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在抑制性消减杂交技术基础上,我们利用cDNA末端快速扩增方法(RACE)从肝癌组织中得到了5条cDNA片段,其中编号为694447-3的cDNA片段,长550 bp(登录号CK730345), 经GenBank检索,发现其与2002年公布的一条新的但功能未知的长1476 bp的基因序列(登录号BC047440)同源性达63%。到目前为止,BC047440基因在肝癌中的表达情况仍不清楚。1.材料与方法:肝癌细胞株HepG2,由第三军医大学西南医院消化科司遂海惠赠。标本取自西南医院肝胆科新鲜手术标本,病理证实为原发性肝细胞肝癌。总RNA的提取参照文献[2]进行,RACE法扩增参照SMARTTM RACE cDNA Ampli- fication试剂盒(美国Clontech公司)的说明书进行。逆转录聚合酶链反应(RT-PCR)法扩增参照日本TaKaRa公司说明进行。cDNA探针制备来自载有BC047440基因片段的重组质粒
Based on the suppression subtractive hybridization, five cDNA fragments were obtained from hepatocellular carcinoma by rapid amplification of cDNA ends (RACE). The cDNA fragment numbered 694447-3 was 550 bp (accession number CK730345) It was found by GenBank that it shared 63% homology with a new, but unknown, 1476 bp long gene (accession number BC047440) published in 2002. So far, the expression of BC047440 gene in HCC remains unclear. 1. Materials and Methods: Hepatocellular carcinoma cell line HepG2 was donated by Suihai Department of Gastroenterology, Southwest Hospital, Third Military Medical University. Specimens taken from the Southwest Hospital of hepatobiliary fresh surgical specimens, the pathology confirmed as primary hepatocellular carcinoma. Total RNA extraction was performed according to [2]. RACE amplification was performed according to the instructions of SMART ™ RACE cDNA Ampli-fication Kit (Clontech, USA). Reverse transcription polymerase chain reaction (RT-PCR) amplification with reference to Japan TaKaRa company instructions. cDNA probes were prepared from recombinant plasmids carrying the BC047440 gene fragment