鼠李糖乳杆菌D-(+)-乳酸脱氢酶基因的克隆及在大肠杆菌中的表达

利用PCR的方法从鼠李糖乳杆菌基因组DNA中扩增到D-(+)-乳酸脱氢酶基因(ldhD),并连接到载体pSE380上,构建表达质粒pSE-ldhD,将重组质粒pSE-ldhD转化大肠杆菌BL21(DE3),重组菌株经IPTG诱导表达,SDS-PAGE电泳分析表明ldhD在大肠杆菌中实现了表达,表达产物的分子量约为37kD。同时采用紫外分光光度法测定D-乳酸脱氢酶的酶活,测得重组菌株的D-乳酸脱氢酶活力为5.4U/mL,最适反应温度为35℃,最适pH为5.6。     

黑曲霉α-葡萄糖苷酶cDNA的克隆及表达

研究黑曲霉(Aspergillus niger)M-1菌株的α-葡萄糖苷酶基因在大肠杆菌中的克隆及表达。以M-1菌株的总RNA为模板,利用RT-PCR扩增α-葡萄糖转苷酶的cDNA,重组到Trc启动子控制下的表达载体pSE380中,构建重组质粒pSE-αtg,转入大肠杆菌BL21(DE3)进行IPTG诱导表达。初步研究表明:重组蛋白具有葡萄糖苷酶活性,最适pH为6.0,最适温度为45℃,金属离子Cu2 和Mn2 对酶活力有明显的促进作用。添加1.6mmol/L IPTG对重组菌的诱导作用最大,在培养中添加麦芽糖,对重组菌产酶有显著的促进作用。     

家蝇伴侣蛋白TCP-1基因的序列分析、克隆及诱导表达

旨在对EST筛选得到的家蝇伴侣蛋白TCP-1(MD-TCPⅠ)基因进行序列分析,克隆其cDNA序列并在大肠杆菌中诱导表达。采用EST测序技术从已构建的家蝇幼虫cDNA质粒文库中筛选到MD-TCPⅠ基因,对其进行序列测定和分析。以该基因的cDNA文库质粒为模板,通过PCR的方法进行扩增,以pET-28a(+)为载体构建重组质粒,再转化到表达宿主大肠杆菌BL21(DE3)中,IPTG诱导表达。表达产物通过SDS-PAGE进行鉴定。结果显示,MD-TCPⅠ基因ORF全长753 bp,编码250个氨基酸,理论分子量27.07 kD;等电点5.92,该序列编码的蛋白属于热休克蛋白60家族的TCP。构建了正确基因序列MD-TCPⅠ重组表达质粒,重组蛋白在大肠杆菌BL21(DE3)中诱导表达。     

枯草杆菌脂肪酶基因在大肠杆菌中的诱导分泌表达

借助生物信息学对已克隆的枯草杆菌脂肪酶LipB2全长基因序列进行比对分析。结果显示该脂肪酶基因全长635bp,编码包括31个氨基酸分泌型信号肽在内的211个氨基酸,与NCBIGenBank中已报道的枯草杆菌属脂肪酶核苷酸序列有94.0%的一致性。将该基因克隆到pET-28a(+)表达载体上,转化大肠杆菌BL21(DE3),利用枯草杆菌脂肪酶的信号肽序列进行了分泌表达。SDS-PAGE电泳显示分泌表达的脂肪酶分子质量约为21kD。对表达条件优化后,在30℃、大肠杆菌菌液OD600值为1.8、乳糖诱导浓度为1.5mM、摇瓶发酵10h后大肠杆菌分泌表达26.0U/mL重组脂肪酶,相比较IPTG的诱导,既实现了脂肪酶的高效表达,又节省了成本。     

A亚群禽白血病病毒囊膜基因gp85片段在大肠杆菌中的表达

将RAV-1囊膜基因gp85片段亚克隆到表达质粒pET-21d(+)中得到重组表达质粒pET-21d-RAV-1env(BglII/SalI),序列分析表明该插入片段的核苷酸序列和阅读框都与RAV-1囊膜基因相应序列相同.用其转化大肠杆菌BL21(DE3)并经IPTG诱导,SDS-PAGE分析表明RAV-1囊膜基因融合蛋白表达产物约20kD,与理论值相符;IPTG诱导起始时间比诱导持续时间对表达量的影响更大.     

炭疽芽孢杆菌保护性抗原在大肠杆菌中的表达及纯化

按照炭疽芽孢杆菌保护性抗原（PA）基因成熟肽编码序列设计引物,从炭疽杆菌pOX1质粒中扩增出PA基因片段,将该片段定向插入到原核表达载体pET-28a中,获得了pET-PA原核表达重组质粒,限制性酶切分析和DNA序列测定均证实该克隆插入片段为PA基因的成熟呔编码序列。将该重组质粒转化大肠杆菌BL21（DE3）,经IPTG诱导,重组蛋白在大肠杆菌表达系统中获得了高效表达;Western印迹分析表明表达产物具有良好的免疫学活性。     

一种不依赖钙离子的酸性α淀粉酶基因的克隆表达

以产酸性淀粉酶菌株Bacillus sp.CN7的基因组DNA为模板,PCR扩增到α淀粉酶成熟肽基因,将该基因插入表达质粒pSE380中,构建重组质粒pSE380-cn7a。将重组质粒导入到Escherichia coli JM109中,IPTG诱导表达。重组酶经Sephacryl S300、Ni-NTA纯化后测定其酶学性质。重组酶CN7A的最适温度为65°C,最适pH为5.5?6.0,对可溶性淀粉的Km值为3.784g/L,最大反应速度为101.2mg/(L·min),该酶的热稳定性不依赖钙离子。     

丁酸梭菌1，3-丙二醇氧化还原酶基因dhaT的克隆及在大肠杆菌中的表达

以丁酸梭菌（Clostridium butyricum）基因组DNA为模板,利用PCR技术扩增得到1,3-丙二醇氧化还原酶基因dhaT,将它连接到pMD18一T载体上,得到重组质粒pMD—dhaT,对此重组质粒进行序列测定,对其DNA序列分析表明,dhaT基因全长为1 158bp。将dhaT基因插入表达载体pSE-380中,构建成重组子pSE—dhaT,并在大肠杆菌JMl09中进行诱导表达。研究表明,以1,3-丙二醇为底物时,基因工程菌在37℃下,以1．0mmol／L IPTG诱导14h,酶活力达到16．28U／mL,比原始菌株提高5、6倍。     

A亚群禽白血病病毒囊膜基因gp85片段在大肠杆菌中的表达

将RAV－1囊膜基因gp85片段来克隆到表达质粒pET-21d( )中得到重组表达质粒pET-21d-RAV-1env(BelⅡ/SalⅠ）序列分析表明该插入片段的核苷酸序列和阅读框都与RAV－1囊膜基因相应序列相同。用其转化大肠杆菌BL21（DE）3并经IPTG诱导,SDS－PAGE分析表明RAV－1囊膜基因融合蛋白表达产物约20kD,与理论值相符;IPTG诱导起始时间比诱持续时间对表达量的影响更大。     

小鼠Nanog基因的克隆及其在大肠杆菌中的表达

按照nanog基因编码序列设计合成引物,利用RT-PCB从小鼠的囊胚期胚胎中扩增得到该 基因,并将该基因克隆到pET-28b(+)载体上,获得pET-28b(+)-nanog原核表达重组质粒,限制 性酶分析和DNA序列测定均证实该克隆插入片段为nanog基因编码序列。重组质粒转化大肠杆 菌BL21(DE3),经IPTG诱导表达,在大肠杆菌表达系统中获得了高效表达,western杂交证实该 蛋白具有6-His抗原活性,从而证实目的蛋白为Nanog蛋白。     

丙酮丁醇梭菌ldhL基因的克隆及其在大肠杆菌中的表达

首次从丙酮丁醇梭菌(Clostridium acetobutylicum ATCC824)中克隆得到L-乳酸脱氢酶(L-lactate dehydrogenase,ldhL)基因,并将其连接到pSE380表达载体上,得到重组质粒pSE380ldhL,将重组质粒转化到乳酸脱氢酶和丙酮酸裂解酶缺陷的Escherichia coli FMJl44大肠杆菌中进行表达。SDS-PAGE分析表达产物的分子量约为34kD,摇瓶发酵后用HPLC检测分析L-乳酸产量为2.4g/L,纯度达到99.9%,不需要再进行手性分离,为以后在工业上生物法生产高纯度的L-乳酸打下基础。     

酿酒酵母gpd1和hor2基因在大肠杆菌中的共表达

利用途径工程的方法,在大肠杆菌中构建一条新的产甘油的代谢途径。从酿酒酵母(Saccharomyces cerevisiae)克隆3-磷酸甘油脱氢酶基因(gpd1)和3-磷酸甘油酯酶基因(hor2),并将两个基因串连到启动子trc的下游,构建由trc启动子控制的能高效表达的多顺反子重组质粒pSE-gpd1-hor2,将重组质粒导入大肠杆菌BL21菌株中,构建得到的重组菌株GxB-gh能将葡萄糖转化为甘油。结果表明重组菌株GxB-gh以葡萄糖为底物进行发酵,甘油产量为46．67g／L,葡萄糖的转化率为42.87％。这为利用工程菌绿色生产甘油进行了前期的探索,也为进一步构建能生产1,3-丙二醇的工程菌打下了良好的基础。     

Plasmid pSE21 of Streptomyces erythraeus strains

Crossing of S. erythraeus 4, a laboratory strain (NRRL 2338) containing a family of plasmids with S. erythraeus 1, a plasmid-free strain resulted in formation of strain 6. A multi-copy plasmid pSE21 11.5 kb in length was isolated from S. erythraeus 6. A detailed restriction map of plasmid pSE21 was constructed. Its cloning to E. coli YM83 on vector pUC19 showed that plasmid pSE21 was not stable in E. coli. It was found that the status of plasmid pSE21 changed in relation to the physiological state of S. erythraeus 6. Southern hybridization of the plasmid pSE21 DNA with the total DNA of the cultures of S. erythraeus 1 maintained for various periods at 4 degrees C demonstrated that plasmid pSE21 was present in S. erythraeus 1 in an autonomous state in 0.1 to 0.2 copies per genome. The number of the plasmid pSE21 copies could be decreased. The chromosomal DNA of S. erythraeus 1 contained the DNA sequences highly homologous to those of plasmid pSE21. It was assumed that during the crossing of S. erythraeus 4 with S. erythraeus 1 the genetic element from the donor strain was transferred to the recipient strain which in some way changed the plasmid pSE21 status and imparted the multicopy pattern to it. Further investigation of the plasmid pSE21 properties and construction of a vector for S. erythraeus on the plasmid basis are under way.     

利用冰核蛋白N末端结构域在大肠杆菌表面展示粘质沙雷氏菌脂肪酶

【目的】利用细胞表面工程技术将活性脂肪酶展示于大肠杆菌细胞表面并对展示脂肪酶的酶学性质进行研究。【方法】将丁香假单胞菌冰核蛋白N末端结构域序列与粘质沙雷氏菌脂肪酶编码基因融合,构建成脂肪酶表面展示载体,并转化大肠杆菌BL21(DE3)。【结果】重组菌以终浓度0.05 mmol/L异丙基硫代-D-半乳糖苷(IPTG)、25°C条件下诱导培养,16 h后表面展示脂肪酶活力达到最大值1 852 U/g细胞干重。表面展示酶的最适pH为9.0,最适反应温度为40°C,表面展示酶热稳定性较游离酶有较大提高,在40°C孵育1 h后仍能保持90%以上的酶活力。【结论】以上结果表明细菌表面展示技术为脂肪酶固定提供了一个很有前景的替代方法。     

橡胶树内生沙雷氏菌脂肪酶基因SmLipase1的克隆和表达分析

脂肪酶在洗涤剂、食品、纸浆以及生物柴油等多个工业领域被广泛应用。不同的应用领域对脂肪酶的酶学特性有不同的要求,因此挖掘脂肪酶基因资源具有重要意义。本研究报道从橡胶树内生菌Serratiamarcescens ITBB5-1中克隆得到一个脂肪酶基因,命名为Sm Lipase1,编码662个氨基酸,pI 4.82,能分泌脂肪酶到培养基中。构建重组表达载体pET22b-Sm Lipase-1,转化大肠杆菌菌株BL21(DE3)。本研究表明该转基因菌株能高效表达脂肪酶,并在平板培养基上形成较大的透明圈,转基因菌株发酵培养所获得的培养液酶活性达到26 U/mL。此外,该酶还具有催化油脂与甲醇反应生产脂肪酸甲酯的活性,在生物柴油领域具有一定的开发价值。     

In vivo functional expression of a screened P. aeruginosa chaperone-dependent lipase in E.coli

ABSTRACT: BACKGROUND: Microbial lipases particularly Pseudomonas lipases are widely used for biotechnological applications. It is a meaningful work to design experiments to obtain high-level active lipase. There is a limiting factor for functional overexpression of the Pseudomonas lipase that a chaperone is necessary for effective folding. As previously reported, several methods had been used to resolve the problem. In this work, the lipase (LipA) and its chaperone (LipB) from a screened strain named AB which belongs to Pseudomonas aeruginosa were overexpressed in E.coli with two dual expression plasmid systems to enhance the production of the active lipase LipA without in vitro refolding process. RESULTS: In this work, we screened a lipase-produced strain named AB through the screening procedure, which was identified as P. aeruginosa on the basis of 16S rDNA. Genomic DNA obtained from the strain was used to isolate the gene lipA (936 bp) and lipase specific foldase gene lipB (1023 bp). One single expression plasmid system E.coli BL21/pET28a-lipAB and two dual expression plasmid systems E.coli BL21/pETDuet-lipA-lipB and E.coli BL21/pACYCDuet-lipA-lipB were successfully constructed. The lipase activities of the three expression systems were compared to choose the optimal expression method. Under the same cultured condition, the activities of the lipases expressed by E.coli BL21/pET28a-lipAB and E.coli BL21/pETDuet-lipA-lipB were 1300U/L and 3200U/L, respectively, while the activity of the lipase expressed by E.coli BL21/pACYCDuet-lipA-lipB was up to 8500U/L. The lipase LipA had an optimal temperature of 30[degree sign]C and an optimal pH of 9 with a strong pH tolerance. The active LipA could catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meant that LipA was able to catalyze esterification reaction. The most suitable fatty acid and alcohol substrates for esterification were octylic acid and hexanol, respectively. CONCLUSIONS: The effect of different plasmid system on the active LipA expression was significantly different. pACYCDuet-lipA-lipB was more suitable for the expression of active LipA than pET28a-lipAB and pETDuet-lipA-lipB. The LipA showed obvious esterification activity and thus had potential biocatalytic applications. The expression method reported here can give reference for the expression of those enzymes that require chaperones.     

Genetic mobility of plasmids of Streptomyces erythraeus

Grossing of S. erythraeus 4 with S. erythraeus 1 resulted in transfer of genetic elements from strain 4 to strain 1 as evidence by the 20 and 18 kb fragments in the experiments on DNA-DNA hybridization. The presence of the genetic elements in strain 1 was the cause of plasmid pSE 21 mobility. In strain 6, a derivative of S. erythraeus 1 plasmid pSE 21 was accompanied by other extrachromosomal DNAs characterized by high instability. During storage of the strain at a temperature of 4 degrees C for more than 1 or 2 months the number of the plasmid pSE 21 copies decreased. When the strain was stored for longer periods (6 months or more) the plasmid DNA was not detectable even with the DNA-DNA hybridization procedure. The results of hybridization of a fraction of the extrachromosomal DNA of S. erythraeus 6, the Bam HIB fragment of plasmid pSE 21 with the total DNA of strains 1, 4, 5, 6 and BTCC 2 of S. erythraeus and hybridization of DNA of plasmid pSE 21 with the total DNA of S. erythraeus 6 and 1 showed that (1) strains 1, 5 and BTCC 2 had the same hybridization patterns, (2) the other extrachromosomal DNAs present in the fraction were homologous with the Bam HIA fragment of plasmid pSE 21, (3) chromosomes of strains 1, 4, 5, 6 and BTCC 2 of S. erythraeus also contained DNA homologous to the plasmid Bam HIA fragment. It was suggested that plasmid pSE 21 could be used as a basis for constructing the integrative vector for S. erythraeus.     

富集宏基因组DNA中α淀粉酶全长基因的克隆及重组表达

利用反向-巢式PCR（IN-PCR）从富集土壤宏基因组DNA中克隆到一种α-淀粉酶基因的全序列,其基因登录号为GU045523,测序分析显示与来自Bacillus sp. KR-8104耐酸淀粉酶不完整基因同源性为99%。将获得的α-淀粉酶成熟肽基因与表达载体pSE380连接,导入Escherichia coli JM109中,IPTG诱导表达。粗酶液经Ni-NTA、Sephacryl S-200纯化后测定酶学性质：重组酶GXAA的最适作用pH为7.0,最适作用温度为75℃,对可溶性淀粉的Km值为11.6g/L。构建突变子E27G、A450T、E27G-A450T,其酶学性质与原始酶没有显著差别。