CDH22基因克隆及其真核表达载体的构建与表达

Cloning of human CDH22 gene and construction of its eukaryotic expression vectors

目的克隆人CDH22基因并构建其真核表达载体。方法设计CDH22特异性引物,提取人大肠癌细胞系SW480总RNA,用RT-PCR方法获取人CDH22cDNA,经T-A克隆,通过双酶切及测序鉴定,将CDH22克隆至pCDNA3/HA,构建人CDH22基因的真核表达载体pCDNA3/HA-CDH22,转染HEK293A细胞,用WesternBlot及免疫荧光细胞化学技术检测目的蛋白的表达。结果成功扩增出人CDH22全长cDNA,双酶切鉴定证实成功构建pCDNA3/HA-CDH22真核表达载体,测序结果表明CDH22全长cDNA与GeneBank中CDH22序列完全一致,转染HEK293A细胞后,可检测出Mr约为86KD的目的蛋白,而免疫荧光细胞化学技术也检测到蛋白表达。结论获得人CDH22基因全长cDNA并成功构建了CDH22真核表达载体,证实pCDNA3/HA-CDH22转染HEK293A细胞后可表达HA-CDH22蛋白,为深入研究CDH22基因在肿瘤发生发展中的作用奠定了基础。

Objective To obtain entirely coding human CDH22 gene and construct its eukaryotic expression vectors.Methods With total RNA extracted from the human colorectal carcinoma cell sw480 as template,CDH22 gene was amplified by RT-PCR with the designed primers based on the public sequence of GeneBank,and then was inserted into pGEM-T Easy vector.The recombinant plasmid pGEM-T-CDH22 was identified by Restriction endonuclease analysis and DNA sequencing.Digested by restriction endonuclease,the CDH22 was cloned into multiclone sites of the eukaryotic expression vector pCDNA3/HA.The expression of CDH22 gene was detected by Western Blot and immunofluorescence cytochemistry using mouse anti-HA antibody after the recombinant plasmid pCDNA3/HA-CDH22 was transfected into HEK293A cells.Results The entirely coding human CDH22 gene was cloned.Recombinant pCDNA3/HA-CDH22 were successfully constructed and expressed.Conclusion The successfully constructed pCDNA3/HA-CDH22 would provide a basis for a further study of the relationship between the tumorigenesis and CDH22 gene.