胰腺癌转移相关基因C14orf166的真核表达及其蛋白相互作用的蛋白质组学筛选

目的旨在对胰腺癌转移相关基因C14orf166进行真核重组表达,并在此基础上结合蛋白质组学方法初步筛选其相互作用蛋白,为进一步研究C14orf166在肿瘤转移中的作用提供研究基础。方法构建人C14orf166的带双标签的真核表达载体pcDNA3.1-Flag-C14orf166-His,通过脂质体将其转染至人胚肾293T细胞。采用Ni-agrose进行His标签蛋白的pull-down纯化,分离C14orf166的蛋白结合复合体,对蛋白混合物进行SDS-PAGE分析,选择差异条带,进行MALDI-TOF-TOF质谱鉴定。结果在293T细胞中重组表达了C14orf166蛋白,对C14orf166蛋白复合体进行分离鉴定后,筛选出RS8、EFCB9和NRAP3个可能与C14orf166相互作用的蛋白。结论基因重组表达结合pull-down和蛋白质组学方法可以发现新的相互作用蛋白,为进一步了解C14orf166在肿瘤发病机制中的作用提供了新的线索。

The eukaryotic expression of pancreatic cancer relted gene C14orf166 and screening of its interacting proteins by Proteomics methods

Objective The aim of this study was to express C14orf166 in eukaryotic cells,and to screen and identify proteins interacting with C14orf166,which will provide foundation for further study on its function on tumor metastasis.
Methods Constructed eukaryotic expression vector of human C14orf166 gene pcDNA3.1- Flag-C14orf166-His with two tags,then the recombinated vector were transfected into 293T cells by Lipofectamine2000.Using Ni-agrose to pull-down His labeled Proteins,then C14orf166 compouds were obtained.SDS-PAGE was used to separate the compouds,then different straps were selected and identified by MALDI-TOF- TOF.
Results C14orf166 was successfully overexpressed in 293T cells,three poteintial interaction proteins of C14orf166 were identified.
Conclusion Gene recombination expression combined with pull-down and Proteomics methods are eligible to find out novel interacting proteins,which provide new clues to investigate the effect of C14orf166 on tumor pathogenesis.