利用大肠杆菌克隆在原核生物中有活性的油菜基因启动子

利用本室构建的基因启动子探针型载体ｐＳＵＰＶ４直接在大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ（Ｍｉｇｕｌａ）ＣａｓｔｅｌａｎｉｅｔＣｈａｌｍｅｒｓ）中分离油菜（ＢｒａｓｉｃａｎａｐｕｓＬ．）基因启动子片段,获得卡那霉素抗性重组子３３个。对重组子ｐＲＰ１０做了进一步鉴定：Ｓｏｕｔｈｅｒｎ杂交表明ＲＰ１０片段来自油菜基因组,并与基因组中的另一些序列具有同源性;ＲＰ１０片段５′端区域的缺失可使卡那霉素抗性水平从１００ｍｇ／Ｌ降至２５ｍｇ／Ｌ,说明缺失的序列可能影响基因转录效率;序列分析发现ＲＰ１０片段内存在几个真核基因启动子的保守序列;用根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｍｅｆａｃｉｅｎｓ（ＳｍｉｔｈｅｔＴｏｗｎｓｅｎｄ）Ｃｏｎｎ）转化法将ＲＰ１０ＧＵＳ基因转入油菜,组织化学分析显示ＲＰ１０在愈伤组织中可以启动融合的ＧＵＳ基因表达     

Cloning and expression in Escherichia coli of dextranase genes from Bacteroides thetaiotaomicron

A genomic bank was constructed in Escherichia coli HB101, consisting of DNA fragments from Bacteroides thetaiotaomicron strain 489 inserted within the vector pBR322. By screening on complex medium containing blue dextran, 10 stable dextranase-positive (Dex+) clones were isolated. Seven groups of Dex+ inserts were identified on the basis of their restriction maps and hybridization responses. Dextanase activity of the recombinant clones was weak, and was revealed on the selection medium after 15 days. Subcloning of a Sau3AI partially digested 3.2-kb insert in the expression vector pDR720 greatly enhanced dextranse activity on blue dextran plates in one clone, but the delay remained unaltered. This suggested that the enzyme was released by cell lysis. Expression of this 0.7-kb subcloned insert was dependent on the promoter region of tryptophan operon carried by pDR720.     

Cloning of the wide spectrum amidase gene from Brevibacterium sp. R312 by genetic complementation. Overexpression in Brevibacterium sp. and Escherichia coli

Abstract The amiE gene of Brevibacterium sp. R312 encoding wide spectrum amidase was isolated by complementation of a Brevibacterium sp. mutant using a plasmid gene bank of chromosomal DNA. The amiE structural gene and its promoter were localized on a 1.8-kb fragment by subsequent subcloning and complementation studies. In Brevibacterium sp., the investigation of amidase activities related to one copy of the gene suggested that the regulation of the amiE gene expression was under negative control. High expression levels have been obtained in Brevibacterium sp. and, after substitution of the amiE promoter by the tac promoter, in Escherichia coli .     

一株高锰氧化活性大肠杆菌的分离鉴定和多铜氧化酶基因的克隆与结构特性

锰氧化物是Mn(Ⅱ)经生物和化学氧化后形成的矿物成分,在元素生物地球化学循环过程中起着重要作用,而不同种类的细菌对Mn(Ⅱ)的氧化作用是自然界中氧化锰矿物形成的主要成因.从山东崅峪采集的铁锰结核棕壤中分离得到一株具有高锰氧化活性的土壤杆菌,其对Mn(Ⅱ)的氧化作用活性明显高于其它分离菌株,达到65 μmol/L.通过个...     

Heterologous pyc gene expression under various natural and engineered promoters in Escherichia coli for improved succinate production

In this study, the expression level of the pyc gene from Lactococcus lactis was fine tuned to improve succinate production in Escherichia coli SBS550MG. IPTG induction in the cultures of SBS550MG with pHL413, a positive control plasmid previously constructed (Sanchez et al., 2005), gave drastically decreased PYC activity and succinate yield. We constructed several plasmids for the expression of pyc to change copy number and variant promoters. Among the constructs, as compared to pHL413, the PYC activity dropped significantly with the Plac, Ptac, Ptrc or native Ppyc promoters in medium or high copy vectors, which resulted in a decrease in succinate yield. Three constructs pThio12, pHL413-Km, and pHL413-Km(lacIq-)N showed considerable PYC activity and improved succinate production in E. coli SBS550MG. The native Ppyc promoter was also modified in order to vary pyc expression levels by site-directed mutagenesis of the −10, −35, −44 regions, and the spacer regions between −10 to −35 and −35 to −44 regions. Out of 9 native promoter variants, the MIII variant resulted in a 20% increase in PYC activity, and improved succinate yield in SBS550MG. We also determined the copy number and stability of pHL413 and pHL413-Km. The two plasmids showed roughly the same copy number, but the pHL413-Km plasmid was relatively more stable. This study provides more understanding of the plasmid characteristics and fine tuning of the expression level of pyc for optimization of the succinate production processes.     

重组基因表达对大肠杆菌生理的影响

重组基因在表达外源蛋白质时常常会耗用大量的宿主细胞资源,从而对宿主造成代谢负荷,代谢负荷使得宿主的生化和生理产生很大的变化,甚至损害宿主正常的代谢功能。而过重的代谢负荷会影响目标蛋白的表达量和表达质量。综述了产生代谢负荷的原因,宿主细胞对代谢负荷的应激反应、以及减轻代谢负荷的策略。     

The xylA promoter of Bacillus megaterium mediates constitutive gene expression in Escherichia coli

In the present study, we demonstrate that the Escherichia coli–Bacillus megaterium shuttle vector pHIS1522 can be used as a versatile expression vector. Recombinant genes under the control of the xylA promoter are constitutively expressed at a high level in E. coli strains, whereas their expression is strongly induced by the addition of xylose in B. megaterium. The utilization of D ‐xylose is known to be dependent on the xylAB genes in a number of bacteria. For B. megaterium a XylA‐based expression system was established that allows tightly regulated and highly efficient heterologous gene expression. The open reading frame (ORF) of the fluorescent protein turboRFP was cloned under the control of the xylA promoter of B. megaterium in the shuttle vector pHIS1522. Unexpectedly, tRFP expression was not only observed in B. megaterium, but also in E. coli. Based on fluorescence measurements and Western blot analysis, expression was comparable or slightly higher compared with the commonly used pET vectors. Therefore, pHIS1522 can be used as a versatile expression vector in both, B. megaterium and E. coli.     

Isolation and characterization of Escherichia coli strains producing microcins of B and C types

Abstract A screening of 11956 enterobacteria isolates resulted in selection of seven active microcin-producing strains. The microcins were shown to be peptides or their derivatives with a rather broad spectrum of activity, mainly against Gram-negative bacteria. According to cross-immunity criteria, the microcins studied belonged to two of the previously suggested types, B (five strains) and C (two strains). Those of type B could be further classified into two subtypes on the account of differences in the spectrum of antibacterial activity. In five cases out of seven the microcin-producing ability has been attributed to plasmids that the strains harboured. The effect of microcins on sensitive cells was shown to depend on ompR and ompF gene products.     

Characterization of the eaeA gene from rabbit enteropathogenic Escherichia coli strain RDEC-1 and comparison to other eaeA genes from bacteria that cause attaching-effacing lesions

Abstract A number of enteric pathogens, including enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli , Hafnia alvei , a strain of Citrobacter freundii , and rabbit EPEC strain RDEC-1 cause attaching-effacing (AE) lesions in the gut mucosa. These bacteria have a pathogenicity cassette (locus of enterocyte effacement or LEE) containing the eaeA gene. This gene encodes intimin, an outer membrane protein required for production of AE lesions. RDEC-1, a non-invasive enteropathogen in young rabbits, produces AE lesions morphologically indistinguishable from lesions caused by human AE bacterial strains. The RDEC-1 example of E. coli diarrhea in rabbits is an important model for studying the pathogenesis of AE bacteria in a natural infection and for analyzing specific roles of the components of LEE. In order to better understand the role of intimin in the development of AE lesions, a portion of DNA within RDEC-1 LEE, containing the eaeA gene and an upstream open reading frame (ORF), was sequenced. The RDEC-1 eaeA gene shared 87%, 92%, and 93% DNA sequence identity and > 80% amino acid sequence identity with the eaeA genes of C. freundii biotype 4280, EHEC O157:H7, and EPEC O127:H6, respectively. The carboxy-terminal 280 amino acid residues of intimin has 80%, 56%, and 54% identity with C. freundii , EHEC O157:H7, and EPEC O127:H6 intimins, respectively. The predicted protein encoded by the upstream ORF (156 amino acids) shares 95%, 97%, and 99% amino acid identity with predicted proteins from C. freundii , EHEC O157:H7, and EPEC O127:H6, respectively. The high degree of sequence homology of the ORF and the eaeA gene of RDEC-1 with those of other AE bacteria suggests an evolutionary relationship of LEE and supports and facilitates the use of the RDEC-1 model for studying the role of LEE in pathogenesis.     

绵羊肺源性致病性大肠杆菌的分离与鉴定

【目的】近年来,羊呼吸系统疾病日益频发,由肠外致病性大肠杆菌感染引起的呼吸道疾病也日渐增多,给养羊业带来了一定经济损失。本文旨在确定新疆石河子地区某羊场表现为呼吸道感染症状的病死羔羊的细菌性病原及其特性。【方法】采用细菌常规分离鉴定结合16S rRNA基因序列分析的方法从发病羔羊的肺脏中分离鉴定细菌,并对分离株进行药敏试验、特异基因PCR检测、小鼠致病性试验及肺脏组织的病理学观察。【结果】从病死羔羊肺脏组织分离得到一株致病性大肠杆菌,该分离株呈多重耐药现象,检测到iutA、fyuA和ireA三种毒力基因;病变肺脏肺泡壁毛细血管充血,界限不清,支气管管腔充血,周围淋巴细胞浸润、增生。【结论】从病死羔羊肺中分离的细菌性病原是肠外致病性大肠杆菌(ExPEC)。     

Cloning and expression in Escherichia coli of the homoserine kinase (thrB) gene from Brevibacterium lactofermentum

Summary Five DNA fragments carrying the thrB gene (homoserine kinase E.C. 2.7.1.39) of Brevibacterium lactofermentum were cloned by complementation of Escherichia coli thrB mutants using pBR322 as vector. All the cloned fragments contained a common 3.1 kb DNA sequence. The cloned fragments hybridized among themselves and with a 9 kb BamHI fragment of the chromosomal DNA of B. lactofermentum but not with the DNA of E. coli. None of the cloned fragments were able to complement thrA and thrC mutations of E. coli. Plasmids pULTH2, pULTH8 and pULTH11 had the cloned DNA fragments in the same orientation and were very stable. On the contrary, plasmid pULTH18 was very unstable and showed the DNA inserted in the opposite direction. E. coli minicells transformed with plasmids pULTH8 or pULTH11 (both carrying the common 3.1 kb fragment) synthesize a protein with an M _r of 30,000 that is similar in size to the homoserine kinase of E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - SDS sodium dodecyl sulphate - TSB tripticase soy broth - m-DAP meso-diaminopimelic acid - Sm^r, Cp^r, Km^r, Am^r, Ap^r, Tc^r, MA15^r resistance to streptomycin, cephalotin, kanamycin, amykacin, ampicillin, tetracycline and microcin A 15, respectively     

抗辐射菌属-大肠杆菌间的穿梭载体的构建及荧光基因的表达

摘要：【目的】构建抗辐射菌属一大肠杆菌间的穿梭载体,通过此载体使荧光素酶基因在大肠杆菌中得到表达。【方法】以质粒pUE30、pGBM5及pKatCAT为基础,构建抗辐射菌属一大肠杆菌间的穿梭载体,将groEL启动子和荧光素酶基因lux+插入到构建的穿梭载体中得到穿梭表达载体,并将该载体转化大肠杆菌诱导荧光素酶基因的表达。【结果】成功构建了大小约为5.8 kb的抗辐射菌属一大肠杆菌间的穿梭载体pZT17,该载体在没有抗生素的非选择性培养基中能稳定存在。在穿梭载体pZT17的EcoRV部位插入含有groEL启动子和荧光素酶基因lux+的DNA片段,构建得到了穿梭表达载体pZTGL2;利用该表达载体在大肠杆菌中可诱导表达荧光素酶基因。【结论】构建的穿梭表达载体为以后用大肠杆菌高效表达来源于抗辐射菌的基因、特别是DNA损伤修复蛋白基因,提供了可能。     

Microarray analysis of differential gene expression in sensitive and resistant pig to Escherichia coli F18

In this study, Agilent two‐colour microarray‐based gene expression profiling was used to detect differential gene expression in duodenal tissues collected from eight full‐sib pairs of Sutai pigs differing in adhesion phenotype (sensitivity and resistance to Escherichia coli F18). Using a two‐fold change minimum threshold, we found 18 genes that were differentially expressed (10 up‐regulated and eight down‐regulated) between the sensitive and resistant animal groups. Our gene ontology analysis revealed that these differentially expressed genes are involved in a variety of biological processes, including immune responses, extracellular modification (e.g. glycosylation), cell adhesion and signal transduction, all of which are related to the anabolic metabolism of glycolipids, as well as to inflammation‐ and immune‐related pathways. Based on the genes identified in the screen and the pathway analysis results, real‐time PCR was used to test the involvement of ST3GAL1 and A genes (of glycolipid‐related pathways), SLA‐1 and SLA‐3 genes (of inflammation‐ and immune‐related pathways), as well as the differential genes FUT1, TAP1 and SLA‐DQA. Subsequently, real‐time PCR was performed to validate seven differentially expressed genes screened out by the microarray approach, and sufficient consistency was observed between the two methods. The results support the conclusion that these genes are related to the E. coli F18 receptor and susceptibility to E. coli F18.     

Hsh载体对外源基因在E. coli中高效表达的机制探讨

pHsh是根据大肠杆菌的热休克反应构建而成的新型表达载体, 受σ32调控。正常E. coli细胞的整个热休克反应持续时间约12 min, 而在携带有外源基因的高拷贝pHsh的E. coli细胞中, 外源基因却能持续高效表达4?10 h。为探求外源基因高效表达的机制, 以一个编码木聚糖酶的外源基因为代表, 首先研究了质粒拷贝数对木聚糖酶表达的影响, 接着通过Western-blot检测了携带质粒pHsh-xynIII和对照组携带pLac-xynIII的E. coli细胞在非诱导条件下(30 °C)和诱导条件下(30 °C→42 °C)胞内σ32的差异, 最后测定了不同温度下(30 °C、37 °C、42 °C、30 °C→42 °C)携带质粒(pHsh-xynIII)的E. coli细胞内稳定状态下热休克的水平(以木聚糖酶活性表征)。研究结果表明外源基因在pHsh中的高效表达是与3个方面密切相关的: pHsh质粒的高拷贝数增加了外源基因的剂量; pHsh的存在使E. coli细胞内σ32的水平较正常E. coli细胞显著增加了, 并最终增强了E. coli的热休克反应; 诱导状态下带有pHsh重组质粒的E. coli细胞内稳定状态下的热休克水平明显高于其它温度的水平。     

Cloning and expression of the Bacteroides fragilis YCH46 neuraminidase gene in Eschirichia coli and Bacteroides uniformis

Abstract A neuraminidase-encoding gene nanH of Bacteroides fragilis strain YCH46 was cloned into the cosmid vector pHC79. The nanH gene was subcloned from the cosmid and was located within a 2.2-kb Xho I- Kpn I fragment. Southern hybridization experiments demonstrated that the gene was present as a single copy on the bacterial chromosome. Neuraminidase activity expressed in the initial Escherichia coli clone was approximately 3600-fold lower than that expressed in B. fragilis YCH46. However, when nanH was transferred from E. coli to B. uniformis by mobilization of a shuttle plasmid, the transconjugant expressed 1100-fold higher activity than the E. coli donor did. These results suggest that modes of nanH expression in E. coli and Bacteroides are heterologous.     

大肠杆菌细胞周质底物结合蛋白gsiB基因的克隆及其表达条件的优化

为深入研究大肠杆菌谷胱甘肽转运系统的蛋白质结构和功能, 对该系统中的gsiB基因进行了克隆和表达条件的优化。根据大肠杆菌谷胱甘肽转运系统中底物结合蛋白gsiB基因序列, 利用PCR方法扩增到该基因的编码区序列, 利用SLIC (Sequence and ligation–independent cloning)方法直接将其插入pWaldo-GFPe中, 成功构建了重组表达质粒pWaldo-GFP-GsiB。将重组质粒转化不同的大肠杆菌表达菌株进行诱导表达, 通过改变培养温度和IPTG浓度等条件, 得到了能够大量表达目标蛋白的重组子。结果表明: 大肠杆菌BL21(DE3)是 gsiB基因表达的最佳宿主菌; 18℃低温诱导培养有利于gsiB基因的大量表达; 0.1 mmol/L IPTG足够诱导gsiB基因表达, 增加IPTG浓度(0.1 mmol/L~1.0 mmol/L)并不能明显地促进gsiB基因的表达。Western blotting结果显示目标蛋白质有表达, 其分子量大小与预期相符。     

大肠杆菌O23 O-抗原基因簇序列的破译及UDP-N-乙酰葡萄糖-C4异构酶的鉴定

采用鸟枪法破译大肠杆菌O23标准株的O-抗原基因簇序列,并用生物信息学的方法进行了基因注释和分析;采用基因缺失和互补的方法鉴定了O23的UDP-GlcNAc C4异构酶(Gne);用同源建模的方法构建了O23 Gne的高级结构并对其活性位点进行了分析;分析了不同血清型大肠杆菌O-抗原基因簇中gne基因的多样性;根据O23O-抗原基因簇中的特异基因筛选出了可用于大肠杆菌O23快速检测的特异DNA序列。