表达猪圆环病毒2d型Cap蛋白的重组猪伪狂犬病病毒的构建

doi:10.16431/j.cnki.1671-7236.2025.05.031

摘要/Abstract

摘要： 【目的】利用同源重组技术和CRISPR/Cas9基因编辑技术，获得表达猪圆环病毒2d型(PCV2d)Cap蛋白的重组猪伪狂犬病病毒(PRV)，为开发预防和控制PCV2d和PRV感染的疫苗提供技术支持。【方法】基于PCV2d KF1株ORF2基因序列设计1对特异引物，以PCV2d KF1株DNA为模板扩增ORF2基因。经限制性内切酶BamHⅠ酶切后将ORF2基因引入到含有绿色荧光蛋白(EGFP)基因的PRV转移载体pG中，获得重组质粒pG-PCV2d-EGFP。运用转染试剂ZLip2000将质粒pG-PCV2d-EGFP和PRV三基因缺失毒株gE^-/gI^-/TK^- PRV NY DNA共转染至ST单层细胞中拯救重组病毒，并利用CRISPR/Cas9 EGFP基因双敲除质粒敲除重组病毒中EGFP基因。经EGFP基因测序、PCR和Western blotting对获得的重组病毒进行鉴定。【结果】经8轮绿色荧光蚀斑筛选，纯化得到重组病毒rPRV-PCV2d-EGFP。CRISPR/Cas9 EGFP基因双敲除质粒敲除EGFP基因后，经3轮筛选没有绿色荧光的蚀斑，获得重组病毒rPRV-PCV2d。EGFP基因测序结果表明，rPRV-PCV2d中EGFP基因被剪截了1 251 bp，PCR和Western blotting结果显示，rPRV-PCV2d携带的ORF2基因能在ST细胞中转录与表达。【结论】本研究成功构建表达PCV2d Cap蛋白的重组病毒rPRV-PCV2d，为进一步研制重组活载体疫苗奠定了基础。

关键词: 猪圆环病毒2型; 猪伪狂犬病病毒; 重组病毒活载体疫苗

Abstract: 【Objective】 The purpose of this study was to obtain recombinant Porcine pseudorabies virus (PRV) expressing Porcine circovirus type 2d (PCV2d) Cap protein using homologous recombination technology and CRISPR/Cas9 gene editing technology,providing data support for the development of vaccines to prevent and control PCV2d and PRV infection.【Method】 According to the ORF2 gene sequence of the PCV2d KF1 strain,a pair of primers was designed and synthesized.The ORF2 gene was amplified by PCR from DNA extracted from the PCV2d KF1 strain,and was introduced into the PRV eukaryotic expression vector pG carrying the green fluorescent protein (EGFP) gene through BamH Ⅰ to construct a recombinant plasmid pG-PCV2d-EGFP.The recombinant virus was constructed by transfecting the plasmid pG-PCV2d-EGFP and DNA of the parental strain rPRV-gE^-/gI^-/TK^-PRV into ST cells using the transfection reagent ZLip2000,and the CRISPR/Cas9 EGFP knockout plasmid pX459-gRNA1-EZ-gRNA2 was used to knockout the EGFP gene of the recombinant virus.And the recombinant virus was identified by EGFP gene sequencing,PCR and Western blotting.【Result】 The recombinant virus strain rPRV-PCV2d-EGFP was obtained by eight rounds of the green fluorescent plaque screening.After EGFP gene was knocked out using the CRISPR/Cas9 EGFP knockout plasmid pX459-gRNA1-EZ-gRNA2,the recombinant virus rPRV-PCV2d was harvested after three rounds of the plaque screening without green fluorescent.The EGFP gene of recombinant virus rPRV-PCV2d was sequenced,and the results showed that EGFP gene was truncated by 1 251 bp.The results of PCR and Western blotting showed that the ORF2 gene of rPRV-PCV2d could be transcribed and expressed in ST cells.【Conclusion】 The recombinant virus rPRV-PCV2d expressing Cap protein of PCV2d was successfully constructed,which laid a foundation for further development of recombinant live vector vaccine.

Key words: Porcine circovirus type 2; Porcine pseudorabies virus; recombinant virus live vector vaccine

中图分类号:

S852.65⁺1

焦显芹, 马晓, 田润博, 刘颖, 马世杰, 陈红英. 表达猪圆环病毒2d型Cap蛋白的重组猪伪狂犬病病毒的构建[J]. 中国畜牧兽医, 2025, 52(5): 2278-2286.

JIAO Xianqin, MA Xiao, TIAN Runbo, LIU Ying, MA Shijie, CHEN Hongying. Construction of Recombinant Porcine Pseudorabies Virus Expressing Cap Protein of Porcine Circovirus Type 2d[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(5): 2278-2286.

参考文献

[1] SEGALÉS J,ALLAN G M,DOMINGO M.Porcine circovirus diseases[J].Animal Health Research Reviews,2005,6(2):119-142.
[2] OPRIESSNIG T,MENG X J,HALBUR P G.Porcine circovirus type 2 associated disease:Update on current terminology,clinical manifestations,pathogenesis,diagnosis,and intervention strategies[J]. Journal of Veterinary Diagnostic Investigation,2007,19(6):591-615.
[3] SEGALÉS J,DOMINGO M,CHIANINI F,et al.Immunosuppression in postweaning multisystemic wasting syndrome affected pigs[J].Veterinary Microbiology,2004,98(2):151-158.
[4] FRANZO G,SEGALÉS J.Porcine circovirus 2 genotypes,immunity and vaccines:Multiple genotypes but one single serotype[J].Pathogens,2020,9(12):1049.
[5] WANG Y,NOLL L,LU N,et al.Genetic diversity and prevalence of Porcine circovirus type 3(PCV3) and type 2(PCV2) in the Midwest of the USA during 2016-2018[J].Transboundary and Emerging Diseases,2020,67(3):1284-1294.
[6] DEI GIUDICI S,LO PRESTI A,BONELLI P,et al.Phylogenetic analysis of Porcine circovirus type 2 in Sardinia,Italy,shows genotype 2d circulation among domestic pigs and wild boars[J]. Infection Genetics and Evolution,2019,71:189-196.
[7] YANG S,YIN S,SHANG Y,et al.Phylogenetic and genetic variation analyses of Porcine circovirus type 2 isolated from China[J].Transboundary and Emerging Disease,2018,65(2):e383-e392.
[8] POMERANZ L E,REYNOLDS A E,HENGARTNER C J.Molecular biology of Pseudorabies virus:Impact on neurovirology and veterinary medicine[J].Microbiology and Molecular Biology Reviews,2005,69(3):462-500.
[9] TAN L,YAO J,YANG Y,et al.Current status and challenge of Pseudorabies virus infection in China[J].Virological Sinica,2021,36(4):588-607.
[10] YANG Y,XU Z,TAO Q,et al.Construction of recombinant Pseudorabies virus expressing PCV2 Cap,PCV3 Cap,and IL-4:Investigation of their biological characteristics and immunogenicity[J].Frontiers in Immunology,2024,15:1339387.
[11] TAO Q,XU L,ZHANG Y,et al.The construction and immunogenicity analyses of a recombinant Pseudorabies virus with Senecavirus A VP3 protein co-expression[J].Veterinary Microbiology,2024,290:110011.
[12] SUN Y Y,LIU K S,ZHANG C,et al.Recombinant Pseudorabies virus (PRV) expressing stabilized E2 of Classical swine fever virus (CSFV) protects against both PRV and CSFV[J].Antiviral Research,2023,211:105548.
[13] MA Z,HAN Z,LIU Z,et al.Epidemiological investigation of Porcine pseudorabies virus and its coinfection rate in Shandong province in China from 2015 to 2018[J].Journal of Veterinary Science,2020,21(3):e36.
[14] TANG Q,GE L,TAN S,et al.Epidemiological survey of four reproductive disorder associated viruses of sows in Hunan province during 2019-2021[J].Veterinary Science,2022,9(8):425.
[15] 张刘辉,刘颖,马晓,等.表达高致病性猪繁殖与呼吸综合征病毒GP5蛋白的伪狂犬病病毒拯救与纯化[J].畜牧与兽医,2023,55(12):91-97.ZHANG L H,LIU Y,MA X,et al.Construction and purification of recombinant Porcine pseudorabies virus expressing GP5 protein of highly pathogenic Porcine reproductive and respiratory syndrome virus[J].Animal Husbandry & Veterinary Medicine,2023,55(12):91-97.(in Chinese)
[16] MEAS S,CHAIMONGKOLNUKUL K,NARKPUK J,et al.Humoral and cellular immune responses induced by bivalent DNA vaccines expressing fusion capsid proteins of Porcine circovirus genotypes 2a and 2b[J].Vaccines (Basel),2024,12(3):324.
[17] KANG S J,BAE S M,LEE H J,et al.Porcine circovirus (PCV) genotype 2d-based virus-like particles (VLPs) induced broad cross-neutralizing antibodies against diverse genotypes and provided protection in dual-challenge infection of a PCV2d virus and a type 1 Porcine reproductive and respiratory syndrome virus (PRRSV)[J]. Pathogens,2021,10(9):1145.
[18] WANG Y,XU F,YUAN C,et al.Comparison of immune effects of Porcine circovirus type 2d (PCV2d) capsid protein expressed by Escherichia coli and Baculovirus-insect cells[J].Vaccine,2024,42(11):2848-2857.
[19] KIM K,CHOI K,SHIN M,et al.A Porcine circovirus type 2d-based virus-like particle vaccine induces humoral and cellular immune responses and effectively protects pigs against PCV2d challenge[J].Frontiers in Microbiology,2024,14:1334968.
[20] CHEN Y Y,YANG W C,CHANG Y K,et al.Construction of polycistronic Baculovirus surface display vectors to express the PCV2 Cap(d41) protein and analysis of its immunogenicity in mice and swine[J].Veterinary Research,2020,51(1):112.
[21] LIU C,LIU Y,FENG H,et al.PCV cap proteins fused with calreticulin expressed into polymers in Escherichia coli with high immunogenicity in mice[J].BMC Veterinary Research,2020,16(1):313.
[22] DUAN J,YANG D,CHEN L,et al.Efficient production of Porcine circovirus virus-like particles using the nonconventional yeast Kluyveromyces marxianus[J].Applied Microbiology and Biotechnology,2019,103(2):833-842.
[23] ILHA M,NARA P,RAMAMOORTHY S.Early antibody responses map to non-protective,PCV2 capsid protein epitopes[J].Virology,2020,540:23-29.
[24] RAMOS N,PORLEY D,MIRAZO S,et al.Molecular study of Porcine circovirus type 2 in wild boars and domestic pigs in Uruguay from 2010 to 2014:Predominance of recombinant circulating strains[J].Gene,2017,637:230-238.
[25] XIAO C T,HALBUR P G,OPRIESSNIG T.Global molecular genetic analysis of Porcine circovirus type 2(PCV2) sequences confirms the presence of four main PCV2 genotypes and reveals a rapid increase of PCV2d[J].Journal of General Virology,2015,96(7):1830-1841.
[26] YU X,ZHOU Z,HU D,et al.Pathogenic Pseudorabies virus,China,2012[J].Emerging Infectious Diseases,2014,20(1):102-104.
[27] VAN OIRSCHOT J T,KAASHOEK M J,RIJSEWIJK F A,et al.The use of marker vaccines in eradication of Herpesviruses[J].Journal of Biotechnology,1996,44(1-3):75-81.
[28] ZHAO Y,WANG L Q,ZHENG H H,et al.Construction and immunogenicity of a gE/gI/TK-deleted PRV based on Porcine pseudorabies virus variant[J].Molecular and Cellular Probes,2020,53:10160.
[29] FENG Z,CHEN J,LIANG W,et al.The recombinant Pseudorabies virus expressing African swine fever virus CD2v protein is safe and effective in mice[J].Virology Journal,2020,17(1):180.
[30] HUANG Y,XU Z,GU S,et al.The recombinant Pseudorabies virus expressing porcine Deltacoronavirus spike protein is safe and effective for mice[J].BMC Veterinary Research,2022,18(1):16.
[31] LI R,SHAO G,XIE Z,et al.Construction and immunogenicity of a recombinant Pseudorabies virus expressing SARS-CoV-2-S and SARS-CoV-2-N[J].Frontiers in Veterinary Science,2022,9:920087.
[32] ZHENG L L,GUO X Q,ZHU Q L,et al.Construction and immunogenicity of a recombinant Pseudorabies virus co-expressing Porcine circovirus type 2 capsid protein and interleukin 18[J].Virus Research,2015,201:8-15.
[33] JU C,FAN H,TAN Y,et al.Immunogenicity of a recombinant Pseudorabies virus expressing ORF1-ORF2 fusion protein of Porcine circovirus type 2[J].Veterinary Microbiology,2005,109(3-4):179-190.
[34] SONG Y,JIN M,ZHANG S,et al.Generation and immunogenicity of a recombinant Pseudorabies virus expressing Cap protein of Porcine circovirus type 2[J].Veterinary Microbiology,2007,119(2-4):97-104.
[35] CHI J N,WU C Y,CHIEN M S,et al.The preparation of Porcine circovirus type 2(PCV2) virus-like particles using a recombinant Pseudorabies virus and its application to vaccine development[J].Journal of Biotechnology,2014,181:12-19.