鸭MHC-Ⅰα链胞外区成熟肽基因的克隆、序列分析及表达载体构建  

Gene cloning,sequence analysis and prokaryotic expression vector construction of duck MHC-Ⅰα chain mature peptide of the extracellular domain

作　　者：孙凯[1,2] 李新生[1,2] 崔保安[1,2] 陈红英[1,2] 魏战勇[1,2] 张蕾[1,2] 宋亚鹏[1,2] 阮武营[1,2] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南省动物性食品安全重点实验室,河南郑州450002

出　　处：《西北农林科技大学学报（自然科学版）》2010年第2期57-62,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家“十一五”科技支撑计划专项(2006BAD06A08)

摘　　要：【目的】克隆鸭MHC-Ⅰα链胞外区成熟肽基因cDNA序列,构建并鉴定其原核表达质粒pET/DuMHC-Ⅰ。【方法】依据GenBank/DDBJ/EMBL基因库中公布的鸭MHC-Ⅰ基因序列设计1对引物,采用RT-PCR技术从鸳鸯鸭脾脏组织中扩增MHC-Ⅰα链胞外区成熟肽基因cDNA,T-A克隆到pGEM-T Easy载体中,并进行测序。通过PCR和酶切的方法,将鸭MHC-Ⅰα链胞外区成熟肽基因亚克隆入pET-28a(+)载体,构建重组表达质粒pET/Du MHC-Ⅰ,并进行PCR、NcoⅠ和NotⅠ双酶切及测序鉴定。【结果】测序结果表明,获得了MHC-Ⅰα链胞外区成熟肽基因,其长度为813 bp,编码271个氨基酸。MHC-Ⅰα链胞外区成熟肽基因与GenBank中登录的鸭MHC-Ⅰα链胞外区成熟肽基因序列的同源性为89.4%~91.7%,氨基酸同源性为78.2%~83.7%;与鸡、鹅、鹌鹑、草鱼、狗、猪、鼠和人的MHC-Ⅰα链胞外区成熟肽氨基酸序列的同源性分别为64.6%,79.3%,59.4%,41.0%,26.6%,45.0%,12.5%和22.1%,这表明MHC-Ⅰα链胞外区成熟肽基因存在着种属多样性,且...【Objective】The study was done to clone duck MHC-Ⅰα chain mature peptide gene cDNA sequence of the extracellular domain and construct a prokaryotic expression plasmid.【Method】The cDNA of duck MHC-Ⅰα chain extracellular domain of mature peptide gene amplified with RT-PCR from healthy duck spleen tissue was cloned into pGEM-T Easy vector by T/A Ligation.After double digestion,MHC-Ⅰα chain extracellular domain of mature peptide cDNA fragment was subcloned into pET-28a(+) vector to construct a prokaryotic expres...

关 键 词：鸭 MHC-Ⅰα链 胞外区成熟肽基因 原核表达载体 

分 类 号：S834[农业科学—畜牧学]