猪圆环病毒4型Cap蛋白的原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2025.10.035

摘要/Abstract

摘要： 【目的】通过原核表达系统表达猪圆环病毒4型(Porcine circovirus type 4,PCV4)衣壳蛋白(Cap)，并制备其多克隆抗体，为PCV4的防控提供技术支撑。【方法】基于PCV4开放阅读框2(ORF2)基因的免疫原区域设计1对特异性引物，通过PCR扩增获得PCV4 ORF2基因序列，并将其连接至pET-28a(+)载体，构建重组表达质粒pET-28a-ORF2；将经PCR和测序鉴定正确的重组质粒pET-28a-ORF2转化大肠杆菌BL21(DE3)感受态细胞，经1 mmol/L IPTG诱导表达重组Cap蛋白，利用SDS-PAGE分析诱导的重组蛋白产物。利用尿素纯化重组Cap蛋白并通过Western blotting鉴定；将纯化的重组Cap蛋白免疫新西兰大白兔制备多克隆抗体并进行Western blotting鉴定，应用间接ELISA法检测兔血清抗体效价。【结果】PCR扩增出大小为414 bp的PCV4 ORF2基因片段，成功构建重组表达质粒pET-28a-ORF2。SDS-PAGE分析结果显示，诱导表达的重组Cap蛋白大小为16 ku；Western blotting结果表明制备的兔源多克隆抗体具有良好的免疫原性。间接ELISA检测显示，制备的兔血清抗体效价达1∶12 800。【结论】本研究利用原核表达系统成功表达了PCV4 Cap蛋白并制备了多克隆抗体，试验结果为PCV4 Cap蛋白的深入研究以及后续开发PCV4基因工程亚单位疫苗、血清学诊断试剂等提供了物质基础。

关键词: 猪圆环病毒4型(PCV4); Cap蛋白; 原核表达; 多克隆抗体

Abstract: 【Objective】 This experiment aimed to express the capsid protein (Cap) of Porcine circovirus type 4 (PCV4) through the prokaryotic expression system and prepare its polyclonal antibody,providing technical support for the prevention and control of PCV4.【Method】 In this study,a pair of specific primers was designed based on the immunogenic region of the PCV4 open reading frame 2(ORF2) gene.The PCV4 ORF2 gene sequence was obtained through PCR amplification and ligated to the pET-28a(+) vector to construct the recombinant expression plasmid pET-28a-ORF2. The recombinant plasmid pET-28a-ORF2 identified by PCR amplification and sequencing was transformed into E.coli BL21(DE3) competent cells,and 1 mmol/L IPTG was added to induce the expression of recombinant Cap protein.Then the induced recombinant protein products were analyzed by SDS-PAGE.The recombinant protein was purified using urea and identified by Western blotting.The purified recombinant Cap protein was used to immunize New Zealand White rabbits to prepare polyclonal antibodies and Western blotting was performed for identification.The titer of rabbit serum antibodies was detected by indirect ELISA.【Result】 PCV4 ORF2 gene fragment with a size of 414 bp was amplified by PCR,and the recombinant expression plasmid pET-28a-ORF2 was successfully constructed.SDS-PAGE analysis results showed that the recombinant Cap protein induced for expression was 16 ku.The results of Western blotting indicated that the prepared rabbit-derived polyclonal antibodies had good immunogenicity.Indirect ELISA detection showed that the titer of the prepared rabbit serum antibody reached 1∶12 800.【Conclusion】 In this study,the PCV4 Cap protein was successfully expressed using the prokaryotic expression system and polyclonal antibodies were prepared.The results provided a material basis for the in-depth study of PCV4 Cap protein and the subsequent development of PCV4 genetic engineering subunit vaccines,serological diagnostic reagents,etc.

Key words: Porcine circovirus type 4 (PCV4); Cap protein; prokaryotic expression; polyclonal antibody

中图分类号:

S852.65⁺9.2

文英会, 崔鸿博, 陈曦艋, 陈红英, 李金磊, 赵丽, 刘占通, 闫志浩. 猪圆环病毒4型Cap蛋白的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2025, 52(10): 4904-4911.

WEN Yinghui, CUI Hongbo, CHEN Ximeng, CHEN Hongying, LI Jinlei, ZHAO Li, LIU Zhantong, YAN Zhihao. Prokaryotic Expression and Polyclonal Antibody Preparation of Cap Protein of Porcine Circovirus Type 4[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(10): 4904-4911.

参考文献

[1] OPRIESSNIG T，KARUPPANNAN A K，CASTRO A M M G，et al.Porcine circoviruses：Current status，knowledge gaps and challenges[J].Virus Research, 2020, 286:198044.
[2] ZHANG H H,HU W Q,LI J Y,et al.Novel Circovirus species identified in farmed pigs designated as Porcine circovirus 4,Hunan province,China[J].Transboundary and Emerging Diseases,2020,67(3):1057-1061.
[3] 陈曦艋.河南省猪圆环病毒4型流行毒株遗传进化分析及重组病毒rPRV-Cap-EGFP的构建[D].郑州:河南农业大学，2024. CHEN X M.Genetic evolution analysis of Porcine circovirus type 4 epidemic strains in Henan province and construction of recombinant virus rPRV-Cap-EGFP[D].Zhengzhou:Henan Agricultural University,2024.(in Chinese)
[4] 田润博,徐通,侯承尧，等.PCV4 PCR检测方法的建立及河南地区PCV4的遗传进化分析[J].中国预防兽医学报,2021,43(1):54-59. TIAN R B,XU T,HOU C Y,et al.Establishment of PCR detection method for PCV4 and genetic evolution analysis of PCV4 in Henan province[J].Chinese Journal of Preventive Veterinary Medicine,2021,43(1):54-59.(in Chinese)
[5] 芮萍，王妍瑾，严世杰，等.河北北部地区PCV4流行株的遗传变异分析[J].中国预防兽医学报,2021,43(11)：1219-1222. RUI P,WANG Y J,YAN S J,et al.Genetic variation analysis of PCV4 strains in Northern Hebei province[J].Chinese Journal of Preventive Veterinary Medicine,2021,43(11):1219-1222.(in Chinese)
[6] SUN W,DU Q,HAN Z,et al.Detection and genetic characterization of Porcine circovirus 4 (PCV4) in Guangxi,China[J].Gene,2021,773:145384.
[7] CHEN N，XIAO Y，LI X，et al.Development and application of a quadruplex Real-time PCR assay for differential detection of Porcine circoviruses (PCV1 to PCV4) in Jiangsu province of China from 2016 to 2020[J].Transboundary and Emerging Diseases，2021，68(3)：1615-1624.
[8] 刘建奎,李娜，吴熠丹,等.福建省猪圆环病毒4型PCR检测及全基因组序列分析[J].动物医学进展,2021,42(8)：33-38. LIU J K,LI N，WU Y D,et al.PCR detection and whole genome sequence analysis of Porcine circovirus type 4 in Fujian province[J].Progress in Veterinary Medicine,2021,42(8):33-38.(in Chinese)
[9] NGUYEN V G,DO H Q,HUYNH T M,et al.Molecular-based detection,genetic characterization and phylogenetic analysis of Porcine circovirus 4 from Korean domestic swine farms[J].Transboundary and Emerging Diseases,2022,69(2):538-548.
[10] SIRISEREEWAN C,NGUYEN T C,PIEWBANG C,et al.Molecular detection and genetic characterization of Porcine circovirus 4 (PCV4) in Thailand during 2019—2020[J].Scientific Reports,2023,13(1):5168.
[11] TAN C Y,THANAWONGNUWECH R,ARSHAD S S,et al.First molecular detection of Porcine circovirus type 4 (PCV4) in Malaysia[J].Tropical Biomedicine,2023,40(3):301-306.
[12] HOLGADO-MARTÍN R,ARNAL J L,SIBILA M,et al.First detection of Porcine circovirus 4 (PCV4) in Europe[J].Virology Journal,2023,20(1):230.
[13] ROSARIO K,BREITBART M,HARRACH B,et al.Revisiting the taxonomy of the family Circoviridae:Establishment of the genus Cyclovirus and removal of the genus Gyrovirus[J].Archives of Virology,2017,162(5):1447-1463.
[14] HOU C Y,XU T,ZHANG L H,et al.Simultaneous detection and differentiation of Porcine circovirus 3 and 4 using a SYBR Green-based duplex quantitative PCR assay[J]. Joournal of Virological Methods,2021,293：114152.
[15] NIU G,ZHANG X，JI W，et al.Porcine circovirus 4 rescued from an infectious clone is replicable and pathogenic in vivo[J].Transboundary and Emerging Diseases,2022,69(5):1632-1641.
[16] CAO X,HUANG M,WANG Y,et al.Immunogenicity analysis of PCV3 recombinant capsid protein virus-like particles and their application in antibodies detection[J].International Journal of Molecular Sciences，2023,24(12):10377.
[17] 祝羊，梁海英，曾智勇，等.猪圆环病毒4型研究进展[J].中国动物传染病学报,2023,31(2)：197-202. ZHU Y,LIANG H Y,ZENG Z Y,et al.Research progress of Porcine circovirus 4[J].Chinese Journal of Animal Infectious Diseases,2023,31(2):197-202.(in Chinese)
[18] BLANCHARD P,MAHE D,CARIOLET R,et al.Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by Porcine circovirus type 2 (PCV2) proteins[J].Vaccine,2003,21(31):4565-4575.
[19] WU M,CHEN Y,LANG W,et al.Genetic variation analysis of Porcine circovirus type 4 in South China in 2019 to 2021[J].Viruses,2022,14(8)：1736.
[20] NAWAGITGUL P,MOROZOV I,BOLIN S R,et al.Open reading frame 2 of Porcine circovirus type 2 encodes a major capsid protein[J].Journal of General Virology,2000,81(Pt 9):2281-2287.
[21] LARRICK J W,THOMAS D W.Producing proteins in transgenic plants and animals[J].Current Opinion in Biotechnology,2001,12(4):411-418.
[22] 于成东，哈卓，王政，等.猪圆环4型病毒Cap蛋白的原核表达[J].中国兽医学报，2021,41(7):1229-1233. YU C D,HA Z,WANG Z,et al.Prokaryotic expression of Porcine type 4 Cap protein[J].Chinese Journal of Veterinary Science，2021,41(7):1229-1233.(in Chinese)
[23] 阮青婷，张琦，龚紫凤，等.猪圆环病毒4型Cap蛋白的原核表达及其单克隆抗体的制备[J].中国兽医科学,2021,51(12)：1554-1560. RUAN Q T,ZHANG Q,GONG Z F,et al.Prokaryotic expression and monoclonal antibody preparation of Cap protein of Porcine circovirus type 4[J].Chinese Veterinary Science,2021，51(12):1554-1560.(in Chinese)
[24] 戴贝凝，周贝怡，周琳怡，等.猪圆环病毒4型Cap蛋白多克隆抗体的制备[J].中国兽医科学,2025,55(1)：33-38. DAI B N,ZHOU B Y,ZHOU L Y,et al.Preparation of polyclonal antibody of Porcine circovirus type 4 Cap protein[J].Chinese Veterinary Science,2025，55(1):33-38.(in Chinese)
[25] 姚晓慧，连拯民，袁丽莉，等.猪圆环病毒4型Cap蛋白单克隆抗体的制备与鉴定[J].畜牧与兽医,2022,54(4)：67-71. YAO X H,LIAN Z M,YUAN L L,et al.Preparation and identification of monoclonal antibodies against Cap protein of Porcine circovirus 4[J].Animal Husbandry & Veterinary Medicine,2022，54(4):67-71.(in Chinese)
[26] GAO Y Y,WANG Q,LI H W,et al.Genomic composition and pathomechanisms of Porcine circoviruses:A review[J].Virulence,2024,15(1):2439524.
[27] TISCHER I,GELDERBLOM H,VETTERMANN W,et al.A very small Porcine virus with circular single-stranded DNA[J].Nature,1982,295(5844):64-66.
[28] OUANG T,ZHANG X,LIU X,et al. Co-infection of swine with Porcine circovirus type 2 and other swine viruses[J].Viruses,2019,11(2):185.
[29] PALINSKI R，PINEYRO P，SHANG P，et al.A novel Porcine circovirus distantly related to known Circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure[J].Journal of Virology，2017,91(1):e01879-16.
[30] WU H,HOU C,WANG Z,et al.First complete genomic sequence analysis of Porcine circovirus type 4 (PCV4) in wild boars[J].Veterinary Microbiology, 2022,273:109547.
[31] WANG Y J,YAN S J,JI Y T,et al.First identificationg and phylogeneticanalysis of Porcine circovirus type 4 in fur animals in Hebei,China[J].Animals (Basel)，2022,12(23):3325.
[32] XU T,CHEN L,HUANG B Z,et al.The first dog-origin Porcine circovirus type 4 complete genomic sequence have high homology with that of pig-derived strains[J].Frontiers in Microbiology,2023,14:1121177.
[33] WEBER J,PENG H,RADER C.From rabbit antibody repertoires to rabbit monoclonal antibodies[J].Experimental and Molecular Medicine,2017,49(3):e305.