具超强富集U(Ⅵ)能力工程菌E.coli的构建

以鼠伤寒沙门氏菌基因组DNA为模板,PCR扩增得到非特异性酸性磷酸酶基因（phoN）,将其克隆到pMD18T-Vector中。用Spe I、Nde I限制性内切酶对重组转移载体T-VectorphoN与穿梭载体pRADZ3分别进行双酶切,再将phoN片段和穿梭载体pRADZ3中的大片段通过T4DNA连接酶连接。经PCR与双酶切双重鉴定,证实重组穿梭载体pRADZ3phoN构建成功。转化Escherichia coli DH5α感受态细胞,使其在正常情况下表达PhoN蛋白,经Western blot 证实phoN基因在DH5α中成功表达。利用含pRADZ3phoN的工程菌进行富集U(Ⅵ)实验,结果表明该工程菌对U(Ⅵ)的富集量较宿主菌提高约4倍,达46.16mg/g,去除率为92.32%。     

Biochemical characterization of a mutant of the yeast Pichia anomala derepressed for malic acid utilization in the presence of glucose

Abstract Myxococcus xanthus cells move over surfaces by gliding motility. The frz signal transduction system is used to control the reversal frequency, and thus the overall direction of movement of M. xanthus cells. We analyzed the behavior of wild-type and frz mutant cells in response to prey bacteria ( Escherichia coli ). Wild-type cells of M. xanthus did not respond to microcolonies of E. coli until they made physical contact. Cells which penetrated a colony remained in the colony until all of the prey cells were digested. Cells of frz mutants also penetrated E. coli microcolonies and digested some of the E. coli cells, but they invariably abandoned the microcolony leaving their food source behind. These observations illustrate the importance of the frz system of signal transduction for the feeding behavior of M. xanthus cells.     

Complementation of argG and hisA mutations of Escherichia coli by DNA cloned from the archaebacterium Methanococcus voltae

DNA derived from the methanogenic archaebacterium Methanococcus voltae was digested with PstI restriction endonuclease and cloned into the PstI site of pBR322. The recombinant plasmids generated were used to transform a multiply auxotrophic strain of Escherichia coli with selection for tetracycline resistance. Plasmids complementing the argG(pAW1) or hisA(pAW2) mutations were isolated and characterized. Nick-translated pAW1 and pAW2 hybridized to the predicted M. voltae PstI fragments but not to digested E. coli DNA. A novel 55,000-dalton protein was synthesized in UV-irradiated cells by pAW1, whereas pAW2 synthesized a novel 26,000-dalton protein. Derivatives of pAW1 carrying insertion elements no longer complemented the argG mutation and failed to produce the 55,000-dalton protein. When an AccI fragment was deleted from pAW2, complementation of hisA did not occur and no 26,000-dalton protein was synthesized. The effect of orientation of the cloned DNA within the vector on complementation and polypeptide synthesis was examined.     

水稻线粒体26S rRNA基因在大肠杆菌JM101中的克隆及分子鉴定

用BT型水稻喜峰A黄化苗为材料,按本实验过去报道经修改后的方法提取线粒体DNA,经EcoR1完全酶切后,随机克隆到pUC19载体上,转化大肠杆菌,在含氨苄青霉素(50μg/m1)和X-gal的LB固体平板上筛选白色转化子。随机提取重组子DNA,以玉米26S rRNA基因为探针,经Southern分子杂交鉴定,一个插入1.3kb水稻线粒体DNA片段的重组质粒杂交结果为阳性,并将这个含有26S rRNA基因片段的重组质粒命名为pXMT1。     

Subcloning of K88ac antigen gene of enterotoigenic E. coli and the restriction map of recombinant plasmid

We had reported a recombinant E. coli RR1(pNZ8801) which was obtained from a wild strain E. coli 79-1454. The recombinant plasmid was digested by EcoRI and generated three segments, medium segment (3.2Md) was removed, the largest and the smallest segment was ligased, then the mixture was transformed into E. coli RRI, screening Ap(r) Tc(s) clones, one of recombinants was named E. coli RR1(pNZ8802). The recombinant plasmid molecular weight is smaller, but expression of K88ac antigen is higher than first cloning. Subcloning can adhere to mucosae of piglet's intesting. Therefore, the recombinant can be use for oval living vaccine.     

New method for the synthesis and cloning of cDNA

A simple method for cloning cDNA has been suggested. The plasmid pUC18 was digested with Pst1. A plasmid primer for cDNA synthesis was prepared by dT tailing with terminal transferase. After synthesis of cDNA, dG tails were added and then 3' ends blocked with rG. The plasmid was digested with Kpn1 and dC tails were added, after which annealing took place and RNA:DNA hybrids were used for Escherichia coli transformation. The efficiency of approx. 10(4) transformants per microgram of starting mRNA has been obtained.     

The primary structure of human EGF produced by genetic engineering, studied by high-performance tandem mass spectrometry

The primary structure of human epidermal growth factor (hEGF), which was produced by Escherichia coli using recombinant DNA technique, has been studied by tandem mass spectrometry. The molecular weight of hEGF (about 6200 amu) was determined by fast atom bombardment mass spectrometry. Then reduced and carboxymethylated hEGF was digested by chymotrypsin into seven peptides which could cover the whole sequence of hEGF. The amino acid sequences of five of these seven peptides could be confirmed by tandem mass spectrometry with or without isolation by high-performance liquid chromatography (HPLC). After isolation by HPLC, the other two peptides were digested with trypsin or thermolysin into small peptides, and sequenced by tandem mass spectrometry.     

The genetic diversity of predominant Escherichia coli strains isolated from cattle fed various amounts of hay and grain(1)

When the 16S rDNA of predominant Escherichia coli strains from cattle was digested with HhaI and HaeIII, the strains could be sub-divided into four operational taxonomic units. When genomic DNA was digested with XbaI, strains could be grouped into 24 pulsed-field gel electrophoresis genotypes (>95% Dice similarity) and five clades (>20% Dice similarity). Diet (hay versus grain) and gastrointestinal compartment (rumen versus colon) did not have a large impact on diversity. However, both analyses indicated that the cows (n=2) had different E. coli populations. When all 22 colonic strains were inoculated into a maltose-limited chemostat, only a single genotype persisted. Based on these results, the genetic diversity of E. coli in the cattle is very great and this bacterium can occupy different niches.     

人抑癌基因PTEN的原核表达载体的构建及融合表达

为研究抑癌因子PTEN蛋白的抑癌机理,掏建了PTEN cDNA的原核表达载体并进行融合表达。将含有PTEN cDNA的质粒pMD-PTEN经EcoR Ⅰ和Sal Ⅰ双酶切,回收PTEN基因片段与经相同酶切的高效原核表达载体pET-44a连接,经序列测定,证实融合型表达载体pET-Nus-PTEN构建成功。转化表达宿主BL21(DE3)后,IPTG诱导表达。经12％SDS-PAGE凝胶电泳,获得118kD的特异蛋白条带。目的蛋白占细菌总蛋白的17％。结果表明：PTEN基因和Nus基因融合表达成功,获得可溶性Nus-PTEN蛋白。该研究为PTEN蛋白的抑癌机理和基因工程药物的研究打下了基础,这是国内PTEN蛋白在原核细胞中成功表达的首次报道。     

Synthesis of recombinant human single-chain urokinase-type plasminogen activator variants resistant to plasmin and thrombin

Single-chain urokinase-type plasminogen activator (scu-PA), a potential therapeutic reagent for thrombosis, is activated in plasma by plasmin. The activated enzyme is further digested by plasmin to generate low-molecular-weight urokinase (LMW-UK), which has no affinity for fibrin. To circumvent this dual effect of plasmin, we synthesized in Escherichia coli a variant of scu-PA, which is not converted to LMW-UK on treatment with plasmin. In another variant, the activation cleavage site was modified such that activation by plasmin was slowed down and that inactivation by thrombin was greatly diminished. The combination of these variants may be applicable as an effective thrombolytic reagent for clinical use.     

腐蹄病致病因子节瘤拟杆菌蛋白酶在大肠杆菌...

Bacteroides nodosus is the essential causative agent of ovine footrot. It produces extracellular proteases which involved in pathogenesis of footrot. In this paper, we report the subcloning of Bacteroides nodosus protease, its overexpression in E. coli and its N-terminal polypeptide sequence. The subclone library was constructed in E. coli using SphI digested original clone (15 kb) and plasmid PTZ18R and screened using immunological assay. The expression was observed using SDS-PAGE. The subcloned DNA fragment was then cut with Sau3AI, cloned into pKK232-8 vector to perform promoter isolation and analysis. The promoter strength was determined using spectrophotometric assay.     

Expression of mature pokeweed antiviral protein with or without C-terminal extrapeptide in Escherichia coli as a fusion with maltose-binding protein.

Genomic clones encoding the mature pokeweed antiviral protein with or without C-terminal extrapeptide (PAPMC and PAPM), which have been reported to be highly toxic to E. coli cells, were inserted into the expression vector pMAL-p2. The recombinant PAPs (rPAPMC and rPAPM) were successfully expressed in E. coli at 25 degrees C, being exported to the periplasm as soluble fusions with maltose-binding protein (MBP). The rPAPs were cleaved from MBP by treatment with factor Xa and subsequently purified with final yields of 4.0 mg/liter (rPAPMC) and 5.5 mg/liter (rPAPM). rPAPM was resistant to protease digestion, but the C-terminal extrapeptide appeared to be susceptible and was partially digested by some protease in E. coli. Both rPAPMC and rPAPM were as active as the native PAPM from pokeweed leaves in depurinating rat liver and E. coli ribosomes, while the activities of rPAPMC on both ribosomes were decreased at least 60-fold by fusion with MBP.     

alpha-Carboxyl-linked glutamates in the folylpolyglutamates of Escherichia coli

Folate cofactors in most cells contain polyglutamate side chains, which since the late 1940s have been assumed to be linked via their gamma-COOH groups. We report here an investigation of the structure of the polyglutamate chain attached to the folates of Escherichia coli. Folates were extracted from E. coli grown with [7-14C] p-aminobenzoate and cleaved to p-aminobenzoyl polyglutamates of varying chain lengths (pAB(Glu)n) by the method of Foo et al. (Foo, S. K., Cichowicz, D. J., and Shane, B. (1980) Anal. Biochem. 107, 109-115). The pAB(Glu)n derived from E. coli did not co-chromatograph with chemically synthesized pAB(gamma-Glu)n-Glu on several high performance liquid chromatography (HPLC) systems, except for the triglutamate which did elute with pAB(gamma-Glu)2-Glu. E. coli-derived pAB(Glu)3-8 were purified by HPLC on C18 columns eluted with acetonitrile/trifluoroacetic acid, and the structures were determined through mass spectrometry, chiral amino acid analysis, and peptidase digestion experiments. Molecular weight determinations on the methyl ester derivatives of E. coli-derived pAB(Glu)n by liquid secondary ion mass spectrometry and sequence analysis using collision-activated dissociation on a tandem mass spectrometer confirmed the structures as pAB(Glu)3-8. Chiral HPLC of hydrolyzed and dansylated E. coli-derived materials, on a beta-cyclodextrin column, identified the glutamate as the L-enantiomer. pAB(Glu)n were digested with carboxypeptidase Y, which specifically cleaved glutamates linked at their alpha-carboxyls; E. coli-derived pAB(Glu)4-8 (but not synthetic pAB(gamma-Glu1-6-Glu) were sequentially digested to pAB(gamma-Glu)2-Glu. Thus, in E. coli folylpolyglutamates, glutamate residues 4-8 were each linked to the polyglutamate chain at the alpha-carboxyl of the preceding glutamate.     

Presence of methylated adenine in GATC sequences in chromosomal DNAs from Campylobacter species.

We digested chromosomal DNAs from 12 Campylobacter strains (C. jejuni, 4 strains; C. coli, 2 strains; C. fetus subsp. fetus, 2 strains; C. hyointestinalis, 2 strains; and C. upsaliensis, 2 strains) and from 4 Helicobacter strains (H. pylori, 2 strains; and H. mustelae, 2 strains) with HindIII, SstI, BamHI, DpnI, MboI, and Sau3AI. Restriction fragments were then separated by electrophoresis in 1% agarose or 10% polyacrylamide gels. Only DNAs from three Campylobacter species (C. jejuni, C. coli, and C. upsaliensis) were digested with DpnI (an enzyme that recognizes only methylated adenine in GATC sequences). We used MboI and Sau3AI to confirm these findings.     

林可链霉菌中的同源重组

为研究链霉菌中的同源整合频率和机制 ,采用不能在链霉菌中复制的大肠杆菌质粒转化链霉菌StreptomyceslincolnensisB48。质粒pYYE0 4a1上携带的被硫链丝菌素抗性基因灭活的林可霉素生物合成基因与染色体DNA上的同源基因发生重组 ,经过低抗筛选 ,得到两个突变子S .lincolnensisYY1和S .lincolnensisYY2。进一步以硫链丝菌素抗性基因为探针杂交染色体DNASmaⅠ片段 ,S .lincolnensisYY1和S .lincolnensisYY2都得到 1 5kb的阳性条带 ;而以缺失的lacZ基因为探针杂交染色体DNAHindⅢ和SmaⅠ联合酶切片段 ,只有S .lincolnensisYY2得到 4 4kb的阳性条带。Southern杂交结果表明S .lincolnensisYY1是由同源交换或二次重组产生的 ,而S .lincolnensisYY2为同源整合的结果。为验证同源整合子上大肠杆菌复制子和氨苄抗性基因的存在 ,用SphⅠ酶切染色体DNA后连接 ,连接液转化E .coliJM83感受态细胞 ,在氨苄抗性板上得到 2个转化子 ,命名为pSLE1。对…     

Cloning of the contiguous 165-kilobase-pair region around the terminus of Escherichia coli K-12 DNA replication.

Escherichia coli K-12 chromosomal DNA was partially digested with either EcoRI or HindIII, and cosmid libraries were constructed. By screening these libraries, a series of partially overlapping clones which covered the terC region was isolated. The cloned area spanned about 165 kilobase pairs, corresponding to the 29.7-to-33.2-min region of the genetic map of the E. coli chromosome.     

鸡传染性贫血病毒VP2基因的表达及其免疫活性分析

利用特异性引物从组织病料中提取的鸡传染性贫血病毒核酸经PCR扩增得到VP2基因,BamHⅠ和SalⅠ双酶切处理,纯化后,克隆至BamHⅠ和SalⅠ双酶切处理的表达载体pET-28a中,构建了原核表达质粒pET-28-VP2。将pET-28-VP2转化至感受态E.coliBL21(DE3)中,经IPTG诱导和SDS-PAGE分析,可见约31kDa的目的蛋白以可溶性形式表达于上清中。Western blot分析发现,表达产物与鸡传染性贫血的阳性血清发生特异性反应。纯化蛋白免疫小鼠后制得的多抗可以与全病毒发生反应,证明其具有免疫原性,为进一步研究VP2蛋白的功能及开展CIAV疫苗及诊断试剂的研制奠定了基础。     

Genotyping of clinical and chicken isolates of Campylobacter jejuni and Campylobacter coli

Genomic DNA from 58 strains of Campylobacter made up of 48 Campylobacter jejuni and ten Campylobacter coli were digested with Sma I and analysed by pulsed-field gel electrophoresis (PFGE). The cleavage of DNA by Sma I gave 22 distinct hybridization patterns. The two Campylobacter species were subtyped by PFGE. The average genomic size for C. jejuni by Sma I digestion was 1.73 Mb, while that of C. coli gave 1.7 Mb. Results from this study indicate that PFGE analysis by Sma I digested genomic DNA provides a reliable means of differentiating between and within species of Campylobacter and provides a practical approach to epidemiological studies of Campylobacter.     

Use of bioluminescent Escherichia coli O157:H7 to study intra-protozoan survival of bacteria within Tetrahymena pyriformis

A method was developed that enabled real-time monitoring of the uptake and survival of bioluminescent Escherichia coli O157 within the freshwater ciliate Tetrahymena pyriformis. Constitutively bioluminescent E. coli O157 pLITE27 was cocultured with T. pyriformis in nutrient-deficient (Chalkley's) and in nutrient-rich (proteose peptone, yeast extract) media. Non-internalised bacteria were inactivated by addition of colistin, indicated by a decline in bioluminescence. Protozoa were subsequently lysed with Triton X-100 which lead to a further drop in bioluminescence, consistent with release of live internal bacteria from T. pyriformis into the colistin-containing environment. Bioluminescence measurements for non-lysed cultures indicated that internalised E. coli O157 pLITE27 cells were only slowly digested by T. pyriformis, in both media, over the time period studied. The results suggest that bioluminescent bacteria are useful tools in the study of bacterial intra-protozoan survival.     

FtsH recognizes proteins with unfolded structure and hydrolyzes the carboxyl side of hydrophobic residues

FtsH of Escherichia coli is an essential membrane-integrated ATP-dependent protease. We cloned a gene for an FtsH homolog (T. FtsH) from Thermus thermophilus HB8, expressed it in E. coli, and purified the expressed protein. ATPase activity of T.FtsH was activated by proteins with unfolded structure ( alpha-casein and pepsin), and T.FtsH digested these proteins in an ATP-, Zn(2+)-dependent manner. alpha-Lactalbumin was digested by T.FtsH when it was largely unfolded, but not in its native form. Analysis of the proteolytic products revealed that, in most cases, T.FtsH cleaved the C-terminal side of hydrophobic residues and produced a characteristic set of small peptides (<30 kDa) without releasing a large intermediate. Thus, T.FtsH recognizes the unfolded structure of the proteins and progressively digests them at the expense of ATP. A soluble domain of T.FtsH, which lacked the N-terminal two transmembrane helices, was also prepared but was found to retain neither ATPase nor protease activities. Thus, the membrane segment appeared to be indispensable for these activities of T.FtsH.