粘膜系统鳞状细胞癌(SCC)相关蛋白(DCUN1D1)的表达、纯化和晶体生长

目的:SCCRO/RP42/DCUN1D1是粘膜系统鳞片状细胞癌(SCC)发生时人类基因组3q区域扩增的潜在靶标之一,其蛋白作用机制尚不清楚,本文拟通过表达并大量纯化SCC相关蛋白DCUN1D1用于蛋白结晶以求获得其三维结构。方法:使用人肝脑组织RNA反转录产物为模板扩增出DCUN1D1基因cDNA片断并将其克隆至原核表达载体PGEX-6P-1中,通过IPTG诱导获得大量可溶性表达,再经过GST亲和层析和Sephadex G-200层析柱纯化。结果:获得了纯度95%以上的蛋白,采用悬滴气相扩散法筛选蛋白晶体,获得显微镜下可见的微晶。结论:初步得出DCUN1D1晶体生长条件及范围,为解析DCUN1D1的三维结构并进一步认识其生物功能奠定了基础。

Expression,Purification and Crystallization of DCUN1D1 (SCC-related oncogene) in Mucosal System

Objective:To discuss squamous cell carcinomas(SCC)in mucosal system,including tumors arising from the head and neck,lung,esophagus,and cervix,which accounts for over one third of all cancers and cancer-related deaths annually in the United States.SCC-related oncogene(SCCRO/RP42/DCUNID1)has been proved exclusively to be amplified in cancer cell lines and involved in Hughog signaling pathway,which is amplified along the 3q26.3 region in human SCC.Methods:In order to know more about the functions of DCUN1D1,firsdy the cDNA fragment of DCUN1D1 was amplified from human livers' and brains' RNA by using RT-PCR.The protein was expressed in soluble form in E.coli and was purified by GST-Sepharose col- umn and Sephadex G-200.Results:The protein DCUN1D1 was crystallized by the hanging-drop vapour-diffusion method and over 95% purity of protein was obtained as well as the visible crystals under microscope.Conclusions:The growing conditions of the protein crystal DCUN1D1 has been initially found out,which laid a foundation for the analysis of tri-dimensional structure of DCUN1D1 as well as further understanding its biological function.