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Background Resistin, a newly discovered cysteine-rich hormone secreted mainly by adipose tissues, has been proposed to form a biochemical link between obesity and type 2 diabetes. However, the resistin receptor has not yet been identified. This study aimed to identify resistin binding proteins/receptor.Methods Three cDNA fragments with the same 11 bp 5’ sequence were found by screening a cDNA phage display library of rat multiple tissues. As the reading frames of the same 11 bp 5’ sequence were interrupted by a TGA stop codon, plaque lift assay was consequently used to prove the readthrough phenomenon. The stop codon in the same 11 bp 5’ sequence was replaced by tryptophan, and the binding activity of the coded peptide [AWIL, which was designated as resistin binding peptide (RBP)] with resistin was identified by the confocal microscopy technique and the affinity chromatography experiment. pDual GC-resistin and pDual GC-resistin binding peptide were co-transfected into 3T3-L1 cells to confirm the function of resistin binding peptide.Results Three cDNA fragments with the same 11 bp 5’ sequence were found. The TGA stop codon in reading frames of the same 11 bp 5’ sequence was proved to be readthroughed. The binding activity of RBP with resistin was consequently identified. The expression of the resistin binding peptide in 3T3-L1 preadipocytes expressing pDual GC-resistin significantly inhibited the adipogenic differentiation.Conclusion RBP could effectively rescue the promoted differentiation of resistin overxepressed 3T3-L1 preadipocyte.  相似文献   

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The integral mature peptide gene of human growth differentiation factor-5 (GDF-5) was出人物cloned to provide the essential foundation for study on the biological characteristics of GDF-5 at gene and protein levels. Two primers were chemosynthesized according to the hGDF-5 sequence reported in Genbank. The hGDF-5 gene was gained by RT-PCR methods from the total RNA extracted from human fetus cartilage tissue, and was cloned into vector pMD18-T. The sequence of recombinant plasmid pMD18-T-hGDF-5 was analyzed by sequence analysis. DNA agarose gel electrophoresis showed that the product of RT-PCR was about 380bp, and double enzyme digestion of the recombinant plasmid corresponded with it. The result of sequence assay was in agreement with the reported hGDF-5 sequence in Genbank. Our results showed that the integral mature peptide gene of human GDF-5 was cloned successfully from human fetal cartilage tissue, and totally identified with the seouence of human GDF-5 in Genhank.  相似文献   

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Gold(Au) nanoparticle HBV DNA or HCV cDNA gene probes were prepared and were used to detect HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection directly by transmission electron microscopy(TEM).PCR identifying HBV and HCV in serum of patients with HBV and HCV coinfection was established.Alkanethiol-modified oligonucleotide was bound with self-made Au nanoparticles to form nanoparticle HBV DNA or HCV cDNA gene probes through covalent binding of Au-S.HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection was added to the detection system composed of nanoparticle HBV DNA and(or) HCV cDNA gene probes.The results showed that HBV DNA and HCV RNA could be specifically amplified by PCR.The zones of DNA amplification appeared in 431 bp and 323 bp respectively.When HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection were added to the detection system,TEM displayed the nanoparticles self-assembled into large network aggregates.It was concluded that the detection of HBV and HCV coinfection by TEM was convenient and efficient with high specificity and sensitivity.  相似文献   

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Objective To identify the egg antigens related to the formation of hepatic granulomas and fibrosis of Schistosomiasis japonica.Methods The egg cDNA library of Schistosoma japonicum (S. japonicum) was constructed and screened by immunological methods with the pooled sera of advanced schistosomiasis patients. The inserted foreign DNA fragments of positive clones were sequenced. The sequence data were analyzed using Wdnasis 2.5 and compared with Genebank data using blast software.Results Eighty-one clones containing recombinant DNA fragments were obtained from the egg cDNA library of S. japonicum by immunological screening. The DNA sequences of all clones belonged to the miracidial antigen family. The longest cDNA fragment was 1604 bp, which contained an open reading frame of 351 bp, which encoded a protein of 1 2913.35 daltons.Conclusion The cDNA sequence of the miracidial antigen of S. japonicum (Chinese strain) was obtained for the first time.  相似文献   

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AIM: Identify the candidate peptides used for anti-angiogenic therapy of esophageal cancer by in vivo screening C7C peptide library for peptides binding specifically to blood vessels of human esophageal cancer. METHODS: The phage displayed C7C peptide library was injected intravenously into mice bearing human esophageal tumor xenografts under renal capsule. After 5 rounds of screening, 13 clones were picked up individually and sequenced. During each round of screening, titers of phage recovery were calculated from tumor xenograft and control tissues. Homing of these 9 peptides to tumor vessel was detected by calculating phage titers in the tumor xenograft and control tissues(lung and spleen) after each phage was injected into mice model, and compared with the distribution of phage M13 and Ⅷ-related antigen in tumor xenograft by immuno- histochemical staining. Comparisons among groups of data were made using one-way analysis of variance (ANOVA), followed by the Bonferroni multiple comparisons test. RESULTS: The number of phage recovered from tumor tissue of each round increased gradually and the number of the fifth round was about 10.63 folds than it is in the first round (P<0.01). At the same time, the titers of spleen and lung used as control tissues decreased (P<0.01in spleen & P<0.05 in lung) by using analysis of variance of repeated measurement data. Immunohistochemical staining showed similar staining pattern with M13 antibody or Ⅷ-related antigen antibody, suggesting that phages displaying the selected peptides could home to blood vessel of human esophageal cancer. According to their DNA, 9 corresponding peptide sequences were deduced. And the homing ability to blood vessel of phages displaying the selected peptides was confirmed by comparing with their recovery in tumor and control tissues There was significant statistical difference in sequence YSFNSWM,PNPNNST,YSINDWH,LPAMPNS,YPTPYDI ,PMNADNL,SRHDLNS and STVATSQ between tumor group and the two control group as using analysis of variance. Two motifs,YSXNXW and PXNXXN,were also obtained by analyzing the homology of these peptide sequences. The staining distribution of phage with the sequence of PNPNNST was similar to that of the blood vessel marker Factor Ⅷ-related antigen staining. After sequencing, each phage with the selected peptide of PNPNNST with 1011 pfu was injected intravenously into mice. The homing ability to tumor vessel of these 9 kinds of peptides in the xenograft was higher than control tissues(lung and spleen). CONCLUSION: Nine peptides obtained from in vivo screening homed to the blood vessel of human esophageal cancer, and the two motifs of YSXNXW and PXNXXN are the possible biochemical recognition units binding to vascular endothelial cells of esophageal cancer. CONCLUSION: Nine peptides obtained from in vivo screening homed to the blood vessel of human esophageal cancer, and the two motifs of YSXNXW and PXNXXN are the possible biochemical recognition units binding to vascular endothelial cells of esophageal cancer.  相似文献   

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Objective: To generate eukaryotic expression vector of pcDNA3.1-BACE and obtain its transient expression in COS-7 cells and high expression in the neuroblastoma SK-N-SH cells. Methods: A 1503 bp cDNA fragment was amplified from the total RNA of human neuroblastoma by RT-PCR method and cloned into plasmid pcDNA3.1. The vector was identified by digestion with restriction enzymes BamHI and XhoI and sequenced by Sanger-dideoxy:mediated chain termination. The expression of BACE gene was detected by immunocytochemistry method. Results: The results showed that the cDNA fragment included 1503 bp total coding region. The recombinant eukaryotic cell expression vector of pcDNA3.1-BACE was constructed successfully, and the sequence of insert was identical to the published sequence. The COS-7 cells and the neuroblastoma SK-N-SH cells transfected with the pcDNA3.1-BACE plasmid expressed high level of BACE protein in cytoplasm. Conclusion: The recombinant plasmid pcDNA3.1-BACE can provide very useful tool for researching the mason of Alzheimer's disease and lays the important foundation for preventing the AD laterly.  相似文献   

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A hepatitis C virus(HCV)cDNA fragment,534bp in length and designated asQ534,was obtained by PCR amplification with self-designed primers.Q534 was cloned in-to Hinc Ⅱ site of pUC18 and the recombinant plasmid pQ534 was then selected from thebacterial transformants.The sequence analysis indicated that Q534 was a cDNA fragmentof HCV core gene,and located in HCV genome from positions 320 to 853 incorrespondence with Chiron's prototype sequence.The homologies between Q534 and theprototype at the levels of nucleotides and amino acids were 90.0% and 97.6%,respectively.The homologies of Q534 with Japanese HCV-J and HCV-BK strains were 96.6% and97.0% at the nucleotide level,and 98.2% and 98.8% at the amino acid level.In terms ofthe sequence,this Chinese HCV isolate should belong to HCV group Ⅱ.  相似文献   

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目的克隆获得树鼩卵磷脂胆固醇酰基转移酶cDNA序列并进行结构分析。方法以树鼩肝脏mRNA逆转录出的Ⅰ链cDNA为模板,运用SMARTRACEPCR扩增技术,扩增得到树鼩卵磷脂胆固醇酰基转移酶(LCAT)cDNA序列,并推导出LCAT氨基酸序列。利用分子生物学软件DNAMAN对氨基酸序列的一、二级结构及主要结构域进行分析和比较。结果树鼩LCATcDNA序列由1340个核苷酸构成,开放阅读框架1320bp,编码440个氨基酸的LCAT前体(含24个氨基酸构成的信号肽和416个氨基酸组成的成熟蛋白)熏3'非翻译区18bp,终止密码TAA,加尾信号AATAAA和一个25bp的poly(A)尾。树鼩LCATcDNA序列已作为新基因被GenBank接受,登记号AF272861。树鼩与人和狒狒LCATcDNA的同源性分别为90%和89%。结论树鼩LCATcDNA序列与人及其他实验动物高度同源。  相似文献   

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 【目的】构建载有小鼠抵抗素(resistin)基因及其反义核酸的重组真核表达质粒,探讨抵抗素对3T3-L1脂肪细胞葡萄糖摄取的影响。【方法】用RT-PCR技术从小鼠脂肪组织扩增抵抗素基因cDNA,A/T克隆于pGEM-T载体,再亚克隆于pcDNA3.1(+)和pcDNA3.1(-)真核表达载体,构建载有小鼠抵抗素(resistin)基因及其反义核酸的重组真核表达质粒pcDNA3.1^(+)resistin和pcDNA3.1^(-)-resistin^(-)。将pcDNA3.1^(-)resistin^(-)转染小鼠3T3-L1脂肪细胞,通过G418筛选稳定表达的细胞株。3T3-L1脂肪细胞分为对照、瞬时、载体和稳定4个细胞组(每组n=6),用^3H-2-脱氧葡萄糖进行非胰岛素和胰岛素介导的葡萄糖摄取试验。【结果】插入pcDNA3.1^(+)的DNA片段的核苷酸序列与小鼠抵抗素基因mRNA编码区序列完全一致.且方向正确:插入pcDNA3.10^(+)的DNA片段的核苷酸序列与抵抗素基因mRNA编码区核苷酸序列完全一致,且方向相反。对照组、瞬时组、载体组和稳定组的3T3-L1脂肪细胞非胰岛素和胰岛素介导的葡萄糖摄取率的无显著性差别(P〉O.05)。【结论】成功构建了载有抵抗素基因和载有抵抗素基因反义核酸的重组真核表达质粒。抵抗素对3T3-L1脂肪细胞的葡萄糖摄取均无明显影响。  相似文献   

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目的克隆不易感动脉粥样硬化动物树(treeshrew,TS)载脂蛋白AI(apolipoproteinAI,apoAI)的基因,比较分析其结构特点。方法以提取的肝组织mRNA为模板,反转录构建了TS肝组织cDNA文库。利用制备的兔抗TS载脂蛋白AI多抗血清为探针筛选该文库,对阳性克隆进行测序、序列分析及组织表达实验。结果克隆获得了TSapoAIcDNA序列,全长序列由900个核苷酸构成,包括28bp组成的5′非翻译区;795bp组成的一个完整开放阅读框架,编码翻译265个氨基酸的TSapoAI前体(含18个氨基酸构成的信号肽、6个氨基酸的原肽片段及241肽的成熟蛋白);两个终止密码TGA、TAA和随后72bp的3′非翻译区。新基因已被GenBank接受。对编码蛋白质的亲疏水性分析表明TSapoAI的疏水性强于人的相应蛋白。Northernblot显示TSapoAI基因主要在肝脏和小肠组织表达。结论获得了不易感动脉粥样硬化动物TSapoAI的新基因,编码的蛋白质结合胆固醇及胆固醇酯的能力可能大于人的apoAI。  相似文献   

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克隆不易感动脉粥样硬化动物树Ju(treeshrew,TS)载脂蛋白A-I(apolipoproteinAI,apoAI)的基因,比较分析其结构特点,方法以提取的肝组织mRNA为模板,反转录构建了TS肝组织cDNA文库。利用制备的兔抗TS载脂蛋白AI多抗血清为探针筛选该文库,对阳性克隆进行测序,序列分析及组织表达实验。结果克隆获得了TSapoAIcDNA序列,全长序列由900个核苷酸构成,包括28  相似文献   

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210 .seq是一个在大鼠心肌缺血预适应中表达上调的表达序列标签 (expressedsequencetag ,EST) ,用GENSCAN预测、Blast(Basiclocalalignmentsearchtool)比对、序列拼接、多序列比对等生物信息学方法克隆了该EST代表的大鼠MIP3基因的cDNA序列。大鼠MIP3基因的开放阅读框为 1332bp ,起始密码子前同一相位存在一个终止密码子 ,起始密码子邻近的序列符合Kozak规则 ;推测编码 4 4 3个氨基酸 ,与小鼠和人的MIP3基因or tholog的同源性很高 ,但是与数据库中其它蛋白质的同源性不高。TMpred分析显示该多肽有一个跨膜区域 ,氨基端位于膜内。Motifscan分析仅发现一个脯氨酸富集区域。以上结果显示MIP3基因是一个功能未知的新基因  相似文献   

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目的 克隆和鉴定一个新的阴道毛滴虫Rab1-like基因(TvRab1-like)及其内含子.方法 我们从一阴道毛滴虫cDNA表达文库中分离出一个cDNA克隆,它与各物种的Rab家族蛋白有较高的同源性,因此我们进一步用BLASTP、RPS-BLAST、ClustalW和MEGA3等分析软件对该cDNA克隆进行了序列分析和进化树分析;用PCR和RT-PCR等技术分别对该基因组和mRNA进行了扩增和测序分析.结果 序列分析结果表明该cDNA克隆长705 bp,开放阅读框具603 bp,推测肽链含有200个氨基酸.序列比较分析结果提示该cDNA克隆所推测的蛋白质是一个Rab1亚家族的亚型.进化树分析也表明它属于阴道毛滴虫Rab1亚家族.基因组PCR扩增和测序分析表明该基因包含一个25 bp的内含子,该内含子具有阴道毛滴虫和其它真核生物较大内含子所具备的典型的5'GT-AG-3'和分支位点基序.RT-PCR产物及其测序分析表明在该基因的转录本中存在着未剪切和剪切后的mRNA,说明确实有内含子的存在.结论 TvRab1-like基因属于阴道毛滴虫Rab1亚家族,该基因含有一个25 bp的内含子.该内含子是至今发现的最小的阴道毛滴虫基因内含子之一,很可能也是真核生物中最小的内含子.对诸类最低等真核生物内含子的研究将有助于我们理解真核生物内含子的起源和进化.  相似文献   

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Dnmt3b cDNA及其在小鼠胚胎组织中选择剪接异构体的克隆   总被引:1,自引:1,他引:0  
目的 克隆与小鼠胚胎发育相关的新的新的全长cDNA。方法 从小鼠胚胎cDNA文库中筛选出一个新的EST,以8~9d齿小鼠胚胎组织mRNA为模板进行RACE(rapid amplification of cDNA ends),结合PCR筛选cDNA文库,获得共全长cDNA序列后进行序列同源检索、结构分析和功能预测。整合后的cDNA两上物进行RT-PCR,克隆PCR产物,并对产物进行全序列测定,验证整  相似文献   

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目的定点突变法扩增制备完整hHGF-α cDNA,克隆并构建其原核表达载体。方法与结果以含有人HGF cDNA全序列的质粒pRC/CMV-hHGF为模板,设计合成一对特异引物进行聚合酶链反应(PCR),扩增hHGF-α cDNA基因,琼脂糖凝胶电泳检测,结果显示扩增得到了1.34 kb长的目的基因产物。将扩增产物以限制性内切酶Xho Ⅰ酶将其切为386 bp、954;bp两个片段,分别以EcoR Ⅰ/Xho Ⅰ、Xho Ⅰ/BamH Ⅰ与pBSKS载体连接重组,对目的基因分段克隆后进行序列测定,结果表明除通过定点突变引入的起始密码ATG、终止密码子TAA、TGA及EcoR Ⅰ、BamH Ⅰ识别位点外,扩增得到的PCR产物序列与Nakamura等报道的HGF cDNA中不包括前体序列的α链部分完全相同。将以EcoR Ⅰ/Xho Ⅰ、Xho Ⅰ/BamH Ⅰ从pBSKS-HGF-α 386、pBSKS-HGF-α 954中切出的386 bp、954 bp两个片段以EcoR Ⅰ/BamH Ⅰ与pBV220连接构建重组表达质粒pBV220-HGF-α,限制性内切酶图谱分析显示HGF-α cDNA插入到载体pBV220的EcoR Ⅰ/BamH Ⅰ位点,插入方向正确。温度诱导表达,SDS-PAGE显示一分子量与单体hHGF-α链吻合的蛋白带,证明表达质粒构建成功。结论设计制备了hHGF-α链cDNA,克隆建立了hHGF-α链原核表达质粒。  相似文献   

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