人神经生长因子基因在大肠杆菌中的克隆、表达及产物纯化

从人胎盘组织中提取总DNA, 经PCR扩增编码人β神经生长因子（β-NGF）成熟肽的基因,并克隆到大肠杆菌表达载体pET15b中。重组质粒pET15b-NGF经测序与报道的完全一致。重组质粒转化大肠杆菌BL21(DE3)pLysS,经IPTG诱导表达得到16kD的目的蛋白带,与预期的大小一致,NGF表达量约占全菌总蛋白的25%.经过亲和层析柱（Ni2+-charged IDA his-bind column）纯化后得到了单一的NGF蛋白条带,蛋白纯度可达90%以上,从每升表达菌液中可以得到4.56mgNGF。表达产物的Western 印迹鉴定结果显示：重组人神经生长因子能与兔抗人β-NGF的多克隆抗体发生特异性结合反应,在16kD处出现单一的条带,表明诱导表达的重组NGF具有免疫学活性。鸡胚背根神经节感觉神经元鉴定试验表明,本实验表达的重组NGF具有良好的生物学活性。     

Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome

To search for new cold-active lipases, a metagenomic library was constructed using cold-sea sediment samples at Edison Seamount and was screened for lipolytic activities by plating on a tricaprylin medium. Subsequently, a fosmid clone was selected, and the whole sequence of 36 kb insert of the fosmid clone was determined by shotgun sequencing. The sequence analysis revealed the presence of 25 open reading frames (ORF), and ORF20 (EML1) showed similarities to lipases. Phylogenetic analysis of EML1 suggested that the protein belonged to a new family of esterase/lipase together with LipG. The EML1 gene was expressed in Escherichia coli, and purified by metal-chelating chromatography. The optimum activity of the purified EML1 (rEML1) occurred at pH 8.0 and 25°C, respectively, and rEML1 displayed more than 50% activity at 5°C. The activation energy for the hydrolysis of olive oil was determined to be 3.28 kcal/mol, indicating that EML1 is a cold-active lipase. rEML1 preferentially hydrolyzed triacylglycerols acyl-group chains with long chain lengths of ≥8 carbon atoms and displayed hydrolyzing activities toward various natural oil substrates. rEML1 was resistant to various detergents such as Triton X-100 and Tween 80. This study represents an example which developed a new cold-active lipase from a deep-sea sediment metagenome.     

粘质沙雷氏菌脂肪酶基因的克隆表达和酶学性质的研究

目的:克隆粘质沙雷氏菌脂肪酶基因(lipA)使其在大肠杆菌B121(DE3)中实现高效表达,并对重组酶进行酶学性质研究.方法:以产脂肪酶粘质沙雷氏菌总DNA为模板,PCR扩增脂肪酶基因lipA,构建重组表达载体pET-lipA,并将其导入大肠杆菌进行诱导表达,对表达产物进行SDS-PAGE和酶学性质的测定.结果:经过优化培养条件,脂肪酶活力最高能达到104U/mL.重组脂肪酶的最适反应温度为40～45℃,最适pH为7.0～7.5,在50℃保温1h下仍能保持80%的酶活力,Ca2+、Sr2+、Mn2+和Mg2+对脂肪酶酶活有较强的激活作用,尤其是Ca2+使脂肪酶酶活提高了1倍多,而Ni2+、Fe2+、Fe3+、Cu2+、Zn2+和Al3+对酶活具有较强的抑制作用,尤其是Zn2+和Al3+使酶活力几乎完全丧失.该酶对一些有机溶剂有较好的耐受性,与50%甲醇混合24h,仍能保持84%的酶活力.结论:该脂肪酶具有较好的热稳定性和甲醇耐受力,作为生产生物柴油的催化剂具有很大的应用价值,为基因工程酶法生产生物柴油打下良好的基础.     

玫瑰链霉菌海藻糖合成酶基因克隆、表达及功能鉴定

目的:克隆玫瑰链霉菌海藻糖合成酶基因(Srt)使其在大肠杆菌XL10-Gold中高效表达,并对重组酶的酶学特性进行研究。方法:利用PCR技术从玫瑰链霉菌中克隆到一段长1 704bp的海藻糖合成酶基因(Srt),构建重组表达质粒pSE380-Srt-treS,将其转化大肠杆菌XL10-Gold中诱导表达,对重组纯酶进行SDS-PAGE分析及酶学特性测定。结果:SDS-PAGE显示在65kDa处有明显单一蛋白条带。该酶可催化麦芽糖和海藻糖之间的可逆反应,海藻糖得率达82%,且含有很低的副产物葡萄糖(5%左右)。最适反应温度和pH分别为30℃、7.5,Cu2+、Zn2+和Tris能明显抑制酶活力。该酶还可催化蔗糖生成一种无龋齿,适合糖尿病患者食用的糖类-海藻酮糖。结论:成功克隆表达了一个海藻糖合成酶基因,该酶转化率高,副产物较少,为工业酶法生产海藻糖奠定基础。     

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。     

Cloning,Expression and Characterization of a Gene from Earthworm Eisenia fetida Encoding a Blood-Clot Dissolving Protein

A lumbrokinase gene encoding a blood-clot dissolving protein was cloned from earthworm (Eisenia fetida) by RT-PCR amplification. The gene designated as CST1 (GenBank No. {"type":"entrez-nucleotide","attrs":{"text":"AY840996","term_id":"56718408","term_text":"AY840996"}}AY840996) was sequence analyzed. The cDNA consists of 888 bp with an open reading frame of 729 bp, which encodes 242 amino acid residues. Multiple sequence alignments revealed that CST1 shares similarities and conserved amino acids with other reported lumbrokinases. The amino acid sequence of CST1 exhibits structural features similar to those found in other serine proteases, including human tissue-type (tPA), urokinase (uPA), and vampire bat (DSPAα1) plasminogen activators. CST1 has a conserved catalytic triad, found in the active sites of protease enzymes, which are important residues involved in polypeptide catalysis. CST1 was expressed as inclusion bodies in Escherichia coli BL21(DE3). The molecular mass of recombinant CST1 (rCST) was 25 kDa as estimated by SDS–PAGE, and further confirmed by Western Blot analysis. His-tagged rCST1 was purified and renatured using nickel-chelating resin with a recovery rate of 50% and a purity of 95%. The purified, renatured rCST1 showed fibrinolytic activity evaluated by both a fibrin plate and a blood clot lysis assay. rCST1 degraded fibrin on the fibrin plate. A significant percentage (65.7%) of blood clot lysis was observed when blood clot was treated with 80 mg/mL of rCST1 in vitro. The antithrombotic activity of rCST1 was 912 units/mg calculated by comparison with the activity of a lumbrokinase standard. These findings indicate that rCST1 has potential as a potent blood-clot treatment. Therefore, the expression and purification of a single lumbrokinase represents an important improvement in the use of lumbrokinases.     

麻疯树脂酶全长基因克隆、表达及其蛋白质结构预测

脂酶(Lipase,EC3.1.1.3)是普遍应用于皮革、饲料及生物柴油工业的工业酶制剂,具有广泛的应用价值。目前对植物来源的脂酶研究较少。本研究用在生物柴油中具有应用前景的油料植物——麻疯树(Jatrophacurcas)作为研究对象,克隆了该物种的脂酶基因(JcLIP)。通过多序列比对并结合物种的亲缘关系设计了具有较高特异性的简并引物,通过使用RT-PCR和RACE技术,最终获得了麻疯树脂酶基因的全长序列并成功地在大肠杆菌中表达,酶活测定结果表明,麻疯树脂酶在大肠杆菌中表达在包涵体中,但是能产生具有活力的蛋白质,酶活约为0.8U.mL-1。结构预测和比较表明,JcLIP蛋白质具有脂酶的结构核心和催化活性中心,而在非核心区具有较毛霉脂酶更多的插入和随机卷曲,这可能是决定二者之间酶活差异的重要原因。     

人IL-18的基因克隆、表达及纯化

目的 :克隆人IL 1 8基因 ,并构建高表达人IL 1 8的工程菌 ,纯化获得重组人IL 1 8。方法 :从人肿瘤组织内提取总RNA ,利用逆转录聚合酶链反应扩增人IL 1 8基因 ,在基因的 5′和 3′端分别加上NcoI和EcoRI酶切位点 ,酶切后直接克隆至质粒表达载体pET2 8a( + )内 ,转化大肠杆菌BL2 1 (DE3) ,挑选转化子 ,IPTG诱导后SDS PAGE筛选高表达人IL 1 8的工程菌。提取表达菌质粒进行DNA序列分析。表达菌大量培养及IPTG诱导后 ,超声破菌收集包涵体 ,十二烷基肌苷酸钠溶解后用阳离子柱和分子筛纯化。结果 :克隆的人IL 1 8基因序列完全正确 ,工程菌表达的人IL 1 8约占菌体蛋白 30 % ,以包涵体形式存在 ,经阳离子柱和分子筛纯化获得了较纯的人IL 1 8。结论 :可用构建的工程菌大量制备人IL 1 8,为开展人IL 1 8药物的临床前研究奠定了基础。     

温泉宏基因组来源的D-来苏糖异构酶性质研究*

目的: 从高温温泉宏基因组中挖掘能够高效催化合成稀有糖的新耐高温D-来苏糖异构酶。方法: 从云南昌宁鸡飞温泉底泥中提取宏基因组DNA并进行高通量测序,经基因注释及序列比对鉴定D-来苏糖异构酶基因,构建大肠杆菌异源表达载体并诱导表达,通过亲和层析纯化重组蛋白并对其性质研究。结果: 从温泉底泥宏基因组测序结果中鉴定得到8个D-来苏糖异构酶基因,选择4个基因进行异源表达,其中JF-LI1和JF-LI4在大肠杆菌中成功表达并检测到酶活性。研究表明,JF-LI1和JF-LI4的最适温度分别为70℃和75℃。JF-LI4具有较宽的作用温度和良好的热稳定性,在30~100℃的温度范围内剩余40%以上的酶活力。JF-LI1和JF-LI4的最适pH分别为7.0和7.5,在中性偏酸性条件下具有较高的活力和较强的稳定性。重组JF-LI1和JF-LI4具有较宽的底物谱,除了对D-来苏糖活性最高外,对L-核糖、L-核酮糖、D-果糖和D-甘露糖均具有活性。重组JF-LI1和JF-LI4对L-核糖的催化效率分别为0.56 L/(mmol·s^-1)和0.61 L/(mmol·s^-1),是目前已知的D-来苏糖异构酶中最高的。结论: 从高温温泉宏基因组中获得8个新的D-来苏糖异构酶基因,对JF-LI1和JF-LI4进行异源表达和性质研究,具有pH稳定性好、热稳定性强以及底物特异性宽泛的特点,在制药、食品、化妆品等工业领域有重要的应用潜力。     

Characterization and its Potential Application of Two Esterases Derived from the Arctic Sediment Metagenome

Two esterase genes (designated as estAT1 and estAT11, respectively) were cloned by activity-based screening of a fosmid library constructed with seashore sediment sample of the Arctic. The sequence analysis of the genes revealed that these esterase genes encoded proteins of 303 and 312 amino acids, respectively, and showed 40-50% identities to members of the hormone-sensitive lipase (HSL) family retaining a catalytic triad with a conserved GDSAG sequence and an oxyanion hole (HGGG). The esterases genes were overexpressed in Escherichia coli by co-expressing GroEL-GroES chaperonine, and the recombinant proteins (rEstAT1 and rEstAT11) were purified to homogeneity. The purified EstAT1 and EstAT11 were active in a broad range of temperature from 20 to 40 degrees C with an optimum temperature at 30 degrees C. The activation energies of rEstAT1 and rEstAT11 to hydrolyze p-nitrophenyl esters of butyrate were determined to be 12.65 kcal/mol and 11.26 kcal/mol, respectively, indicating that they are cold-adapted esterases. The purified EstAT1 and EstAT11 could hydrolyze racemic ofloxacin esters, and further rEstAT11 hydrolyzed preferentially (S)-racemic ofloxacin butyl ester with an enantiomeric excess (ee(p)) value of 70.3%. This work represents an example that develops enzymes from the Arctic using metagenomic approach, potentially applicable to chiral resolution of heat-labile substrates.     

链霉菌UDP-葡萄糖脱氢酸编码基因Ste6的克隆表达和性质研究

链霉菌139能够产生一种全新的胞外多糖——依博素（139A）,该多糖体内具有显著抗类风湿性关节炎活性。其生物合成基因簇（GenBank Accession Number：AYl31229）已被鉴定约31．3kb,包含22个开放阅读框（ste1—ste22）。以pET-30a为载体,克隆并在大肠杆菌BL21（DE3）中进行了ste6基因的表达,对该基因的克隆、表达与性质进行了研究。亲和层析法证实,纯化后重组蛋白具有催化UDP-葡萄糖脱氢变成UDP-葡萄糖醛酸的活性。这表明ste6编码产物是葡萄糖脱氢酶。为了证实ste6基因与依博素生物合成的关系,采用单交换基因破坏策略构建了ste6基因阻断突变株。结果初步显示ste6和依博素生物合成相关。     

水牛瘤胃宏基因组的一个新的b-葡萄糖苷酶基因umcel3G的克隆、表达及其表达产物的酶学特性

以木质纤维素为原料、应用同步糖化共发酵工艺发酵生产酒精时需要酸性中低温高活力纤维素酶包括b-葡萄糖苷酶。本工作分6次构建了水牛瘤胃未培养微生物宏基因组文库, 获得1.26×105个克隆, 文库含外源DNA的总长度 约为4.8×106 kb。从文库中筛选到118个表达b-葡萄糖苷酶活性的独立克隆。发现其中8个克隆表达的b-葡萄糖苷酶在pH5.0、37oC条件下活性较强。对其中一个克隆进行了亚克隆, 序列分析发现一个2223 bp的潜在的编码b-葡萄糖苷酶基因(umcel3G)的开放阅读框(ORF), 其编码产物的氨基酸序列与来自于 Bacillus sp.的一个b-葡萄糖苷酶同源性最高, 具有60%的一致性和73%的相似性。该ORF在E.coli中的表达产物Umcel3G的分子量与预测大小相似, 酶谱分析表明该表达产物具有b-葡萄糖苷酶活性, 证实该基因为一个b-葡萄糖苷酶基因。测定了用Ni-NTA纯化的Umcel3G的酶学特性, 其最适pH和最适温度分别为6.0~6.5和45oC。一些金属离子如Ca2+、Zn2+能显著提高该酶的酶活, 而另外一些金属离子如Fe3+、Cu2+能抑制Umcel3G的活性。在pH4.5、35oC和5 mmol/L的 Ca2+存在的条件下, 用Ni-NTA纯化的重组酶的比活为22.8 IU/mg, 说明该酶在用SSCF工艺发酵生产酒精中有潜在的应用价值。     

水牛瘤胃宏基因组的一个新的b-葡萄糖苷酶基因umcel3G的克隆、表达及其表达产物的酶学特性

以木质纤维素为原料、应用同步糖化共发酵工艺发酵生产酒精时需要酸性中低温高活力纤维素酶包括b-葡萄糖苷酶。本工作分6次构建了水牛瘤胃未培养微生物宏基因组文库, 获得1.26×105个克隆, 文库含外源DNA的总长度 约为4.8×106 kb。从文库中筛选到118个表达b-葡萄糖苷酶活性的独立克隆。发现其中8个克隆表达的b-葡萄糖苷酶在pH5.0、37oC条件下活性较强。对其中一个克隆进行了亚克隆, 序列分析发现一个2223 bp的潜在的编码b-葡萄糖苷酶基因(umcel3G)的开放阅读框(ORF), 其编码产物的氨基酸序列与来自于 Bacillus sp.的一个b-葡萄糖苷酶同源性最高, 具有60%的一致性和73%的相似性。该ORF在E.coli中的表达产物Umcel3G的分子量与预测大小相似, 酶谱分析表明该表达产物具有b-葡萄糖苷酶活性, 证实该基因为一个b-葡萄糖苷酶基因。测定了用Ni-NTA纯化的Umcel3G的酶学特性, 其最适pH和最适温度分别为6.0~6.5和45oC。一些金属离子如Ca2+、Zn2+能显著提高该酶的酶活, 而另外一些金属离子如Fe3+、Cu2+能抑制Umcel3G的活性。在pH4.5、35oC和5 mmol/L的 Ca2+存在的条件下, 用Ni-NTA纯化的重组酶的比活为22.8 IU/mg, 说明该酶在用SSCF工艺发酵生产酒精中有潜在的应用价值。     

日本血吸虫信号转导蛋白Sjwnt-4基因的克隆、表达及功能分析

由Wnt基因家族产物与其它相关基因产物构成的Wnt信号通路,是细胞发育和生长调节的一个关键途径,对动物的发育特别是生殖系统的发育起重要的调节作用。在人类和小鼠中,Wnt4蛋白是性腺分化过程中主要调节因子,在胚胎发育中起着关键作用。利用RACE技术从日本血吸虫19d童虫中首次扩增到一个Wnt家族基因,序列分析表明该基因的完整编码框含1311bp,编码436个氨基酸,理论分子量49.6kD。同源性分析结果表明,该基因的氨基酸序列具有典型Wnt家族蛋白特征,与日本三角涡虫、人Wnt4的氨基酸序列相似性分别达43%、37%,推测为血吸虫的Wnt4基因,命名为Sjwnt4(GenBank登陆号DQ643829)。实时定量PCR分析显示该基因在14d童虫、19d童虫、31d虫体、44d雌虫及44d雄虫中均有表达,其中19d童虫中的表达量明显高于其它发育阶段,44d雌虫中的表达量明显高于雄虫。构建了该基因的原核表达载体pGEX-4T-2-Sjwnt4,应用大肠杆菌系统进行了表达,表达蛋白以包涵体形式存在,Western印迹显示表达产物能被日本血吸虫成虫粗抗原免疫血清所识别。Sjwnt4基因及其表达产物的获得,为探索Wnt信号通路对血吸虫发育、生殖的调节提供了重要基础。     

Cloning, Expression and Characterization of a Thiolase Gene from Clostridium pasteurianum

A thl gene encoding the thiolase (EC 2.3.1.9) of Clostridium pasteurianum was cloned by thermal asymmetric interlaced (TAIL) PCR. It consists of 1179 bp with 36.8% GC content and encodes 392 amino acids with a deduced molecular mass of 40,954 Da and shows 77% identity and 88% similarity to that of Clostridium tetani E88 and should be classified as a biosynthetic thiolase with three conserved residues Cys89, Cys382 and His352. The gene was over-expressed in Escherichia coli and the thiolase was purified with Ni-NTA agarose column to homogeneity. The K _m of this thiolase for acetoacetyl-CoA is 0.13 mM with 0.06 mM CoASH at pH 8.2, 25°C and a V _max value of 46 μmol min⁻¹ mg⁻¹.     

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。     

金银花丙酮酸激酶基因的克隆与表达特性分析

采用RT-PCR和RACE技术,首次从金银花中克隆丙酮酸激酶基因全长cDNA,命名为LjPkc(GenBank登录号JQ621946.1),LjPkc基因编码区序列全长为1 533bp,共编码510个氨基酸。基于基因序列亲缘关系分析结果显示,LjPkc基因与烟草Pkc基因亲缘关系最近,属于双子叶植物Pkc基因家族。蛋白质亚细胞定位分析认为LjPkc在线粒体或叶绿体中发挥作用。LjPkc蛋白质的三维空间结构预测结果显示,LjPkc蛋白主要由α-螺旋、不规则盘绕组成。定量PCR检测发现,LjPkc基因在金银花不同组织中广泛表达,但表达水平各有差异,黄花中表达量最高。该研究丰富了Pkc基因库资源,为揭示LjPkc基因在金银花糖代谢方面的生物学作用奠定了基础。     

Molecular Cloning, Expression, and Pharmacological Characterization of humEAA1, a Human Kainate Receptor Subunit

Abstract: Kainate is a potent neuroexcitatory agent; its neurotoxicity is thought to be mediated by an ionotropic receptor with a nanomolar affinity for kainate. In this report, we describe the cloning of a cDNA encoding a human glutamate ionotropic receptor subunit protein from a human hippocampal library. This cDNA, termed humEAA1, is most closely related to rat and human cDNAs for kainate receptor proteins and, when expressed in COS or Chinese hamster ovary cells, is associated with high-affinity kainate receptor binding. We have successfully established cell lines stably expressing humEAA1. This is the first report of establishment of stable cell lines expressing a glutamate receptor subunit. The relative potency of compounds for displacing [³H] kainate binding of humEAA1 receptors expressed in these stable cell lines was kainate > quisqualate > domoate > L-glutamate > ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid > dihydro-kainate > 6, 7-dinitroquinoxaline-2, 3-dione > 6-cyano-7-nitroquinoxaline-2, 3-dione. Homooligomeric expression of humEAA1 does not appear to elicit ligand-gated ion channel activity. Nevertheless, the molecular structure and pharmacological characterization of high-affinity kainate binding of the humEAA1 expressed in the stable cell line (ppEAA1–16) suggest that the humEAA1 is a subunit protein of a human kainate receptor complex.     

蜂房哈夫尼菌植酸酶基因appA的克隆、表达及酶学性质研究

从蜂房哈夫尼菌(Hafniaalvei)中克隆获得一个植酸酶编码基因appA,该基因全长1335bp,编码444个氨基酸,其中前33个氨基酸为信号肽,成熟蛋白的理论分子量为45.2kD。将基因appA克隆到大肠杆菌E.coli表达载体pET-22b( ),并在大肠杆菌中表达,表达产物具有植酸酶活性。对表达的酶蛋白进行纯化,并初步研究了该酶的酶学性质,结果表明:酶的作用最适pH值为4.5;在pH2.0~10.0范围内,酶活性保留80%以上;酶的作用最适温度为60℃;酶的比活性为356.7U/mg,酶动力学分析表明其Km为0.49mmol/L,Vmax为238U/mg;该酶对胰蛋白酶和胃蛋白酶有一定的抗性。该研究为哈夫尼菌属来源植酸酶的首次报道。