豇豆钙调蛋白cDNA的克隆及序列分析

从豇豆成熟叶片中提取总RNA,反转录合成cDNA第一链,根据钙调蛋白结构基因两端保守序列设计引物,PCR扩增豇豆钙调蛋白基因,克隆到T-easy载体上并测定了其全序列.序列分析结果表明,豇豆钙调蛋白基因由450个核苷酸组成,编码150个氨基酸.与已知的多种植物钙调蛋白基因相比有很高的相似性,核苷酸序列相似性在80%以上,编码的氨基酸序列相似性在90%以上.     

鲫血清转铁蛋白cDNA克隆及其序列分析

从GenBank数据库查询发表的鱼类转铁蛋白cDNA或基因序列，根据铁离子结合转运功能位点，设计并合成了两对引物P1、P4以及P2、P3，克隆出鲫血清转铁蛋白cDNA中的核心片段，长度为866bp。再根据克隆出的核心片段分别设计上游及下游两对引物P5、P6以及P7、P8，随后用RACE方法分别克隆出鲫血清转铁蛋白cDNA的5’端(787bp)和3’端(1081bp)以及全长cDNA，最后用计算机程序排列出鲫血清转铁蛋白全长cDNA，长度为2444bp。比较了12种鱼类血清转铁蛋白cDNA序列的同源性。     

鲫血清转铁蛋白cDNA克隆及其序列分析

从GenBank数据库查询发表的鱼类转铁蛋白cDNA或基因序列,根据铁离子结合转运功能位点,设计并合成了两对引物P1、P4以及P2、P3,克隆出鲫血清转铁蛋白cDNA中的核心片段,长度为866 bp.再根据克隆出的核心片段分别设计上游及下游两对引物P5、P6以及P7、P8,随后用RACE方法分别克隆出鲫血清转铁蛋白cDNA的5'端(787 bp)和3'端(1 081 bp)以及全长cDNA,最后用计算机程序排列出鲫血清转铁蛋白全长cDNA,长度为2 444 bp.比较了12种鱼类血清转铁蛋白cDNA序列的同源性.     

人乳铁蛋白(hLF)cDNA的克隆及序列分析

从正常人外周血中分离中性粒白细胞（WBC）,提取总RNA,用RT—PCR的方法扩增人乳铁蛋白（hLF）cDNA．cDNA分hLF1．5、hLF0．8两段扩增并连入pMD18-T载体上,再利用限制酶连入巴氏毕赤酵母表达载体pPICZα—A中,构成完整的hLF基因．序列测定表明,所克隆的hLF基因序列全长为2,136bp,与Gene Bank中登录的序列相比,同源性达99％以上．     

两个东亚钳蝎神经毒素cDNA的克隆和序列分析

构建了东亚钳蝎毒腺组织ｃＤＮＡ库，用ＰＣＲ筛选到两个新的神经毒素全长ｃＤＮＡ，其开放阅读框（ＯＲＦ）包括：信号肽、成熟毒素和／或额外氨基酸序列，它们编码的成熟毒分别命名为ＢｍａＴＸ９和ＢｍＩＴ（ｃｐ）２。序列分析表明，ＢｍａＴＸ９为抗哺乳动物神经毒素；ＢｍＩＴ（ｃｐ）２为痉挛型昆虫毒素。章还对东亚钳蝎神经毒素的命名，分类以及进化等问题进行了初步的探讨。     

豌豆cop1cDNA的克隆,序列分析及其在大肠杆菌中的表达

以拟南芥ｃｏｐ１ｃＤＮＡ为探针,从豌豆ＣＤＮＡ文库中克隆到了豌豆ｃｏｐ１ｃＤＮＡ。序列分析表明,它全长为２８６３ｂｐ,其中包括６０４ｂｐ５‘非编码区,２４３ｂｐ３’非编码区和２０１６ｂｐ编码区,编码６７２个氨基酸。在大肠杆菌中实现了豌豆ｃｏｐ１基因的高效表达。对拟南芥、豌豆和番茄３种植物ｃｏｐ１的序列同源性比较表明,ｃｏｐ１可能是一种进化上很保守的蛋白质。     

水貂朊蛋白前体基因的原核表达

 构建了水貂朊蛋白前体基因的表达载体,所筛选出的阳性克隆质粒经转化表达菌BL21(DE3)后,收获表达菌,纯化鉴定表明获得了目的产物.     

水稻黑条矮缩病毒基因组第九组分cDNA的克隆及序列分析

从我国发病的玉米材料中提取水稻黑条矮缩病毒,抽提病毒RNA,利用RT-PCR等手段,获得了病毒基因组第九组分（S9）cDNA克隆。序列分析结果表明：S9全长1900bp,含有2个不重叠的ORF,编码蛋白的分子质量分别为39.9,24.2ku,与日本株S9核苷酸序列同源性为89%,与意大利株MRDV S8同源性为86%。     

人类RING型锌指蛋白DTX2的全长cDNA克隆与序列分析

鉴定了一个人类WWE与RING型锌指基因DTX2,位于人染色体7q11.23. 通过RACE获得的全长cDNA显示,DTX2编码包含两个WWE区域与一个RING锌指区域的蛋白.     

t-PA cDNA基因的克隆及测序

从基因工程菌株里氏木霉中提取基因组DNA,根据t-PAcDNA序列设计一对引物,利用PCR法扩增目的基因,将其与pMD18-T-Vector连接后转入大肠杆菌,通过Amp抗性和蓝白斑筛选出重组菌株,PCR法和双酶切法鉴定后再进行测序鉴定,测序结果表明:克隆的t-PAcDNA的第34位、450位和521位基因发生了突变。     

九龙牦牛CAST基因部分序列的克隆及其表达差异研究

根据牛钙蛋白酶抑制蛋白（Calpastatin, CAST） 设计并合成一对引物,从九龙牦牛肌肉组织提取总RNA,利用RT-PCR扩增获得九龙牦牛CAST基因部分片段,利用SQRT-PCR技术检测九龙牦牛各组织中CAST mRNA的表达差异．结果,成功克隆九龙牦牛CAST部分cDNA序列485bp,获得GenBank登录号为FJ483833;用DNAman软件分析发现九龙牦牛CAST基因与普通牛的核苷酸同源性依次为99％;SQRT-PCR组织表达检测表明,CAST基因在心脏、肝脏、脾脏、肺脏、肾脏、肌肉、脂肪中均表达,在心脏中表达最高,在脂肪组织中表达最低．     

秀丽小杆线虫Caenorhabditis elegans pkc-2基因cDNA的克隆和表达

蛋白激酶C在秀丽小杆线虫中具有调节肌细胞渐进性萎缩的功能.为了揭示它的调节机制,本研究克隆了秀丽小杆线虫中蛋白激酶C pkc-2基因的cDNA pkc-2-c,构建了含该pkc-2 基因cDNA亚型的重组质粒pPD 118.20-pKG 63;揭示了该cDNA在秀丽小杆线虫体壁肌细胞中的定位.     

Cloning and sequencing of Gtx-2 homeobox sequence

Murine genomic DNA was surveyed by polymerase chain reaction (PCR) to detect homeobox sequence of mammal. The PCR product (413 bp) was cloned and sequenced. The nucleotide sequence and the deduced amino acid sequence of a homeobox, which was isolated in this study, designated Gtx-2 were obtained. The result of sequence analysis showed that there is an intron between the nucleotides at position 138 and 139 in the Gtx-2 homeobox. Southern blot analysis revealed that Gtx-2 is likely to exist as a single copy in the murine genome. Comparison of the encoded polypeptide sequence with other homeodomains reveals that Gtx-2 has 96% and 58% identity to that of Gtx-1 and Tcl-3, respectively. Ma Xiaojun: born in 1941, Associate professor.     

Cloning and analysis of a cDNA encoding acetohydroxy acid isomeroreductase from G2 pea

Using cDNA representational difference analysis (cDNA RDA) method, we have successfully isolated a gene fragment whose expression was specifically induced by external GA₃ application. Screening a G2 pea cDNA library using this fragment as a probe, we obtained a 2036 bp full-length cDNA. It contains a 1746 bp open reading frame and encodes a protein of 581 amino acids with a theoretical molecular weight of 64 ku. It shares high-level sequence identity withAAIR genes from other plant species. This cDNA was cloned into expression vector and recombinantE. coli DH5α cells with remarkable AAIR enzyme activity were obtained.     

牛蛙生长激素基因cDNA克隆及在大肠杆菌中的表达

以成体牛蛙脑垂体总RNA为模板进行RT-PCR,克隆到牛蛙生长激素基因(bullfrog growth hormone, bfGH)cDNA编码序列,其长度为651 bp,编码的前体GH蛋白序列经BLAST分析,与已报道的牛蛙GH蛋白质序列AAB24792、AAB19428、CAA31038的同源性分别为98.1%、96.3%、95.3%,其在Genbank的登录号为AY251538;将bfGH亚克隆到原核表达载体pGEX1-λt构建成牛蛙GH原核表达载体VGBfGH,转化BL21（DE3）E.coli得     

兔生长激素cDNA克隆和表达

从兔脑下垂体中的mRNA中,利用合成引物逆转录PCR,获得了前体和成熟生长激素的cDNA,成熟生长激素的cDNA克隆在pET-3a表达载体并转化到大肠杆菌BL21(DE3 plys)中,获得了高效表达,表达量达到大肠杆菌总蛋白的40%。     

Cloning, construction of prokaryotic expression vector and expression ofEscherichia coli cytosine deaminase gene

Cytosine deaminase gene ofEscherichia coli strain H-30 was cloned, and its initiation codon of ‘GTG’ was mutated to ‘ATG’ by PCR. Prokaryotic recombinant expression vector pBV220-CD was constructed. Clone with high enzyme activity were selected by detecting their specific activity of cytosine deaminase. 5-FC(5-FC, 5-fluorocytosine) could induce the lethal toxicity to cells containing active CD gene. DNA sequence analysis indicated that there were 16 altered bases and 5 of them resulted in the alteration of amino acids in predicted peptide by comparing DNA sequence of the clone H-30-CD-11 with high enzyme activity with CD gene reported in Gene Bank.     

绵羊防御素sBD-1 cDNA的克隆及序列分析

从成年绵羊舌表皮组织分离提取总RNA，经反转录PCR（RT-PCR)扩增出绵羊防御素sBD-lcDNA，将扩增片段回收，重组插入克隆载体T—vector，经限制性酶切鉴定和DNA序列测定，结果扩增出cDNA215bp，与发表的绵羊sBD-lcDNA序列完全相同。该cDNA编码以个氨基酸，其中信号肽与前片段相同，成熟肽位于前片段的羧基端，由38个氨基酸残基组成。     

Cloning and expression analysis ofMBLL cDNA

Thembl (muscleblind) gene ofDrosophila encodes a nuclear protein which contains two Cys₃His motifs. The mutation ofmbl gene will disturb the differentiation of all theDrosophila’s photoreceptors. Primers have been designed according to human EST086139, which is highly homologous tombl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designatedMBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys₃His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology toDrosophila’s mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show thatMBLL is a widely expressed gene, but the expression amounts differ in these tissues.