摘要
为了给非洲猪瘟的临床诊断技术和实验室研究提供物质基础,本研究以我国首次分离的非洲猪瘟病毒(ASFV)Pig/HLJ/2018株基因组为模板,扩增pA104R基因,并将其克隆至pET-30a载体,构建重组原核表达质粒pET-30a-A104R,表达并纯化重组pA104R蛋白(rpA104R),以其为抗原免疫BALB/c小鼠制备单克隆抗体(MAb),并采用ELISA、western blot、间接免疫荧光法(IFA)和免疫组化(IHC)对所获得的MAb进行鉴定。结果显示,正确构建p ET-30a-A104R重组质粒,并获得大小约为12 ku的可溶性蛋白。通过间接ELISA方法筛选到2株阳性杂交瘤细胞株为4G2和4E10,并且2株MAb的重链均为IgG1,轻链为Kappa链。Western blot和IFA结果显示,MAb 4G2株能够特异性地识别ASFV pA104R蛋白和病毒感染的猪肺泡巨噬细胞中表达的pA104R蛋白。IHC结果表明,MAb 4G2可以与病毒感染的阳性猪组织发生特异性反应。本研究也为ASFV基因缺失疫苗的开发提供了技术储备。
In order to provide material basis for the clinical diagnosis development technologies and laboratory research on ASF,the p A104 R gene was amplified by using the first isolated African swine fever virus strain Pig/HLJ/2018 in China and cloned into the pET-30 a vector to construct a recombinant prokaryotic expression plasmid pET-30 a-A104 R.The recombinant pA104 R protein was expressed and purified,and BALB/c mice were then immunized with the purified protein to prepare monoclonal antibodies(MAbs).The obtained MAbs were identified by enzyme linked immunosorbent assay(ELISA),western blot,indirect immunofluorescent assay(IFA)and immunohistochemistry(IHC).The results showed that the recombinant pET-30 a-A104 R plasmid was successfully constructed and a soluble protein with a size of about 12 ku was obtained.Two positive hybridoma cell lines were selected by indirect ELISA,named as 4 G2 and 4 E10,respectively.The heavy chain of the two MAbs were determined as IgG1,and the light chain was Kappa chain.The results of western blot and IFA showed that MAb 4 G2 specifically recognizedthe purified pA104 R protein and the expressed pA104 R protein in ASFV-infected porcine alveolar macrophages.IHC results showed that MAb 4 G2 specifically reacted with ASFV-infected tissues.This research provides not only material basis for the clinical diagnosis development technologies and laboratory research on ASFV,but also technical reserve for the development of gene-deleted vaccines.
作者
王露露
皇甫皓月
席飞
张振江
Weldu Tesfagaber
张纪文
黄炼榆
李芳
步志高
赵东明
WANG Lu-lu;HUANG FU Hao-yue;XI Fei;ZHANG Zhen-jiang;Weldu Tesfagaber;ZHANG Ji-wen;HUANG Lian-yu;LI Fang;BU Zhi-Gao;ZHAO Dong-ming(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2021年第11期1214-1218,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
黑龙江省应用技术研究与开发计划(GA19B301)
