辽宁绒山羊朊蛋白基因克隆与原核表达  

Cloning and prokaryotic expression of the prion protein gene in Liaoning cashmere goat

作　　者：龚商羽[1] 方永辉[1] 林荣峰[1] 王金旭[2] 赵军[2] 李旋亮[1] 吴长德[1] 

机构地区：[1]沈阳农业大学畜牧兽医学院,沈阳110161 [2]沈阳市畜牧兽医科学研究所,沈阳110161

出　　处：《黑龙江畜牧兽医》2011年第12期23-26,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：辽宁省博士启动基金项目(20071057)

摘　　要：为了构建辽宁绒山羊朊蛋白(PrP)基因原核表达质粒,试验根据已报道的哺乳动物朊病毒基因序列及pEASY-E1载体多克隆位点设计引物,采用PCR方法扩增了辽宁绒山羊的朊病毒基因,将基因克隆到pEASY-E1载体中构建重组原核表达质粒,用重组质粒转化E.coli BL21(DE-3)感受态细胞,并用IPTG诱导表达,通过SDS-PAGE电泳鉴定表达的重组蛋白。结果表明:所得到的辽宁绒山羊朊病毒基因完整开放阅读框(ORF)全长为771个核苷酸,编码256个氨基酸的前体蛋白,核苷酸序列同源性为99.6%;共发现了5个碱基突变,即G126A、C414T、A428G、A652C和T718C,其中G126A、C414T为同义突变,A428G、A652C和T718C为异义突变;在pEASY-E1载体中成功克隆了PrP基因,并在E.coli中成功表达了重组的蛋白。说明辽宁绒山羊PrP基因可以在E.coli中表达。To construct eukaryotic expression plasmid of the prion protein（PrP） gene in Liaoning cashmere goat,the PrP genes from forty Liaoning cashmere goats were amplified by PCR and inserted into the expression vector pEASY-E1.The results showed that the complete length of their PrP gene with 771 bp could be correctly inserted and encoded 256 amino acids,and the homology was 99.6%,screening the sequences,five single nucleotide polymorphisms were found,namely,G126A,C414T,A428G,A652C and T718C,among which,G126A and C414T were synonymous mutations,the others three were non-synonymous mutations.The PrP gene was cloned successfully in the vector pEASY-E1,and the PrP proteins was found to be expressed in E.coli BL21（DE3）cells and demonstrated by SDS-PAGE analysis.It is evident that the prion protein gene in Liaoning cashmere goat can be expressed in E.coli cells.

关 键 词：辽宁绒山羊 朊病毒基因 多态性 克隆 原核表达 

分 类 号：S827[农业科学—畜牧学]