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| 牛病毒性腹泻病毒E2基因在毕赤酵母GS115中的表达与鉴定 | |
| 引用本文： | 赵月兰,左玉柱,范京惠,王安忠,杨汉春,秦建华.牛病毒性腹泻病毒E2基因在毕赤酵母GS115中的表达与鉴定[J].中国兽医学报,2010,30(1). |
| 作者姓名： | 赵月兰 左玉柱 范京惠 王安忠 杨汉春 秦建华 |
| 作者单位： | 1. 河北农业大学中兽医学院,河北,定州,073000 2. 河北农业大学动物科技学院,河北,保定,071000 3. 中国农业大学动物医学院,北京,100094 |
| 基金项目： | 国家科技支撑计划，河北省重大技术创新专项计划，河北农业大学校科研和教改项目 |
| 摘 要： | 将牛病毒性腹泻病毒(Bovine viral diarrhoea-mucosal disease virus,BVDV)河北分离株HB-bd毒株E2基因去除跨膜区获得sE2基因,将sE2基因克隆入巴斯德毕赤酵母(P.pastoris)分泌型表达载体pPIC9K中,筛选培养后提取阳性重组质粒,经酶切和PCR鉴定,命名为pPIC9K-sE2。重组质粒pPIC9K-sE2经SalⅠ酶切后,电穿孔导入P.pastorisGS115基因进行整合,使外源基因sE2稳定地整合到P.pastoris染色体中,G418筛选得到阳性高拷贝转化子GS115-pPIC9K-sE2。经甲醇诱导表达后,sE2融合蛋白获得了表达,表达产物经SDS-PAGE、Western-blot和Dot-ELISA分析,确定其表达的sE2融合蛋白相对分子质量大小为37 000,且具有天然蛋白的抗原特异性。免疫活性研究证明,P.pastoris表达的sE2蛋白能刺激动物产生抗体。 |
| 关 键 词： | BVDV E2基因 毕赤酵母 分泌型表达 |
Expression of E2 gene of bovine viral diarrhoea-mucosal disease virus in Pichia Pastoris and identification of its expression product |
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| ZHAO Yue-lan,ZUO Yu-zhu,FAN Jing-hui,WANG An-zhong,YANG Han-chun,QIN Jian-hua.Expression of E2 gene of bovine viral diarrhoea-mucosal disease virus in Pichia Pastoris and identification of its expression product[J].Chinese Journal of Veterinary Science,2010,30(1). | |
| Authors: | ZHAO Yue-lan ZUO Yu-zhu FAN Jing-hui WANG An-zhong YANG Han-chun QIN Jian-hua |
| Abstract: | The sE2 gene of HPrbd strain isolated of BVDV was obtained from E2 gene removed C-terminal trans-mernbrane domain by PCR. The sE2 gene was subcloned into the expression vector pPIC9K and then transfected into DH5a. The recombinant plasmids were extracted and identificated by enzyme digestion and PCR. The positive recom-binant plasmids was designated pPIC9K-sE2. The recombination expression vector, pPIC9K-sE2 was linearized by Sal Ⅰ and stransformed into P. pastoris GS115 by electroporation. The sE2 genes were integrated stably into chro-mosome of P. pastoris. High-copied transformants GS115-pPIC9K-sE2 were obtained by G418 screening. Expression of sE2 fusion protein was induced in recombinant P. pastoris GSll5 by the addition of 1% methanol at 30℃ ,shaking at 200 r/min. The results of SDS-PAGE and Western-blot showed that expressed fusion proteins were provided with antigenic characteristic of BVDV. The results of studies on immunological activity indicated that the sE2 protein ex-pressed in P. pastoris could induce animals to produce BVDV specific antibodies against E2 protein. |
| Keywords: | bovine viral diarrhea virus(BVDV) E2 genes Pichia pastoris expression |
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