激光生物学报摘要, 更新时间: 2006年10月29日
  由美国生科集团 (BVTech, Inc.) 主办
  
KCTD10基因的表达、抗体制备及其在常见细胞系中的表达
激光生物学报摘要 2006-5

张文峰 , 周爱东 , 杨利平 , 李红, 张 健* (蛋白质化学与发育生物学教育部重点实验室,湖南师范大学生命科学学院 410081)

摘 要: 人类KCTD10(channel tetramerisation domain-containing 10)基因属于PDIP1(polymerase delta-interacting protein 1)基因家族。最近的研究表明,小鼠KCTD10能同时和PCNA以及DNA聚合酶δ相互作用,并受肿瘤坏死因子α诱导;可能在诸如DNA修复、DNA复制、细胞周期调控以及人类的多种疾病中发挥重要作用,但对其确切功能和作用机制研究不多。本文克隆人的KCTD10基因到pQE-N3原核表达载体,在原核细胞株BL21(DE3)中表达并获得融合表达产物。获得的融合蛋白产物通过免疫新西兰大白兔获得兔抗KCTD10多克隆抗体。采用Western印迹技术,用该抗体检测KCTD10基因的原核和真核表达产物,证明该抗体有较好的针对KCTD10蛋白的专一性,可用于对KCTD10的结构与功能研究。同时利用实时定量PCR的方法检测该基因在几种常见细胞系的表达情况,发现在NIH3T3细胞系中表达较高,而在HeLa、HepG2、SW480、PanC1、MCF7等癌细胞系中表达较低,由此推测KCTD10可能在癌症的发生中起到一定的作用。


Expression and preparation of antiserum of human KCTD10 and its expression profile in the cell lines

ZHANG Wen-Feng, ZHOU Ai-Dong, YANG Li-Ping, LI Hong , ZHANG Jian* (Key laboratory of Protein Chemistry and Developmental Biology,State Education Ministry of China, College of Life Science , Hunan Normal University , Changsha 410081, China )

Abstract: Human channel tetramerisation domain-containing 10(KCTD10) gene belongs to polymerase delta-interacting protein 1 (PDIP1) gene family. Recent studies demonstrated that mouse KCTD10 can interact with PCNA and the small subunit of polymerase δ,also can be induced by tumor necrosis factor α(TNF-α),and may play roles in DNA repair, DNA replication ,cell cycle control and human diseases. But its real role and mechanism has not been reported. In this paper,the coding region of human KCTD10 gene was successfully subcloned into the prokaryotic expression vector pQE-N3,The vector was then introduced into E. coli BL21(DE3)cells for highly efficient expression.His-fusion protein was used to immunize New Zealand white rabbit to produce polyclonal antibodies.The specificity of the anti-KCTD10 antibody was successfully proved by Western blotting and analysis on both of the prokaryotic and eukaryotic expressed products of KCTD10 protein. Moreover, the expression of KCTD10 was determined in a number of cell lines by Real-time PCR. We found that the expression levels of KCTD10 are high in NIH3T3 cell lines but low in the cancer cell lines. These suggest that KCTD10 may be involved in the process of cancer.


 

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