EID3基因在大肠癌传代细胞mRNA的表达及其克隆  

EID3 gene cloning and mRNA expression analysis in colon cancer cell lines

作　　者：王钦富[1] 李志 牟红梅[1] 徐娜[1] 张雪梅[1] 韩慧[1] 

机构地区：[1]大连大学免疫学研究所,大连116622 [2]大连中心医院检验科

出　　处：《中国癌症防治杂志》2012年第1期38-42,共5页CHINESE JOURNAL OF ONCOLOGY PREVENTION AND TREATMENT

基　　金：辽宁省高等学校科研项目计划(No200802919)

摘　　要：目的研究新的大肠癌相关性抗原EID3基因的克隆及其诊断价值。方法用SEREX技术筛选睾丸组织cDNA噬菌体表达文库,发现了一类似于肿瘤抑制基因(E1A基因)的EID3基因(NM_001008394.1),构建原核表达载体pET32k-EID3,IPTG诱导重组EID3蛋白质的表达,并利用Ni NTA His Bind树脂纯化该重组蛋白质。用RT-PCR技术研究EID3mRNA在正常组织和大肠癌传代细胞的表达。结果经PCR扩增成功获得1002bp的人EID3基因,测序正确。在大肠杆菌中目的蛋白质的表达量占菌体总蛋白质的10%,SDS-PAGE及Western blot分析显示,其相对分子质量为39000KD,纯化后的6xHisEID3纯度可达92%。通过RT-PCR分析发现,EID3基因在43例大肠癌传代细胞株中有39例阳性,阳性率为90.7%。在正常组织中,除睾丸组织不表达或有极低水平转录外,余均不表达。结论 EID3基因克隆、表达及纯化的成功,为其今后的肿瘤诊断和肿瘤多肽疫苗治疗研究奠定了基础。EID3m RNA表达检测用于诊断大肠癌,可能具有高特异性和高敏感性的特点。EID3蛋白在大肠癌患者中能够诱导机体的抗体免疫应答,可能成为一个新的大肠癌相关性抗原分子,其用于诊断大肠癌的可行性,有待进一步深入研究。Objective To clone the gene encoding the novel colon cancer-associated antigen EID3 and investigate the possibility of using EID3 mRNA expression to diagnose patients with colon cancer.Methods Screening of a testis cDNA library using sera from patients with colon cancer led to discovery of the EID3 gene（NM_001008394.1）,similar to tumor suppressor gene E1A.The pattern of EID3 mRNA expression was investigated in normal tissue and colon cancer cell lines using reverse transcription-PCR（RT-PCR）.Results The EID3 gene（1 002 bp） was cloned by PCR and overexpressed in E.coli at levels reaching 10% of total protein.The mole-cular weight was confirmed to be 39 kDa by SDS-PAGE and Western blot,and it was purified to 92% homogeneity.The gene encoding EID3 is located on chromosome 19q13.2,which contains one exon.RT-PCR analysis detected the gene in 39 of 43（90.7%） colon can-cer cell lines,while EID3 mRNA was undetectable in all normal tissues examined except testis.Conclusions EID3 mRNA detection may prove to be a highly sensitive and specific technique to diagnose colon cancer.EID3 protein has been reported to initiate a hu-moral immune response in patients with colon cancer.Further studies are needed to explore the diagnostic potential of EID3 in colon cancer.

关 键 词：大肠癌 EID3 克隆 MRNA 

分 类 号：R734.38[医药卫生—肿瘤]