全长及细胞内磷酸化位点缺失的人组织因子真核表达重组质粒的构建及其生物学功能
Construction of eukaryotic expression plasmid for human TF full-length，TF truncated and characterization of their biological function
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摘要: 目的 ： 研究组织因子(tissue factor，TF)细胞内区域的功能，分析TF细胞内区域对人乳腺癌细胞MCF-7迁移能力的影响。 方法 ： Trizol一步法抽提人乳腺癌细胞MDA-MB-231总RNA，RT-PCR扩增TF全长(TF full-length)基因和TF细胞内磷酸化位点缺失(TF truncated)基因，分别克隆入pGEM-T载体，再构建人TF full-length基因和TF truncated基因的真核表达质粒pcDNA3.1(+)-TF full-length和pcDNA3.1(+)-TF truncated。将构建好的pcDNA3.1(+)-TF full-length和pcDNA3.1(+)-TF truncated瞬时转染MCF-7细胞，RT-PCR和Western blotting鉴定重组质粒的表达，并用发色底物法检测瞬时转染后TF的促凝血功能。利用Transwell小室检测转入人TF full-length基因和TF truncated基因后MCF-7细胞的迁移能力。 结果 ： 扩增出人TF full-length基因和TF truncated基因，成功克隆入真核表达载体pcDNA3.1(+)，并且在MCF-7细胞中表达后具有促凝血活性。瞬时转染pcDNA3.1(+)-TF truncated不能增加MCF-7 细胞的迁移能力，而转染 pcDNA3.1(+)-TF full-length后可以显著增强MCF-7细胞的迁移能力。 结论 ： 成功构建了带有人TF full-length，TF truncated基因的真核表达质粒pcDNA3.1(+)-TF full-length和pcDNA3.1(+)-TF truncated，为研究TF以及其细胞内磷酸化位点区域在肿瘤学中的作用奠定了基础。
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关键词:
- 组织因子 /
- 磷酸化位点缺失 /
- 真核表达载体pcDNA3.1(+) /
- 细胞迁移
Abstract: Aim ：To investigate the function of tissue factor(TF) cytoplasmic domain，and study its biological functions in breast cancer migration. Methods ：Total RNA was isolated from human breast cells(MDA-MB-231) with Trizol，and the full-length TF and truncated TF genes were amplified by RT-PCR，and then inserted into pGEM-T vectors.DNA sequencing was performed before the amplified products were cloned into the eukaryotic expression vector pcDNA3.1(+).The recombinant plasmid was transiently transfected into MCF-7 cells by means of liposome and the expression levels were examined by RT-PCR and Western blotting.And the TF procoagulant activities were detected by chromogenic substrate assay after being transfected with TF full-length，TF truncated cDNA and empty vector.A transwell assay was employed to examine the over-expression of TF full-length and TF truncated on breast cancer cells migration. Results ：The amplified products were confirmed as the cDNA of TF full-length and TF truncated by DNA sequencing.Positive clones pcDNA3.1(+)-TF full-length and pcDNA3.1(+)-TF truncated were verified by endonuclease digestion.With the increased TF expression，procoagulant activity was also augmented.TF truncated had no effect on MCF-7 cells migration，but over-expression of TF full-length significantly increased MCF-7 cell migration. Conclusion ：The eukaryotic expression plasmids for TF full-length and TF truncated were constructed successfully，which provides a basis for further investigation on the role of TF and its cytoplasmic phosphorylation domain in cancers. -
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