大肠杆菌MalQ基因的克隆与原核融合表达

为了构建用于表达麦芽糖转糖基酶的基因工程菌,用PCR方法获得大肠杆菌(Escherichia coli)K1 MalQ基因.将该基因插入原核表达栽体pET-DsbA中,对质粒所含外源片段进行双向测序,并经局部相似性基本查询工具(BLAST)比较分析,发现其与GenBank中报道的大肠杆菌K12 MalQ基因的同源性高达99%.重组质粒转化E.coliBL21(DE3)plysS,经异丙基硫代-β-D-半乳糖(IPTG)诱导后以融合蛋白形式表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示MalQ基因与DabA表达的融合蛋白在目标位置相对分子质量约为103000处有明显条带.经薄层层析分析粗酶液酶处理的麦芽三糖溶液,证实粗酶液具有麦芽糖转糖基活性.     

大肠杆菌RsmH基因克隆及表达

以大肠杆菌全基因组DNA为模版,PCR扩增目的基因rsmh,构建pMD18-T-RsmH重组克隆载体和pET-28aRsmH重组表达载体,转化大肠杆菌BL21。经IPTG诱导表达RsmH蛋白,利用重组His-tag,通过Ni-NTA亲和层析对RsmH蛋白进行纯化。SDS-PAGE对RsmH蛋白进行定性分析,考马斯亮蓝法测定RsmH蛋白含量,确认获得质量浓度为3.91 g/L纯化的rsmh表达蛋白。     

Cloning and characterization of a new actin gene from Oryza sativa L.

Using Rho family member osRACD as bait, a new member of actin gene family -Act was isolated from Oryza sativa by yeast two-hybrid system. The full-length cDNA was cloned with 5' RACE technology, which contains an open reading frame of 1134 bp with a predicted protein of 377 amino acids. Sequence alignment revealed 96% to 81.8% identities with some known actin proteins in plants. The method of bioinformatics was used to analyze the protein modification sites, structure and evolution of the gene. Southern blot analysis showed that Act is a single-copy gene in the genome. The result of RT-PCR showed it is ubiquitously expressed in root, shoot, callus and panicle in a temporal fashion. The relationship between Rho family and actin family in evolution and function was also studied.     

Cloning of a down-regulated gene encoding small GTP binding protein in hybrid wheat

Previous studies showed that differential gene expression between wheathybrids and their parents was responsible for the heterosis. To provide an insight into the molecular basis of wheat heterosis, one cDNA, designated TaRab, was identified from the cDNA library of wheat seedling leaves. The sequence comparison in GenBank revealed that TaRab is homologous to a group of genes encoding Rab-GTP binding protein. Semi-quantitative RT-PCR analysis indicated that TaRab was expressed in all plant tissues examined, but at slightly higher level in leaves. Further analysis exhibited that TaRab displayed lower expression in hybrid than in its patents in both roots and leaves, which was in agreement with the original results of suppression subtractive hybridization. TaRab was located on chromosome 7B and C-7DS5-0.36 by in silico mapping. The relationship between differential expression of TaRab and the molecular basis of wheat heterosis was also discussed.     

大肠杆菌K12碱性磷酸酶基因的克隆表达

大肠杆菌碱性磷酸酶是同二聚体金属酶，被phoA基因编码．采用PCR方法从大肠杆菌K12中扩增到碱性磷酸酶基因（phoA）．用限制性内切酶BamHI和HindⅢ将片段插入克隆载体pETBlue-2．该基因被重组、转化后在大肠杆菌中经IPTG诱导表达，而后进行SDS-PAGE检测及紫外吸收分析测OD410．克隆得到了phoA基因并实现了蛋白表达，碱性磷酸酶的活性提高了18，3倍，为进行组织化学、免疫印迹、突变分析等工作打下了坚实的基础．     

人血管抑素AK(1-3)基因的克隆及在大肠杆菌中的表达

用6月龄大月份流产的新鲜肝脏组织为材料,提取总RNA,通过反转录得到cDNA,并以此为模板,经PCR得到人Angiostatin基因的AK(1-3)片段.将此PCR产物直接与载体pMD18-T连接,转化大肠杆菌DH5α.经蓝白菌落筛选,PCR和酶切检测,筛选出阳性克隆,命名为pMDA,测序证明克隆序列与GenBank发表序列一致,大小为858bp.用Sal 和Bgl 酶切将该基因插入载体pQE40,并转化宿主菌M15[pREP4].经质粒电泳PCR和酶切检测筛选阳性克隆,得到表达载体pQEA,插入基因与载体上小鼠DHFR基因重组形成嵌合基因.在30℃,2×YT培养基(Kan25μg/mL Amp200μg/mL),2mmol/LIPTG条件下诱导pQEA/M15[pREP4]表达.所得菌体总蛋白经SDS-PAGE分析,结果表明,在预期的61kD处得到一条特异带,为AK(1-3)cDNA与载体固有基因DHFR-6хHis的重组蛋白表达产物.实验结果为抗肿瘤药物的研制提供了条件.     

纳豆激酶原基因的克隆及表达

从纳豆菌中克隆具有溶血栓性质的纳豆激酶原的基因，与质粒PBV220连接，转化大肠杆菌E.coli DH5α。挑取阳性克隆用EcoR I/BamH I双酶切鉴定得到一条约1000bp的条带。阳性克隆经温度诱导表达，用SDS－PAGE电泳鉴定，在38kD处有一条明显的带，占菌体蛋白的20％左右，同时还表明该蛋白以包涵体的形式蛋白分子量为存在。包涵体经收集、蛋白变性及复性后显示溶血栓活性。     

人胰岛素样生长因子—Ⅰ的基因克隆,融合表达及产物纯化

利用聚合酶链式反应（ＰＣＲ）方法,以ＩＧＦ－ＩｃＤＮＡ为模板,扩增ＩＧＦ－Ｉ的基因。在序列测定确证后,将其定向克隆到载体ｐＥｘＳｅｃＩ上,构建了ＩＧＦ－Ｉ融合表达载体ｐＥｘＩＧＦ,经ＩＰＴＧ诱导表达后,ＳＤＳ－ＰＡＧＥ分析表明：含有重组表达质粒的菌株可表达出约３２．５８％的２６ＫＤ融合蛋白,且主要以可溶性形式存在。经ＩｇＧ－Ｓｅｐｈａｒｏｓｅ亲和层析一步纯化后,可获得纯度约９０％的融合蛋白,经Ｗｅｓｔｅｒｎ－Ｂｌｏｔ分析,免疫学活性呈阳性反应。     

小鼠OB基因的克隆及在大肠杆菌中的高效表达

利用RT-PCR技术从小鼠脂肪组织中扩增了OB基因,并利用定向克隆技术克隆至原核表达载体pTO-T7.将构建好的质粒导入表达型大肠杆菌BL21(DE3),IPTG诱导表达,并对表达产物进行Western-blot检测.表达产物利用亲和层析技术进行纯化.结果显示:表达的带有his标签的小鼠leptin融合蛋白,分子量约为20 ku.0.5 mmol/L的IPTG在37℃诱导2.5 h时,leptin融合蛋白的表达量达到最大,达菌体总蛋白50%以上.表达产物经过纯化,纯度可达90%以上.Western-blot杂交显示,小鼠leptin融合蛋白有很强的抗原特异性.     

人肠激酶轻链基因的克隆及其在大肠杆菌中的表达

研究目的：克隆表达人肠激酶轻链编码基因,以期应用于融合蛋白的切割与纯化．方法：从人的全基因组中扩增出编码人肠激酶轻链的5个外显子基因片段,经过酶切、连接后得到正确编码的人肠激酶轻链完整基因序列;随后,将目的基因片段插入原核表达载体pBV220中,构建成融合型表达载体pBV—EKL,转化大肠杆菌BL21（DE3）,调节温度至42℃诱导重组蛋白表达;通过镍亲和层析纯化得到重组蛋白．结果：所表达的重组蛋白经SDS—PAGE分析,相对表观分子质量约为31kDa,表达量占菌体总可溶蛋白量的46％;通过镍亲和层析纯化得到的重组蛋白经复性后显示出具有催化切割活性．结论：成功构建了能大量表达重组人肠激酶轻链的菌株,为进一步进行重组人肠激酶活性的研究及其在生产和医学方面的应用研究奠定了基础．     

大肠杆菌核苷磷酸化酶的重组表达和活性

将大肠杆菌K-12菌株来源的嘌呤核苷磷酸化酶、尿苷磷酸化酶和胸苷磷酸化酶基因分别克隆到pET-11a载体并转化大肠杆菌BL21（DE3）宿主菌表达,通过SDS-PAGE分析和酶的活性测定,重组菌诱导表达后目的蛋白的表达量占菌体总蛋白的37%以上,与产核苷磷酸化酶的野生菌比较,酶的活性也有显著提高。在特异反应条件下,构建的工程菌可以用来高效催化核苷的转糖基反应。