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人朊蛋白基因的克隆与原核表达
引用本文:姜海霞,刘永生,杨孝朴,陈豪泰,朱小玲,张杰,谢庆阁.人朊蛋白基因的克隆与原核表达[J].甘肃农业大学学报,2006,41(3):11-15.
作者姓名:姜海霞  刘永生  杨孝朴  陈豪泰  朱小玲  张杰  谢庆阁
作者单位:1. 甘肃农业大学动物医学院,甘肃,兰州,730070;中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,农业部畜禽病毒学重点开放实验室,甘肃,兰州,730046
2. 中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,农业部畜禽病毒学重点开放实验室,甘肃,兰州,730046
3. 甘肃农业大学动物医学院,甘肃,兰州,730070
基金项目:甘肃省科技攻关重点项目(2GS042-A41-001-09) 甘肃省自然科学基金(3ZS051-A25-065) 中国博士后科学基金 (20040350585).
摘    要:以人血液中提取的总DNA为模板,克隆朊蛋白(prion protein,PrP)基因Prnp户的开放阅读框(ORF),与pMD -18T载体连接后转入E.coli JM109,用Blast软件比较重组质粒序列,结果表明获得的人Prnp的ORF与Genbank登录的相应核苷酸序列最高同源性为99.6%,推导的氨基酸序列同源性为99.8%,将编码人成熟PrP的基因定向克隆到 pET-30a(+)表达载体,将重组质粒pET-homPrP转化E.coli BL21(DE3),在37℃下用1.0 mmol/L IPTG诱导,重组融合蛋白获得高效表达,SDS-PAGE显示表达产物以包涵体形式存在,占菌体总蛋白的20%~25%.用Ni-NTA 树脂亲和层析纯化了表达产物.Western-blotting分析表明,融合蛋白被抗PrP的单克隆抗体特异识别.因此,在大肠杆菌中表达的人成熟PrP融合蛋白可以作为制备PrP抗体的免疫原.

关 键 词:  朊蛋白基因  克隆  原核表达
文章编号:1003-4315(2006)03-0011-05
收稿时间:2005-10-31
修稿时间:2005-10-31

Cloning and prokaryotic expression of human prion gene
JIANG Hai-xia,LIU Yong-sheng,YANG Xiao-pu,CHEN Hao-tai,ZHU Xiao-ling,ZHANG Jie,XIE Qing-ge.Cloning and prokaryotic expression of human prion gene[J].Journal of Gansu Agricultural University,2006,41(3):11-15.
Authors:JIANG Hai-xia  LIU Yong-sheng  YANG Xiao-pu  CHEN Hao-tai  ZHU Xiao-ling  ZHANG Jie  XIE Qing-ge
Institution:1. College of Veterinary Medieine,Gansu Agricultural University,Lanzhou 730070; 2. Key Laboratory of Animal Virology, Ministry of Agriculture State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Scienees,Lanzhou 730046, China
Abstract:The open reading frame (ORF) of prion protein gene,Prnp,was amplified by PCR using total DNA extracted from human blood as template. The PCR product was ligated with pMD-18T vector and then transformed into E. coli JM109. The recombinant plasmid was sequenced and analyzed with Blast software. It shared 99.6 % homology at their nucleotide level and 99.8 % identity at deduced amino acid level with the corresponding sequences deposited in Genbank. The gene encoding mature human prion protein was cloned into pET-30a(+) vector and then transformed by electroporation into E. coli BL21(DE3) competent cells. The expected protein was expressed in E. coli BL21(DE3) harboring the recombinant plasmid pET-homPrP induced by 1.0 mmol/L IPTG at 37 ℃. The expressed fusion protein was purified by Ni—NTA affinity resin according to the manufacture's directions. SDS—PAGE analysis showed that the fusion protein accounted for 20 %-25 % of the total bacterial proteins and accumulated as inclusion bodies. Western blotting demonstrated that the fusion protein could be recognized by the anti-PrP monoclonal antibody and used as antigen for preparing the monoclonal antibody against PrP.
Keywords:human  prion protein gene  clone  prokaryotic expression
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