南方农业学报2024，Vol.55Issue(6)：1798-1806,9.DOI:10.3969/j.issn.2095-1191.2024.06.024
猪德尔塔冠状病毒N蛋白原核表达及其多克隆抗体制备
Prokaryotic expression and polyclonal antibody preparation of porcine deltacoronavirus N protein
摘要
Abstract
[Objective]A prokaryotic expression system for the nucleocapsid(N)protein of porcine deltacoronavirus(PDCoV)was constructed,and polyclonal antibodies were prepared to provide reference for the development of novel vaccines,diagnostic reagents,and in-depth research into the pathogenic mechanisms of PDCoV.[Method]RNA from the PDCoV-HeN17 strain was extracted and amplified by RT-PCR,and the N gene was cloned into the pGEX-6P-l prokaryo-tic expression vector to construct a recombinant prokaryotic expression plasmid.This plasmid was transformed into Esche-richia coli BL21(DE3)competent cells,and expression was induced with IPTG.The recombinant protein was purified using Glutathione Sepharose 4B affinity chromatography,and the tag was removed with 3C protease.Polyclonal antibo-dies were prepared using the purified N protein as the immunogen to immunize 3-month-old New Zealand white rabbits.The antibody titer was determined by indirect enzyme-linked immunosorbent assay(ELISA),and the antibodies were characterized by Western blotting,indirect immunofluorescence(IFA)and immunoprecipitation(IP)assays.[Result]The prokaryotic expression vector pGEX-6p-1-PDCoV-N was successfully constructed,and the obtained PDCoV-N-GST fusion protein was primarily expressed in a soluble form,with an approximate size of 69 kD.After the GST tag was re-moved,a single band of the target protein was obtained,with a concentration determined to be 12.6 mg/mL.The pro-duced PDCoV-N protein could react with PDCoV positive serum to produce a single specific band.The prepared poly-clonal antibody against PDCoV-N from rabbits reached a titer of 1:4096000 and could specifically recognize the Flag-N protein expressed in HEK 293T cells transfected with pCAGGS-Flag-N and the PDCoV-N protein expressed in LLC-PK1 cells infected with PDCoV.It could also detect the PDCoV-N protein expressed in HEK 293T cells transfected with the eu-karyotic expression vector pCAGGS-Flag-N.[Conclusion]An efficient prokaryotic expression system for PDCoV-N is successfully constructed,yielding PDCoV-N protein with high purity,concentration and good immunogenicity.The pre-pared anti-PDCoV-N polyclonal antibodies from rabbits exhibited high titers,strong affinity,and specificity,making them suitable for applications in Western blotting,IFA,and IP assays.关键词
猪德尔塔冠状病毒(PDCoV)/N蛋白/原核表达/多克隆抗体Key words
porcine deltacoronavirus(PDCoV)/Nprotein/prokaryotic expression/polyclonal antibody分类
农业科技引用本文复制引用
赵梓桥,张昆丽,张春红,翟少伦,康欣桐,黄淑坚,张建峰..猪德尔塔冠状病毒N蛋白原核表达及其多克隆抗体制备[J].南方农业学报,2024,55(6):1798-1806,9.基金项目
National Key Research and Development Program of China(2022YFD1800801-02) （2022YFD1800801-02）
General Project of Guangdong Natural Science Foundation(2023A1515012153) （2023A1515012153）
Special Project for Science and Technology Innovation Strategy(Construction of High-level Academy of Agriculture Sciences)-Talent Project(R2019YJ-YB2005,R2023PY-QY013) 国家重点研发计划项目(2022YFD1800801-02) （Construction of High-level Academy of Agriculture Sciences）
广东省自然科学基金面上项目(2023A1515012153) （2023A1515012153）
科技创新战略专项(高水平农科院建设)—人才项目(R2019YJ-YB2005,R2023PY-QY013) （高水平农科院建设）
