中国兽医科学2025，Vol.55Issue(2)：171-181,11.DOI:10.16656/j.issn.1673-4696.2025.0003
非洲猪瘟病毒B602L基因编码蛋白多克隆抗体的制备及其在PRRSV活载体中的表达
Polyclonal antibody preparation of African swine fever virus B602L gene encoding protein and its expression in PRRSV live vector
摘要
Abstract
To further investigate the encoding gene B602L of African swine fever virus(ASFV),the primers were designed for synthesizing the full-length sequence of the 1 593 bp B602L gene based on B602L gene sequence of ASFV-SY18 strain(MH766894.3)to construct the pCold-TF-B602L prokaryotic recombinant plasmid.Following identification,the recombinant plasmid was transformed into competent cells.Then the recombinant protein B602L was induced by IPTG as well as verified and analyzed by SDS-PAGE.Polyclonal antibody was prepared by immunizing rabbits with purified pB602L protein.Then,the titer of the antibody was determined,and its specificity was identified by Western-blot.Meanwhile,based on previous studies,a full-length infectious clone of recombinant porcine reproductive and respiratory syndrome virus(PRRSV)expressing pB602L protein was constructed.Specifically,the full-length sequence of B602L gene was inserted between PRRSV ORF1b and ORF2a.Then the recombinant virus rPRRSV-B602L was rescued after transfecting the infectious clone plasmid,and it's virological characteristics were analyzed.Additionally,the recombinant virus and protein exptessed in eukaryotic cells were verified by Western-blot and IFA tests using prepared antibody.The results demonstrated that pCold-TF-B602L prokaryotic plasmid was successfully constructed,and the recombinant protein pB602L could be reacted specifically with His labeled monoclonal antibody and the prepared anti-pB602L polyclonal antibody,as well as the titer of polyclonal antibody was 1∶16 000.In addition,the recombinant virus rPRRSV-B602L was successfully constructed and rescued with similar virological properties to the parental virus.The recombinant virus rescued in this study can stably express pB602Lprotein.This anti-pB602L polyclonal antibody can specifically recognize both the protein expressed by the recombinant virus and the protein expressed in the eukaryotic cells,demonstrating good specificity and reactivity.This provides a solid research foundation for further exploration of the pB602L protein's function and the development of a recombinant PRRSV live vector vaccine expressing the ASFV-specific antigen protein.关键词
非洲猪瘟病毒/pB602L蛋白/多克隆抗体/病毒活载体Key words
African swine fever virus/pB602L protein/polyclonal antibody/viral live vector分类
农业科技引用本文复制引用
罗梦,丛雁方,韦祖樟,高飞,郭子强,李佳乐,帅文娜,李丽薇,周艳君,姜一峰,童武,童光志..非洲猪瘟病毒B602L基因编码蛋白多克隆抗体的制备及其在PRRSV活载体中的表达[J].中国兽医科学,2025,55(2):171-181,11.基金项目
国家重点研发计划项目(2022YFD1800800,2021YFD1801401) （2022YFD1800800,2021YFD1801401）
上海市自然基金项目(21ZR1476900) （21ZR1476900）
国家自然科学基金项目(32373050,32072861) （32373050,32072861）
中国农业科学院科技创新工程项目(CAAS-CSLPDCP-202402) （CAAS-CSLPDCP-202402）
上海市兽医生物技术重点实验室开放基金项目(BD1500010) （BD1500010）
山东省科技型中小企业创新能力提升工程项目(2022TSGC1142) （2022TSGC1142）
