B群脑膜炎球菌lpxL2基因敲除突变株的构建及初步鉴定

 应用基因敲除技术的方法和原理,通过PCR扩增B群脑膜炎球菌lpxL2基因及载体pGBK T7上的Kan抗性片段,lpxL2片段与puc-18载体连接得到重组质粒msb-puc,以重组质粒msb-puc为基础,分别通过反向PCR和酶切2种方法构建lpxL2基因中间片段的缺失,并在缺失位点连入Kan抗性表达盒,从而得到重组质粒mpK,mpK转化B群脑膜炎球菌,并用PCR的方法对转化子进行初步筛选鉴定,初步确定突变株1株.本研究通过基因敲除MenB中LPS合成途径相关基因lpxL2的方法,降低LPS毒性,为B群脑膜炎球菌OMV疫苗的研发做了铺垫.     

茄青枯假单胞菌毒性基因的功能分析

分离自花生的茄青枯假单胞菌(Ralstonia solanacearum,R.s)T2015的一段4.5 kb DNA中ORF2和ORF3位于同一个操纵子中.ORF2和ORF3 DNA序列与GeneBank同源性比较分析和T2015及其突变体T2136(ORF2)、T2135/R(ORF3)、T2135(ORF3极性突变体)和互补株T2136/R(ORF2)、T2145(T2135)的脂多糖(lipopolysaccharide,LPS)SDS-PAGE带型比较分析表明:ORF2参与LPS完整的核心的合成;ORF3编码产物在LPS完整的生物合成中起脂质A核心区—O-antigen连接酶作用.菌落形态和植株实验结果表明LPS与病菌菌落形态和其对寄主的致病性有关,而与其在非寄主上引起过敏反应能力无关.     

红景天苷对LPS诱导大鼠心肌损伤的保护作用及机制研究

研究红景天苷(Salidroside)对脂多糖(LPS)致大鼠心肌损伤的保护作用及机制。雄性清洁级SD大鼠50只,随机分组,分为对照组(NS)10只、LPS模型组(10 mg/kg)10只、红景天苷小剂量(Sal 5 mg/kg)组10只,红景天苷中剂量(Sal 20 mg/kg)组10只,红景天苷大剂量(Sal 50 mg/kg)组10只。腹腔注药后6 h测定左心室收缩压(LVSP)、左心室内压上升及下降最大变化速率(±dp/dtmax)等心功能指标。采用ELISA法和Western blot法测定TNF-α、Toll样受体4和核因子κB蛋白表达。结果显示:与对照组比较,LPS可明显抑制大鼠左室功能,显著增加大鼠心肌TNF-α、TLR4和NF-κB的表达。而与模型组比较,红景天苷可剂量依赖性地下调心肌TLR4、NF-κB及TNF-α表达,改善大鼠左室功能。可见,红景天苷对LPS诱导的大鼠心肌损伤具有一定的保护作用,其机制与抑制TLR4-NFκB-TNFα炎症信号转导通路密切相关。     

元宝枫叶黄酮抑制脂多糖诱导的小胶质细胞激活的作用

 探讨枫叶黄酮对脂多糖(Lipopolysaccharide,LPS)诱导的小胶质细胞株BV-2细胞炎性因子释放的抑制作用.用LPS刺激BV-2细胞构建炎症模型,采用免疫荧光双标和RT-PCR方法检测不同浓度枫叶黄酮(5,10,15μmol/L)对细胞炎性蛋白酶诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和环氧合酶-2 (cyclooxygenase-2,COX-2)、细胞炎性因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白介素-1β (interleukin-1β,IL-1β)、炎性信号蛋白核因子-κB(nuclear factor-κB,NF-κB)蛋白与mRNA的表达变化.结果发现:不同浓度的枫叶黄酮在翻译水平和转录水平上明显抑制了LPS诱导的细胞炎性蛋白酶iNOS和COX-2,细胞炎性因子TNF-α和IL-1β与炎性信号蛋白NF-κB的上调.上述结果表明枫叶黄酮可通过调控LPS诱导的小胶质细胞株BV-2细胞炎性因子释放从而抑制小胶质细胞激活,发挥抗神经炎症的作用.     

Characterization of the Expression of CTRP9, a Paralog of Adiponectin

Adiponectin is one of the hormones secreted exclusively by the adipose tissue with anti-diabetic anti-inflammatory, anti-atherogenic, anti-atherosclerotic, and insulin-sensitizing effects. Adiponectin has structural features including a signal peptide at the N terminus, a short variable region, a collagenous domain, and a C-terminal globular domain homologous to Clq. The family of proteins containing this globular domain is called the C1q/tumor necrosis factor-a-related proteins （CTRP）. Among all the CTRPs characterized so far, the CTRP9 protein has the highest homology to adiponectin. However, little is known about the functions of CTRP9. This study examines the tissue expression of CTRP9 as well as the expression of CTRP9 during mouse development and 3T3-L1 preadipocyte differentiation. The effects of LPS, TNFα, and starvation on the expression of CTRP9 were also investigated. CTRP9 mRNA is shown to be expressed in a variety of tissues including the adipose tissues. The expression profile of CTRP9 differs from that of adiponectin, implying that they are functionally diverse although structurally homologous.     

Membrane protein folding on the example of outer membrane protein A of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The biophysical principles and mechanisms by which membrane proteins insert and fold into a biomembrane have mostly been studied with bacteriorhodopsin and outer membrane protein A (OmpA). This review describes the assembly process of the monomeric outer membrane proteins of Gram-negative bacteria, for which OmpA has served as an example. OmpA is a two-domain outer membrane protein composed of a 171-residue eight-stranded -barrel transmembrane domain and a 154-residue periplasmic domain. OmpA is translocated in an unstructured form across the cytoplasmic membrane into the periplasm. In the periplasm, unfolded OmpA is kept in solution in complex with the molecular chaperone Skp. After binding of periplasmic lipopolysaccharide, OmpA insertion and folding occur spontaneously upon interaction of the complex with the phospholipid bilayer. Insertion and folding of the -barrel transmembrane domain into the lipid bilayer are highly synchronized, i.e. the formation of large amounts of -sheet secondary structure and -barrel tertiary structure take place in parallel with the same rate constants, while OmpA inserts into the hydrophobic core of the membrane. In vitro, OmpA can successfully fold into a range of model membranes of very different phospholipid compositions, i.e. into bilayers of lipids of different headgroup structures and hydrophobic chain lengths. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers. Their formation was monitored by time-resolved distance determinations by fluorescence quenching, and they were structurally distinguished by the relative positions of the five tryptophan residues of OmpA in projection to the membrane normal. Recent studies indicate a chaperone-assisted, highly synchronized mechanism of secondary and tertiary structure formation upon membrane insertion of -barrel membrane proteins such as OmpA that involves at least three structurally distinct folding intermediates.     

小鼠脂多糖应答蛋白mlrpS的载体构建及融合蛋白表达

为构建小鼠mlrpS-cDNA基因原核、真核表达载体，大肠杆菌表达其融合蛋白。采用反转录-聚合酶链反应从经脂多糖刺激的鼠NIH3T3细胞cDNA中，扩增出编码mlrpS的cDNA。用限制性内切酶KpnⅠand XhoⅠ消化后，插入原核表达载体pTAT中，经酶切鉴定与测序证实后，转化大肠杆菌BL21（DE3）菌株。异丙基β—D硫代半乳糖苷（IPTG）诱导产生融合蛋白。经KpnⅠ和XhoⅠ酶切回收mlrpS-cDNA，插入pcDNA3．1载体中；pcDNA3．1-mlrpS再经KpnⅠ和ApaⅠ酶切后，插入到pEGFP—c1载体上，构建pEGFP-mlrpS融合的真核表达载体。构建mlrpS表达载体经测序证实，与GenBank登录的序列完全一致；双酶切鉴定证实，克隆基因正确插入载体pEGFP及pTAT；SDS—PAGE证实融合蛋白表达成功。说明：成功地构建了mlrpS原核、真核表达载体，成功正确表达了6His／mlrpS融合蛋白。     

微囊藻毒素和脂多糖对鲢鱼和草鱼肝脏去毒相关基因活体诱导表达的影响

采用50μg·(kg体质量)-1的微囊藻毒素·LR(MC-LR)、2 mg·(kg体质量)-1的脂多糖(LPS)和50μg·(kg体质量)-1 MC-LR+2 mg·(kg体质量)-1 LPS分别活体腹腔注射鲢鱼、草鱼,采用实时荧光定量PCR方法测定MC-LR和LPS对鲢鱼、草鱼肝脏alpha-谷胱甘肽S-转移酶(GSTA)、rho-谷胱甘肽S-转移酶(GSTR)、谷胱甘肽过氧化物酶(GPX)和解偶联蛋白2(UCP2)等去毒相关基因活体诱导表达的影响.结果表明,鲢鱼、草鱼GSTA和GSTR的不同诱导变化除与两种淡水鱼类对毒素的耐受性相关外,还与毒索剂量、诱导时间等因素相关.LPS对GSTA和GSTR基因组成型、诱导型的不同作用,可能与调制因子LPS对不同生态习性淡水鱼类肝脏GST基因表达水平的调制作用不同有关.UCP2与GPX也分别在抑制过量ROS发生方面与肝脏抗氧化胁迫等方面起重要作用.     

Antibody to tumor necrosis factor (TNF) reduces endotoxin fever

Summary Antibody to tumor necrosis factor (TNF), injected intravenously, reduced endotoxin fever in the rabbit. The fever-reducing effect was apparent in the latter half of the febrile response.This work was supported by a Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (Grant No. 62480112).