势。     

健康仔猪肠道乳杆菌黑龙江地方株的鉴定与种属分析

为了从健康仔猪肠道中分离筛选用于研制微生态制剂的乳酸杆菌菌株,选择黑龙江省大庆、哈尔滨及宝泉岭地区部分猪场,采集12-60日龄健康仔猪肠道粪样64份。选用MRS乳酸杆菌专用培养基分离培养乳酸杆菌,通过分离株的形态特征和培养特性筛选出革兰阳性,厌氧,无芽胞杆菌48株。再通过生化试验鉴定和PCR种属分析,确定18株为乳酸杆菌。其中,罗伊乳杆菌7株,嗜酸乳杆菌5株,约氏乳杆菌2株,短乳杆菌1株,干酪乳杆菌假植物亚种1株,植物乳杆菌1株,詹氏乳杆菌1株。     

鼠李糖乳杆菌菌毛SpaA亚基的表达、抗体制备及其种属特异性研究

【目的】制备鼠李糖乳杆菌菌毛亚基Spa A多克隆抗体,研究其种属特异性。【方法】应用PCR方法从鼠李糖乳杆菌GG的基因组扩增出spa A,并连接到质粒p ET-28α(+)中。将重组质粒转化大肠杆菌BL21(DE3),经IPTG诱导表达和镍柱纯化制备重组SpaA。通过免疫BALB/c小鼠获得多克隆抗体,利用全菌ELISA、Western和Dot-blot分析了SpaA在18株乳酸菌(12个种)中的分布特征。【结果】表达的重组SpaA分子量为36 k D,与预期大小一致;获得的Spa A抗体效价为1:12 800。Western结果显示抗体与天然Spa A具有良好的反应性。在测定的18株乳酸菌中,鼠李糖乳杆菌、干酪乳杆菌、副干酪乳杆菌3个种属菌株的spa A基因PCR和RT-PCR检测均为阳性。但全菌ELISA和Dot-blot结果显示,只有3株鼠李糖乳杆菌的全菌细胞与SpaA抗体呈特异性反应,而其它种属的菌株没有明显的交叉反应。【结论】尽管spa A基因在鼠李糖乳杆菌、干酪乳杆菌、副干酪乳杆菌中具有高度同源性,但SpaA蛋白只特异性地呈现在鼠李糖乳杆菌细胞表面。本研究中获得的Spa A抗体,为高黏附性鼠李糖乳杆菌的免疫磁珠分离及菌毛功能研究提供了工具。     

罗伊氏乳杆菌的益生功能

罗伊氏乳杆菌是目前已报道的几乎可存在于所有脊椎动物和哺乳动物肠道内的乳酸杆菌,是具有益生功效的肠道益生菌.通过对罗伊氏乳杆菌良好的肠道定植能力和其产生的罗伊氏菌素的介绍,阐明其可能的益生作用机理.重点论述了罗伊氏乳杆菌促进人类和动物健康功能的研究进展,并探讨了今后罗伊乳杆菌益生菌制剂的工业化发展趋势.     

苏云金芽胞杆菌CcpA蛋白对几丁质酶基因chiA和chiB的表达调控作用

【目的】研究苏云金芽胞杆菌Bti75中糖代谢蛋白Ccp A对两种几丁质酶基因chi A和chi B的表达调控。【方法】利用PREDetector软件分析Bti75 chi A和chi B的基因上游区序列,EMSA方法在体外验证Ccp A是否能与chi A和chi B基因的启动子区域片段特异性结合。构建ccp A基因敲除载体以获得敲除突变株Δccp A,运用实时荧光定量PCR技术和Western blot技术比较有无葡萄糖存在的情况下,Ccp A对chi A和chi B基因表达的影响。【结果】计算机分析显示,chi B上游启动子区存在一个潜在的Ccp A结合位点crechi B,而chi A上游启动子区未发现类似序列。体外实验表明,Ccp A蛋白在共阻遏蛋白Hpr-Ser45-P的参与下可与chi B基因启动子区特异性结合,而与chi A基因启动子区没有特异性结合;实时荧光定量PCR和Western blot结果均显示,Bti75中ccp A基因敲除后,同样在葡萄糖存在下chi B的表达量提高而chi A的表达量变化不明显。【结论】在葡萄糖存在的情况下,Ccp A蛋白能抑制苏云金芽胞杆菌中几丁质酶chi B的表达,而chi A的表达不受Ccp A调控。     

重组GFP干酪乳杆菌的构建及其在小鼠肠道内的定植分布

【目的】研究重组干酪乳杆菌(Lactobacillus casei)在小鼠肠道内定植能力及分布规律。【方法】利用绿色荧光蛋白(GFP)基因作为报告基因,构建pgsA基因与GFP的融合基因载体pLA-GFP,电转化到乳酸杆菌中,得到阳性重组菌。将重组菌以每只109mL-1的量,口服接种SPF级BALB/c小鼠,分别于口服后的1.5h、3h、12h、1d、3d、5d、6d、7d之后取其十二指肠、空肠、回肠、盲肠的肠道冲洗液,通过平板菌落计数法检测肠道内的重组干酪乳杆菌。【结果】Western blot结果显示约69kDa的融合蛋白在乳酸菌中得到了正确的表达;重组菌在蓝紫光激发下,发出绿色荧光。小鼠口服重组菌后能在肠道黏膜的不同部位以一定的比例存活并附着在肠黏膜表面,口服6d后达到定植高峰期,7d后在十二指肠、空肠、回肠和盲肠定植率分别占第1天的16.49%、25.08%、47.71%、41.03%。【结论】GFP在干酪乳杆菌中得到了稳定的表达,且在小鼠肠道内具有良好的定植能力,定植规律回肠盲肠空肠十二指肠,这为研究乳酸杆菌作为口服疫苗抗原递送载体及其对小鼠肠道免疫机理提供试验基础。     

炭疽芽胞杆菌BslA(260－652)蛋白的表达纯化与黏附功能鉴定

【目的】克隆表达炭疽芽胞杆菌BlsA的功能区片段并对其生物学功能进行鉴定。【方法】以炭疽芽胞杆菌A16R基因组DNA为模板PCR扩增bslA(260-652)基因片段,克隆至pET-28a(+)载体。将成功构建的重组质粒转化入大肠杆菌Rosetta(DE3)中,诱导表达后收集菌体经超声破碎后,对可溶表达部分用镍柱进行亲和层析纯化。以纯化后的蛋白为抗原,免疫BALB/c小鼠制备该蛋白的多抗,用ELISA和Western blot检测抗血清;使用间接免疫荧光实验和细菌黏附实验研究目标蛋白及其抗体的生物学功能。【结果】BslA(260-652)获得了可溶性表达,纯化后纯度约为87.4%。以纯化蛋白为抗原,免疫BALB/c小鼠制备的抗血清ELISA效价可达1∶20000。将BslA(260-652)蛋白与Hela细胞共孵育后,能够直接和Hela的细胞膜结合。细菌黏附实验表明BslA(260-652)蛋白及其相应的多抗血清都能够显著地抑制炭疽芽胞杆菌A16R对Hela细胞的黏附。【结论】大肠杆菌表达得到的炭疽芽胞杆菌BslA(260-652)蛋白具有与天然蛋白相似的生物活性,为深入研究BslA蛋白在炭疽芽胞杆菌致病过程中的作用奠定实验基础。     

3株罗伊氏乳杆菌生物学特性的分析比较

【目的】对3株罗伊氏乳杆菌的生物学特性进行分析比较,为后期生产应用提供一定的参考。【方法】对实验室保藏的3株罗伊氏乳杆菌的生长曲线、pH曲线、耐受人工胃液能力、耐受猪胆盐能力、黏附能力、抑菌能力和对抗生素的耐药性等特性进行了分析比较。【结果】3株菌生长趋势大致相同;3株菌对人工胃液均具有良好的耐受性,且可以有效地抑制大肠杆菌和金黄色葡萄球菌的生长;菌株L0和L2对高胆盐的环境耐受性较差,菌株L1则对高胆盐环境具有极强的耐受性;菌株L1和L2具有很强的黏附能力;3株菌对20种抗生素表现出不同的耐受性。【结论】菌株L1的生物学特性明显优于其他两株菌株,有利于后期的生产应用。     

空肠弯曲菌细胞致死性肿胀毒素单克隆抗体的制备与鉴定

【目的】原核表达空肠弯曲菌细胞致死性肿胀毒素B蛋白(CdtB),制备其单克隆抗体(mAb),并研究mAb抗毒性作用。【方法】扩增空肠弯曲菌cdtB基因并将其构建到pET-30a(+)和pGEX-6p-1表达载体,以原核表达的GST-CdtB蛋白为免疫原,应用杂交瘤技术进行细胞融合;采用间接ELISA方法测定细胞上清和mAb腹水效价,Dot-ELISA、Western blot分析mAb特异性,并以CaCo-2和HD-11细胞为模型,鉴定mAb抗毒性能力。【结果】成功构建重组原核表达质粒pET-30a(+)-cdtB和pGEX-6p-1-cdtB,并融合表达rHis-CdtB和rGST-CdtB蛋白。获得5株稳定分泌CdtB抗体的杂交瘤细胞株,分别命名为1F3,1F5,2E4,2E11,2F2。抗体Ig类和亚类检测显示2E11 Ig亚类为IgG2b,其他4株均为IgG1。抗体效价高达1:(1×108)。Dot-ELISA试验表明5株mAb均能与空肠弯曲菌标准株发生特异性反应,与非空肠弯曲菌呈阴性反应;Western blot法分析表明5株mAb均能与纯化蛋白rGST-CdtB有良好的反应性。基于CaCo-2细胞的黏附和侵袭实验表明mAb能显著降低细菌的黏附和侵袭能力(P0.01)。【结论】成功制备了针对空肠弯曲菌CdtB蛋白的mAb。为进一步研究空肠弯曲菌致病机制,以及为研制治疗性类药物奠定了基础。     

猪源乳酸杆菌与益生元组合筛选及其体外发酵特性

【目的】对3株乳酸杆菌和4种寡糖类益生元进行组合筛选,并探究其对猪结肠微生物体外发酵特性的影响。【方法】将3株乳酸杆菌(罗伊氏乳杆菌L45、植物乳杆菌L47和罗伊氏乳杆菌L63)分别添加至以菊粉(inulin)、低聚果糖(FOS)、低聚半乳糖(GOS)或乳果糖(lactulose)为唯一碳源的培养基中,结合菌株24 h的生长活性和产酸特性,筛选出最优组合;进一步利用体外发酵技术探究所筛选合生元组合对微生物和发酵特性的影响。【结果】L45和L63分别以FOS和Lactulose为碳源发酵时的生长曲线与葡萄糖相似,L47发酵Inulin和FOS的ΔOD600显著高于葡萄糖(P<0.05),且L47发酵Inulin产生的乳酸是葡萄糖的1.20倍,L47+FOS和L47+Inulin组合效应较好。体外发酵结果表明,与对照组相比,L47+FOS和L47+Inulin降低了发酵液中螺旋体门(Spirochaetes)的相对丰度,提高了乳杆菌属(Lactobacillus)和双歧杆菌属(Bifidobacterium)的相对丰度;L47+Inulin和L47+FOS显著提高了总短链脂肪酸含量(P<0.05);与L47+Inulin组相比,L47+FOS组乙酸和丁酸含量更高(P<0.05)。【结论】L47+FOS与L47+Inulin具有较好的组合效应,且具有改善猪结肠体外发酵的能力,提示L47+FOS和L47+Inulin作为合生元的发展潜力,两种组合在体内情况的效果有待深入研究。     

Integrating statistical pair potentials into protein complex prediction

The biophysical study of protein-protein interactions and docking has important implications in our understanding of most complex cellular signaling processes. Most computational approaches to protein docking involve a tradeoff between the level of detail incorporated into the model and computational power required to properly handle that level of detail. In this work, we seek to optimize that balance by showing that we can reduce the complexity of model representation and thus make the computation tractable with minimal loss of predictive performance. We also introduce a pair-wise statistical potential suitable for docking that builds on previous work and show that this potential can be incorporated into our fast fourier transform-based docking algorithm ZDOCK. We use the Protein Docking Benchmark to illustrate the improved performance of this potential compared with less detailed other scoring functions. Furthermore, we show that the new potential performs well on antibody-antigen complexes, with most predictions clustering around the Complementarity Determining Regions of antibodies without any manual intervention.     

Symmetric primary and tertiary structure mutations within a symmetric superfold: a solution, not a constraint, to achieve a foldable polypeptide

In previous studies designed to increase the primary structure symmetry within the hydrophobic core of human acidic fibroblast growth factor (FGF-1) a combination of five mutations were accommodated, resulting in structure, stability and folding kinetic properties similar to wild-type (despite the symmetric constraint upon the set of core residues). A sixth mutation in the core, involving a highly conserved Met residue at position 67, appeared intolerant to substitution. Structural analysis suggested that the local packing environment of position 67 involved two regions of apparent insertions that distorted the tertiary structure symmetry inherent in the beta-trefoil architecture. It was postulated that a symmetric constraint upon the primary structure within the core could only be achieved after these insertions had been deleted (concomitantly increasing the tertiary structure symmetry). The deletion of these insertions is now shown to permit mutation of position 67, thereby increasing the primary structure symmetry relationship within the core. Furthermore, despite the imposed symmetric constraint upon both the primary and tertiary structure, the resulting mutant form of FGF-1 is substantially more stable. The apparent inserted regions are shown to be associated with heparin-binding functionality; however, despite a marked reduction in heparin-binding affinity the mutant form of FGF-1 is surprisingly approximately 70 times more potent in 3T3 fibroblast mitogenic assays. The results support the hypothesis that primary structure symmetry within a symmetric protein superfold represents a possible solution, rather than a constraint, to achieving a foldable polypeptide.     

Protein microarrays as a discovery tool for studying protein–protein interactions

Exploring the function of the genome and the encoded proteins has emerged as a new and exciting challenge in the postgenomic era. Novel technologies come into view that promise to be valuable for the investigation not only of single proteins, but of entire protein networks. Protein microarrays are the innovative assay platform for highly parallel in vitro studies of protein–protein interactions. Due to their flexibility and multiplexing capacity, protein microarrays benefit basic research, diagnosis and biomedicine. This review provides an overview on the basic principles of protein microarrays and their potential to multiplex protein–protein interaction studies.     

The heat capacity of proteins

The heat capacity plays a major role in the determination of the energetics of protein folding and molecular recognition. As such, a better understanding of this thermodynamic parameter and its structural origin will provide new insights for the development of better molecular design strategies. In this paper we have analyzed the absolute heat capacity of proteins in different conformations. The results of these studies indicate that three major terms account for the absolute heat capacity of a protein: (1) one term that depends only on the primary or covalent structure of a protein and contains contributions from vibrational frequencies arising from the stretching and bending modes of each valence bond and internal rotations; (2) a term that contains the contributions of noncovalent interactions arising from secondary and tertiary structure; and (3) a term that contains the contributions of hydration. For a typical globular protein in solution the bulk of the heat capacity at 25°C is given by the covalent structure term (close to 85% of the total). The hydration term contributes about 15 and 40% to the total heat capacity of the native and unfolded states, respectively. The contribution of non-covalent structure to the total heat capacity of the native state is positive but very small and does not amount to more than 3% at 25°C. The change in heat capacity upon unfolding is primarily given by the increase in the hydration term (about 95%) and to a much lesser extent by the loss of noncovalent interactions (up to ～5%). It is demonstrated that a single universal mathematical function can be used to represent the partial molar heat capacity of the native and unfolded states of proteins in solution. This function can be experimentally written in terms of the molecular weight, the polar and apolar solvent accessible surface areas, and the total area buried from the solvent. This unique function accurately predicts the different magnitude and temperature dependences of the heat capacity of both the native and unfolded states, and therefore of the heat capacity changes associated with folding/unfolding transitions. © 1995 Wiley-Liss, Inc.     

A large scale test of computational protein design: folding and stability of nine completely redesigned globular proteins

A previously developed computer program for protein design, RosettaDesign, was used to predict low free energy sequences for nine naturally occurring protein backbones. RosettaDesign had no knowledge of the naturally occurring sequences and on average 65% of the residues in the designed sequences differ from wild-type. Synthetic genes for ten completely redesigned proteins were generated, and the proteins were expressed, purified, and then characterized using circular dichroism, chemical and temperature denaturation and NMR experiments. Although high-resolution structures have not yet been determined, eight of these proteins appear to be folded and their circular dichroism spectra are similar to those of their wild-type counterparts. Six of the proteins have stabilities equal to or up to 7kcal/mol greater than their wild-type counterparts, and four of the proteins have NMR spectra consistent with a well-packed, rigid structure. These encouraging results indicate that the computational protein design methods can, with significant reliability, identify amino acid sequences compatible with a target protein backbone.     

Lethal mutations in the major homology region and their suppressors act by modulating the dimerization of the rous sarcoma virus capsid protein C‐terminal domain

An infective retrovirus requires a mature capsid shell around the viral replication complex. This shell is formed by about 1500 capsid protein monomers, organized into hexamer and pentamer rings that are linked to each other by the dimerization of the C‐terminal domain (CTD). The major homology region (MHR), the most highly conserved protein sequence across retroviral genomes, is part of the CTD. Several mutations in the MHR appear to block infectivity by preventing capsid formation. Suppressor mutations have been identified that are distant in sequence and structure from the MHR and restore capsid formation. The effects of two lethal and two suppressor mutations on the stability and function of the CTD were examined. No correlation with infectivity was found for the stability of the lethal mutations (D155Y‐CTD, F167Y‐CTD) and suppressor mutations (R185W‐CTD, I190V‐CTD). The stabilities of three double mutant proteins (D155Y/R185W‐CTD, F167Y/R185W‐CTD, and F167Y/I190V‐CTD) were additive. However, the dimerization affinity of the mutant proteins correlated strongly with biological function. The CTD proteins with lethal mutations did not dimerize, while those with suppressor mutations had greater dimerization affinity than WT‐CTD. The suppressor mutations were able to partially correct the dimerization defect caused by the lethal MHR mutations in double mutant proteins. Despite their dramatic effects on dimerization, none of these residues participate directly in the proposed dimerization interface in a mature capsid. These findings suggest that the conserved sequence of the MHR has critical roles in the conformation(s) of the CTD that are required for dimerization and correct capsid maturation. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

蛋白质相互作用研究的新技术与新方法

目前,蛋白质相互作用已成为蛋白质组学研究的热点. 新方法的建立及对已有技术的改进标志着蛋白质相互作用研究的不断发展和完善．在技术改进方面,本文介绍了弥补酵母双杂交的蛋白定位受限等缺陷的细菌双杂交系统;根据目标蛋白特性设计和修饰TAP标签来满足复合体研究要求的串联亲和纯化技术,以及在双分子荧光互补基础上发展的动态检测多个蛋白质间瞬时、弱相互作用的多分子荧光互补技术．还综述了近两年建立的新方法：与免疫共沉淀相比,寡沉淀技术直接研究具有活性的蛋白质复合体;减量式定量免疫沉淀方法排除了蛋白质复合体中非特异性相互作用的干扰;原位操作的多表位－配基绘图法避免了样品间差异的影响,以及利用多点吸附和交联加固研究弱蛋白质相互作用的固相蛋白质组学方法.     

Human dihydrofolate reductase and thymidylate synthase form a complex in vitro and co-localize in normal and cancer cells

Enzymes involved in thymidylate biosynthesis, thymidylate synthase (TS), and dihydrofolate reductase (DHFR) are well-known targets in cancer chemotherapy. In this study, we demonstrated for the first time, that human TS and DHFR form a strong complex in vitro and co-localize in human normal and colon cancer cell cytoplasm and nucleus. Treatment of cancer cells with methotrexate or 5-fluorouracil did not affect the distribution of either enzyme within the cells. However, 5-FU, but not MTX, lowered the presence of DHFR-TS complex in the nucleus by 2.5-fold. The results may suggest the sequestering of TS by FdUMP in the cytoplasm and thereby affecting the translocation of DHFR-TS complex to the nucleus. Providing a strong likelihood of DHFR-TS complex formation in vivo, the latter complex is a potential new drug target in cancer therapy. In this paper, known 3D structures of human TS and human DHFR, and some protozoan bifunctional DHFR-TS structures as templates, are used to build an in silico model of human DHFR–TS complex structure, consisting of one TS dimer and two DHFR monomers. This complex structure may serve as an initial 3D drug target model for prospective inhibitors targeting interfaces between the DHFR and TS enzymes.