摘要
为表达非洲猪瘟病毒B602L重组蛋白,制备相应的多克隆抗体,根据NCBI网站上公布的非洲猪瘟病毒(Pig/HLJ/2018株)B602L基因序列进行密码子优化和基因合成,随后克隆至杆状病毒转移载体pFastBac1中,获得重组杆状病毒质粒,再把重组杆粒转染昆虫细胞Sf9,拯救重组B602L基因的杆状病毒。经间接免疫荧光和western blots鉴定结果显示,B602L重组蛋白与His标签抗体、非洲猪瘟标准阳性血清均能发生特异性反应,并且B602L重组蛋白可以细胞分泌上清的形式进行表达。通过His镍株亲和纯化的方式纯化重组蛋白,B602L蛋白大小约70 ku,与预期相符。将B602L重组蛋白与弗氏佐剂配比免疫BALB/c小鼠,制备小鼠多克隆抗体,经间接ELISA检测,该抗体效价可达1∶512000,并且与B602L重组蛋白具有良好的反应性。本研究可为进一步研究非洲猪瘟B602L蛋白的结构与功能、研制非洲猪瘟诊断制品提供生物材料。
To express the recombinant protein of African swine fever virus B602L,the corresponding polyclonal antibody was prepared.In this experiment,codon optimization and gene synthesis were carried out according to the B602L gene sequence of African swine fever virus(Pig/HLJ/2018 strain)published on NCBI website,subsequently B602L gene was cloned into the baculovirus transfer vector pFastBac1,and obtain a recombinant baculovirus plasmid.Then transfect the baculovirus plasmid into insect cell Sf9 to rescue the recombinant B602L gene baculovirus.The results of IFA and western blots identification showed that the B602L recombinant protein can specific react with His labeled antibodies and African swine fever standard positive serum,and the B602L recombinant protein can be expressed in the form of cell secreted supernatant.The recombinant protein was purified by affinity purification of His nickel strain,and the size of B602L protein was approximately 70 ku,which was consistent with expectations.Immunize BALB/c mice with the combination of B602L recombinant protein and Freund's adjuvant to prepare mouse polyclonal antibodies.Indirect ELISA detection showed that the antibody had a titer of 1∶512000 and showed good reactivity with the B602L recombinant protein.This study can provide biomaterials for further studying the structure and function of African swine fever B602L protein and developing diagnostic products for African swine fever.
作者
刘郁夫
林晓慧
郝文茜
冯夏宁
欧阳征亮
陈瑞爱
LIU Yu-fu;LIN Xiao-hui;HAO Wen-qian;FENG Xia-ning;OUYANG Zheng-liang;CHEN Rui-ai(School of Life Sciences,Zhaoqing University,Zhaoqing,Guangdong 526060,China;Zhaoqing Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology,Zhaoqing,Guangdong 526238,China;Key Laboratory of Biotechnology and Bioproducts Development for Animal Epidemic Prevention,Ministry of Agriculture and Rural Affairs/Guangdong Enterprise Key Laboratory of Biotechnology R&D of Veterinary Biologics/Zhaoqing Dahuanong Biology Medicine Co.,Ltd,Zhaoqing,Guangdong 526238,China)
出处
《中国兽药杂志》
2023年第10期1-8,共8页
Chinese Journal of Veterinary Drug
基金
广东省科技计划项目(2020B1212070015)
肇庆市科技项目(2021C001)
肇庆学院大学生创新训练项目(X202310580122)。
关键词
非洲猪瘟
B602L
真核表达
多克隆抗体
African swine fever virus
B602L
eukaryotic expression
polyclonal antibody
