微信公众号

官网二维码
中国癌症防治杂志

中国癌症防治杂志 ›› 2012, Vol. 4 ›› Issue (1): 38-42.doi: 10.3969/j.issn.1674-5671.2012.01.09

• 基础研究 • 上一篇    下一篇

EID3基因在大肠癌传代细胞mRNA表达及其克隆

  

  1. 大连大学免疫学研究所；大连中心医院检验科
  • 出版日期:2012-03-25 发布日期:2012-04-16
  • 通讯作者: 王钦富 E-mail:qinfuwang@126.com
  • 基金资助:

    广西自然科学基金资助项目（2011GXNSFA018307）；广西硕士研究生科研创新项目（2011105981002M232）

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

  • Online:2012-03-25 Published:2012-04-16

PDF (PC)

581

赞

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EID3 mRNA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1 002 bp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39 000 KD， 纯化后的6xHis EID3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。EID3 mRNA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。

关键词: 大肠癌, EID3, 克隆, mRNA

Abstract: 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 molecular 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 cancer 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 humoral immune response in patients with colon cancer.Further studies are needed to explore the diagnostic potential of EID3 in colon cancer.

Key words: Colon cancer, EID3, Gene cloning, mRNA