牛传染性鼻气管炎病毒gC结构蛋白的真核表达及其反应原性分析  

Eukaryotic expression and reactogenicity of infectious bovine rhinotracheitis virus gC structural protein

作　　者：张帆[1] 刘行波[1] 倪宏波[1] ZHANG Fan;LIU Xing-bo;NI Hong-bo(College of A nimal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,Heilongjiang Province,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院

出　　处：《中国生物制品学杂志》2018年第12期1333-1335,1339,共4页Chinese Journal of Biologicals

基　　金：黑龙江省农垦总局攻关课题(HNK125B-11-10A,HNK125B-11-08A)

摘　　要：目的真核表达牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)gC结构蛋白,并对其反应原性进行鉴定。方法以GenBank中登录的IBRV基因序列合成gC基因全长,构建重组真核表达质粒p CDNA4-gCHis,并进行双酶切鉴定。将鉴定正确的质粒转染MDBK细胞,收集上清,镍柱纯化带有His标签的重组gC蛋白,经Dot blot和间接免疫荧光法鉴定其反应原性。结果质粒p CDNA4-gC-His经双酶切鉴定证明构建正确,表达的重组gC蛋白相对分子质量约54 000,纯化后的浓度为0. 03 mg/mL,可与IBRV免疫的兔多抗发生反应。结论成功构建了真核表达质粒pCDNA4-gC-His,并能在MDBK细胞系中正确表达,表达的蛋白与天然IBRV gC蛋白具有相同的反应原性,为后续免疫学诊断方法的建立奠定了基础。Objective To express the infectious bovine rhinotracheitis virus(IBRV) gC structural protein in eukaryotic cells and identify its immunogenicity. Methods Full-length gC gene was synthesized according to the IBRV gC gene sequence in GenBank,based on which recombinant plasmid p CDNA4-gC-His was constructed,identified by restriction analysis and transfected to MDBK cells. The supernatant was collected,from which the recombinant gC protein with His tag was purified by nickel ion affinity chromatography and identified for reactogenicity by dot blot and indirect immunofluorescent assay(IFA). Results Restriction analysis proved that recombinant plasmid pCDNA4-gC-His was constructed correctly. The expressed recombinant gC protein,with a relative molecular mass of about 54 000,reached a concentration of 0. 03 mg/mL and showed specific reaction with rabbit polyclonal antibody against IBRV. Conclusion Eukaryotic expression vector p CDNA4-gC-His was successfully constructed and expressed in MDBK cells. The expressed protein showed the same reactogenicity as that of natural IBRV gC protein,which laid a foundation of further development of immunological diagnostic method.

关 键 词：牛传染性鼻气管炎病毒 gc蛋白 真核表达 反应原性 

分 类 号：R852.653[医药卫生—航空、航天与航海医学]