肺癌转移相关蛋白1相互作用蛋白的筛选及验证

Screening and verification of LCMR1 interacting protein

  • 摘要: 目的 筛选肺癌转移相关蛋白1(lung cancer metastasis related protein 1,LCMR1)的相互作用蛋白并进行验证。 方法 以人肺癌95D细胞RNA作为模板,构建95D细胞cDNA文库。将LCMR1质粒及人肺癌95D细胞cDNA文库共转化酵母菌AH109,初步筛选LCMR1相互作用蛋白。应用融合蛋白沉降技术和免疫共沉淀技术对酵母双杂交结果进行验证,确定蛋白质相互作用。 结果 成功构建了人肺癌95D细胞的cDNA文库。应用酵母双杂交技术筛选出6种LCMR1相互作用蛋白,选取其中DEK原癌基因进行验证。使用融合蛋白沉降技术及免疫共沉淀技术证实,LCMR1蛋白与DEK蛋白在体外及细胞内均可以特异结合,特异结合发生在DEK蛋白N端功能区。 结论 LCMR1与DEK为相互作用蛋白,DEK蛋白N端功能区与细胞凋亡密切相关,为LCMR1基因功能研究提供线索和相应分子基础。

     

    Abstract: Objective To detect and verify the interaction proteins of LCMR1 (Lung Cancer Metastasis Related Protein 1) through protein-protein interaction analysis, and provide clues for the study of LCMR1 gene function. Methods RNA of human lung cancer 95D cells was extracted as a template to construct a cDNA library. The LCMR1 plasmid and human lung cancer 95D cell cDNA library were co-transformed into yeast AH109 to screen the protein interacting with LCMR1. The interacting proteins of LCMR1 were confirmed by co-immunoprecipitation in vivo and further confirmed by GST pull-down in vitro assay. Results The 95D cell library was successfully constructed with the total RNA of human lung cancer 95D cells as a template. The LCMR1 plasmid could express the LCMR1 fusion protein correctly in yeast. Six kinds of LCMR1 interacting proteins were screened by yeast two-hybrid technique and proto-oncogene DEK was selected for validation. Co-immunoprecipitation and GST pull-down assays confirmed that DEK protein and LCMR1 protein could bind specifically in vitro and in vivo. GST pull-down assay confirmed the specific binding site of LCMR1 protein located in the N-terminal functional region of DEK. Conclusion LCMR1 interacts with DEK, and the interaction is mediated by N-terminal functional region of DEK.

     

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