1型鸭甲肝病毒VP3蛋白的抗血清中和活性分析及B细胞表位鉴定

doi:10.11843/j.issn.0366-6964.2016.01.019

Abstract

Abstract:

This study aimed at researching neutralizing activity of VP3 antiserums and determining B-cell epitopes of VP3 protein from duck hepatitis A virus type 1(DHAV-1).The pGEX-4T-1 expression plasmid was used to express VP3 gene of DHAV-1 in E.coli BL21(DE3).The expressed recombinant VP3 protein was purified by gel extraction and was used as immunogen to rabbit to prepare polyclonal antibodies.Chicken embryo neutralization test was conducted to detect the neutralizing titer of polyclonal antibodies.Moreover，Karplus&Schulz，Emini，Jameson-Wolf and Parker method were used to analyze the flexibility，surface accessibility，antigenicity and hydrophilicity of the VP3 protein，respectively.This contributed to obtain four probable B-cell epitopes for VP3 protein.The rabbit anti-VP3 polyclonal antibody was used as the first antibody of indirect ELISA to identity the synthetic peptides.Furthermore，a panel of clinical duck serum samples was used to evaluate capacity of the identified B-cell epitopes for antibodies detection.The results showed that the recombinant VP3 protein was expressed as inclusion body in E.coli BL21(DE3) with a molecular weight about 54 kD，and proved to be with good reactogenicity by Western blot analysis.The prepared polyclonal antibodies reached an titer of 1∶16 by AGP test，it could neutralize DHAV-1 and reached a titer of 1∶39.Furthermore，the B-cell epitopes identified by indirect ELISA were GKRKPCRRPIHKPKNPPQEP(1-20 aa)，FNTGRYQMSWYPIADGEQSL(131-150 aa) and VNSSAPSNID(200-209 aa).The test for antibody detection ability revealed the epitopes were capable of detecting clinical duck antiserums of DHAV-1.This study proved antiserums to VP3 protein of DHAV-1 with neutralizing activity and identified VP3’s three promising B-cell epitopes，1-20 aa，131-150 aa and 200-209 aa，for clinical use.

CLC Number:

SHEN You-lin,WANG Ming-shu，CHENG An-chun，JIA Ren-yong，ZHU De-kang，CHEN Shun，LIU Ma-feng，LIU Fei，YANG Qiao，SUN Kun-feng，CHEN Xiao-yue. Neutralizing Activity Analysis of VP3 Antiserums and B-cell Epitopes Identification of VP3 Protein form Duck Hepatitis A Virus Type 1[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(1): 141-148.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2016.01.019

https://www.xmsyxb.com/EN/Y2016/V47/I1/141

References

［1］WANG L，PAN M，FU Y，et al.Classification of duck hepatitis virus into three genotypes based on molecular evolutionary analysis［J］.Virus Genes，2008，37(1)：52-59.
［2］KING A M Q,ADAMS M J,CARSTENS E B，et al.Virus taxonomy：classification and nomenclature of viruses：ninth report of the international committee on taxonomy of viruses［M］.San Diego:Elsevier,2012：855-880.
［3］TODD D，SMYTH V J，BALL N W，et al.Identification of chicken enterovirus-like viruses，duck hepatitis virus type 2 and duck hepatitis virus type 3 as astroviruses［J］.Avian Pathol，2009，38(1)：21-30.
［4］SWAYNE D E，GLISSON J R，MCDOUGALD L R，et al.Diseases of poultry［M］.13ed.Wiley & Sons，2013：422-431.
［5］KIM M C，KWON Y K，JOH S J，et al.Molecular analysis of duck hepatitis virus type 1 reveals a novel lineage close to the genus Parechovirus in the family Picornaviridae［J］.J Gen Virol，2006，87(Pt 11)：3307-3316.
［6］JIN X，ZHANG W，ZHANG W，et al.Identification and molecular analysis of the highly pathogenic duck hepatitis virus type 1 in Hubei province of China［J］.Res Vet Sci，2008，85(3)：595-598.
［7］TSENG C H，KNOWLES N J，TSAI H J.Molecular analysis of duck hepatitis virus type 1 indicates that it should be assigned to a new genus［J］.Virus Res，2007，123(2)：190-203.
［8］刘家森，甘一迪，姜骞，等.鸭肝炎病毒Ⅰ型VP3基因的克隆及原核表达［J］.中国兽医科学，2008，38(7)：587-590.
LIU J S，GAN Y D，JIANG Q，et al.Cloning and prokaryotic expression of VP3 gene of duck hepatitis virus typeⅠ［J］.Chinese Veterinary Science，2008，38(7)：587-590.(in Chinese)
［9］廖俊伟，张小飞，黄显明，等.Ⅰ型鸭肝炎病毒VP3基因的克隆与原核表达［J］.动物医学进展，2009，30(7)：49-52.
LIAO J W，ZHANG X F，HUANG X M，et al.Cloning and prokaryotic expression of VP3 gene of duck hepatitis virus typeⅠ［J］.Progress in Veterinary Medicine，2009，30(7)：49-52.(in Chinese)
［10］高慎阳，查恩辉，王坤，等.一种 “高性价比” 切胶纯化原核表达蛋白的方法［J］.中国农学通报，2010，26(22)：24-26.
GAO S Y，ZHA E H，WANG K，et al.A ‘Cost-Effective’ method for purification of prokaryotic expression proteins in gel slices［J］.Chinese Agricultural Science Bulletin，2010，26(22)：24-26.(in Chinese)
［11］LIU M，ZHANG T，ZHANG Y，et al.Development and evaluation of a VP1-ELISA for detection of antibodies to duck hepatitis type 1 virus［J］.J Virol Methods， 2010，169(1)：66-69.
［12］LI C，CHEN Z，MENG C，et al.High yield expression of duck hepatitis A virus VP1 protein in Escherichia coli，and production and characterization of polyclonal antibody［J］.J Virol Methods，2013，191(1)：69-75.
［13］ZHANG T，LI X，WU X，et al.Characterization of monoclonal antibodies against duck hepatitis type 1 virus VP1 protein［J］.J Virol Methods，2014，208：166-170.
［14］WANG C，LI X K，WU T C，et al.Recombinant VP1 protein of duck hepatitis virus 1 expressed in Pichia pastoris and its immunogenicity in ducks［J］.Acta Virol，2014，58(4)：333-339.
［15］杨发龙，张焕荣，程方明，等.基于基因C型鸭甲肝病毒VP1重组蛋白的鸭甲肝病毒抗体ELISA检测方法的建立［J］.畜牧兽医学报，2014，45(7)：1148-1153.
YANG F L，ZHANG H R，CHENG F M，et al.Development of an indirect ELISA for detecting antibodies against genotype A and C duck hepatitis A virus based on recombinant VP1 protein［J］.Acta Veterinaria et Zootechnica Sinica，2014，45(7)：1148-1153.(in Chinese)
［16］ZHANG Z W，ZHANG Y G，WANG Y L，et al.Screening and identification of B cell epitopes of structural proteins of foot-and-mouth disease virus serotype Asia1［J］.Vet Microbiol，2010，140(1-2)：25-33.
［17］YAUCH R L，KEREKES K，SAUJANI K，et al.Identification of a major T-cell epitope within VP3 amino acid residues 24 to 37 of Theiler’s virus in demyelination-susceptible SJL/J mice［J］.J Virol，1995，69(11)：7315-7318.
［18］李露，程英杰，李传峰，等.Ⅰ型鸭肝炎病毒VP3基因的克隆及原核表达［J］.中国畜牧兽医，2013，40(10)：33-37.
LI L，CHENG Y J，LI C F，et al.Cloning and prokaryotic expression of VP3 gene of duck hepatitis virusⅠ［J］.Chinese Animal Husbandry and Veterinary Medicine，2013，40(10)：33-37.(in Chinese)
［19］徐建生，唐波，成大荣，等.禽脑脊髓炎病毒结构蛋白 VP3 基因真核表达载体的构建及其免疫［J］.中国兽医学报，2006，26(4)：367-368，372.
XU J S，TANG B，CHENG D R，et al.Construction and immunization of recombinants of the structural protein VP3 gene of avian encephalomyelitis virus［J］.Chinese Journal of Veterinary Science，2006，26(4)：367-368，372.(in Chinese)
［20］郭春艳，赵向绒，胡军.B细胞抗原表位的研究进展及其应用［J］.生物技术通讯，2013，24(2)：266-270.
GUO C Y，ZHAO X R，HU J.Research and application of B cell epitope［J］.Letters in Biotechnology，2013，24(2)：266-270.(in Chinese)
［21］JOKI-KORPELA P，ROIVAINEN M，LANKINEN H，et al.Antigenic properties of human parechovirus 1［J］.J Gen Virol，2000，81(Pt 7)：1709-1718.
［22］SHEN Y,CHENG A,WANG M,et al.Development of an indiret ELISA method based on the VP3 protein of duck hepatitis A virus type 1(DHAV-1) for dual detection of DHAV-1 and DNAV-3 antibodies［J］.J Virol Methods,2015,225:30-34.

Neutralizing Activity Analysis of VP3 Antiserums and B-cell Epitopes Identification of VP3 Protein form Duck Hepatitis A Virus Type 1

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 1

Recommended Articles

Metrics

Comments