BVDV中E^(rns)、N^(pro)蛋白真核表达质粒的构建  

Construction of Eukaryotic Expression Plasmids for E^(rns) and N^(pro) Proteins in BVDV

作　　者：巩转娣[1] 裴梦源 拜小强 袁肇方 李殿玉 魏锁成[1,3] GONG Zhuandi;PEI Mengyuan;BAI Xiaoqiang;YUAN Zhaofang;LI Dianyu;WEI Suocheng(College of Life Science and Engineering,Northwest MInzu University,Lanzhou 730030,China;Rural Agriculture Bureau of Hezheng County,Hezheng 731200,China;Biomedical Center,Northwest MInzu University,Lanzhou 730030,China)

机构地区：[1]西北民族大学生命科学与工程学院,甘肃兰州730030 [2]和政县农村农业局,甘肃和政731200 [3]西北民族大学生物医学研究中心,甘肃兰州730030

出　　处：《西北民族大学学报（自然科学版）》2024年第2期46-53,共8页Journal of Northwest Minzu University(Natural Science)

基　　金：国家自然科学基金项目(31460684);西北民族大学高原动物疾病创新团队项目(2020-88-07-3)。

摘　　要：目的:试验旨在构建牛病毒性腹泻病毒(BVDV)结构蛋白(糖基化囊膜蛋白E^(rns))和非结构蛋白(N^(pro))重组表达载体,利用哺乳动物真核表达系统对E^(rns)、N^(pro)进行表达.方法:参考GenBank中公布的BVDV参考株核苷酸序列分别设计引物N^(pro)-F/R和E^(rns)-F/R,用BVDV病毒的cDNA进行PCR扩增,将回收纯化的N^(pro)和E^(rns)目的基因和真核表达载体pCMV-Myc进行双酶切.利用琼脂糖凝胶电泳进行回收纯化,将回收纯化产物采用T4 DNA连接酶进行连接并转化至BL-21感受态细胞中,构建真核表达质粒,将重组质粒命名为pCMV-Myc-N^(pro)和pCMV-Myc-E^(rns).双酶切及测序鉴定正确后转染HEK-293细胞,采用PCR及Western blotting检测N^(pro)和E^(rns)在细胞内的表达.结果:pCMV-Myc-N^(pro)和pCMV-Myc-E^(rns)真核表达质粒构建成功,N^(pro)和E^(rns)在HEK293细胞中成功表达.结论:N^(pro)、E^(rns)蛋白的成功表达可为探究蛋白质生物学功能、研究病毒感染机制及研制亚单位疫苗提供理论参考.Objective The experiment studies aimded to construct recombinant expression vectors for the structural protein(glycosylated envelope protein E^(rns))and non structural protein(N^(pro))of bovine viral diarrhea virus(BVDV),and to express E^(rns) and N^(pro) using a mammalian eukaryotic expression system.Methods The primers N^(pro)-F/R and E^(rns)-F/R were designed referring to the nucleotide sequence of the BVDV reference strain published in GenBank.The cDNA of BVDV virus was used for PCR amplification.The recovered and purified N^(pro) and E^(rns) target genes and the eukaryotic expression vector pCMV-Myc were digested with double enzyme cleavage.The agarose gel electrophoresis was used for the recovery and purification.The harvested and purified products were linked with T4 DNA ligase and transformed into BL-21 competent cells,and the eukaryotic expression plasmid was constructed.The recombinant plasmids were named pCMV-Myc-N^(pro) and pCMV-Myc-E^(rns).After the double enzyme digestion and sequencing identification were correct,HEK-293 was transfected.The expression of N^(pro) and E^(rns) was detected in the cells by PCR and Western blotting.Results The pCMV-Myc-N^(pro) and pCMV Myc-E^(rns) eukaryotic expression plasmids was successfully constructed,and N^(pro) and E^(rns) were successfully expressed in HEK293 cells.Conclusion The successful expression of N^(pro) and E^(rns) proteins will provide theoretical references for exploring protein biological functions,studying viral infection mechanisms,and developing subunit vaccines.

关 键 词：牛病毒性腹泻病毒 结构蛋白 真核表达 

分 类 号：Q95[生物学—动物学] R373.25[医药卫生—病原生物学]