【作者】 吕海鹏； 史俊南； 赵守亮； Athony J．Smith；

【Author】 LV Hai-peng, SHI Jun-nan, ZHAO Shou-liang, Athony J, Smith.( School of Stomatology , Fourth Military Medical University. Xi’ an 710032 , Shanxi Province ,China;Oral Biology,School of Dentistry,University of Birmingham. B4 6NN,UK)

【机构】 第四军医大学口腔医学院； 英国伯明翰大学牙科学院；

【摘要】 目的:获得原核表达的人成牙本质细胞样细胞L型钙离子通道α1亚基D亚型特异性蛋白,并制备其多克隆抗体。方法: 采用RT-PCR技术从人成牙本质细胞样细胞系中获得cDNA,亚克隆至PUC18载体中,经测序确证后,将该基因插入原核表达载体pProEXHTb中,通过电穿孔技术转化E．coli表达菌DH5α,以IPTG诱导蛋白的表达,并经Ni-NTA亲和柱层析纯化,SDS- PAGE鉴定。用纯化的抗原制备多克隆抗体并进行Weatern印迹检测。结果:获得的人成牙本质细胞样细胞L型钙离子通道α1 亚基D亚型特异性cDNA序列与GenBank登录的cDNA序列一致。成功克隆并纯化了目的蛋白和多克隆抗体。结论:成功地克隆人成牙本质细胞样细胞L型钙离子通道α1亚基D亚型特异性基因,建立了原核表达、纯化体系,制备了纯化的人成牙本质细胞样细胞L型钙离子通道α1亚基D亚型特异性蛋白。提示人成牙本质细胞样细胞细胞膜上含有L型钙离子通道α1亚基D亚型。更多还原

【Abstract】 PURPOSE: To clone and sequence L type calcium channel α1D subunit specific gene of human odontoblast-like cells, the protein was expressed and purified and polyclonal antibody was prepared. METHODS: cDNA isolated from the human odontoblast-like cell line using RT-PCR was cloned into PUC18.After sequencing, the cDNA was inserted into prokaryotic expression vector pProEXHTb. The recombinant plasmid was transformed into DH5α.L type calcium channel α1D subunit specific gene of human odontoblast expression was then induced by IPTG.The protein was purified through Ni -NTA affinity chromatography column and characterized by SDS -PAGE. Polyclonal antibodies against the purified protein were produced and analyzed by Western blot. RUSULTS: The cloned cDNA sequence was identical with the submitted one.The target protein and polyclonal antibody was purified and produced successfully. CONCLUSION: L type calcium channel α1D subunit specific genes in human odontoblast-like cell line have been cloned and expressed successfully. It suggested that there was L type calcium channel α1D subunit in human odontoblast-like cell membrane.更多还原