马动脉炎病毒主要结构蛋白基因酵母工程菌的构建及鉴定  

Secretion vector construction and identification of the major structural protein of equine arteritis virus in Hansenula polymorpha Yeast

作　　者：杜建[1] 王志亮[1] 宋厚辉[2] 金宁一[3] 张念祖[4] 

机构地区：[1]农业部动物检疫所国家外来动物疫病诊断中心 [2]中国科学院微生物研究所,北京海淀100088 [3]解放军军需大学病毒基因工程重点实验室,吉林长春130062 [4]农业部热带亚热带动物病毒学重点实验室,云南昆明650224

出　　处：《中国兽医杂志》2005年第11期3-5,共3页Chinese Journal of Veterinary Medicine

基　　金：农业部诊断技术标准和农业部热带亚热带动物病毒学重点开放实验室;基金项目(200205)

摘　　要：目的:构建马动脉炎病毒读码框ORF5、ORF6、ORF7主要结构蛋白基因全长片段的克隆载体及汉逊酵母穿梭载体,并获得重组工程菌,为下一步酵母表达重组目的蛋白奠定基础。方法:应用RT-PCR方法从病毒核酸中扩增ORF5、ORF6和ORF7读码框的全长基因,产物回收后分别与PUC18和PMD18-T载体连接并转化DH5αE.coli,经氨苄筛选及测序,建立克隆载体PUC18-GL、PUC18-M和PMD18T-ORF7;以构建的克隆载体为模板,用重新设计的引物构建了汉逊酵母分泌性穿梭载体,然后电转化至汉逊酵母基因组中。结果:所有重组质粒经PCR和酶切鉴定均出现目的片段,测序结果证实正确,目的基因ORF5、ORF6与酵母重组成功。结论:构建了含有目的基因ORF5、ORF6的重组酵母工程菌,为实现分泌表达目的蛋白,进而研制病毒亚单位疫苗及诊断试剂提供了物质基础。Objective： To construction the cloning and yeast expression vectors containing ORF5, ORF6, ORF7 structural protein gene of equine arteritis virus and lay foundation for expressing these recombinant protein. Methods： The whole sequence of ORF5, ORF6, ORF7 were amplified from EAV RNA genome by RT transformation, screen PCR, and their products were cloned into the PUC18 and and sequencing,these cloning vectors PUC18-GL PUC18-M PMD18T. Through and PMD18T-ORF7 were constructed. The new primers were designed, then these cloning vector as template, The yeast expression plasmids were constructed. Results： All of these expression plasmids were confirmed by using PCR, enzyme digestion and sequence analysis. Conclusion： These recombinant cloning vector and yeast Secret vectors of structural protein to Equine Arteritis Virus were constructed successfully.

关 键 词：马动脉炎病毒 结构蛋白基因 克隆 汉逊酵母菌 

分 类 号：Q78[生物学—分子生物学]