用SEREX方法筛选HCC抗原及hcct-19表达谱的检测  

Identification of hepatocellular carcinoma antigens by serological analysis of recombinant cDNA expression library and detection of hcct-19 expression in human tissues and cell lines

作　　者：陈晓红[1] 陈智[1] 陈峰[1] 姚航平[1] 夏琦[1] 焦婧婧[1] 

机构地区：[1]浙江大学医学院附属第一医院传染病研究所,浙江省杭州市310003

出　　处：《世界华人消化杂志》2006年第11期1064-1070,共7页World Chinese Journal of Digestology

摘　　要：目的:用SEREX方法鉴定HCC表达的肿瘤抗原,检测hcct-19的表达谱．方法:将HCC表达文库铺板,IPTG诱导蛋白质表达,BSA封闭．同1:1 000稀释的预吸收 HCC患者血清反应后,与1:5 000稀释的羊抗人抗体反应,在BCIP／NBT的作用下显色,挑取阳性克隆噬菌斑块,重复筛选和铺板3次,直至得到一致的单克隆免疫阳性重组噬菌体．挑取阳性克隆噬菌斑,用载体克隆位点两端的通用引物进行PCR扩增,PCR产物纯化测序, 序列结果用BLAST软件同GenBank中的已知基因进行对比分析．检测阳性克隆抗原基因 hcct-19在部分正常组织、肿瘤组织及肿瘤细胞株中的表达,半定量RT-PCR方法检测阳性克隆抗原基因在肝癌及癌旁组织中的表达．结果:共得到31个阳性克隆,代表14个不同的 cDNA序列,其中10个为已知功能的基因,4 个为未知功能的基因．在已知功能的基因中, RFC2、NDUFA4及MCART1首次被发现与 HCC有关．hcct-19在部分正常组织、肿瘤组织及肿瘤细胞株中表达,在肝癌组织中的表达强度明显高于癌旁组织(13．2±2．7vs2．9±0．3, P<0．05)．结论:本实验筛选出的HCC抗原有助于进一步阐明HCC的形成过程．hcct-19可能作为一个过量表达的基因参与了HCC的发病过程．AIM： To identify hepatocellular carcinoma （HCC） antigens by serological analysis of recombinant cDNA expression library （SEREX） and observe the expression of hcct-19 in human tissues and cell lines. METHODS： Firstly, a high-quality cDNA library from human HCC tissue was constructed successfully. E.coli transfected with recombinant ;λTripleX2 phages were plated onto LBagar plates. The expression of recombinant proteins was induced with isopropyl-beta-Dthiogalactopyranoside （IPTG）. The membranes were blocked with BSA and incubated with a 1 ： 1000 dilution of patient＇s serum, which had been preabsorbed with transfected E.coli phage lysate. Then they were incubated with a 1 ： 5000 dilution of the alkaline phosphatase-conjugated antibody specific for human IgG. Reactive clones were visualized by staining with BCIP/NBT, and these clones were subcloned three times to obtain monoclonality. Secondly, positive clone plaques were randomly picked and tested using polymerase chain reaction （PCR） with universal primers derived from the sequence flanking the vector. PCR products were purified and sequenced, and the results were analyzed with BLAST software in comparison with the known genes in GenBank. Thirdly, the positive clone of hcct-19 gene expression was detected by PCR in normal human tissues of stomach, colon, liver, pancreas, kidney, uterus, testicle and bladder, human tumor tissues of colon carcinoma, renal carcinoma, pancreatic cancer, gastric carcinoma, prostatic carcinoma, HCC, ovarian cancer and lung cancer, and tumor cell lines of HepGv, Hela, MGC-803, PANC-1, PC3, W-549 and SW-480, and semi-quantitative reverse transcription PCR was used to detect hcct-19 expression in HCC and cancer-adjacent tissues. RESULTS： Firstly, thirty-one positive clones were obtained, representing fourteen different cDNA sequences （lengths from 324 to 805 bp）. Ten cDNAs were genes with known functions, of which replication factor C 2, NADH dehydrogenase 1 alpha subcomplex 4 and mitochondrial carrier t

关 键 词：SEREX 肝细胞癌 抗原 hcct-19 表达谱 人组织 细胞株 

分 类 号：R735.7[医药卫生—肿瘤] R554.1[医药卫生—临床医学]