牛朊蛋白27-30基因的克隆与表达  

Cloning and expression of bovine prion protein 27-30 gene

作　　者：路伟[1] 张鹏[2] 张杰[1] 刘永生[1] 卫广森[1] 陈豪泰[1] 姜海霞[3] 朱小玲[3] 

机构地区：[1]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 [2]辽宁省动物疫病预防控制中心,辽宁沈阳110161 [3]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《中国预防兽医学报》2007年第4期277-281,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：甘肃省科技攻关重点项目(2GS042-A41-001-09);甘肃省自然科学基金暨中青年科技基金项目(3ZS051-A25-065);国家博士后科学基金(20040350585)

摘　　要：利用PCR技术,从含有牛朊蛋白(Prion protein,PrP)基因Prnp的开放阅读框的克隆质粒BoPrnp-T中扩增出约420 bp的目的基因(PrP^(27-30)基因)。将PrP^(27-30)基因和载体pPIC9K分别用限制性核酸内切酶EcoR I和Not I进行双酶切,T4 DNA连接酶作用后,转化至E.coli JM109中,构建重组表达载体pPIC9K-boPrP^(27-30) pPIC9K-boPrP^(27-30)经限制性核酸内切酶Sal I线性化后电转至毕赤酵母GS115中,经G418筛选后得到高拷贝的重组菌株GS115/pPIC9K-boPrP^(27-30)。GW115/pPIC9K_boPrP^(27-30)经1.0%甲醇诱导后,表达产物用SDS-PAGE和Western blot分析,结果表明牛PrP^(27-30)基因在毕赤酵母细胞中获得表达,表达产物的分子量约为27 Ku。能够被单克隆抗体SAF-70识别。A 420 bp PrP^27-30 gene fragment of the bovine Prop gene was amplified by PCR from plasmid BoPmp-T containing the ORF of bovine Prop gene. The PCR product was cloned into pPIC9K vector to produce recombinant plasmid pPIC9K-boPrP^27-30 and transformed into E.coli JMI09. The pPIC9K-boPrP^27-30 was linearized with Sal I and then transfected into Pichia Pastoris GS115 by electroporation. A recombinant sWain GS115/pPIC9K-boPrP^27-30 was obtained by G418 selection. Expression of the recombinant PrP was induced with 1.0 % methanol and the expression products were identified by SDS-PAGE and Western blot analysis. The results demonstrated that bovine PrP^27-30 gene was successfully expressed in Pichia Pastoris as a 27 Ku protein that could be specifically recognized by monoclonal antibody SAF-70.

关 键 词：牛PrP^27-30基因 克隆 表达 

分 类 号：Q78[生物学—分子生物学] S852.659.7[农业科学—基础兽医学]