鸭肠炎病毒gB抗原优势区的表达及其多克隆抗体的反应特性

doi:10.11843/j.issn.0366-6964.2015.03.012

Abstract

Abstract:

This experiment was conducted to prepare the antiserum of antigenic domain region of duck enteritis virus (DEV) gB gene，and showed its initial application as detection reagent to detect DEV.Specific primers were designed for DEV C-KCE ul27 gene according to the cloned gene sequence of our laboratory，then the antigenic domain region of DEV gB gene （331-570 bp）was cloned by PCR which named gB-AD.Recombinant gB-AD region protein was expressed in prokaryotic expression system，pET-32a(+) / Rosetta(DE3)pLysS，by induction of IPTG and was purified by Ni-NTA column affinity chromatography，and the antigenicity of recombinant protein was identified by using the DEV positive serum.The purified recombinant protein was retrieved by cutting the gel slices that contain the right bands，and was used to immunize rabbits as immunogen to prepare the rabbit anti-gB-AD region serum.Results were as follows：The recombinant protein could be identified by DEV positive serum specifically，and the titer of polyclonal antibody for immunizing rabbit was 1∶10 240 by I-ELISA with neutralizing activity.The quality of the DEV gB molecule was about 66 kD reacted to polyclonal antibody in the reducing SDS-PAGE，on the contrary，it was about 120 and 66 kD in the non-reducing SDS-PAGE.I-ELISA and Western blot analysis showed that antibody for immunizing rabbit could identify recombinant gB-AD region protein from the infected cells respectively.In the further，indirect immunofluorescence test and neutralization assay confirmed that the antiserum could identify DEV specifically with neutralization.Above all，the results showed that antiserum for immunizing rabbit can be used as detection reagent to detect DEV，and it afforded significant data for the study on the detection of DEV and function of DEV gB.

CLC Number:

S852.4

ZHANG Shu-dong，CAO Chun-qiu，DONG Jing-quan，GAO Ming-chun，ZHANG Wen-long，ZHENG Tie-xin，MA Bo，WANG Jun-wei. Prokaryotic Expression of Antigenic Domain Region of Duck Enteritis Virus gB Gene and Reactivity of Its Polyclonal Antibody[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(3): 430-437.

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References

［1］殷震，刘景华.动物病毒学［M］.2版.北京：科学出版社，1997：1073-1077.
YIN Z，LIU J H.Animal virology［M］.2ed.Beijing：Science Press，1997：1073-1077.（in Chinese）
［2］LI Y F，HUANG B，MA X L，et al.Molecular characterization of the genome of duck enteritis virus［J］.Vriology，2009，391(2)：151-161.
［3］GUO P X.Characterization of the gene and an antigenic determinant of equine herpesvirus type-1 glycoprotein 14 with homology to gB-equivalent glycoproteins of other herpesviruses［J］.Gene，1990，87(2)：249-255.
［4］SCHLEISS M R，CHOI K Y，ANDERSON J，et al.Glycoprotein B (gB) vaccines adjuvanted with AS01 or AS02 protect female guinea pigs against cytomegalovirus (CMV) viremia and offspring mortality in a CMV-challenge model［J］. Vaccine，2014，32(23)：2756-2762.
［5］SHARMA S，WISNER T W，JOHNSON D C，et al.HCMV gB shares structural and functional properties with gB proteins from other herpesviruses［J］.Virology，2013，435(2)：239-249.
［6］潘华奇.鸭肠炎病毒gB基因的克隆与表达及其产物的初步应用［D］.兰州：甘肃农业大学，2007.
PAN H Q.Cloning and expression of duck enteritis virus gB gene and preliminary application of its product［D］.Lanzhou：Gansu Agricultural University，2007.（in Chinese）
［7］马波.鸭肠炎病毒ul27基因特征及其编码蛋白部分抗原表位优势区的鉴定［D］.哈尔滨：东北农业大学，2008.
MA B.Molecular characterization and identification of partially immunodomiant epitope regions of duck enteritis virus ul27 gene［D］.Harbin：Northeast Agricultural University，2008.（in Chinese）
［8］董广阔.鸭肠炎病毒囊膜蛋白gB和gC主要抗原域的原核表达和单克隆抗体的研制［D］.扬州：扬州大学，2012.
DONG G K.Prokaryotic expression of major antigenic domains and development of monoclonal antibodies for gB and gC of duck enteritis virus［D］.Yangzhou：Yangzhou University，2012.（in Chinese）
［9］BENDER F C，WHITBECK J C，PONCE DE LEON M，et al.Specific association of glycoprotein B with lipid rafts during herpes simplex virus entry［J］.J Virol，2003，77(17)：9542-9552.
［10］于在江，马学恩，周建华.切胶纯化表达蛋白包涵体的可行性分析［J］.生物技术，2007，17(3)：46-48.
YU Z J，MA X E，ZHOU J H.A modified method for purification of inclusion bodies proteins in gel slices［J］.Biotechnology，2007，17(3)：46-48.（in Chinese）
［11］刘晓玫.鸭肠炎病毒US2蛋白细胞定位及感染相关功能的研究［D］.哈尔滨：东北农业大学，2010.
LIU X M.Cellular locationand infeetion related function of duck enieritis virus US2 protein［D］.Harbin：Northeast Agricultural University，2010.（in Chinese）
［12］PESCADOR L S，PAZ P，NAVARRO D，et al.Epitopes of herpes simplex virus type 1 glycoprotein B that bind type-common neutralizing antibodies elicit type-specific antibody-dependent cellular cytoxicity［J］.J Infect Dis，1992，166(3)：623-627.
［13］GOEL N，ZIMMERMAN D H，ROSENTHAL K S.Ligand epitope antigen presentation system vaccines against herpes simplex virus［J］. Front Biosci，2005，10：966-974.
［14］潘华奇，曹瑞兵，刘磊，等.鸭瘟病毒gB蛋白N端主要抗原域的表达及间接ELISA检测［J］.微生物学报，2008，48(1)：98-102.
PAN H Q，CAO R B，LIU L，et al.Prokaryotic expression of N-terminal antigenic domain of duck plague virus gB protein and the establishment of putative indirect ELISA assay［J］.Acta Microbiologica Sinica，2008，48(1)：98-102.（in Chinese）
［15］王文洁，唐泰山，张常印，等.鸭病毒性肠炎gB基因的原核表达及产物的抗原性分析［J］.畜牧与兽医，2008，40(10)：5-9.
WANG W J，TANG T S，ZHANG C Y，et al.Prokaryotic expression of duck enteritis virus gB gene and antigenicity analysis of the expressed product［J］.Animal Husbandry & Veterinary Medicine，2008，40(10)：5-9.（in Chinese）
［16］张小荣，董广阔，成大荣，等.鸭肠炎病毒囊膜蛋白gB和gC主要抗原域的原核表达与鉴定［J］.中国家禽，2011，33(2)：22-25，28.
ZHANG X R，DONG G K，CHENG D R，et al.Prokaryotic expression and identification of major antigenic domain in envelope proteins gB and gC encoded by duck enteritis virus［J］.China Poultry，2011，33(2)：22-25，28.（in Chinese）
［17］POULSEN D J，KEELER C L Jr.Characterization of the assembly and processing of infectious laryngotracheitis virus glycoprotein B［J］.J Gen Virol, 1997，78(11)：2945-2951.
［18］文明，程安春，汪铭书，等.鸭病毒性肠炎病毒的提纯及其结构蛋白SDS-PAGE分析［J］.中国兽医学报，2006，26(3)：243-246.
WEN M，CHENG A C，WANG M S，et al.Purification of duck enteritis virus and its structural proteins analyzed by SDS-PAGE［J］. Chinese Journal of Veterinary Science，2006，26(3)：243-246.（in Chinese）
［19］姜龙，刘会娟，程安春，等.疱疹病毒gB基因及其编码蛋白研究进展［J］.病毒学报，2010，26(5)：414-417.
JIANG L，LIU H J，CHENG A C，et al.Herpes virus gB genes and the research progress of encoded proteins［J］.Chinese Journal of Virology，2010，26(5)：414-417.（in Chinese）

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Prokaryotic Expression of Antigenic Domain Region of Duck Enteritis Virus gB Gene and Reactivity of Its Polyclonal Antibody

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