鹅源副黏病毒融合蛋白在毕赤酵母细胞中的表达  

Expression of Fusion Protein of Goose Paramyxovirus in Pichia pastoris

作　　者：冯新[1] 宋战昀[2] 刘阳[2] 张琼[3] 姜永莉[2] 丁壮[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]吉林出入境检验检疫局,吉林长春130062 [3]辽宁出入境检验检疫局,辽宁大连116001

出　　处：《动物医学进展》2010年第9期7-11,共5页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(30771606;30901069)

摘　　要：应用特异性引物,从鹅源副黏病毒NA-1株中扩增出F蛋白基因,PCR产物纯化后克隆入pGEM-T载体,得到重组质粒pT-F。用EcoRⅠ和NotⅠ双酶切pT-F,回收目的基因F片段,并将其定向克隆到pPICZαA中,构建重组质粒pPICZαA-F。用PmeⅠ酶切pPICZαA-F使其线性化,电击转化至感受态毕赤酵母GS115菌中。PCR法鉴定阳性重组子,10 mL/L甲醇诱导表达后,进行SDS-PAGE及Westernblot分析。结果表明,在酵母菌培养基上清中检测到相对分子质量为63 ku的重组蛋白,该重组蛋白可与NA-1株鹅源副黏病毒多克隆抗体发生特异性血清学反应。To express F protein of NA-1 in Pichia pastoris,F gene was amplified by RT-PCR from NA-1 with a pair of specific primers.Then PCR product was purified and cloned into pGEM-T vector to obtain the plasmid pT-F.The gene fragment was recovered after the double enzyme digestion with EcoRⅠ and NotⅠ,then was subcloned into pPICZα A.The recombinant pPICZαA-F was linearized with PmeⅠ and then transformed into GS115 yeast cells for expression.The recombinant strains were screened by PCR technique.The expression products were identified by SDS-PAGE and Western blot.The results showed that there was a molecular weight of 63 ku protein specifically recognized by polyclonal antibody against NA-1.It suggested that the F protein of NA-1 was obtained in Pichia pastoris expression system.

关 键 词：鹅源副黏病毒 F蛋白 毕赤酵母 表达 

分 类 号：S852.659.5[农业科学—基础兽医学]