肺炎克雷伯菌菌毛粘附素克隆表达及其对体外培养细胞的粘附活性

摘要：【目的】肺炎克雷伯菌(K.pn)与宿主细胞的粘附是致病的首要条件,粘附过程主要通过菌毛粘附素MrkD蛋白介导。为了进一步分析MrkD蛋白与宿主细胞间的粘附机制,进一步确定MrkD蛋白的粘附阻断作用。【方法】构建肺炎克雷伯菌菌毛粘附素融合蛋白原核表达质粒pGEX-4T-mrkD,转入大肠杆菌BL21,优化诱导表达条件,表达产物经亲和层析纯化、凝血酶切除融合蛋白GST标签后,进行SDS-PAGE和Western blot鉴定。激光共聚焦显微镜定位MrkD蛋白在宿主细胞上的结合部位;通过粘附活性试验与粘附动力学实验研究了MrkD蛋白的生物活性。【结果】实验得到了分子量为35 kDa的MrkD蛋白,定位了MrkD蛋白在宿主细胞上的结合部位,并证明了MrkD蛋白可以显著影响肺炎克雷伯菌对宿主细胞的粘附力。【结论】本试验首次证实了MrkD蛋白的粘附阻断作用并观察到了其与宿主细胞的作用位点,为研究肺炎克雷伯菌的致病机制,寻找粘附素功能表位奠定了基础。     

盐穗木HcUKPP原核表达及重组菌非生物胁迫耐受性研究

该研究采用RT-PCR技术,从盐穗木cDNA文库中克隆获得未知功能多肽HcUKPP基因,构建了大肠杆菌Escherichia coli BL21∷pET30a-HcUKPP重组菌株,并检测了重组菌株在不同非生物胁迫下的耐受性。结果显示:HcUKPP基因开放阅读框为243bp,融合His的HcUKPP蛋白的分子量约为15kD。在37℃条件下,不同浓度的异丙基-β-D硫代半乳糖苷(IPTG)诱导4h后His-HcUKPP融合蛋白均可表达,且E.coli BL21∷pET30aHcUKPP重组菌在不同浓度NaCl(100~900mmol/L)、聚乙二醇(2.5%~20%,PEG 6000)和甲基紫精(25~200μmol/L)胁迫处理下,其生长均具有明显优势。尤其是在500mmol/L NaCl、10%PEG 6000和75μmol/L甲基紫精胁迫12h后,重组大肠杆菌BL21呈现出极显著的优势,分别达到了对照菌的1.81、1.47和3.48倍。研究表明,盐穗木HcUKPP可以显著提高重组大肠杆菌对不同非生物胁迫的耐受性,证明HcUKPP是一类新发现的能够响应非生物胁迫的多肽。     

酸胁迫下短乳杆菌NCL912蛋白质的差异表达及其作用

【目的】本文从蛋白质组水平,对本实验室分离的一株高产γ-氨基丁酸的短乳杆菌NCL912(Lactobacillus brevis)在酸胁迫下蛋白质的差异表达及其应激机理进行探讨。【方法】利用双向凝胶电泳技术对pH 5.0和pH 4.0条件下,不含L-谷氨酸钠的培养物的蛋白质组电泳图谱进行了分析,并对酸胁迫下差异表达的蛋白进行了比较。利用质谱检测技术和生物信息学技术对这些差异表达的蛋白进行了鉴定、功能分类和代谢途径分析等。【结果】通过双向凝胶电泳技术,可以得到均匀、背景清晰、分辨率高、重复性好的Lb.brevis NCL912的双向凝胶电泳图谱。对pH 5.0和pH 4.0条件下培养的该菌总蛋白质电泳图谱进行比较,发现有25个差异表达的蛋白点。对这25个差异表达的蛋白进行了质谱鉴定。由于缺乏短乳杆菌NCL912的全基因组,所以其中只有8个蛋白点被质谱鉴定和分析得到。它们分别参与了蛋白质的合成、核苷酸的合成、糖酵解代谢、细胞能量水平的调节等。【结论】酸应激下这些表达蛋白质可通过其相应的功能来保护细胞耐受酸胁迫,从而使菌能够在酸性环境下生存增值。这可能就是Lb.brevis NCL912的酸胁迫应激机理之一。     

酮古龙酸菌细胞色素c基因的克隆及表达

目的:从酮古龙酸菌SCB329中分离细胞色素c(Cytc)相关基因,在大肠杆菌中进行表达并验证。方法:根据酮古龙酸菌SCB329基因组序列设计引物,通过PCR从SCB329基因组中扩增cytc基因,酶切后连接pET22b表达载体,转化至大肠杆菌DH5α后提取质粒,经PCR、质粒双酶切及测序鉴定后,转入大肠杆菌BL21(DE3),并对表达条件进行考察;用Chelating Sepharose珠粒对可溶性的Cytc-His融合蛋白进行纯化;经光谱扫描和血红素染色等方法对表达蛋白定性分析。结果:PCR扩增的cytc基因长513 bp;重组菌在IPTG浓度为0.025 mmol/L的条件下,于28℃诱导10 h后,SDS-PAGE分析可见表达条带,相对分子质量约为18×103;Ni柱亲和层析纯化得到目的蛋白,纯化蛋白经光谱扫描呈现Cytc特征峰,血红素染色呈现阳性结果。结论:从酮古龙酸菌SCB329中分离得到一种cytc基因,并表达纯化了融合蛋白Cytc-His,纯化蛋白呈现Cytc特性,为研究酮古龙酸菌中产酸关键酶的电子传递机制奠定了基础。     

龟源肺炎克雷伯菌SYBR-GreenⅠ荧光定量PCR检测方法的建立

本研究在黄喉拟水龟中分离的肺炎克雷伯菌基础上,根据肺炎克雷伯菌Ⅲ型菌毛的Mrk D基因序列设计引物,通过对SYBR-GreenⅠ荧光定量PCR反应条件、特异性、灵敏性实验进行调节优化,建立了肺炎克雷伯菌的SYBR-GreenⅠ荧光定量PCR检测方法。研究结果表明,建立的肺炎克雷伯菌荧光定量PCR方法,检测时间短,用时73 min;特异性强,对非肺炎克雷伯菌无交叉反应;灵敏度高,检测肺炎克雷伯菌DNA的最低检测量为2.78 fg。该方法的建立,为肺炎克雷伯菌的辅助诊断、流行病学调查、毒力基因的分析提供科学借鉴。     

新型植生克雷伯菌生物学特性及药物敏感性分析

从肺结核患者伴感染痰液中检出９株植生克雷伯菌。经生物学特性、药物敏感性分析以及与其它克雷伯菌型比较后的结果表明：植生克雷伯菌具有克雷伯菌属的生物学共性,但与肺炎、产酸、土生、解鸟氨酸克雷伯菌生化特性有别;血清学与动物实验证明,分离株为致病患者临床感染的病原株;其对青霉素、氨苄青霉素、红霉素、四环素、氯霉素、庆大霉素、氧哌嗪青霉、卡那霉素等耐药率较高,对亚胺培南、环丙沙星、诺氟沙星敏感,对二、三代头孢效果也较好。     

肺炎克雷伯氏菌VBNC状态转化突变株的筛选与特性研究

采用双亲本接合法对从文山湖富营养化水体中分离得到肺炎克雷伯氏菌进行Tn5转座子插入诱变,在含四环素和卡那霉素的LB培养基上获得接合子,利用饥饿冷冻高渗透压法对接合子进行细菌VBNC状态转化突变株的筛选和特性的研究。结果表明,带有卡那霉素基因的Tn5成功地插入到了肺炎克雷伯氏菌的染色体中,在双抗性培养基上获得约2.3×104 cell/mL的接合子,转座效率为6.58×10-4。对多个接合子和对照肺炎克雷伯氏菌的诱导、筛选及比较,发现KPQT-7是最快进入VBNC状态的突变株,该突变株在胁迫诱导9d后超过30%的细胞都进入了VBNC状态,而对照的肺炎克雷伯氏菌至少要在诱导21d后才大部分进入VBNC状态。在此基础上,对VBNC转化突变株KPQT-7作进一步的遗传学分析将会为细菌VBNC状态分子机理的研究奠定坚实的基础。     

龟裂链霉菌RsigB基因提高大肠杆菌对环境胁迫的耐受性

龟裂链霉菌RsigB是与天蓝色链霉菌与枯草芽孢杆菌中的sigmaB基因同源的一个σ因子,其也可能为一种全局压力调控蛋白,在菌体遇到生长环境改变时起着重要的调控作用。本文以龟裂链霉菌基因组为模版,PCR扩增出RsigB基因,以pET28a为载体,构建重组质粒pET28aRsigB,并且转化至Escherichia coli BL21(DE3)中。将对照菌与重组子置于不同环境胁迫条件下培养,并用IPTG诱导蛋白表达,测其生长曲线,研究RsigB对于大肠杆菌对环境胁迫的耐受性的作用。结果表明,RsigB在大肠杆菌中的表达能够提高其对温度,高渗透压以及氧化还原压力的耐受性。RsigB的成功表达以及对于逆境的耐受性能为龟裂链霉菌中RsigB因子的功能以及机理研究提供实验基础。     

肠道细菌产酸克雷伯氏菌及其挥发性物质对斑翅果蝇成虫的引诱效果

【目的】明确肠道细菌产酸克雷伯氏菌Klebsiella oxytoca对斑翅果蝇Drosophila suzukii的引诱效果,并鉴定和验证该菌挥发性物质对斑翅果蝇成虫的引诱效果。【方法】在无寄主植物和有寄主植物（巨蜂葡萄）的条件下,测定并分析产酸克雷伯氏菌和NB培养基(对照)的上清液对斑翅果蝇成虫的诱集比例以及被引诱成虫的雌雄性比;利用气相色谱质谱联用法(HS-SPME-GC-MS)分别鉴定产酸克雷伯氏菌和NB培养基(对照)上清液中的挥发性物质,并检测浓度较高的4种产酸克雷伯氏菌挥发性物质对斑翅果蝇成虫的引诱效果。【结果】产酸克雷伯氏菌上清液对斑翅果蝇成虫具有引诱作用。其中,无寄主植物条件下,产酸克雷伯氏菌上清液对雄虫引诱作用强于对雌虫,但是在有寄主植物条件下产酸克雷伯氏菌上清液诱集的雌雄成虫数量差异不显著。在产酸克雷伯氏菌上清液中检测到21种挥发性物质,其中浓度较高的为3-甲基-1-丁醇、2-苯乙醇、乙酸异戊酯和吲哚,斑翅果蝇成虫对3-甲基-1-丁醇、2-苯乙醇和吲哚具有趋向性,而对乙酸异戊酯则有趋避性,且3-甲基-1-丁醇可吸引更多的雄虫。【结论】肠道微生物产酸克雷伯氏菌上清液可用于引诱斑翅果蝇成虫,3-甲基-1-丁醇是产酸克雷伯氏菌代谢物中引诱雄虫的重要物质。本研究为斑翅果蝇的防治提供了参考依据。     

扁蓿豆MrLEA2基因的克隆和原核表达

从扁蓿豆(Medicago ruthenica L.)幼苗中克隆到一个编码晚期胚胎发生丰富蛋白的基因MrLEA2,Pfam数据库检索表明其编码产物属于LEA_2蛋白家族。半定量RT-PCR分析发现MrLEA2在幼苗中表达水平不受非生物胁迫(脱水、高盐和低温)和脱落酸诱导。利用MrLEA2基因构建原核表达载体,在大肠杆菌中实现了过量表达。通过斑点试验和菌落计数实验,对过量表达MrLEA2蛋白的大肠杆菌细胞在高盐(0.5mol/L NaCl和0.5mol/L KCl)、55℃高温和-20℃冷冻胁迫处理下的生长存活情况检测发现,MrLEA2蛋白过量表达能够明显提高大肠杆菌对上述胁迫的耐受性。研究表明,MrLEA2蛋白对高盐和温度胁迫引起的细胞损伤具有保护作用。     

The priB gene of Klebsiella pneumoniae encodes a 104-amino acid protein that is similar in structure and function to Escherichia coli PriB

Primosome protein PriB is a single-stranded DNA-binding protein that serves as an accessory factor for PriA helicase-catalyzed origin-independent reinitiation of DNA replication in bacteria. A recent report describes the identification of a novel PriB protein in Klebsiella pneumoniae that is significantly shorter than most sequenced PriB homologs. The K. pneumoniae PriB protein is proposed to comprise 55 amino acid residues, in contrast to E. coli PriB which comprises 104 amino acid residues and has a length that is typical of most sequenced PriB homologs. Here, we report results of a sequence analysis that suggests that the priB gene of K. pneumoniae encodes a 104-amino acid PriB protein, akin to its E. coli counterpart. Furthermore, we have cloned the K. pneumoniae priB gene and purified the 104-amino acid K. pneumoniae PriB protein. Gel filtration experiments reveal that the K. pneumoniae PriB protein is a dimer, and equilibrium DNA binding experiments demonstrate that K. pneumoniae PriB's single-stranded DNA-binding activity is similar to that of E. coli PriB. These results indicate that the PriB homolog of K. pneumoniae is similar in structure and in function to that of E. coli.     

An improved heuristic for haplotype inference

We cloned a gene, kexD, that provides a multidrug-resistant phenotype from multidrug-resistant Klebsiella pneumoniae MGH78578. The deduced amino acid sequence of KexD is similar to that of the inner membrane protein, RND-type multidrug efflux pump. Introduction of the kexD gene into Escherichia coli KAM32 resulted in a MIC that was higher for erythromycin, novobiocin, rhodamine 6G, tetraphenylphosphonium chloride, and ethidium bromide than that of the control. Intracellular ethidium bromide levels in E. coli cells carrying the kexD gene were lower than that in the control cells under energized conditions, suggesting that KexD is a component of an energy-dependent efflux pump. RND-type pumps typically consist of three components: an inner membrane protein, a periplasmic protein, and an outer membrane protein. We discovered that KexD functions with a periplasmic protein, AcrA, from E. coli and K. pneumoniae, but not with the periplasmic proteins KexA and KexG from K. pneumoniae. KexD was able to utilize either TolC of E. coli or KocC of K. pneumoniae as an outer membrane component. kexD mRNA was not detected in K. pneumoniae MGH78578 or ATCC10031. We isolated erythromycin-resistant mutants from K. pneumoniae ATCC10031, and some showed a multidrug-resistant phenotype similar to the drug resistance pattern of KexD. Two strains of multidrug-resistant mutants were investigated for kexD expression; kexD mRNA levels were increased in these strains. We conclude that changing kexD expression can contribute to the occurrence of multidrug-resistant K. pneumoniae.     

Posttranslational modification of Klebsiella pneumoniae flavodoxin by covalent attachment of coenzyme A, shown by 31P NMR and electrospray mass spectrometry, prevents electron transfer from the nifJ protein to nitrogenase. A possible new regulatory mechanism for biological nitrogen fixation.

A strain of Escherichia coli (71-18) that produces ca. 15% of its soluble cytoplasmic protein as a flavodoxin, the Klebsiella pneumoniae nifF gene product, has been constructed. The flavodoxin was purified using FPLC and resolved into two forms, designated KpFldI and KpFldII, which were shown to have identical N-terminal amino acid sequences (30 residues) in agreement with that predicted by the K. pneumoniae nifF DNA sequence. 31P NMR, electrospray mass spectrometry, UV-visible spectra, and thiol group estimations showed that the single cysteine residue (position 68) of KpFldI is posttranslationally modified in KpFldII by the covalent, mixed disulfide, attachment of coenzyme A. KpFldII was inactive as an electron carrier between the K. pneumoniae nifJ product (a pyruvate-flavodoxin oxidoreductase) and K. pneumoniae nifH product (the Fe-protein of nitrogenase). This novel posttranslational modification of a flavodoxin is discussed in terms of the regulation of nitrogenase activity in vivo in response to the level of dissolved O2 and the carbon status of diazotrophic cultures.     

肺炎克雷伯氏菌特异性抗原的筛选与鉴定

目的寻找肺炎克雷伯氏菌（Klebsiella pneumoniae）血清学检测用特异性抗原。方法双向电泳分离K.pneumoniae总蛋白,通过免疫印迹（Western blotting）与常见病原菌的多抗反应筛选特异性抗原蛋白,原核表达该蛋白并用ELISA法验证。结果获得K.pneumoniae的双向电泳图谱。寻找Western blotting中与K.pneumoniae自身多抗反应而不与与其它病原菌多抗反应的蛋白,质谱鉴定为酸性磷酸酶（Acid phosphatase,GI：238894261）。经表达纯化并以酶联免疫吸附试验（ELISA）验证,证明该蛋白作为包被抗原的灵敏度高,特异性强。结论 K.pneumoniae酸性磷酸酶是适用于该菌感染检测特异性抗原蛋白。     

Propanediol oxidoreductases of Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium. Aspects of interspecies structural and regulatory differentiation.

The enzyme propanediol oxidoreductase, which converts the lactaldehyde formed in the metabolism of fucose and rhamnose into propane-1,2-diol under anaerobic conditions, was investigated in Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium. Structural analysis indicated that the enzymes of E. coli and K. pneumoniae have the same Mr and pI, whereas that of Salm. typhimurium also has the same Mr but a slightly different pI. One-dimensional peptide mapping showed identity between the E. coli and K. pneumoniae enzymes when digested with alpha-chymotrypsin, Staphylococcus aureus V8 proteinase or subtilisin. In the case of Salm. typhimurium, this held only for the subtilisin-digested enzymes, indicating that the hydrophobic regions were preserved to a considerable extent. Anaerobically, the three species induced an active propanediol oxidoreductase when grown on fucose or rhamnose. An inactive propanediol oxidoreductase was induced in Salm. typhimurium by either fucose or rhamnose under aerobic conditions, and this was activated once anaerobiosis was established. An inactive propanediol oxidoreductase was also induced in E. coli under aerobic conditions, but only by growth on fucose. The inactive enzyme was not induced by either of the sugars in K. pneumoniae.     

Cloning and expression of Klebsiella pneumoniae genes coding for citrate transport and fermentation.

Three Escherichia coli clones (DH1/Cit1, DH1/Cit2 and DH1/Cit3) capable of utilizing citrate as a sole carbon source were isolated from a cosmid bank of Klebsiella pneumoniae wild-type DNA. Two of these clones (DH1/Cit1 and DH1/Cit2) only grew aerobically on citrate minimal medium, the third clone (DH1/Cit3) could also be cultured under fermentative conditions. The aerobic as well as the anaerobic generation times of the three clones were from 4.5 to 7 h. Whereas clone DH1/Cit3 showed a pronounced lag phase on citrate when the cells were pre-grown in medium without citrate, clone DH1/Cit1 immediately started growth, while with clone DH1/Cit2 a short lag phase could be observed upon transfer to citrate minimal medium. Restriction analyses of the three plasmids showed that no common fragments had been cloned. The length of the inserts were 13 and 6 kb for the aerobic Cit+ clones and 27 kb (10 kb) for the anaerobic one. Cultures of the anaerobic Cit+ clone were analyzed by immunoblotting techniques and shown to contain oxaloacetate decarboxylase, which confers citrate utilization under anaerobic conditions to K. pneumoniae. Enzyme assays demonstrated the active state of this biotin-containing membrane protein. The specific activity in vesicle preparations from the E. coli clone was 30% of the wild-type K. pneumoniae vesicles. Citrate acts as an inducer of enzyme protein synthesis in the E. coli clone as it does in K. pneumoniae.     

Citrate transport in Klebsiella pneumoniae

Sodium ions were specifically required for citrate degradation by suspensions of K. pneumoniae cells which had been grown anaerobically on citrate. The rate of citrate degradation was considerably lower than the activities of the citrate fermentation enzymes citrate lyase and oxaloacetate decarboxylase, indicating that citrate transport is rate limiting. Uptake of citrate into cells was also Na+ -dependent and was accompanied by its rapid metabolism so that the tricarboxylic acid was not accumulated in the cells to significant levels. The transport could be stimulated less efficiently by LiCl. Li+ ions were cotransported with citrate into the cells. Transport and degradation of citrate were abolished with the uncoupler [4-(trifluoromethoxy)phenylhydrazono]propanedinitrile (CCFP). After releasing outer membrane components and periplasmic binding proteins by cold osmotic shock treatment, citrate degradation became also sensitive towards monensin and valinomycin. The shock procedure had no effect on the rate of citrate degradation indicating that the transport is not dependent on a binding protein. Citrate degradation and transport were independent of Na+ ions in K. pneumoniae grown aerobically on citrate and in E. coli grown anaerobically on citrate plus glucose. An E. coli cit+ clone obtained by transformation of K. pneumoniae genes coding for citrate transport required Na specifically for aerobic growth on citrate indicating that the Na-dependent citrate transport system is operating. Na+ and Li+ were equally effective in stimulating citrate degradation by cell suspensions of E. coli cit+. Citrate transport in membrane vesicles of E. coli cit+ was also Na+ dependent and was energized by the proton motive force (delta micro H+). Dissipation of delta micro H+ or its components delta pH or delta psi by ionophores either totally abolished or greatly inhibited citrate uptake. It is suggested that the systems energizing citrate transport under anaerobic conditions are provided by the outwardly directed cotransport of metabolic endproducts with protons yielding delta pH and by the decarboxylation of oxaloacetate yielding delta pNa+ and delta psi. In citrate-fermenting K. pneumoniae an ATPase which is activated by Na+ was not found. The cells contain however a proton translocating ATPase and a Na+/H+ antiporter in their membrane.     

血管抑制素功能结构域K1与PKA底物磷酸化基序的融合表达及磷酸化标记

通过 RT- PCR,从人肝组织中扩增出血管形成抑制素 ( angiostatin) c DNA的 K1片段 ,经DNA序列分析证实其正确性 ;将 K1与 GST融合并带上 1 7个氨基酸的 PKA底物磷酸化基序 ,IPTG诱导表达 ,以还原型谷胱甘肽偶联的琼脂糖凝胶亲合层析直接从细菌裂解上清中纯化融合蛋白 ;以 PKA催化单位将 3 2 P通过磷酸化作用标记至纯化的蛋白 ,再用凝血酶切去 GST,进行SDS- PAGE.放射自显影结果显示 ,GSTag- K1和 Tag- K1分别在 40 k D和 1 7k D处有信号强而特异的显影条带 ,表明带有磷酸化序列的蛋白能够被 PKA特异地磷酸化标记