硫氧还蛋白Lmo1903介导单增李斯特菌抵抗氧化应激的生物学功能研究

【目的】以单增李斯特菌(Listeria monocytogenes, LM)硫氧还蛋白Lmo1903为研究对象,研究其在细菌环境适应过程中的抗氧化应激生物学作用。【方法】使用生物信息学方法分析Lmo1903的进化关系和关键活性位点,使用酶切连接的方法构建Lmo1903蛋白表达载体,获得纯化的重组蛋白,以胰岛素为底物分析其氧化还原酶学活性;同时制备鼠源多克隆抗体,分析其在细胞内的定位;采用核苷酸定点突变技术构建CX1X2C基序中的半胱氨酸点突变蛋白,分析关键位点半胱氨酸对Lmo1903酶活的影响;采用同源重组原理构建lmo1903基因缺失株Δlmo1903和回补株CΔlmo1903,研究lmo1903在单增李斯特菌生长、运动和抗氧化应激方面发挥的功能。【结果】生物信息学分析显示,Lmo1903含有CX1X2C基序,与枯草芽孢杆菌(Bacillussubtilis)的TrxA的亲缘关系较近,属于硫氧还蛋白家族成员,主要定位在细菌细胞质中,具有较强的还原酶学活性,突变CX1X2C基序中的半胱氨酸残基会显著降低Lmo1903的还原酶活能力。缺失lmo1903不影响单增李斯特菌的生长能力,但显...     

单增李斯特菌LPXTG蛋白Lmo0880在感染致病中的作用

【目的】通过构建单核细胞增生李斯特菌(单增李斯特菌) LPXTG蛋白Lmo0880的基因缺失菌株和回补菌株,探究Lmo0880在细菌生长、细胞感染和宿主感染等方面发挥的作用。【方法】利用同源重组原理构建lmo0880的基因缺失株及回补株,比较野生株、缺失株和回补株在生长能力、细胞黏附与侵袭和胞内增殖能力等方面的差异,从而鉴定Lmo0880在单增李斯特菌感染宿主中的作用。【结果】缺失lmo0880基因后,单增李斯特菌在生长能力上无明显变化;对细胞的黏附能力无显著差异,但对细胞侵袭能力、胞内增殖能力、小鼠致病力和小鼠组织定殖能力显著降低。【结论】本研究阐明了单增李斯特菌LPXTG蛋白Lmo0880在细胞侵袭、胞内增殖和组织定殖等方面发挥的重要作用。     

单核细胞增多性李斯特菌硫氧还蛋白Lmo1609的应激生物学功能研究

【目的】本研究旨在构建单核细胞增多性李斯特菌(Listeria monocytogenes)硫氧还蛋白Lmo1609的基因缺失株,分析Lmo1609的氧化还原酶学活性,及其在细菌生长、运动过程中发挥的作用,并探究了Lmo1609参与细菌抗氧化应激和致病的生物学基础。为阐明其抗应激生物学作用以及完善李斯特菌的感染机制奠定分子基础。【方法】利用同源重组原理构建lmo1609基因缺失株及回补株。通过分子生物学、应激生物学和感染生物学等手段,对Lmo1609的生物学功能进行探索。以胰岛素为底物分析其氧化还原酶学活性;通过构建lmo1609缺失株和回补株,比较野生株和突变株在运动性、生长能力、抗氧化应激、细胞黏附、侵袭和增殖能力等方面的差异,进而鉴定Lmo1609的生物学功能。【结果】缺失lmo1609后,单增李斯特菌在生长能力上无明显变化,而运动能力明显减弱;对H2O2的敏感性增强;对细胞的黏附侵袭能力没有差异;对小鼠的致病力没有显著影响。【结论】本研究首次证实了单增李斯特菌硫氧还蛋白Lmo1609具有还原酶学活性,参与调控细菌的运动和对H2O2的氧化应激耐受,不介导单增李斯特菌的致病性。     

原核表达MurA蛋白并制备其多克隆抗体用于免疫磁珠快速检测单增李斯特菌

【目的】制备MurA多抗,结合免疫磁珠与选择平板进行单增李斯特菌的快速检测,建立单增李斯特菌的免疫磁珠快速检测方法。【方法】构建MurA的原核表达载体,转化大肠杆菌进行优化表达。镍柱纯化表达产物,质谱鉴定重组蛋白,再免疫小鼠,制备其多克隆抗体。用所获多抗制备免疫磁珠,建立单增李斯特菌免疫磁珠-选择性培养基检测方法,并对人工污染牛奶样品进行检测。【结果】在大肠杆菌中高效表达了分子量约为72 kD的可溶性融合蛋白,质谱鉴定其为MurA蛋白;免疫小鼠获得的抗血清效价达1:10 000,与伤寒沙门氏菌、副溶血弧菌、大肠杆菌及属内其它病原菌均无交叉;所建立的免疫磁珠-选择性培养基检测法可检出浓度为103 CFU/mL及以上的单增李斯特菌,仅与英诺克李斯特菌存在一定交叉反应;牛奶样品单次仅需9 h增菌就能被检出,较常规增菌时间缩短39 h;检测限为0.4 CFU/mL。【结论】表达并纯化得到高纯度的单增李斯特菌MurA蛋白,制备的鼠源多克隆抗体亲和力高,特异性好;建立了快速检测单增李斯特菌的免疫磁珠联合选择性培养基法,在灵敏度不变的情况下,实现24 h内成功对牛奶样品的检测,较国标法减少42 h以上。     

单增李斯特菌InternalinA的表达、纯化及多克隆抗体的制备

【目的】克隆表达单增李斯特菌膜表面蛋白InternalinA(InlA),经免疫家兔获得多克隆抗体,为建立其免疫磁珠富集快速检测方法奠定基础。【方法】利用生物软件设计单增李斯特菌inlA基因的引物,通过PCR扩增出inlA基因,并将其克隆至pET28a()原核表达载体,转化大肠杆菌BL21进行优化表达。镍柱纯化表达产物,质谱鉴定重组蛋白,ELISA分析其免疫原性。免疫家兔,制备其多克隆抗体。间接ELISA检测多抗的效价及交叉性,免疫荧光分析多抗与单增李斯特菌菌体结合的特异性。【结果】成功表达了InlA蛋白,融合表达产物分子量约为92 kD,质谱鉴定其为InlA蛋白;免疫家兔获得的抗血清效价为1:100 000,除与金黄色葡萄球菌约20%的交叉外,与副溶血弧菌等其它病源菌均无交叉;免疫荧光证实该多抗特异性结合于单增李斯特菌膜表面,与同种属的威尔斯李斯特菌不结合。【结论】成功制备了单增李斯特菌特异性的兔多克隆抗体,为单增李斯特菌免疫磁珠富集快速检测方法的建立奠定了基础。     

乳腺癌和卵巢癌易感基因2及2a编码蛋白N端的原核表达

目的:分别表达乳腺癌和卵巢癌易感基因BRCA2编码蛋白的N端500个氨基酸残基(nBRCA2)及其缺乏第3个外显子编码的蛋白(nBRCA2a),为分析比较含与不含第3个外显子所表达蛋白的功能差异准备条件。方法:采用RT-PCR,从BT-474细胞系的RNA扩增获得nBRCA2(编码1～500氨基酸残基)和nBRCA2a(编码1～18-105～500氨基酸残基)的基因片段,通过体外重组技术将目的基因片段连接到原核表达载体pGEX-2TK中,将经酶切和测序鉴定正确的重组质粒转化大肠杆菌BL21(DE3)pLysS受体菌,用IPTG诱导重组融合蛋白(与GST融合)的表达并进行纯化。结果和结论:构建了含有和不含有第3个外显子的重组原核表达质粒pGEX-2TK/nBRAC2和pGEX-2TK/nBRCA2a;融合表达产物经SDS-PAGE鉴定,并采用十二烷基肌氨酸钠进行了可溶性纯化,获得了相对分子质量分别为81000和71000的融合表达蛋白。     

拟南芥Antiquitin基因的原核表达和生物信息学分析

将拟南芥Antiquitin基因重组到原核表达载体pMAL-c4x和pET41中,在T7 Express菌株中诱导表达,经Amylose和Ni-NTA亲和层析柱纯化获得重组蛋白.SDS-PAGE结果表明:MBP和His-tag融合的拟南芥Antiquitin主要以可溶性形式存在,表达量分别占细胞总蛋白的25.1%和39.4%.以乙醛和NAD~+为底物测定融合蛋白活性,结果显示:His-tag融合的Antiquitin具有醛脱氢酶活性,比活力为8.98 U/mg,乙醛的表观K_m和V_(max)值分别为0.98 mmol/L和12.75 U/mg.序列比对和结构预测结果显示:拟南芥Antiquitin包含该家族蛋白典型的催化结构域、NADH结合结构域和寡聚化结构域,活性中心残基为Gly238、Gly291、Glu391、Phe393.     

香港海鸥菌OsmC蛋白的原核表达、纯化及活性研究

[目的]原核表达纯化香港海鸥菌OsmC蛋白,并检测其抗氧化功能。[方法]通过PCR法扩增香港海鸥菌OsmC基因,将目的片段进行双酶切后连接到pET28a,构建重组质粒pET28a-OsmC并转化大肠杆菌,诱导OsmC蛋白表达,亲和层析纯化目的蛋白并利用氧化铁二甲酚橙实验检测其过氧化物酶活性。[结果]克隆得到全长基因,大小为441bp,编码147个氨基酸。得到重组表达质粒pET28a-OsmC,表达并纯化获得重组OsmC蛋白,OsmC蛋白能够降解H2O2。[结论]Osm C蛋白具有过氧化物酶活性,为研究香港海鸥菌的抗氧化机制奠定了基础。     

拟南芥阿拉伯糖-5-磷酸异构酶的原核表达、纯化及酶催化特性

阿拉伯糖-5-磷酸异构酶(Kds D)是2-酮-3-脱氧辛糖酸(KDO)生物合成途径的第一个关键限速酶,通过无缝克隆技术将拟南芥Kds D基因构建至原核表达载体p ET-HTT,经过IPTG诱导,在大肠杆菌BL21(DE3)中获得了大量重组蛋白的可溶性表达;表达产物经Ni-NTA亲和层析和分子筛层析(SEC)方法进行酶蛋白的分离纯化步骤,得到纯度85%以上的高纯度酶;分子筛层析结果发现纯化后的目的蛋白Kds D在溶液中主要以多聚体、二聚体和单体形式存在,这同微生物来源Kds D酶在溶液中以四聚体形式存在很大差异;进一步使用Western blotting和MALDI-TOF MASS技术对纯化的蛋白进行鉴定;测定了拟南芥Kds D酶学性质,证明该酶催化反应的最适p H值为8.0,最适作用温度为37℃,各种金属离子在低浓度均对酶活性存在不同程度的抑制作用,其中以Co~(2+)、Cd~(2+)对酶活性的抑制作用最强,而5 mmol/L金属螯合剂EDTA对酶有激活作用。此外,以阿拉伯糖-5-磷酸(A5P)为底物时,拟南芥Kds D酶动力学常数Vmax和Km值分别为0.18 mmol/(L·min)、0.16 mmol/L,比较发现该酶与底物的亲和性高于大肠杆菌Kds D。以上研究结果为Kds D蛋白结构与功能及其在新型抗生素研制领域中的工业化应用奠定了基础。     

甜菜夜蛾羧酸酯酶基因cDNA的克隆、表达及序列分析

羧酸酯酶是昆虫体内重要的解毒酶系之一,与昆虫抗药性产生相关。利用粉纹夜蛾Trichoplusia ni (Hübner)肠粘蛋白多克隆抗体免疫筛选甜菜夜蛾Spodoptera exigua (Hübner)中肠cDNA表达文库,得到编码羧酸酯酶的全长cDNA克隆。该cDNA克隆全长1 812 bp(GenBank登录号EF580101),开放阅读框长1 605 bp,编码535个氨基酸。该蛋白活性中心包括3个氨基酸残基（催化三联体）： Ser186, Glu319和His443, 3个N-联糖基化位点,具备羧酸酯酶的结构特征,属羧酸酯酶家族（EC: 3.1.1.-）。将该基因与pQE30载体重组,经IPTG诱导,spot-blot鉴定,蛋白获得了表达;以α-醋酸萘酯为底物,检测表达的羧酸酯酶活性为1.3 nmol/100 μL酶液。     

Two-enzyme systems for glycolipid and polyglycerolphosphate lipoteichoic acid synthesis in Listeria monocytogenes

Lipoteichoic acid (LTA) is an important cell wall polymer in Gram-positive bacteria and often consists a polyglycerolphosphate backbone chain that is linked to the membrane by a glycolipid. In Listeria monocytogenes this glycolipid is Gal-Glc-DAG or Gal-Ptd-6Glc-DAG. Using a bioinformatics approach, we have identified L. monocytogenes genes predicted to be involved in glycolipid ( lmo2555 and lmo2554 ) and LTA backbone ( lmo0644 and lmo0927 ) synthesis. LTA and glycolipid analysis of wild-type and mutant strains confirmed the function of Lmo2555 and Lmo2554 as glycosyltransferases required for the formation of Glc-DAG and Gal-Glc-DAG. Deletion of a third gene, lmo2553 , located in the same operon resulted in the production of LTA with an altered structure. lmo0927 and lmo0644 encode proteins with high similarity to the staphylococcal LTA synthase LtaS, which is responsible for polyglycerolphosphate backbone synthesis. We show that both proteins are involved in LTA synthesis. Our data support a model whereby Lmo0644 acts as an LTA primase LtaP and transfers the initial glycerolphosphate onto the glycolipid anchor, and Lmo0927 functions as LTA synthase LtaS, which extends the glycerolphosphate backbone chain. Inactivation of LtaS leads to severe growth and cell division defects, underscoring the pivotal role of LTA in this Gram-positive pathogen.     

Serine/threonine protein kinase PrkA of the human pathogen Listeria monocytogenes: biochemical characterization and identification of interacting partners through proteomic approaches

Listeria monocytogenes is the causative agent of listeriosis, a very serious food-borne human disease. The analysis of the proteins coded by the L. monocytogenes genome reveals the presence of two eukaryotic-type Ser/Thr-kinases (lmo1820 and lmo0618) and a Ser/Thr-phosphatase (lmo1821). Protein phosphorylation regulates enzyme activities and protein interactions participating in physiological and pathophysiological processes in bacterial diseases. However in the case of L. monocytogenes there is scarce information about biochemical properties of these enzymes, as well as the physiological processes that they modulate. In the present work the catalytic domain of the protein coded by lmo1820 was produced as a functional His(6)-tagged Ser/Thr-kinase, and was denominated PrkA. PrkA was able to autophosphorylate specific Thr residues within its activation loop sequence. A similar autophosphorylation pattern was previously reported for Ser/Thr-kinases from related prokaryotes, whose role in kinase activity and substrate recruitment was demonstrated. We studied the kinase interactome using affinity chromatography and proteomic approaches. We identified 62 proteins that interact, either directly or indirectly, with the catalytic domain of PrkA, including proteins that participate in carbohydrates metabolism, cell wall metabolism and protein synthesis. Our results suggest that PrkA could be involved in the regulation of a variety of fundamental biological processes.     

A Listeria monocytogenes RNA helicase essential for growth and ribosomal maturation at low temperatures uses its C terminus for appropriate interaction with the ribosome

Listeria monocytogenes, a Gram-positive food-borne human pathogen, is able to grow at temperatures close to 0°C and is thus of great concern for the food industry. In this work, we investigated the physiological role of one DExD-box RNA helicase in Listeria monocytogenes. The RNA helicase Lmo1722 was required for optimal growth at low temperatures, whereas it was dispensable at 37°C. A Δlmo1722 strain was less motile due to downregulation of the major subunit of the flagellum, FlaA, caused by decreased flaA expression. By ribosomal fractionation experiments, it was observed that Lmo1722 was mainly associated with the 50S subunit of the ribosome. Absence of Lmo1722 decreased the fraction of 50S ribosomal subunits and mature 70S ribosomes and affected the processing of the 23S precursor rRNA. The ribosomal profile could be restored to wild-type levels in a Δlmo1722 strain expressing Lmo1722. Interestingly, the C-terminal part of Lmo1722 was redundant for low-temperature growth, motility, 23S rRNA processing, and appropriate ribosomal maturation. However, Lmo1722 lacking the C terminus showed a reduced affinity for the 50S and 70S fractions, suggesting that the C terminus is important for proper guidance of Lmo1722 to the 50S subunit. Taken together, our results show that the Listeria RNA helicase Lmo1722 is essential for growth at low temperatures, motility, and rRNA processing and is important for ribosomal maturation, being associated mainly with the 50S subunit of the ribosome.     

Structural and functional analysis of the Lmo2642 cyclic nucleotide phosphodiesterase from Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen invading humans and animals with the highest fatality rate among the food-borne pathogens. The Listeria pathogenic processes, such as cell entry and escape from phagosomes, depend on the actions of diverse bacterial factors, including lipoproteins. Here, we report the crystal structure of Lmo2642, a conserved putative lipoprotein containing a Ser/Thr phosphatase domain. The protein consists of two distinct domains: a catalytic domain that belongs to the metallophosphoesterase superfamily and an auxiliary α-helical bundle domain. The active site in the catalytic domain of Lmo2642 contains a dinuclear metal center in which Mn2(+) and Fe3(+) are preferentially positioned at the site1 and site2, respectively. On the basis of the structural analysis and enzymatic assays, we identified the biochemical activity of the protein as a cyclic nucleotide phosphodiesterase toward 2',3'- and 3',5'-cyclic nucleotides. Considering the cNMP phosphodiesterase activity and the putative surface localization of Lmo2642, we speculate that Lmo2642 has some potential roles in the host-pathogen interactions by changing the cAMP concentration of host cells during L. monocytogenes infection.     

绮丽刺毛霉的一种新型甘氨酸氨肽酶的研究

研究了产自于绮丽刺毛霉（Actinomucor elegans）的一种甘氨酸氨肽酶。分子筛层析表明该酶的天然分子的分子量为320kD,SDSPAGE分析表明蛋白质的亚基分子量为565kD。该酶水解含有甘氨酸残基的底物（如glycinenaphthylamine）的效率要较其它氨基酸残基高得多。该酶的最佳反应温度为30℃,最佳pH为8.0。酶的Km和Kcat值分别为0.24mmol/L与1008 s^-1。1.0mmol/L Zn²⁺,Cu²⁺和Cd²⁺可完全抑制该酶的活性。作用于酶巯基的化学物质对酶活性都有抑制作用。根据络合剂反应的实验结果表明该酶是一种含有金属的酶。当与蛋白酶共同作用时该酶除了甘氨酸外还能提高脯氨酸、精氨酸及谷氨酸的水解率。