7号淀粉酶链霉菌M1033木糖异构酶基因的启动子活性检测、转录起始点的确定

分别从重组质粒ｐＵＢ１及其Ｍ（１３）亚克隆Ｓ１中将７号淀粉酶链霉菌（ＳｔｒｅｐｔｏｍｙｃｅｓｄｉａｓｔａｔｉｃｕｓＮｏ．７）Ｍ１０３３（以下简称Ｓ．ｄｉ．Ｍ１０３３）木糖异构酶基因的－１９２－＋５８１ｂｐ片段克隆入链霉菌启动子探测质粒ｐＩＪ４０８３中,转化变铅青链霉菌（Ｓ．ｌｉｖｉｄａｎｓ）ＴＫ２４。通过对其邻苯二酚加双氧酶活性的检测表明,该片段具有启动子活性。应用Ｍ１３亚克隆Ｓ１和合成引物Ｐ６延伸制备放射性标记的单链ＤＮＡ探针;通过Ｓ．ｄｉ．Ｍ１０３３的总ＲＮＡ的Ｓ１核酸酶保护实验,确定了其转录的起始位点,并由此探讨了与木糖异构酶基因表达有关的一些因素。     

7号淀粉酶链霉菌M1033木糖异构酶基因的启动子活性检测,转录起始点…

分别从重组质粒ＰＵＢ１及其Ｍ１３亚克隆Ｓ１中将７号淀粉酶链霉菌（Ｓｔｒｃｐｔｏｍｙｃｅｓ ｄｉａｓｔａｔｉｃｕｓ Ｎｏ．７）Ｍ１０３３（以下简称Ｓ。ｄｉ．Ｍ１０３３）木糖异构酶基因的－１９２－＋５８１ｂｐ片段克隆入链霉菌启动子探测质粒ＰＩＪ４０８３中,转化变铅青链霉菌（Ｓ。ｌｉｖｉｄａｎｓ）ＴＫ２４。通过对其邻苯二酚加双氧酶活性的检测表明,该片段具有启动子活性。应用Ｍ１３亚克隆Ｓ１和合成引物Ｐ６延     

利用同源重组建立地中海拟无枝菌酸菌U32染色体的基因置换/中断系统

地中海拟无枝菌酸菌U32是力复霉素SV的工业产生菌,其遗传操作一直是一个难题。在该菌株DNA高效电转化的基础上,利用同源重组的原理,建立了地中海拟无枝菌酸菌染色体的基因置换/中断系统。通过大肠杆菌重组质粒pDK110构建、转化及两步重组筛选,成功地用α淀粉酶基因（amy）取代了地中海拟无枝菌酸菌U32染色体上的3-氨基-5-羟基苯甲酸合成酶基因（ahbas）。第一步单交换和第二步双交换的频率分别是0.5%～0.7% 和 2%。将质粒pDK110变性后转化可显著提高重组频率,在第二步筛选双交换前对单交换重组子进行电击也能够提高其双交换重组的频率。此外,通过转化构建的两端带同源区段的线性DNA片段及一步重组筛选,我们在地中海拟无枝菌酸菌U32染色体的amrD,rifO基因中间插入了阿普拉霉素抗性基因（apr）,其效率约为30～50转化子/μgDNA。     

大肠杆菌胸腺嘧啶合成酶thyA缺失突变菌株的构建

大肠杆菌K12 DY330菌株的染色体上整合有一种新型的同源重组系统——缺陷型λ原噬菌体同源重组系统。以DY330为出发菌,通过同源重组构建大肠杆菌thyA-株DY330TI,其基因组特点是：thyA基因除保留N端的1～49氨基酸残基相对应的必需核苷酸序列外,将其余部分全部缺失;此外,还敲除了DY330TI中与缺陷型λ原噬菌体同源重组功能相关的基因,从而尽可能避免了通过同源重组产生回复突变的可能性。通过大肠杆菌thyA基因对该突变株的转化实验,检测转化子的回复突变率,进一步证实该突变株的突变性状稳定,为构建以thyA为选择标志的大肠杆菌染色体质粒平衡致死系统提供了合适的缺陷型宿主菌。     

一种快速、精确构建大肠杆菌组氨酸营养缺陷型的方法*

将表达Red体内重组蛋白的质粒pKD46转化大肠杆菌DH5α,用 5′端与组氨酸基因同源 ,3′端与卡那霉素抗性基因同源的引物获得具有卡那霉素抗性基因的PCR产物 ,然后电击转化DH5α,在λRed重组系统的帮助下 ,通过卡那霉素抗性基因两侧的组氨酸基因序列在体内与大肠杆菌染色体上的组氨酸基因发生同源重组 ,置换了DH5α组氨酸操纵元中的hisDCB基因 ,最后利用卡那霉素抗性基因两端的FRT位点 ,通过FTP位点专一性重组将卡那霉素抗性基因去除 ,最终获得了不具抗性的大肠杆菌组氨酸营养缺陷型菌株。为在大     

一种快速、精确构建大肠杆菌组氨酸营养缺陷型的方法

将表达Red体内重组蛋白的质粒pKD46转化大肠杆菌：DH5α,用5′端与组氨酸基因同源,3′端与卡那霉素抗性基因同源的引物获得具有卡那霉素抗性基因的PCR产物,然后电击转化DH5α,在λRed重组系统的帮助下,通过卡那霉素抗性基因两侧的组氨酸基因序列在体内与大肠杆菌染色体上的组氨酸基因发生同源重组,置换了DH5α组氨酸操纵元中的hisDCB基因,最后利用卡那霉素抗性基因两端的FRT位点,通过FTP位点专一性重组将卡那霉素抗性基因去除,最终获得了不具抗性的大肠杆菌组氨酸营养缺陷型菌株。为在大肠杆菌及其他菌株中快速、精确的构建营养缺陷型菌株提供了有益的参考。     

染色体整合系统在多价疫苗构建中的应用研究

通过同源重组将编码异源抗原的DNA整合到减毒的鼠伤寒沙门氏菌的染色体上,获得了表达霍乱毒素B亚单位（CTB）的双价活疫苗候选株。该系统包括两个步骤：首先将GisOG缺失突变的DNA片段整合进鼠伤寒沙门氏菌疫苗候选株SL3261的染色体上,得到His营养缺陷型。然后,用带有CTB抗原基因的完整HisOGDNA片段置换HisOG缺失的DNA片段,获得表达CTB的His回复的SL3261菌株（命名为TT201）。Southern杂交证明,TT201菌株的染色体带有CTB抗原基因。Westernblot分析表明,TT201菌株能表达CTB,且具有很好的稳定性。用重组菌株口服免疫接种小鼠,能够激发抗CTB抗体的产生。TT201菌株是一种潜在的双价疫苗候选株。     

新型高密度发酵基因工程菌G830

运用PCR方法,从磷酸乙酰转移酶（Pta）－乙酸激酶（Ack)代谢途径缺失菌株E.coliPA1染色体上,扩增出天氨酸激酶－1-高丝氨酸脱氢酶－I（thrA）和高丝氨酸激酶（thrB）基因部分序列,构建了整合型重组质粒pVHb-Kan;应用染色体－质粒同源重组的方法,将透明颤菌血红蛋白（Vitreoscila haemoglobin,VHb)基因整合到大杆菌PA1染色体上的thr操纵子,构建了新型整合工程菌G830。在高密度发酵条件下,G830的细胞呼吸强度、能量代谢、最高菌密度和细胞干重,均明显优于对照菌株PA1和BL21;重组蛋白脯氨酰内肽酶在G830和PA1中获得稳定高表达;重组菌生长状况及发酵指标均与空宿主菌基本一致且表达质粒能维持较好的稳定性。整合型vhb的表达及乙酸代谢途径（Pta-Ack）的缺陷,改善了宿主在贫氧条件下的生长,且促进了重组蛋白的表达。该工程菌具有良好的氧耐受力,且乙酸积累得到大幅度降低,可作为适于高密度发酵的基因工程菌。     

生技霉素稳定型基因工程菌的构建

运用同源重组技术将异戊酰基转移酶基因整合至螺旋霉素产生菌（ＳｔｒｅｐｔｏｍｙｃｅｓｓｐｉｒａｍｙｃｅｔｉｃｕｓＦ２１）的染色体上,构建了稳定的生技霉素基因工程菌。在不加压的情况下传代,菌种携带选择性遗传标记情况、生长、发酵效价及发酵产物的ＴＬＣ分析均表明此基因工程菌有较好的遗传稳定性,且发酵效价及产物的组分均得到改善。Ｓｏｕｔｈｅｒｎ杂交证明外源基因在螺旋霉素产生菌染色体上的整合情况。     

产L-乳酸的运动发酵单胞菌代谢工程菌株的构建

以运动发酵单胞菌(Zymomonas mobilis)CP4基因组DNA为模板,采用PCR技术克隆得到其丙酮酸脱氢酶基因(pdc)同源下游p3片段,并连接到广谱宿主载体pBBR1MCS3-Ppdc-ldhL中构建了重组质粒pBBR1MCS3-Ppdc-ldhL-p3,将此重组质粒转化到受体菌Z.mobilis CP4中,分别以Ppdc和p3片段作为同源上游和下游片段,利用同源双交换重组技术将重组质粒中的ldhL基因置换了Z.mobilis染色体中的pdc基因,得到重组菌株Z.mobilis CP4(△pdc∷ldhL).测得重组菌株乳酸产量为10.8g/L,明显高于出发菌株,说明初步成功构建了产L-乳酸的运动发酵单胞菌代谢工程菌株.     

同源重组菌株M53的构建及其作为链霉菌通用克隆受体的可行性

为了发展优良的链霉菌宿主系统 ,以带有硫链丝菌素抗性的同源重组葡萄糖异构酶 (GI)缺陷型菌株M10 33XW78,M10 33XW194为出发菌株 ,利用摇瓶、影印和负筛的方法 ,获得一株既无GI活性又对硫链丝菌素敏感的回复菌株 ,命名为淀粉酶链霉菌M5 3.通过染色体PCR检测、酶切图谱鉴定、序列分析等方法 ,确认M5 3含有和M10 33一致的 1 2kb葡萄糖异构酶基因 ,但在结构基因345～ 10 95bp片段内有 17个碱基发生突变 .这说明在染色体内自发同源重组过程中 ,有低频率的突变位点引入 .酶活力测定和SDS PAGE分析表明 ,该突变的GI基因不表达 4 2 5KD葡萄糖异构酶 ,这为M5 3菌株发展成为优良的链霉菌宿主提供了足够的表达空间 .一系列的转化实验也证明了M5 3菌株很可能是一种新型链霉菌克隆受体     

Construction of new stable strain over-expressing the glucose isomerase of the Streptomyces sp. SK strain

In order to over express the xylA gene of Streptomyces sp. SK strain, it was cloned under the control of the constitutive ermE-up promoter. This construct was integrated through site-specific recombination process into the chromosome of a Streptomyces violaceoniger glucose isomerase deficient strain using the non-replicative vector pTS55. The resulting CBS4 strain shows a perfect stability in the absence of selection pressure. Its glucose isomerase activity was about four and nine-fold greater, than that obtained from Streptomyces sp. SK, respectively fully induced or not by xylose.     

Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function

A recA deletion mutant of Mycobacterium smegmatis has been isolated by homologous recombination using a sacB counterselection strategy. Deletion of the recA gene from the chromosome was demonstrated by Southern hybridizations and by polymerase chain reaction (PCR). Western analysis using anti-RecA antibodies confirmed that the RecA protein was not made by the mutant strain. The recA deletion strain exhibited enhanced sensitivity to UV irradiation and failed to undergo homologous recombination. The results obtained from the recombination assays suggest that in wild-type M. smegmatis the majority of colonies arise from single cross-over homologous recombination events with only a very minor contribution from random integrations. The deficiencies in UV survival and recombination were complemented by introduction of the cloned M. smegmatis recA gene. Overexpression of RecA was found to be toxic in the absence of recX , which is found downstream of and co-transcribed with recA and is thus also affected by the deletion of recA . The M. smegmatis recA deletion strain was also complemented by the M. tuberculosis recA gene with or without its intein; most importantly, the frequency of double cross-over homologous recombination events was identical regardless of whether the M. tuberculosis recA gene contained or lacked the intein. Thus, the low frequency of homologous recombination observed in M. tuberculosis is not due to the presence of an intein-coding sequence in its recA gene per se .     

7号淀粉酶链霉菌M1033木糖异构酶基因序列分析

测定了来自海南的7号淀粉酶M1033木糖异构酶(Ⅺ)基困的DNA序列。：该酶的结构基因由l161bp组成,相当于387个氨基酸残基。其GC含量为72．1克分子％,密码子第三位的Gc利用率达98克分子％。在氨基酸序列上,M1033的木糖异构酶与其它放线菌菌株的相比具有较高的同源性;特别是与3种链霉菌菌株的同源性高达90%左右。     

Construction of Double-Copy Glucose Isomerase Gene Engineering Strain of Streptomyces diastaticus by Homologous Recombination

The plasmid pUT for homologous recombination was constructed by the insertion of the 1.1-kb thiostrepton resistance (tsr ^R) gene into the E. coli plasmid pUB1-GI1. Plasmid pUTK was produced through ligating the cleaved plasmid pUT by KpnI. After pUT and pUTK were introduced into Streptomyces diastaticus No.7 strain M1033 (SM33) by protoplast transformation, a series of tsr^R transformants were obtained, further based on enzyme assays. These results for polymerase chain reaction (PCR), DNA sequencing, restriction enzyme digestion, and recovery of cloned fragments from the transformant chromosome demonstrated the plasmid pUT and pUTK had integrated into the SM33 chromosome in three different patterns of single cross-over by homologous recombination. This directly results in double-copy GI gene in the transformant chromosome, of which one is wild-type GI gene, the other mutant GI (GIG138P, GI1) gene. Among the strains of the three kinds of recombinant patterns, one transformant was chosen and named K1, T2, and T3, respectively. The further identification of the three recombinant strains by PCR, DNA sequencing, restriction enzyme digestion, and Southern hybridization also proved there is a double-copy GI gene within their chromosome. Enzyme activity assay and thermostability analysis indicated that all three engineering strains expressed not only wild-type enzyme but also mutant GI. Received: 9 July 2001 / Accepted: 8 August 2001     

葡萄糖异构酶基因工程菌的改造初探

葡萄糖异构酶（ｇｌｕｃｏｓｅｉｓｏｍｅｒａｓｅ,ＧＩ）是使用量最大的工业酶之一,可用于高果糖浆的生产,也可以用含木聚糖物质及废料为底物发酵生产乙醇,具有重要的经济价值．本文选择了表达载体ｐＢＶ２２０［１］,利用ＰＣＲ方法删除了原表达质粒ｐＴＫＤＧＩ１中ＧＩ结构基因５′端多余的核苷酸,并添加了合适的酶切位点,重新构建了能在大肠杆菌ＤＨ５α中高效表达ＧＩＧ１３８Ｐ的表达质粒ｐＢＺＧＩ１．传代实验表明,新表达体系的稳定性明显优于原表达体系．粗酶液经热处理、ＤＥＡＥＳｅｐｈａｒｏｓｅＦＦ和分子筛Ｓｅ…     

结核分枝杆菌EF4敲除菌株的构建

EF4是一个由 lepA 基因编码的与蛋白质翻译密切相关的延伸因子,在细菌中高度保守,但其确切功能和分子机制尚不清楚,在结核分枝杆菌中的功能至今未见报道。为探索EF4在结核分枝杆菌中的功能,需构建一株结核分枝杆菌 lepA 基因敲除株。本研究以结核分枝杆菌H37Ra全基因组DNA为模板,设计并通过聚合酶链反应(polymerase chain reaction,PCR)扩增 lepA 基因左、右臂,连接到p0004S质粒,构建同源重组质粒p0004S-Δ lepA 。然后,通过噬菌体体外包装,将p0004S-Δ lepA 质粒连接到phAE159质粒,构建phAE159-Δ lepA 噬菌体包装质粒。在耻垢分枝杆菌mc 2155中大量扩增噬菌体并受结核分枝杆菌侵染进行同源重组,筛选阳性克隆,从基因组和蛋白质表达水平检测该突变株中 lepA 基因及EF4蛋白表达。PCR结果显示,敲除株基因组中 lepA 基因已被潮霉素抗性基因成功替换,蛋白免疫印迹结果显示该敲除株中无EF4表达,表明其为成功构建的Ra Δ lepA 。生长曲线分析显示,正常培养条件下,结核分枝杆菌野生株与敲除株生长趋势一致。敲除株与野生株在菌落形态上有一定差异,相比于野生株,Ra Δ lepA 菌落颜色发黄,凸起偏厚,生长过程中生物膜皱褶较少。耐胁迫能力分析显示,与野生株相比,Ra Δ lepA 耐热、抗去垢剂、抗氧化能力无显著差异,但耐酸性环境能力明显增强。本研究利用噬菌体介导的重组法成功构建了结核分枝杆菌 lepA 基因敲除株,为后续研究结核分枝杆菌EF4的功能提供了重要基础。     

利用Red同源重组系统构建兔次黄嘌呤-鸟嘌呤磷酸核糖转移酶基因打靶载体

利用EL350基因工程菌进行同源重组,成功进行基因敲除已有报道,但利用该系统进行兔次黄嘌呤-鸟嘌呤磷酸核糖转移酶(Hypoxanthine guanine phosphoribosyl transferase,HPRT)基因突变和基因打靶方面的研究还没有报道。本实验首先在已经筛选到含有兔全长HPRT基因BAC克隆（LBNL1-304M19）的基础上,利用Red重组系统,通过Gap-Repair方式从此克隆上将一段47Kb无启动子的HPRT基因组片段（不含有第1个外显子）克隆到pBACLinkSp质粒上,产生pBACLinkSp-rHPRT质粒。然后基于pBACLinkSp-rHPRT质粒,设计不同的同源臂,从而删除了HPRT基因的不同编码区,成功构建了三个不同的HPRT基因打靶载体。同时对利用同源重组技术敲除不同大小的DNA片段的效率进行了研究。基于本实验所构建的三个不同的兔HPRT基因打靶载体,为探索兔成纤维细胞和胚胎干细胞基因打靶的适宜条件,及进一步获得兔HPRT基因敲除动物疾病模型奠定了基础。     

Gene replacement by homologous recombination in Mycobacterium bovis BCG

Gene replacement by homologous recombination is a powerful tool for fundamental studies of gene function, as well as allowing specific attenuation of pathogens, but has proved difficult to achieve for Mycobacterium tuberculosis. We have used a plasmid-based test system to demonstrate the occurrence of homologous recombination in the tuberculosis vaccine strain Mycobacterium bovis BCG, and we have successfully replaced a target gene in BCG by homologous recombination, using a shuttle plasmid. Specific inactivation of selected genes will facilitate study of virulence factors and drug resistance as well as allowing rational attenuation of M. tuberculosis for the production of new vaccines.     

Immobilization of a recombinant strain producing glucose isomerase on SiO2-xerogel and properties of prepared biocatalysts

An original method of immobilization of nongrowing microorganism cells on xerogel of silicon dioxide containing insoluble hydroxyl compounds of cobalt(III) has been developed. A recombinant strain producing glucose isomerase has been constructed on the basis of Escherichia coli with the use of a gene of Arthrobacter nicotianae. It was revealed that glucose isomerase activity and stability of biocatalysts prepared on the basis of the recombinant E. coli strain was 3-5 times greater compared with the biocatalysts prepared with the use of the donor strain A. nicotianae. Under conditions of continuous hydrolysis of 3 M fructose at 62-65 degrees C in a fixed bed reactor, time of half-inactivation of the biocatalysts prepared from the recombinant strain and A. nicotianae was -60 and -25 days, respectively.