NA-1株鹅源副黏病毒表面结构蛋白HN的真核表达  

Expression of HN gene of goose paramyxovirus NA-1 in Pichia pastoris

作　　者：冯新[1] 宋战昀[2] 母连志[1] 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

出　　处：《中国兽医学报》2010年第6期709-712,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30771606)

摘　　要：应用特异性引物,从鹅源副黏病毒NA-1株中扩增出HN蛋白基因,PCR产物纯化后克隆入pGEM-T载体,得到重组质粒pTHN。用EcoRⅠ和NotⅠ双酶切pTHN,回收目的基因HN片段并将其定向克隆到pPICZαA中,构建重组质粒pPICZαAHN。用SacⅠ酶切pPICZαAHN使其线性化,并电击转化至感受态毕赤酵母细胞GS115。PCR法鉴定阳性重组子,用1%甲醇诱导表达后,进行SDS-PAGE及Western-blot分析。结果在酵母菌培养基上清中检测到相对分子质量为63000的重组蛋白,且该重组蛋白可与NA-1株病毒多克隆抗体发生特异性血清学反应,表明NA-1的HN蛋白片段在Pichiapastoris中获得成功表达。To express HN protein of NA-1 in Pichia pastoris yeast,HN gene was amplified by TR-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 pTHN.The gene fragment was recovered after the double enzyme digestion with EcoR Ⅰ and Not Ⅰ,then was subcloned into pPICZα A.The recombinant pPICZαA-HN was linearized with Sac Ⅰ and then transformed into GS115 yeast cells for secretory 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 63 000,which could be specifically recognized by polyclonal antibody against NA-1.It suggested that the HN protein of NA-1 was obtained in Pichia expression system.

关 键 词：鹅源副黏病毒 HN蛋白 毕赤酵母 表达 

分 类 号：S852.65[农业科学—基础兽医学]