大肠杆菌trpBA基因的克隆表达

目的:提高大肠杆菌中色氨酸合成酶的表达量和表达活性。方法:利用PCR方法从大肠杆菌K-12的基因组中直接克隆出紧密连锁trpB和trpA基因(简称trpBA),并将其连接到原核表达载体pet22b( )中,得到重组质粒pet22b( )-trp-BA,转化大肠杆菌BL21,IPTG诱导重组蛋白表达,表达产物经SDS-PAGE分析并用比色法测定其活性。结果:凝胶电泳可见PCR扩增产物大小约为2kb,SDS-PAGE鉴定目的蛋白的Mr分别约为29000和44000,色氨酸合成酶α、β亚基分别得到了高效表达,色氨酸合成酶活性提高到对照菌的3.7倍。结论:成功构建了重组质粒pet22b( )-trpBA,色氨酸合成酶的表达量和表达活性在大肠杆菌中得到了提高,为高产色氨酸基因工程菌的构建奠定基础。     

大肠杆菌ppsA和tktA基因的串联表达

ppsA和tktA是芳香族氨基酸生物合成中心途径的两个关键酶基因,在大肠杆菌中,ppsA基因编码磷酸烯醇式丙酮酸合成酶A(PpsA),该酶催化丙酮酸合成磷酸烯醇式丙酮酸;tktA基因编码转酮酶A,该酶在磷酸戊糖途径中生成4-磷酸赤藓糖起主要作用。采用PCR方法从大肠杆菌K-12株中扩增到ppsA和tktA,并实现了两基因的高效表达,其中ppsA活性提高了10．8倍,tktA活性提高了3．9倍,当这两个基因串联在一个质粒上导入大肠杆菌进行表达时,PpsA的活性变化较大(2．1～9．1倍),TktA的活性相对稳定(3．9～4．5倍),且这两个基因单独表达和串联表达都能使芳香族氨基酸生物合成共同途径中关键中间产物DAHP的产量提高,且串联表达比单独表达较高。     

大肠杆菌ppsA和tktA基因的串联表达

ppsA和tktA是芳香族氨基酸生物合成中心途径的两个关键酶基因,在大肠杆菌中,ppsA基因编码磷酸烯醇式丙酮酸合成酶A（PpsA）,该酶催化丙酮酸合成磷酸烯醇式丙酮酸;tktA基因编码转酮酶A,该酶在磷酸戊糖途径中生成4-磷酸赤藓糖起主要作用。采用PCR方法从大肠杆菌K-12株中扩增到ppsA和tktA,并实现了两基因的高效表达,其中ppsA活性提高了10.8倍,tktA活性提高了3.9倍,当这两个基因串联在一个质粒上导入大肠杆菌进行表达时,PpsA的活性变化较大（2.1～9.1倍）,TktA的活性相对稳定（3.9～4.5倍）,且这两个基因单独表达和串联表达都能使芳香族氨基酸生物合成共同途径中关键中间产物DAHP的产量提高,且串联表达比单独表达较高。     

苯丙氨酸生物合成途径关键基因的串联表达

目的:改造大肠杆菌苯丙氨酸生物合成的中心代谢途径,优化关键酶基因pheA、aroF、ppsA、tktA的协同表达,进一步提高苯丙氨酸产量。方法:构建重组质粒pZE12-AFPT,鉴定后通过SDS-PAGE观察其蛋白表达量,并转入缺陷菌大肠杆菌MGΔ中构建工程菌,发酵培养后测量苯丙氨酸产量,与本室保存的重组质粒MGΔpZE12-AF做对比;构建重组质粒pZE21-AF和pZA31-PT,将后者转入感受态pZE12-AF和pZE21-AF中,得到双抗性质粒,并比较转化前后苯丙氨酸的产量。结果:工程菌MGΔpZE12-AFPT的苯丙氨酸产量比对照菌株MGΔpZE12-AF提高了近1.6倍,并且实现了4个串联基因的协同表达;质粒pZA31-PT转入pZE12-AF和pZE21-AF后,苯丙氨酸产量比原质粒pZE12-AF和pZE21-AF分别提高了近0.6倍和2.8倍。结论:实现了4个关键酶基因的串联表达,改造了苯丙氨酸的生物合成途径,使得苯丙氨酸产量有所提高,为进一步得到其高产菌株奠定了基础。     

外源基因在大肠杆菌中的高效表达

为了提高外源蛋白在大杨杆菌中的表达量,人们对大肠杆菌表达系统进行了许多研究。作者综述了有关外源基因在大肠杆菌中高效表达的研究进展。     

外源基因在大肠杆菌中表达研究进展

近年来,基因工程技术的迅速发展,大量有价值的蛋白质大肠杆菌中获得了高表达。多种表达系统的完善与发展,及蛋白质分离纯化技术的提高,异源蛋白的产量与纯度已不再是困扰人们的主要问题,人们开始更多地关注异源蛋白的活性,比活性及异源蛋白的正确性,完整性。随着这些问题的解决,重组蛋白的应用才能真正走向成熟。     

大肠杆菌\%otsA\%基因的克隆和表达

用ＰＣＲ方法扩增了１．５ｋｂ的ｏｔｓＡ基因片段,将该片段连接到多拷贝克隆载体后转化ｏｔｓＢＡ缺失和ｏｔｓＡ缺陷的大肠杆菌菌株,使转化株重新获得ｏｔｓＡ基因功能。生长曲线表明转化株在高渗培养基中生长良好,薄层层析法（ＴＬＣ）检测海藻糖实验说明转化株细胞诱导后合成海藻糖,ｏｔｓＡ基因的克隆和表达为赋予转基因植物抗高渗、耐干旱能力提供了实验依据和材料。     

大肠杆菌中外源基因的表达调节

大肠杆菌已经被广泛地应用于表达各种外源基因,但基因的表达受到多种因素的调节,而且不同的外源基因在大肠杆菌中的表达效率也有很大差异。本文从转录水平调节、翻译水平调节、培养条件调节等方面综述了大肠杆菌中外源基因的表达调节,以便认识其规律,有助于使用有效的方法提高外源基因在大肠杆菌中的表达效率。     

大肠杆菌tktA基因的克隆表达

tktA是芳香族氨基酸生物合成共同途径的关键酶基因之一,在大肠杆菌中,tktA编码转酮酶A,在磷酸戊糖途径生成4－磷酸赤藓糖中起主要作用。采用PCR方法从大肠杆菌K－12株中扩增到tktA,并实现了高效表达,tktA活性提高了3．9倍,并且使芳香族氨基酸生物合成共同途径中关键中间产物DAHP的产量有所提高。     

猪肥胖基因在大肠杆菌中的高效融合表达

采用PCR方法扩增猪肥胖基因编码原成熟蛋白cDNA序列,并在5′端加上BamHⅠ位点,3′端加上EoRⅠ位点,将5′端密码子CCC转变为大肠杆菌常见密码子CCG,扩增得到459bp的片段,克隆于融合表达载体p GEX-2TBamHⅠ和EcoRⅠ位点,酶切、测序正确,经0．1mmol/LIPTG诱导表达出一条约42kD的融合蛋白,其中26kD为pGEX-2T中带有的谷胱苷肽转移酶,16kD是猪肥胖基因表达产物瘦蛋白。利用非融合表达产品制备抗血清,检测融合表达的重组蛋白,Western-blot为阳性。     

Genome diversity at the serA-linked capsule locus in Escherichia coli

Individual isolates of Escherichia coli synthesize one of more than 70 chemically distinct polysaccharides which form the capsule. In this article we review the genetics of capsule production in E. coli and highlight what this is beginning to reveal in terms of the genetic basis of the structural diversity of polysaccharides. The serA-linked capsule locus can take three different allelic forms. Two of these are associated with capsule genes and are themselves internally variant, whilst the third form has not so far been implicated in capsule biogenesis. Thus the serA-linked region of the E. coli genome is strikingly polymorphic.     

重组共表达大肠杆菌细胞从D-葡萄糖一锅法生产D-甘露糖

【背景】D-甘露糖的酶促转化方法已受到相当大的关注。【目的】研究D-葡萄糖异构酶(D-glucoseisomerase,D-GIase)和D-来苏糖异构酶(D-lyxoseisomerase,D-LIase)共表达于大肠杆菌细胞生产D-甘露糖的工艺条件。【方法】将D-GIase和D-LIase基因片段合成后酶切连接到载体p CDFDuet-1上,构建p CDFDuet-Acce-DGI/Peba-DLI重组质粒并导入到大肠杆菌BL21(DE3)中共表达,通过摇瓶培养得到产D-GIase和D-LIase的菌体,测定该共表达细胞体系的反应条件。【结果】添加1 mmol/L Co~(2+),共表达体系酶的最适温度和p H分别为70°C和6.0。以浓度分别为100、300、500 g/L的D-葡萄糖为底物生产D-甘露糖,平衡后D-甘露糖质量浓度分别为13.8、38.1、62.6 g/L,相应的转化率分别为13.8%、12.7%、12.5%,D-葡萄糖、D-果糖和D-甘露糖的平衡比约为50:37.5:12.5。【结论】D-GIase和D-LIase在大肠杆菌细胞中组成的共表达体系通过一锅法可利用D-葡萄糖为底物生产D-甘露糖。     

Shiga toxin-producing Escherichia coli and enteropathogenic Escherichia coli in healthy goats in India: occurrence and virulence properties

AIMS: To describe the occurrence and virulence gene pattern of shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in healthy goats of Jammu and Kashmir, India. METHODS AND RESULTS: A total of 220 E. coli strains belonging to 60 different 'O' serogroups was isolated from 206 local (nonmigratory) and 69 migratory goats. All the 220 strains were screened for the presence of stx(1), stx(2), eaeA and hlyA genes. Twenty-eight E. coli (75.6%) strains from local and nine (24.3%) strains from migratory goats belonging to 18 different serogroups showed at least presence of one virulence gene studied. Twenty-eight strains (16.47%) (belonging to 13 different serogroups) from local goats carried stx(1) gene alone or in combination with stx(2) gene, while as only one strain (2%) from migratory goats possessed stx(2) gene alone. Interestingly in the present study none of the STEC strains carried eaeA gene. Similarly, none of the strains from local goats possessed eaeA and none of the migratory goats possessed stx(1) gene. Eight strains (16%) (belonging to four different serogroups) from migratory goats carried eaeA gene. Twenty-five (14.7%) and seven (14%) strains from local and migratory goats harboured hlyA gene respectively. CONCLUSIONS: Healthy goats of Jammu and Kashmir state serve as a reservoir of STEC and EPEC. Further studies in this direction are needed to work out whether or not they are transmitted to humans in this part of world. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report of isolation of STEC and EPEC strains from healthy goats in Jammu and Kashmir State of India, which could be a source of infection to humans.     

Genes coding for Shiga-like toxin and heat-stabile enterotoxin in porcine strains of Escherichia coli

Besides diarrheagenic enterotoxigenic Escherichia coli (ETEC) that produce classical heat stable and/or heat labile enterotoxins (STs, LTs) and the class of Shiga-like toxin-producing entero-hemorrhagic E. coli (EHEC), a new category of E. coli is defined sharing similarities with ETEC and EHEC. DNA hybridization studies indicate that some E. coli serovars from porcine origin harbor genes encoding cytotonic ST and cytotoxic Shiga-like toxin. The presence of two potent toxins might contribute to the virulence of such strains and should be taken into consideration when bio-assays are performed.     

Impact of microbial diversity on rapid detection of enterohemorrhagic Escherichia coli in surface waters

Enterohemorrhagic Escherichia coli (EHEC) are a physiologically, immunologically and genetically diverse collection of strains that pose a serious water-borne threat to human health. Consequently, immunological and PCR assays have been developed for the rapid, sensitive detection of presumptive EHEC. However, the ability of these assays to consistently detect presumptive EHEC while excluding closely related non-EHEC strains has not been documented. We conducted a 30-month monitoring study of a major metropolitan watershed. Surface water samples were analyzed using an immunological assay for E. coli O157 (the predominant strain worldwide) and a multiplex PCR assay for the virulence genes stx(1), stx(2) and eae. The mean frequency of water samples positive for the presence of E. coli O157, stx(1) or stx(2) genes, or the eae gene was 50%, 26% and 96%, respectively. Quantitative analysis of selected enriched water samples indicated that even in samples positive for E. coli O157 cells, stx(1)/stx(2) genes, and the eae gene, the concentrations were rarely comparable. Seventeen E. coli O157 strains were isolated, however, none were EHEC. These data indicate the presence of multiple strains similar to EHEC but less pathogenic. These findings have important ramifications for the rapid detection of presumptive EHEC; namely, that current immunological or PCR assays cannot reliably identify water-borne EHEC strains.     

改善大肠杆菌胞内氨基酰tRNA池提高外源基因表达水平

大肠杆菌是研究和高效表达异源蛋白的常用宿主细胞。由于大肠杆菌和真核生物及大部分古细菌在同义密码子上的使用有很大的差异,来源于真核或古细菌的外源基因在大肠杆菌中表达时,常常由于其带有稀有密码子而带来翻译方面的问题,如产生移码突变和表达量下降等,从而改变异源蛋白的表达质量和表达水平。在研究常见嗜热菌tRNA结构和组成的基础上,以古细菌α淀粉酶基因为例,采用增加有argU、ileY和leuWtRNA基因的大肠杆菌DE3为表达宿主,改善了胞内氨基酰tRNA池的大肠杆菌表达异源蛋白,表达量提高约9倍。     

大肠杆菌O150 O-抗原基因簇的破译和dTDP-鼠李糖合成酶基因的鉴定

利用鸟枪法对大肠杆菌O150 O-抗原基因簇进行测序,序列全长13551bp,用生物信息学的方法进行序列分析,共发现11个基因,分别为鼠李糖合成酶基因(rmlB、rmlD、rmlA、rmlC)糖基转移酶基因(3个)、O-抗原转运酶基因(wzx)和O-抗原聚合酶基因(wzy),另外还有两个基因功能未知。用PCR的方法筛选出了针对大肠杆菌O150的特异基因,可以用于基因芯片或PCR方法对大肠杆菌O150的快速检测。另外,通过进化分析发现大肠杆菌O150的O-抗原基因簇中携带有典型的大肠杆菌鼠李糖合成酶基因,并且这些基因参与了O-抗原基因簇间的重组以形成新的基因簇的过程。