一种含多个CCP结构域的新型基因的克隆及生物学特性初探

目的克隆并分析新型基因CCP22，研究其结构及初探生物学特性。方法常规分子克隆、生物信息学分析、Western blot、RT—PCR。结果利用酵母双杂交技术从人乳腺文库中分离到一种含有22个补体调控蛋白（complement control protein，CCP）序列元件的新基因，命名为CCP22。生物信息学分析发现，该基因定位于人染色体9q31．2-32，共15个外显子，基因编码序列全长4494bp，编码1497个氨基酸。将CCP22基因全长编码序列克隆于真核载体中，该表达载体转染人胚肾细胞293T后获得的表达产物经Western blot证实CCP22属于分泌蛋白。将CCP22与绿色荧光蛋白（EGFP）标签融合后证实CCP22主要分布在细胞核外。Northern blot证实，内源性CCP22 mRNA转录本全长约6kb。RT-PCR检测表明，CCP22在正常乳腺细胞株中高表达，而在多种乳腺肿瘤细胞株中不表达或低表达。结论CCP22在乳腺肿瘤发生发展中可能发挥重要作用。

Cloning and preliminary functional analysis of a novel gene with many CCP modules

Objective To study the structure and biological function of the novel CCP22 gene.Methods Routine molecular clone, biological information analysis, Western blot assay and RT-PCR technology were applied.Results A novel gene named CCP22 whose coding sequence contains 22 complement control protein(CCP) elements was obtained from a human breast cDNA library. CCP22 gene was mapped on chromosome 9q 31.2-32, including 15 exons. The full length of cDNA had an open reading frame of 4494 bp, which encoded 1497 amino acids. The entire coding sequence of CCP22 was cloned into an eukaryotic vector. The expression vector was transfected into human embryonic kidney 293T cells. Western blot test indicated that the expression product CCP22 was a secreted protein. When fused to enhanced green fluorescence protein(EGFP), CCP22 was located outside of nuclei. RT-PCR analysis showed that higher level of CCP22 was expressed in a normal breast cell line but low or undetectable level was found in many breast cancer cell lines.Conclusion CCP22 may play an important role in the pathogenesis of breast cancer.