人vigilin基因全长编码区的分段克隆及鉴定  

Cloning and Identification of Human Vigilin Full-length Coding Region

作　　者：杨文理[1] 武静[1] 谢晓砚[1] 魏玲[1] 覃扬[1] 

机构地区：[1]四川大学华西基础医学与法医学院生物化学与分子生物学教研室,成都610041

出　　处：《四川大学学报（医学版）》2008年第6期877-881,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(批准号30470792)资助

摘　　要：目的为了探讨全长VIGILIN、VIGILIN的N端、VIGILIN的C端以及不同KH组合的功能,采取5分段扩增的策略克隆全长vigilin基因。方法根据vigilin基因全长编码区内的限制性核酸内切酶位点,将vigilin基因全长编码区分为5段。从意外死亡健康成人肝组织中提取总RNA,RT-PCR分段扩增目的基因,克隆至pUC118载体,测序,并亚克隆至pUC118载体,对接头部位测序。结果获得8个大小不同的人vigilin基因编码区片段和全长编码区。重组全长vigilin质粒pC118/vigilin(12+345)经测序,与GenBank上登陆的人vigilin cDNA(NM:005336)序列完全一致。结论用分段克隆的方法成功获得了人vigilin基因的全长编码区。Objective The full-length of human vigilin encodes 1289 amino acids, containing 14 KH domains. We adopted a five step PCR technique to clone the full-length vigilin coding regions to investigate the functions of full-length vigilin, N-terminal of vigilin, C- terminal of vigilin and different KH. Methods Vigilin was dissected into five fragments according to the restrictive endounclease enzymes site of vigilin. The total RNA was extracted from the livers of healthy adults died from accident. The cDNA was amplified by RT-PCR. Then the amplified PCR product was digested and inserted into pUC118 vector and subcloned into pUC118 vector. Results Eight gene fragments and full-length of human vigilin were obtained. The gene sequence of vigilin was identical with its cDNA sequence reported （NM：005336） in the GenBank. Conclusion The full-length vlgilin coding region has been cloned successfully.

关 键 词：vigilin基因 分段扩增 克隆 

分 类 号：Q78[生物学—分子生物学]