Overexpression of HumanN-Myristoyltransferase Utilizing a T7 Polymerase Gene Expression System

Myristoyl CoA:proteinN-myristoyltransferase catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins. The gene encoding humanN-myristoyltransferase (hNMT) was cloned into the overexpression vector pT7-7 which utilizes the T7 RNA polymerase gene expression system. The hNMT enzyme was purified to near homogeneity with more than 95% recovery using a single-step purification method involving SP–Sepharose fast flow column chromatography. The specific activity of the purified NMT was 220 nmol/min/mg of protein in the presence of oncoprotein-derived peptide substrate pp60^src. The hNMT exhibited an apparent molecular weight of 49 kDa on SDS–polyacrylamide gel electrophoresis. Antibodies toEscherichia coli-expressed hNMT specifically recognize hNMT from crude bacterial lysates. The overexpressed hNMT was homogeneous and showed enzyme activity.     

磷酸烯醇式丙酮酸羧化酶cDNA在T7 RNA多聚酶/启动子系统中专一性表达的研究

用PT7和pGP1—2质粒偶联表达系统,通过温度诱导(30～42℃),使温度敏感基因CI875失活;加入利福霉素选择性抑制E.coliRNA多聚酶的表达,从而使外源PEP羧化酶cDNA得到专一性的表达。产物经SDS—PAGE和Wesern杂交分析,表明该系统表达出两条PEP羧化酶带,分子量分别是78kD和80kD。     

原核表达系统T7RNA聚合酶/启动子在真核细胞中表达目的基因的实验研究

将目前高表达水平强大的原核表达系统之一T7 RNA聚合酶/启动子表达系统通过一系列改进引入真核细胞.通过转染真核细胞实验表明,采用真核启动子CMV调控T7 RNA聚合酶的表达和在T7启动子下游插入EMCV IRES序列两种解决方案能使该原核表达系统在真核细胞高效表达目的基因,且能适应不同的真核细胞环境,是一良好的细胞类型非依赖的表达体系.     

用T7 RNA聚合酶体外转录合成大鼠肝tRNAIle

采用PCR技术从rec-M13mp18中扩增出120 bp的大鼠肝tRNA^Ile合成基因片段,经限制性内切酶BstNⅠ酶切后作为模板,利用T7 RNA聚合酶在体外无细胞体系转录由T7启动子带动的大鼠肝tRNA^Ile基因,生成不含修饰碱基的tRNA^Ile,并对体外转录反应条件进行了优化,回收的tRNA产量可达DNA模板量的40倍.     

Fish as Reservoirs and Vectors of Vibrio cholerae

Vibrio cholerae, the etiologic agent of cholera, is autochthonous to various aquatic environments, but despite intensive efforts its ecology remains an enigma. Recently, it was suggested that copepods and chironomids, both considered as natural reservoirs of V. cholerae, are dispersed by migratory waterbirds, thus possibly distributing the bacteria between water bodies within and between continents. Although fish have been implicated in the scientific literature with cholera cases, as far as we know, no study actually surveyed the presence of the bacteria in the fish. Here we show for the first time that fish of various species and habitats contain V. cholerae in their digestive tract. Fish (n = 110) were randomly sampled from freshwater and marine habitats in Israel. Ten different fish species sampled from freshwater habitats (lake, rivers and fish ponds), and one marine species, were found to carry V. cholerae. The fish intestine of Sarotherodon galilaeus harboured ca. 5×10³ V. cholerae cfu per 1 gr intestine content—high rates compared with known V. cholerae cfu numbers in the bacteria''s natural reservoirs. Our results, combined with evidence from the literature, suggest that fish are reservoirs of V. cholerae. As fish carrying the bacteria swim from one location to another (some fish species move from rivers to lakes or sea and vice versa), they serve as vectors on a small scale. Nevertheless, fish are consumed by waterbirds, which disseminate the bacteria on a global scale. Moreover, V. cholerae isolates had the ability to degrade chitin, indicating a commensal relationship between V. cholerae and fish. Better understanding of V. cholerae ecology can help reduce the times that human beings come into contact with this pathogen and thus minimize the health risk this poses.     

Bile Salts Modulate the Mucin-Activated Type VI Secretion System of Pandemic Vibrio cholerae

The causative agent of cholera, Vibrio cholerae, regulates its diverse virulence factors to thrive in the human small intestine and environmental reservoirs. Among this pathogen’s arsenal of virulence factors is the tightly regulated type VI secretion system (T6SS). This system acts as an inverted bacteriophage to inject toxins into competing bacteria and eukaryotic phagocytes. V. cholerae strains responsible for the current 7^th pandemic activate their T6SS within the host. We established that T6SS-mediated competition occurs upon T6SS activation in the infant mouse, and that this system is functional under anaerobic conditions. When investigating the intestinal host factors mucins (a glycoprotein component of mucus) and bile for potential regulatory roles in controlling the T6SS, we discovered that once mucins activate the T6SS, bile acids can further modulate T6SS activity. Microbiota modify bile acids to inhibit T6SS-mediated killing of commensal bacteria. This interplay is a novel interaction between commensal bacteria, host factors, and the V. cholerae T6SS, showing an active host role in infection.     

用表达T7RNA聚合酶细胞系拯救麻疹病毒微复制子

构建稳定表达T7RNA聚合酶的细胞系,用于提高拯救麻疹病毒微复制子效率.PCR扩增T7RNA聚合酶基因,克隆到真核表达载体,转染Vero细胞,用G418筛选到稳定表达的细胞株Vero/pcDNA3-T7.用Westernblotting证明了T7RNA聚合酶在细胞中的表达.将T7启动子控制绿色荧光蛋白表达的质粒转染该细胞后,绿色荧光蛋白在细胞株中得到表达.反向插入报告基因的微复制子转染感染麻疹病毒的Vero/pcDNA3-T7细胞后,细胞中能够检测到报告基因的表达.用细胞系取代痘苗病毒系统,可以提高拯救效率.     

“Host Shutoff” Function of Bacteriophage T7: Involvement of T7 Gene 2 and Gene 0.7 in the Inactivation of Escherichia coli RNA Polymerase

The "host shutoff" function of bacteriophage T7 involves an inactivation of the host Escherichia coli RNA polymerase by an inhibitor protein bound to the enzyme. When this inhibitor protein, termed I protein, was removed from the inactive RNA polymerase complex prepared from T7-infected cells by glycerol gradient centrifugation in the presence of 1 M KCl, the enzyme recovered its activity equivalent to about 70 to 80% of the activity of the enzyme from uninfected cells. Analysis of the activity of E. coli RNA polymerase from E. coli cells infected with various T7 mutant phages indicated that the T7 gene 2 codes for the inhibitor I protein. The activity of E. coli RNA polymerase from gene 2 mutant phage-infected cells, which was about 70% of that from uninfected cells, did not increase after glycerol gradient centrifugation in the presence of 1 M KCl, indicating that the salt-removable inhibitor was not present with the enzyme. It was found that the reduction in E. coli RNA polymerase activity in cells infected with T7(+) or gene 2 mutant phage, i.e., about 70% of the activity of the enzyme compared to that from uninfected cells after glycerol gradient centrifugation in the presence of 1 M KCl, results from the function of T7 gene 0.7. E. coli RNA polymerase from gene 0.7 mutant phage-infected cells was inactive but recovered a full activity equivalent to that from uninfected cells after removal of the inhibitor I protein with 1 M KCl. E. coli RNA polymerase from the cells infected with newly constructed mutant phages having mutations in both gene 2 and gene 0.7 retained the full activity equivalent to that from uninfected cells with or without treatment of the enzyme with 1 M KCl. From these results, we conclude that both gene 2 and gene 0.7 of T7 are involved in accomplishing complete shutoff of the host E. coli RNA polymerase activity in T7 infection.     

用T7RNA聚合酶体外转录合成大鼠肝tRNA~(Ile)

采用PCR技术从rec M1 3mp1 8中扩增出 1 2 0bp的大鼠肝tRNAIle合成基因片段 ,经限制性内切酶BstNⅠ酶切后作为模板 ,利用T7RNA聚合酶在体外无细胞体系转录由T7启动子带动的大鼠肝tRNAIle基因 ,生成不含修饰碱基的tRNAIle,并对体外转录反应条件进行了优化 ,回收的tRNA产量可达DNA模板量的 4 0倍     

T7 RNA聚合酶表达系统的真核化及其偶联表达系统的建立

为了使T7RNA聚合酶(T7RNAP)表达系统真核化,首先,利用两种方法建立能够表达T7RNAP的真核细胞:(1)共转染表达T7RNAP的真核表达重组质粒于靶细胞;(2)利用稳定表达T7RNAP的BHK-21细胞系。然后,将FMDV内部核糖体进入位点(IRES)序列和增强型绿色荧光蛋白(EGFP)基因定向克隆进原核载体pET-40a-c( )的T7启动子下游,得到的重组质粒pIERS-EGFP-E。用该质粒分别转染上述两种细胞,在紫外显微镜下都能够观察到绿色荧光,表明真核化的T7RNAP偶联表达系统建立。并利用流式细胞仪对偶联表达水平进行了分析。这为利用该系统真核高效表达外源蛋白奠定了坚实的基础。用实验证明了FMDV5′端含有IERS具有介导非帽依赖性的翻译的功能,为以IERS为基础的双顺反子表达系统的建立及深入研究IERS与相关蛋白的功能奠定了基础。T7RNAP偶联表达体系是一种良好的外源基因表达体系。     

痘苗病毒/T7RNA聚合酶辅助的原核基因在真核细胞中的表达

痘苗病毒/T7RNA聚合酶这一瞬时表达系统由于具有很多优于其他表达系统的特点而被广泛地应用于表达外源蛋白.TB-Chen株轮状病毒的VP6 DNA编码片段插入到原核质粒pETL的噬菌体T7启动子和终止子之间,获得重组表达质粒pET-VP6.构建好的重组表达质粒pET-VP6通过脂质体转染到真核细胞MA104中,用携带噬...