牛传染性鼻气管炎病毒gD基因真核表达载体的构建及其在MDBK细胞中的表达  

Construction of eukaryotic expression vector for bovine infectious rhinotracheitis virus gD gene and its expression in MDBK cells

作　　者：贾晓雪 刘慧珍 张帆[1] 武文博 马君艳 倪宏波[1] JIA Xiao-xue;LIU Hui-zhen;ZHANG Fan;WU Wen-bo;MA Jun-yan;NI Hong-bo(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163119,Heilongjiang Province,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163119

出　　处：《中国生物制品学杂志》2020年第8期894-897,共4页Chinese Journal of Biologicals

基　　金：中央引导地方科技发展专项(ZY18C07);黑龙江八一农垦大学校级研究生创新(YJSCX2018-Y27)。

摘　　要：目的构建牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)gD基因的真核表达质粒,并在MDBK细胞中表达。方法参照GenBank中BHV-1-gD基因序列,于上下游分别加入KpnⅠ、BSTBⅠ双酶切位点,连接至真核表达载体pcDNA4-myc-His中,全基因合成重组质粒pcDNA4-gD-His,经双酶切及测序鉴定正确后,转染MDBK细胞,收集上清,镍柱纯化带有His标签的重组gD蛋白,对表达及纯化的蛋白进行间接免疫荧光及Dot blot鉴定。结果质粒pcDNA4-gD-His经双酶切及测序鉴定证明构建正确。表达及纯化的重组gD蛋白相对分子质量约为61000,纯化后的重组gD蛋白浓度为0.85 mg/mL。表达及纯化的重组gD蛋白均可与IBRV-gD单克隆抗体发生反应,具有良好反应原性。结论已成功构建了pcDNA4-gD-His真核表达质粒,并在MDBK细胞系中成功表达,经验证重组蛋白具有良好的反应原性。Objective To construct a eukaryotic expression vector for bovine infectious rhinotracheitis virus(IBRV)gD gene express in MDBK cells.Methods According to the sequence of BHV-1-gD gene in GenBank,the target gene was synthesized with the primers to which KpnⅠ/BSTBⅠrestriction sites were introduced upstream and downstream,and inserted into eukaryotic expression vector pcDNA4-myc-His.The constructed recombinant plasmid pcDNA4-gD-His was identified by restriction analysis and sequencing and transfected to MDBK cells.The culture supernatant was collected,from which the recombinant gD protein with His tag was purified by nickel ion column chromatography.The expressed and purified proteins were identified by indirect IFA and Dot blot.Results Restriction analysis and sequencing proved that recombinant plasmid pcDNA4-gD-His was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 61000,reached a protein concentration of 0.85 mg/m L after purification.Both the expressed and purified recombinant g D protein showed reactions with monoclonal antibody against IBRV-gD,indicating a good reactogenicity.Conclusion Eukaryotic expression plasmid pcDNA4-gD-His was successfully constructed and expressed in MDBK cell line,and the recombinant protein showed good reactogenicity.

关 键 词：牛传染性鼻气管炎病毒 GD基因 MDBK细胞 真核表达 

分 类 号：S852.653[农业科学—基础兽医学] Q786[农业科学—兽医学]