【作者】 崔晓燕； 方定志； 刘秉文；

【Author】 CUI Xiao -yan, FANG Ding - zhi, and LIU Bing - wen. Department of Biochemistry and Molecular Biology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China

【机构】 四川大学华西基础医学与法医学院生物化学与分子生物学教研室；

【摘要】 目的筛选人肝脏高凝状态相关基因的全长cDNA序列。方法采用差异筛选法对本室建立的高凝状态大鼠肝脏差异表达基因消减cDNA文库进行筛选;以获得的差异cDNA克隆为模板,采用PCR方法扩增目的片段;以PCR产物作探针,用噬菌斑原位杂交法筛选人肝脏cDNA文库,经初筛、二筛和三筛后,将获得的阳性克隆转入E.coli BM 25.8,使λTripIEx环化成质粒pTripIEx,经酶切鉴定后进行DNA测序。得到的DNA序列用standard nucleotide-nucleotide BLAST[blastn]软件,与GenBank DNA数据库(non-redundant)中的DNA序列进行同源性分析。结果从人肝脏cDNA文库中筛选获得4条全长cDNA序列,长度分别为:1576 bp、1078 bp、1199 bp和913 bp。生物信息学分析表明,GenBank DNA数据库中与第一条cDNA同源性最高的已知基因是“Homo sapiens fibrinogen,gam-ma polypeptide,transcript variant gamma-A,mRNA"(gi | 13937873),其长度为1594 bp,同源序列长度为1543 bp,Score值为3039 bits,E值为0.0,Identities为99%;与第二条cDNA同源性最高的已知基因是“Homo sapiens LFIRE1(LFIRE1)mRNA”(gi | 22023089),其长度为1270 bp,同源序列长度为1028 bp,Score值为1968 bits,E值为0.0,Identities为99%;与第三条cDNA同源性最高的已知基因是“Homo sapiens chromosome 10 open reading frame 104,mRNA”(gi | 16306924),其长度为1177bp,同源序列长度为1151 bp,Score值为2232 bits,E值为0.0,Identities为99%;与第四条cDNA同源性最高的已知基因是“Homo sapiens carbamoyl-phosphate synthetase 1,mitochondrial,mRNA”(gi |34783937),其长度为1490 bp,同源序列2条,长度分别为491 bp和349 bp,Score值分别为967 bits和682 bits,E值均为0.0,Identities为99%。结论用高凝状态大鼠肝脏差异表达基因cDNA为探针,从人肝脏cDNA文库中筛选获得4条PTS相关基因。其中3条的表达产物分别为纤维蛋白原、LFIRE1、10号染色体开放阅读框架104 mRNA;另1条与氨基甲酰磷酸合成酶1同源。更多还原

【Abstract】 Objective To screen prothrombotic state -related cDNA sequences from human hepatic cD NA library. Methods The subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state (PTS) was constructed by suppression subtractive hybridization. Positive clones were identified by differential screening. The target DNA sequences of positive cDNA clones of differentially expressed genes from rat liver of PTS were amplified by PCR. The PCR products were used as probes to screen a human hepatic cD NA library. After the first, second and third screening, the positive λTripIEx lysate was transduced into E. coli strain BM25. 8 circularization of pTripIEx. The positive circularized plasmids were identified by double enzyme digestion. The cDNA fragments of the positive plasmids were sequenced and analyzed by bioinformatics (blastn) . Results 4 PTS -related cDNA sequences were identified from human hepatic cDNA library. For 3 of them, their products of expression were respectively fibrinogen, LFIRE1, and chromosome 10 open reading frame 104 mRNA. The fourth sequence had homology with carbamoyl - phosphate synthetase 1 gene. Conclusion 4 PTS - related cDNA sequences were identified from human hepatic cDNA library.更多还原