非洲猪瘟病毒EP153R蛋白的原核表达及其多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2020.04.022

Abstract

Abstract: The aim of this study was to construct the prokaryotic expression system of EP153R gene of African swine fever virus (ASFV),express the recombinant protein EP153R (rEP153R),and prepare the polyclonal antibody against rEP153R.EP153R gene sequence was synthesized according to EP153R gene sequence of ASFV Georgia 2007/1 (GenBank accession No.:FR682468.1),and inserted into pET-28a vector to construct pET-28a-EP153R recombinant plasmid.After identification by double enzyme digestion and sequencing,the recombinant plasmid was transformed into E.coli BL21(DE3) competent cells,which were induced by IPTG at 16 and 37 ℃ for 12 h respectively.The recombinant protein was expressed at the optimum temperature and identified by SDS-PAGE and Western blotting.The 25 ku bands of SDS-PAGE protein gel were cut,and the expression of recombinant protein was identified by liquid chromatography-mass spectrometry LC-MS.The polyclonal antibody against rEP153R was prepared by immunizing mice with purified rEP153R.The antibody titer was detected by indirect ELISA,and the specificity was identified by Western blotting.The results showed that the expression level of the recombinant bacteria was high at 16 ℃,and it was expressed in the form of inclusion body,and the product appeared obvious bands at 25 ku,which was consistent with the expected protein size,indicating that rEP153R was successfully expressed.The results of LC-MS showed that the specific peptides of YIGLINK、NESVLLR and KYIGLINK were matched,and the protein was confirmed to be rEP153R.The titer of polyclonal antibody was 1∶128 000 by indirect ELISA.Western blotting results showed that the antibody had good specificity.The above results showed that rEP153R was successfully expressed and its specific antibody was prepared,which provided technical support and material for further study of biological function of the protein and identification of live carrier vaccine of African swine fever.

Key words: African swine fever virus (ASFV); EP153R gene; liquid chromatography; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65⁺1

WANG Zhaoyang, LIN Weidong, LIU Xueting, JIANG Yajun, XIN Ting, HOU Shaohua, GUO Xiaoyu, ZHU Hongfei, JIA Hong. Prokaryotic Expression of EP153R Protein of African Swine Fever Virus and Preparation of Its Polyclonal Antibodies[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(4): 1163-1171.

References

[1] OURA C A,POWELL P P,ANDERSON E,et al.The pathogenesis of African swine fever in the resistant bushpig[J].Journal of General Virology,1998,79(Pt 6):1439-1443.
[2] PENRITH M L,VOSLOO W.Review of African swine fever:Transmission,spread and control[J].Journal of the South African Veterinary Association,2009,80(2):58-62.
[3] NETHERTON C L,GOATLEY L C,REIS A L,et al.Identification and immunogenicity of African swine fever virus antigens[J].Frontiers in Immunology,2019,10:1318.
[4] ZHOU X,LI N,LUO Y,et al.Emergence of African swine fever in China,2018[J].Transboundary and Emerging Diseases,2018,65(6):1482-1484.
[5] DIXON L K,CHAPMAN D A,NETHERTON C L,et al.African swine fever virus replication and genomics[J].Virus Research,2013,173(1):3-14.
[6] GALINDO I,ALMAZAN F,BUSTOS M J,et al.African swine fever virus EP153R open reading frame encodes a glycoprotein involved in the hemadsorption of infected cells[J].Virology,2000,266(2):340-351.
[7] HURTADO C,GRANJA A G,BUSTOS M J,et al.The C-type lectin homologue gene (EP153R) of African swine fever virus inhibits apoptosis both in virus infection and in heterologous expression[J].Virology,2004,326(1):160-170.
[8] HURTADO C,BUSTOS MJ,GRANJA A G,et al.The African swine fever virus lectin EP153R modulates the surface membrane expression of MHC class Ⅰ antigens[J].Archives of Virology,2011,156(2):219-234.
[9] BURMAKINA G,MALOGOLOVKIN A,TULMAN E R,et al.African swine fever virus serotype-specific proteins are significant protective antigens for African swine fever[J].Journal of General Virology,2016,97(7):1670-1675.
[10] GALLARDO C,SANCHEZ E G,PEREZNUNEZ D,et al.African swine fever virus (ASFV) protection mediated by NH/P68 and NH/P68 recombinant live-attenuated viruses[J].Vaccine,2018,36(19):2694-2704.
[11] LOKHANDWALA S,WAGHELA S D,BRAY J,et al.Adenovirus-vectored novel African swine fever virus antigens elicit robust immune responses in swine[J].PLoS One,2017,12(5):e177007.
[12] 宋天琪,金红岩,张通明,等.重组犬α2干扰素的原核表达及其抗病毒活性评价[J].中国畜牧兽医,2018,45(1):14-21. SONG T Q,JIN H Y,ZHANG T M,et al.Prokaryotic expression of recombinant canine interferon alpha 2 and evaluation of their antiviral activities[J].China Animal Husbandry&Veterinary Medicine,2018,45(1):14-21.(in Chinese)
[13] 李洪利,曹金山,王君玮,等.非洲猪瘟病毒实时荧光定量PCR检测方法的建立及应用[J].中国畜牧兽医,2012,39(6):37-40. LI H L,CAO J S,WANG J W,et al.Construction and application of Real-time quantitative PCR for detection of African swine fever virus[J].China Animal Husbandry&Veterinary Medicine,2012,39(6):37-40.(in Chinese)
[14] 哈登楚日亚,樊晓旭,赵永刚,等.非洲猪瘟病毒实时荧光重组酶聚合酶扩增技术(RPA)检测方法的建立[J].中国畜牧兽医,2017,44(11):3270-3277. HADENG C R Y,FAN X X,ZHAO Y G,et al.Establishment of a Real-time fluorescent recombinase polymerase amplification (RPA) for the detection of African swine fever virus[J].China Animal Husbandry&Veterinary Medicine,2017,44(11):3270-3277.(in Chinese)
[15] GIMENEZLIROLA L G,MUR L,RIVERA B,et al.Detection of African swine fever virus antibodies in serum and oral fluid specimens using a recombinant protein 30(p30) dual matrix indirect ELISA[J].PLoS One,2016,11(9):e161230.
[16] ZAKARYAN H,REVILLA Y.African swine fever virus:Current state and future perspectives in vaccine and antiviral research[J].Veterinary Microbiology,2016,185:15-19.
[17] SANCHEZ E G,PEREZ-NUNEZ D,REVILLA Y.Development of vaccines against African swine fever virus[J].Virus Research,2019,265:150-155.
[18] 张艳艳,陈腾,张静远,等.非洲猪瘟病毒基因缺失疫苗株的构建和免疫保护特性[J].中国兽医学报,2019,39(8):1421-1427. ZHANG Y Y,CHEN T,ZHANG J Y,et al.Construction and immunoprotective characterization of gene deleted African swine fever virus vaccine candidates[J].Chinese Journal of Veterinary Science,2019,39(8):1421-142.(in Chinese)
[19] JANCOVICH J K,CHAPMAN D,HANSEN D T,et al.Immunization of pigs by DNA prime and recombinant vaccinia virus boost to identify and rank African swine fever virus immunogenic and protective proteins[J].Journal of Virology,2018,92(8):e02219-17.
[20] SI L,XU H,ZHOU X,et al.Generation of influenza a viruses as live but replication-incompetent virus vaccines[J].Science,2016,354(6316):1170-1173.
[21] MATSUO E,CELMA C C,BOYCE M,et al.Generation of replication-defective virus-based vaccines that confer full protection in sheep against virulent bluetongue virus challenge[J].Journal of Virology,2011,85(19):10213-10221.
[22] TEKLUE T,SUN Y,ABID M,et al.Current status and evolving approaches to African swine fever vaccine development[J].Transboundary and Emerging Diseases,2020,67(2):529-542.
[23] LOKHANDWALA S,WAGHELA S D,BRAY J,et al.Induction of robust immune responses in swine by using a cocktail of adenovirus-vectored African swine fever virus antigens[J].Clinical and Vaccine Immunology,2016,23(11):888-900.
[24] MURGIA M V,MOGLE M R,CERTOMA A,et al.Evaluation of an African swine fever (ASF) vaccine strategy incorporating priming with an alphavirus-expressed antigen followed by boosting with attenuated ASF virus[J].Archives of Virology,2019,164(2):359-370.
[25] YANEZ R J,RODRIGUEZ J M,NOGAL M L,et al.Analysis of the complete nucleotide sequence of African swine fever virus[J].Virology,1995,208(1):249-278.

Prokaryotic Expression of EP153R Protein of African Swine Fever Virus and Preparation of Its Polyclonal Antibodies

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