鲤春病毒血症病毒糖蛋白基因的克隆及其在毕赤酵母中的初步表达  

Clone and construction of the expression strain of Pichia pastoris for glycoprotein gene of spring viremia of carp virus

作　　者：付峰[1] 刘荭[2] 范万红[2] 江育林[2] 黄倢[1] 

机构地区：[1]农业部海洋渔业资源可持续利用重点开放实验室中国水产科学研究院黄海水产研究所 [2]深圳出入境检验检疫局动植食中心水生动物病重点实验室,518001

出　　处：《海洋水产研究》2007年第4期72-76,共5页Marine Fisheries Research

基　　金：国家质检总局科研项目(2002IK063);黄海水产研究所海水养殖生物疾病控制与分子病理学实验室开放课题共同资助

摘　　要：根据SVCV糖蛋白基因序列设计引物,在5’引物和3’引物中分别引入酶切位点。以病毒核酸为模板用RT-PCR方法扩增该段糖蛋白基因,将所得的基因片段(517和521)克隆至穿梭载体pGAPZαA/B之GAP启动子下游位点,构建含α信号肽的重组表达载体。获得的重组质粒pGAPZαA-517和pGAPZαB-521经线性化后,电击法转化毕赤酵母SMD1168菌株,构建分泌型表达SVCV糖蛋白的重组酵母菌株。酵母菌落PCR法检测表明,已成功构建分泌表达SVCV糖蛋白的酵母基因工程菌株,斑点印迹法进一步分析表明有目的蛋白的成功表达。PCR primers were designed according to glycoprotein nucleotide sequence of SVCV, the enzyme recognizing sites being introduced into 5＇- and 3＇- primers. 2 glycoprotein fragments were obtained by means of RT-PCR with SVCV nucleotide taken as template, and then cloned into plasmid pGAPZαA/B. The recombinant plasmids pGAPZαA-517 and pGAPZαB-521 were linearized and transformed respectively into SMD1168 by electroporation. Checking with yeast colony PCR indicated that the gene-engineering strains which could express SVCV glycoprotein had been constructed successfully. Furthermore, analysis of Western-blot also testified that the objective protein was expressed.

关 键 词：鲤春病毒血症病毒 糖蛋白 克隆 毕赤酵母 分泌表达 

分 类 号：S94[农业科学—水产养殖]