摘要
目的:克隆原发性肝癌相关新基因. 方法:利用抑制消减杂交法(suppression subtractive hybridi- zation,SSH)已经发现了1条新的肝癌相关基因片断表达序列标签(EST),长447 bp,经Genebank检索,90%无同源性.在其保守序列区设计了2条用于3’Race扩增的寡聚核苷酸引物(3’GSP2:5’-CGCATAGTACCAGTATCGAC AAAGG-3’,3’NGSP2:5’-TCCACATTACGGACC CGACGGATT-3’),利用cDNA末端快速扩增法(RACE) 进一步克隆该基因的全长cDNA序列.人原发性肝癌细胞株HepG2,体外传代培养,培养基为RPMI1640培养基.提取HepG2总RNA,方法参照SV Total RNA Isolation System 的说明进行.RACE法扩增采用Clontech公司的Smart TM Race cDNA Amplification Kit.将3’RACE-PCR扩增的目的片段以Race自带的产物纯化试剂盒进行纯化、回收,然后将其克隆到PMD 18-T Vector中,提纯质粒后进行酶切鉴定,确认质粒内有插入片段,由宝生物工程(大连)有限公司协助完成测序.将克隆所得cDNA片段用NCBI 提供的BLASTN与GeneBank的dbEST、nr数据库进行同源性比较,确认代表新基因的EST并且登录GeneBank (http://www.ncbi.nlm.nih.Gov/submission).3例病理标本取自西南医院肝胆科,病理证实均为原发性肝细胞肝癌, 分别提取肝癌及远端正常肝组织总RNA.将酶切回收的克隆插入片段分别进行同位素标记获得cDNA探针,利用Clontech公司的ExpressHybTM杂交液通过RNA印记法检测克隆片段在肝癌及正常肝组织中的表达,方法参照ExpressHybTM Hybridization Solution user manual说明进行. 同时,利用互联网的基因表达分析序列数据库(http://www. ncbi.nlm.nih.gov/SAGE)(series analysis of gene expression, SAGE)对基因的表达及其表达水平进行分析,从而确定其组织的分布. 结果:得到5条3’EST(694 447-3,724 447-3,697 447-3,711 447-3,692 447-3;大小500-550 bp),5 条3’EST均已登录GeneBank(登录号:CK730344, CK730345,CK730346,CK730347,CK730348).对其中2条带有poly-A尾的3’EST(694 447-3,724 447-3),进�
AIM: To clone and characterize novel genes that might be involved in hepatocellular carcinoma. METHODS: An EST fragment differentially expressed between hepatocellular carcinoma (HCC) and its adjacent nontumorous liver tissues had been cloned using suppression subtractive hybridization (SSH). With this approach, we identified a novel EST that consisted of 447bp. In order to gain its full-length cDNA fragment, two gene-specific primers were designed for 3'-rapid amplification of cDNA end RACE. One HCC cell line, HepG2, was maintained in the RPMI1640 media and recommended culture conditions. Total RNA was extracted from HepG2 by the SV Total RNA Isolation System (Promega). RACE reactions were prepared using the SMARTTM RACE cDNA amplification kit (Clontech). Initial amplification was carried out with gene-specific primer 3'GSP2(5'-CGCATAGT ACCAGTATCGACAA AGG-30, followed by nested PCR using gene-specific primer 3'NGSP2(5'-TCCACATTACGGACCCGACGGATT-3'). These amplified cDNA fragments obtained from RACE were subcloned into the PMD18-T vector (TaKaRa) and sequenced by ABI PRISM377 DNA sequencer. Basic local aligment search tools were carried out using BLASTN and dbEST and nr database. Northern blot was applied to detect the expression of these cDNA fragments between HCC and its adjacent nontumorous liver tissues. A total of 3 liver specimens were collected from the Southwest Hospital of Chongqing in China. The final diagnosis of HCC was confirmed by histological examination. Total RNA was extracted from either HCC or its adjacent nontumorous liver tissues in the same way. These cDNA fragments were excised from the PMD18-T vector, purified and 32P-labeled as cDNA probes using the random primed labeling method. Northern blot was prepared by using the ExpressHybTM hybridization solution (Clontech) according to the protocol provided by the manufacturer. Combination of Northern blot and virtual Northern (http://www.ncbi.nlm.nih.gov/SAGE) (series analysis of gene expression, SAGE), expression of these cDNA fragments in multi
出处
《世界华人消化杂志》
CAS
2004年第8期1785-1788,共4页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No.30170424~~
