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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、Westernblot、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Westernblot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。Northernblot证实,内源性CCP22mRNA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。
Objective To clone and analyze a novel gene CCP22 and study its structure and biological characteristics. Methods Conventional molecular cloning, bioinformatics analysis, Westernblot, RT-PCR. Results A novel gene containing 22 complement control protein (CCP) sequence elements was isolated from human breast cDNA library by yeast two-hybrid technique and named CCP22. Bioinformatics analysis showed that the gene was located on the human chromosome 9q31.2-32, a total of 15 exons, the gene coding sequence of 4494bp in length, encoding 1497 amino acids. The full-length coding sequence of CCP22 gene was cloned into eukaryotic vector. The expression product of 293T after transfection of the expression vector was confirmed by Western blot to be a secreted protein. The fusion of CCP22 and green fluorescent protein (EGFP) tag confirmed that CCP22 mainly distributed outside the nucleus. Northernblot confirmed that the endogenous CCP22 mRNA transcript is about 6 kb in length. The results of RT-PCR showed that CCP22 was highly expressed in normal breast cell lines but not in many breast cancer cell lines. Conclusion CCP22 may play an important role in the development of breast cancer.