猪痘病毒TK、ORF121、ORF143基因缺失毒株的构建及其生物学特性的测定

doi:10.16431/j.cnki.1671-7236.2020.03.022

Abstract

Abstract: In order to screen the replication non-essential region of swinepox virus (SWPV) and determine the biological characteristics of the deletion strains,primers were designed and synthesized for TK(ORF063),ORF121 and ORF143 genes of SWPV according to the principle of homologous recombination,and the overlap extension PCR technology was used to splice the gene fragments containing enhanced green fluorescent protein (EGFP) and homologous recombination of the left and right arms.The spliced fragments were transfected into PK15 cells infected with SWPV,respectively.Fluorescence microscope was used to select single plaque with EGFP as the reporter gene,and the recombinant strains were identified by PCR and Western blotting.The plaque size of PK15 cells and the pathogenicity of pigs were measured.The results showed that the gene fragment successfully integrated into recombinant strain genome.These recombinant strains were named as rSWPV-TK,rSWPV-121 and rSWPV-143,respectively.The results of Western blotting showed that the recombinant strains stably expressed the foreign protein on PK15 cells.The formation of pox spots on PK15 cells was smaller than that of the parent strain,and the difference of the ORF121 gene deletion strain was extremely significant (P<0.01).All the strains could lead to the formation of pox spots on nursery pigs,in which the ORF121 gene deletion strain caused the lesion for a short time,produced less pox spots,and the virulence was lower than that of wild strains.In summary,in this study,two non-essential replication regions ORF121 and ORF143 in the SWPV genome for foreign protein insertion were selected,laying a foundation for the construction of SWPV gene engineering vectors.

Key words: recombinant swinepox virus (rSWPV); splicing by overlap extension PCR; replication non-essential regions; biological characteristics

CLC Number:

S852.65

LIU Changjin, DENG Shunzhou, LUO Feng, ZHONG Luohua, WANG Zhe, GAO Yingxue, LIU Xiaolan. Construction of Swinepox Virus TK,ORF121 and ORF143 Genes Deletion Strains and Determination of Their Biological Characteristics[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(3): 828-836.

References

[1] AFONSO C L,TULMAN E R,LU Z,et al.The genome of swinepox virus[J].Journal of Virology,2002,76(2):783-790.
[2] BHANUPRAKASH V,HOSAMANI M,VENKATESAN G,et al.Animal poxvirus vaccines:A comprehensive review[J].Expert Review of Vaccines,2012,11(11):1355-1374.
[3] LIN H X,MA Z,HOU X,et al.Corrigendum:Construction and immunogenicity of a recombinant swinepox virus expressing a multi-epitope peptide for porcine reproductive and respiratory syndrome virus[J].Scientific Reports,2017,7:46592.
[4] JEFFREY Z,LOCKE K,ALEJANDRO R,et al.Diseases of Swine[M].10th Edtion.Wiley,2012.
[5] JOHNSTON J B,MCFADDEN G.Technical knockout:Understanding poxvirus pathogenesis by selectively deleting viral immunomodulatory genes[J].Cellular Microbiology,2010,6(8):695-705.
[6] YUAN X M,LIN H X,LI B,et al.Swinepox virus vector-based vaccines:Attenuation and biosafety assessments following subcutaneous prick inoculation[J].Veterinary Research,2018,49(1):14-25.
[7] YUAN X M,LIN H X,LI B,et al.Efficacy and immunogenicity of recombinant swinepox virus expressing the truncated S protein of a novel isolate of porcine epidemic diarrhea virus[J].Archives of Virology,2017,162(12):3779-3789.
[8] 蔺辉星,周红,范红结.032基因缺失猪痘病毒载体的构建及鉴定[J].安徽科技学院学报,2016,30(6):1-6. LIN H X,ZHOU H,FAN H J.Construction and identification of the 032 gene deletion in swinepox virus[J].Journal of Anhui Science and Technology University,2016,30(6):1-6.(in Chinese)
[9] WINSLOW B J,COCHRAN M D,HOLZENBURG A,et al.Replication and expression of a swinepox virus vector delivering feline leukemia virus Gag and Env to cell lines of swine and feline origin[J].Virus Research,2003,98(1):1-15.
[10] 钟罗华,邓舜洲,胡杨,等.表达猪圆环病毒2型衣壳蛋白的重组猪痘病毒的构建及鉴定[J].中国畜牧兽医,2018,45(2):279-290. ZHONG L H,DENG S Z,HU Y,et al.Construction and identification of recombinant swinepox virus expressing porcine circovirus type 2 capsid protein[J].China Animal Husbandry & Veterinary Medicine,2018,45(2):279-290.(in Chinese)
[11] HECKMAN K L,PEASE,LARRY R.Gene splicing and mutagenesis by PCR-driven overlap extension[J].Nature Protocols,2007,2(4):924-932.
[12] 王小凤,李明奇,郭嘉,等.布鲁氏菌vceA基因缺失株的构建及生物学特性研究[J].中国畜牧兽医,2019,46(4):38-45. WANG X F,LI M Q,GUO J,et al.Construction and biological characteristics of Brucella vceA gene deletion strain[J].China Animal Husbandry & Veterinary Medicine,2019,46(4):38-45.(in Chinese)
[13] HALLER S L,PENG C,MCFADDEN G,et al.Poxviruses and the evolution of host range and virulence[J].Infection,Genetics and Evolution,2014,21:15-40.
[14] 胡玉珍,陈普成,丁蕾蕾,等.鸭瘟病毒疫苗株基因组复制非必需区的筛选[J].中国预防兽医学报,2018,40(10):29-33. HU Y Z,CHEN P C,DING L L,et al.Selection of nonessential regions for viral replication of duck enteritis virus (vaccine strain) genome[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(10):29-33.(in Chinese)
[15] HAHN J,PARK S H,SONG J Y,et al.Construction of recombinant swinepox viruses and expression of the classical swine fever virus E2 protein[J].Journal of Virological Methods,2001,93(1):49-56.
[16] 陈轶霞,龙玲,王明明.痘病毒ANK/F-box蛋白的生物学特性及泛素化相关功能[J].病毒学报,2017(4):155-161. CHEN Y X,LONG L,WANG M M.The biological characteristics and ubiquitination-related functions of poxviral ANK/F-box repeat proteins[J].Chinese Journal of Virology,2017(4):155-161.(in Chinese)
[17] TULMAN E R,AFONSO C L,LU Z,et al.The genomes of sheeppox and goatpox viruses[J].Journal of Virology,2002,76(12):6054-6061.
[18] AFONSO C L,DELHON G,TULMAN E R,et al.Genome of deerpox virus[J].Journal of Virology,2005,79(2):966-977.
[19] MOHAMED M R,RAHMAN M M,RICE A,et al.Cowpox virus expresses a novel ankyrin repeat NF-κB inhibitor that controls inflammatory cell influx into virus-infected tissues and is critical for virus pathogenesis[J].Journal of Virology,2009,83(18):9223-9236.
[20] LAMB S A,RAHMAN M M,MCFADDEN G.Recombinant myxoma virus lacking all poxvirus ankyrin-repeat proteins stimulates multiple cellular anti-viral pathways and exhibits a severe decrease in virulence[J].Virology,2014,464-465:134-145.
[21] BLANIÉ,SOPHIE,GELFI J,et al.MNF,an ankyrin repeat protein of myxoma virus,is part of a native cellular SCF complex during viral infection[J].Virology Journal,2010,7(1):56-56.
[22] WILLIS K L,LANGLAND J O,SHISLER J L.Viral double-stranded RNAs from vaccinia virus early or intermediate gene transcripts possess PKR activating function,resulting in NF-κB activation,When the K1 protein is absent or mutated[J].Journal of Biological Chemistry,2011,286(10):7765-7778.
[23] ROPER R L.Characterization of the vaccinia virus A35R protein and its role in virulence[J].Journal of Virology,2006,80(1):306-313.
[24] REHM K E,CONNOR R F,JONES G J B,et al.Vaccinia virus A35R inhibits MHC class Ⅱ antigen presentation[J].Virology,2010,397(1):176-186.
[25] FLEISCHAUER C,UPTON C,VICTORIA J,et al.Genome sequence and comparative virulence of raccoonpox virus:The first North American poxvirus sequence[J].Journal of General Virology,2015,96(9):2806-2821.
[26] 黄彩云,赵志荀,朱学亮,等.表达绿色荧光蛋白的MVA重组毒株的构建及筛选[J].中国畜牧兽医,2019,46(7):2079-2087. HUANG C Y,ZHAO Z X,ZHU X L,et al.Construction and screening of GFP recombinant MVA strain[J].China Animal Husbandry & Veterinary Medicine,2019,46(7):2079-2087.(in Chinese)
[27] YAKUBITSKYI S N,KOLOSOVA I V,MAKSYUTOV R A,et al.Highly immunogenic variant of attenuated vaccinia virus[J].Doklady Biochemistry and Biophysics,2016,466(1):35-38.

Construction of Swinepox Virus TK,ORF121 and ORF143 Genes Deletion Strains and Determination of Their Biological Characteristics

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