猪瘟病毒E2基因部分表位区重组猪伪狂犬病病毒的构建及生物学特性研究

doi:10.16431/j.cnki.1671-7236.2025.04.033

Abstract

Abstract: 【Objective】 Classical swine fever virus (CSFV) and porcine Pseudorabies virus (PRV) are two important pathogens affecting the development of current swine industry.Currently,there is no effective drug treatment,and the prevention mainly depends on vaccination.The aim of this study was to construct a recombinant PRV strain containing part of the epitope region of CSFV E2 gene and explore its biological characteristics,so as to provide reference for the development of dual vaccine against CSFV and PRV simultaneously.【Method】 Firstly,a gene segment containing the 4 epitope regions of B/C/D/A of CSFV E2 protein was inserted into the BamHⅠ site of the pG-EGFP recombinant plasmid,and the recombinant plasmid pG-E2AD-EGFP was constructed.The plasmid was then transfected into ST cells inoculated with PRV tri-gene deletion strain rPRV NY-gE^―/gI^―/TK^― and homologous recombination occurred in ST cells.The recombinant virus rPRV-E2AD-EGFP with green fluorescent protein was rescued by the plaque purification.The EGFP gene was knocked out using CRISPR/Cas9 knockout vector,and the non-fluorescent recombinant virus rPRV-E2AD was purified by plaque.The expression of A-D epitopes of E2 of recombinant virus rPRV-E2AD was detected by PCR amplification and Western blotting.The proliferation of the virus after treatment with different physicochemical properties was detected by 50% tissue culture infective dose (TCID₅₀) method,and the culture and physicochemical properties of the recombinant virus were evaluated.【Result】 Recombinant virus rPRV-E2AD-EGFP expressing both CSFV E2AD antigen and EGFP was obtained through intracellular homologous recombination and viral plaque purification.EGFP gene was knocked out by CRISPR/Cas9 technique,and recombinant virus rPRV-E2AD without EGFP gene expression was obtained.Western blotting results showed that the protein size of this strain was about 27 ku,which was consistent with the CSFV E2AD antigen size.The growth characteristics of rPRV-E2AD and parent strains were basically the same in ST,IPEC-J2,PK-15 and Vero cells,both of which caused cell fusion at 12 h after infection and cell loss at 36 h after infection.Physical and chemical characteristics test results showed that the recombinant strain and the parent strain had similar culture characteristics under the same physical and chemical conditions.【Conclusion】 In this study,recombinant virus rPRV-E2AD was successfully obtained,and the foreign gene did not affect the proliferation characteristics of the parent strain.The results provided the candidate strains for the development of recombinant CSFV E2 gene live vector vaccine,and also provided the research basis for the development of PRV live vector vaccine.

Key words: Classical swine fever virus (CSFV); Pseudorabies virus (PRV); recombinant virus; E2 gene

CLC Number:

S852.65⁺1

WANG Linqing, ZHANG Liuhui, CHEN Ximeng, MA Shijie, SONG Yue, CHEN Hongying. Construction and Biological Characteristics of Recombinant PRV in Partial Epitope Region of CSFV E2 Gene[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(4): 1815-1824.

References

[1] DONG X N,CHEN Y H.Marker vaccine strategies and candidate CSFV marker vaccines[J].Vaccine,2007,25(2):205-230.
[2] LIU Y,BAHOUSSI A N,WANG P H,et al.Complete genome sequences of Classical swine fever virus:Phylogenetic and evolutionary analyses[J].Frontiers in Microbiology,2022,13:1021734.
[3] PAN S,MOU C,CHEN Z.An emerging novel virus:Atypical porcine pestivirus (APPV)[J].Reviews in Medical Virology,2019,29(1):e2018.
[4] YI W,ZHENG F,ZHU H,et al.Role of the conserved E2 residue G259 in Classical swine fever virus production and replication[J].Virus Research,2022,313:198747.
[5] ZHANG H,WEN W,ZHAO Z,et al.Enhanced protective immunity to CSFV E2 subunit vaccine by using IFN-γ as immunoadjuvant in weaning piglets[J].Vaccine,2018,36(48):7353-7360.
[6] WENSVOORT G.Topographical and functional mapping of epitopes on Hog cholera virus with monoclonal antibodies[J].The Journal of General Virology,1989,70(11):2865-2876.
[7] QI Y,ZHANG B Q,SHEN Z,et al.Candidate vaccine focused on a Classical swine fever virus epitope induced antibodies with neutralizing activity[J].Viral Immunology,2009,22(3):205-213.
[8] PARK Y,LEE S,KANG H,et al.A Classical swine fever virus E2 fusion protein produced in plants elicits a neutralizing humoral immune response in mice and pigs[J].Biotechnology Letters,2020,42(7):1247-1261.
[9] ANDREW M,MORRIS K,COUPAR B,et al.Porcine interleukin-3 enhances DNA vaccination against classical swine fever[J].Vaccine,2006,24(16):3241-3247.
[10] LI W,MAO L,ZHOU B,et al.The swine CD81 enhances E2-based DNA vaccination against classical swine fever[J].Vaccine,2015,33(30):3542-3548.
[11] SUN Y,LI H Y,TIAN D Y,et al.A novel Alphavirus replicon-vectored vaccine delivered by Adenovirus induces sterile immunity against classical swine fever[J].Vaccine,2011,29(46):8364-8372.
[12] LIN H,MA Z,CHEN L,et al.Recombinant swinepox virus expressing glycoprotein E2 of Classical swine fever virus confers complete protection in pigs upon viral challenge[J].Frontiers in Vetrinary Science,2017,4:81.
[13] ABID M,TEKLUE T,LI Y,et al.Generation and immunogenicity of a recombinant Pseudorabies virus co-expressing Classical swine fever virus E2 protein and Porcine circovirus type 2 capsid protein based on fosmid library platform[J].Pathogens (Basel, Switzerland),2019,8(4):279.
[14] WANG Y,YUAN J,CONG X,et al.Generation and efficacy evaluation of a recombinant Pseudorabies virus variant expressing the E2 protein of Classical swine fever virus in pigs[J].Clinical and Vaccine Immunology,2015,22(10):1121-1129.
[15] 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.
[16] XIA S L,XIANG G T,LEI J L,et al.Efficacy of the marker vaccine rAdV-SFV-E2 against classical swine fever in the presence of maternally derived antibodies to rAdV-SFV-E2 or C-strain[J].Veterinary Microbiology,2016,196:50-54.
[17] 殷震,刘景华.动物病毒学[M].2版.北京:科学出版社,1997.YIN Z,LIU J H.Virology of Animals[M].2nd ed.Beijing:Science Press,1997.(in Chinese)
[18] 张璐,何慧芬,秦爱建,等.以畜禽疱疹病毒为载体的重组基因工程疫苗研究进展[J].动物医学进展,2022,43(8):88-93.ZHANG L,HE H F,QIN A J,et al.Research progress on recombinant gene engineering vaccines using livestock and poultry Herpesvirus as vectors[J].Progress in Veterinary Medicine,2022,43(8):88-93.(in Chinese)
[19] PAVULRAJ S,PANNHORST K,STOUT R W,et al.A triple gene-deleted Pseudorabies virus-vectored subunit PCV2b and CSFV vaccine protects pigs against PCV2b challenge and induces serum neutralizing antibody response against CSFV[J].Vaccines (Basel),2022,10(2):305.
[20] 张宇,刘芳,田润博,等.猪伪狂犬病病毒变异株gE/gI/TK三基因缺失突变株的构建及纯化[J].中国兽医学报,2020,40(3):476-482.ZHANG Y,LIU F,TIAN R B,et al.Generation and purification of a gE/gI/TK-gene-deleted Pseudorabies virus variant[J].Chinese Journal of Veterinary Science,2020,40(3):476-482.(in Chinese)
[21] HUANG B,HUANG Y,DENG L,et al.Intranasal administration with recombinant vaccine PRVXJ-delgE/gI/TK-S induces strong intestinal mucosal immune responses against PDCoV[J].BMC Veternariy Research,2023,19(1):171.
[22] WEI J,LIU C,HE X,et al.Generation and characterization of recombinant Pseudorabies virus delivering African swine fever virus CD2v and p54[J].International Journal of Mollecular Sciences,2023,25(1):335.
[23] DENG L,GU S,HUANG Y,et al.Immunogenic response of recombinant Pseudorabies virus carrying B646L and B602L genes of African swine fever virus in mice[J].Veterinary Microbiology,2023,284:109815.
[24] 侯晓璇,穆永,王彪,等.表达PEDV S1蛋白重组PRV的构建与鉴定[J].中国兽医科学,2023,53(8):1003-1009.HOU X X,MU Y,WANG B,et al.Construction and characterization of recombinant PRV expressing S1 protein of PEDV[J].Veterinary Science in China ,2023,53(8):1003-1009.(in Chinese)
[25] 侯晓璇,穆永,王同燕,等.表达PEDV S蛋白重组PRV的构建与生物学特性评价[J].中国兽医学报,2024,44(4):657-663.HOU X X,MU Y,WANG T Y,et al.Construction and biological characteristics evaluation of recombinant PRV expressing S protein of PEDV[J].Chinese Journal of Veterinary Science,2024,44(4):657-663.(in Chinese)
[26] 赵丽,吕玉金,徐通,等.共表达TGEV AD蛋白和PEDVS蛋白CS区重组伪狂犬病病毒的构建及纯化[J].中国兽医学报,2024,44(2):237-243.ZHAO L,LYU Y J,XU T,et al.Construction and purification of a recombinant Pseudorabies virus co-expressing the AD protein of the TGEV and the CS region of the PEDV S protein[J].Chinese Journal of Veterinary Science,2024,44(2):237-243.(in Chinese)
[27] 穆艳霞,张金玲,刘春羽,等.基于CRISPR/Cas9技术构建BHV-1 gE缺失株及其免疫生物学特性初步鉴定[J].农业生物技术学报,2018,26(12):2003-2016.MU Y X,ZHANG J L,LIU C Y,et al.Construction of BHV-1 gE deletion strain based on CRISPR/Cas9 technique and preliminary identification of its immunobiological characteristics[J].Journal of Agricultural Blotechnology,2018,26(12):2003-2016.(in Chinese)

Construction and Biological Characteristics of Recombinant PRV in Partial Epitope Region of CSFV E2 Gene

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	HUANG Shilei, LUO Gan, CHENG Peng, NIU Zheng, GUO Jianhua, ZOU Hong. Epidemic Report of Classical Swine Fever Virus in Pigs from 2005 to 2023 of China:Meta-Analysis and Systematic Review [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 2058-2070.
[2]	LIANG Dongge, YAO Chen, CHAI Yajing, CAI Mengpan, CHU Beibei, LU Weifei, WANG Jiang, ZENG Lei, LIU Zhonghu, MING Shengli. Construction of Porcine Derived Rab Gene shRNA Libraries and Stable Cell Lines and the Effects on PRV Proliferation [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(3): 1250-1258.
[3]	YANG Dawei, LIU Shuyi, LIN Yating, CHEN Hu, ZHANG Hongliang, LI Guimei. Preparation of Monoclonal Antibody Against gB Protein of Porcine Pseudorabies Virus and Development of a Competitive ELISA Detection Method [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(12): 5439-5448.
[4]	XIN Ruolan, ZHOU Zhiyu, ZHANG Jiawen, DU Jige, YIN Chunsheng, WANG Fengxue, CHEN Xiaoyun, WEN Yongjun, ZHU Zhen. Construction and Growth Characteristics of Recombinant Lumpy Skin Disease Virus Carrying eGFP Gene [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(12): 5479-5488.
[5]	ZHANG Jingxu, XU Yu, LIANG Haiying, ZENG Zhiyong, TANG Deyuan, WANG Bin, XU Songping, WAN Juan, ZHU Yang. Phylogenetic Evolution and Epitope Analysis of Pseudorabies Virus Guizhou Isolates and Vaccine Strains [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1093-1106.
[6]	YANG Suzhen, LIU Yunchao, XING Yunrui, SUN Yaning, SHANG Yanli, ZHANG Gaiping. Study on Quantum Dots-based Fluorescent Immunochromatographic Strip for gB Antibody of Porcine Pseudorabies Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(10): 4160-4167.
[7]	XIE Sihao, GOU Hongchao, BIAN Zhibiao, LI Bin, CAI Rujian, ZANG Yingan, LI Chunling. Effects of MLKL Gene Knockout on Replication of Pseudorabies Virus [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1934-1941.
[8]	SHANG Yu, LI Lintao, LI Li, XU Yingying, FENG Helong, WANG Hongcai, ZHANG Rongrong, ZENG Zhe, WANG Honglin, LUO Qingping, SHAO Huabin, WEN Guoyuan. Construction of Recombinant Heat-resistant Newcastle Disease Virus Expressing Fiber2 Protein of Fowl Adenovirus Serotype 4 and Evaluation of Its Immune Effect [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 650-659.
[9]	ZHANG Yanmin, ZHOU Yanan, CAO Lei, TIAN Yuan, LIU Xuping, TAN Wensong, ZHAO Liang. Expression and Immunogenicity Analysis of Classical Swine Fever Virus E2-GM-CSF Fusion Protein in HEK293T Cells [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 669-676.
[10]	LIU Huan, ZHANG Mengmeng, CUI Yifang, LIU Jing, ZHANG Jingju, CHEN Dengjin, LI Xiubo, LI Cun, XU Fei. Optimization of Foaming Performance of Compound Quaternary Ammonium Salt Foam Disinfectant and Evaluation of Its Killing Effect on Viruses [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4866-4877.
[11]	XING Jiayou, FAN Wenjie, WANG Yumin, LU Xiuxiang, SONG Yue, CHU Beibei, WANG Jiang, WANG Mengdi. Effect of Tamoxifen on the Proliferation of Porcine Pseudorabies Virus in vitro [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4503-4512.
[12]	YANG Mingzhu, CHEN Xiaochun, SONG Jiacheng, HOU Yaxin, LI Junping. Research Progress on Construction and Application of Recombinant Animal Herpesvirus Live Vector Vaccine [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 338-351.
[13]	WU Tongzhong, NUERSAILIKE Nusufu, HUANG Xin, HAN Mengli, ZHANG Xingxing, ZHANG Qian, WANG Xinhua, HE Yanhua, ZHONG Fagang. Construction of Recombinant Lactococcus Expressing E2 Protein of Bovine Viral Diarrhea Virus and Immunogenicity Analysis of the Protein [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 2975-2981.
[14]	QU Zhehui, ZHANG Xiwen, ZHAO Yu, ZHENG Quanfang, LIAN Huixiang, ZHAO Yunhuan, GUO Xiaoqiu. Genetic Variation Analysis of Major Virulence Genes of Pseudorabies Virus Epidemic Strains in Southern Henan Province [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 3019-3029.
[15]	QU Zhehui, ZHAO Yu, ZHANG Xiwen, LIAN Huixiang, ZHAO Yunhuan, CHEN Bin, GUO Xiaoqiu. Expression of Pseudorabies Virus gE Protein and Preparation of Its Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 631-639.