中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (41): 7687-7690.doi: 10.3969/j.issn.1673-8225.2010.41.021

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

锌指转录因子snai1高表达质粒构建和稳定株的筛选

周传文，李倩君   

  1. 南京医科大学附属淮安第一医院(淮安市第一人民医院)消化科，江苏省淮安市 223300
  • 出版日期:2010-10-08 发布日期:2010-10-08
  • 作者简介:周传文，男，1967年生，江苏省淮安市人，汉族，副主任医师，主要从事消化科方面的研究。 zhou_cw1967@sina.cn

Construction of zinc finger transcription factor snai1 expressing vector and screening of stably transfected cell clone

Zhou Chuan-wen, Li Qian-jun   

  1. Department of Gastroenterology, Huaian First Hospital (the First People’s Hospital of Huai’an), Nanjing Medical University, Huaian  223300, China
  • Online:2010-10-08 Published:2010-10-08
  • About author:Zhou Chuan-wen, Associate chief physician, Department of Gastroenterology, Huaian First Hospital (the First People’s Hospital of Huaian), Nanjing Medical University, Huaian 223300, China zhou_cw1967@sina.cn

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341

被引次数

1

摘要：

背景：研究表明Snai1和E-钙黏素在肿瘤组织中有负向表达关系，且与Snai1与CDH1启动子区box盒结合，抑制CDH1基因转录。
目的：构建针对snai1的高表达载体，建立稳定转染的细胞株。
方法：根据GenBank中snai1的基因序列人工合成snai1全序列，将合成好的序列装入Pmd-18T载体并转化感受态细胞DH5α，进行阳性克隆筛选。测序验证重组克隆中基因序列正确后，用EcoR Ⅰ和Xho Ⅰ双酶切Pmd-18T/snai1质粒，将所得的snai1全基因片段转入pcDNA3.1(+)空质粒，构建pcDNA(+)/snai1真核表达质粒，同时构建阴性对照质粒，并分别对结肠腺癌细胞HT-29进行转染，最后利用G418进行稳定表达株的筛选，并采用RT-PCR和Western blot鉴定宿主细胞内snai1基因的表达情况。
结果与结论：成功构建了真核表达质粒pcDNA(+)/snai1，顺利转染结肠癌细胞HT-29后于600 mg/L G418浓度时筛选 21 d得到snai1基因稳定表达株，构建的snai1基因正义表达质粒pCDNA3.1(+)/snai1转染宿主细胞后可促进snai1基因的表达。

关键词: 锌指转录因子snai1, 表达质粒, 感受态细胞, 结肠癌, 转染　

Abstract:

BACKGROUND: Studies demonstrated that Snai1 has been shown to negative interact with E-cadherin in tumor tissue and combine with CDH1 promoter region box motif, thus, suppress the CDH1 genetic transcription. 
OBJECTIVE: To construct snai1 eukaryotic expression vector and to establish a stable transfective cell line.
METHODS: Complete exon sequence based on the sequence of snai1 in the GenBank was designed and inserted into the plasmid Pmd-18T, recombining the plasmid Pmd-18T/snai1, which was transformed into E.coli DH5 and selected with ampicillin. The positive clones containing the recombinant Plasmid Pmd-18T/snai1 were sequenced and digested by restriction endonucleases EcoRI and XhoI.The digestion product of EcoRI and XhoI was purified and inserted into the eukaryotic expression vector pcDNA3.1(+) to construct pcDNA3.1(+)/Snai1,which was identified by sequencing and transfected into adenocarcinoma of colon HT-29 cells. The HT-29 cells with stable expression of snai1 were selected by G418 and confirmed by RT-PCR and Western blot.
RESULTS AND CONCLUSION: The eukaryotic expression vector pcDNA3.1(+)/snai1 was constructed successfully. HT-29 cells stably expressing snai1 were selected by G418 with 600 mg/L at 21 days. The successful construction of the plasmid of pcDNA3.1(+)/snai1 could significantly up-regulate the expression of snai1in colon neoplasms cells HT-29.

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