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

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (1): 141-148.doi: 10.11843/j.issn.0366-6964.2016.01.019

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

沈友林^1，2，3，汪铭书^{1，2，3 *} ，程安春^1，2，3 ，贾仁勇^1，2，3，朱德康^1，3 ，陈舜^1，2，3 ，刘马峰^1，2，3，刘菲³，杨乔^1，2，3 ，孙昆峰^1，2，3 ，陈孝跃^1，3

（1.四川农业大学动物医学院禽病防治研究中心，成都 611130；2.四川农业大学预防兽医研究所，成都 611130；3.动物疫病与人类健康四川省重点实验室，成都 611130）

收稿日期:2015-04-24 出版日期:2016-01-23 发布日期:2016-01-23
通讯作者: 汪铭书，E-mail：mshwang@163.com
作者简介:沈友林（1989-），男，湖北当阳人，硕士，主要从事禽病学研究，E-mail：youlin_5151luck@sina.com
基金资助:
国家“十二五”科技支撑计划（2015BAD12B05）；国家现代农业（水禽）产业技术体系专项（CARS-43-8）；四川省创新团队项目（12TD005/2013TD0015）

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

SHEN You-lin^1，2，3 ，WANG Ming-shu^1，2，3* ，CHENG An-chun^1，2，3，JIA Ren-yong^1，2，3，ZHU De-kang^1，3，CHEN Shun^1，2，3，LIU Ma-feng^1，2，3，LIU Fei³，YANG Qiao^1，2，3，SUN Kun-feng^1，2，3，CHEN Xiao-yue^1，3

(1.Avian Disease Research Center，College of Veterinary Medicine，Sichuan Agricultural University，Chengdu 611130，China；2.Preventive Veterinary Institute，Sichuan Agricultural University，Chengdu 611130，China；3.Key Laboratory of Animal Disease and Human Health of Sichuan Province，Chengdu 611130，China)

Received:2015-04-24 Online:2016-01-23 Published:2016-01-23

摘要/Abstract

摘要：

旨在探究1型鸭甲肝病毒（DHAV-1）VP3蛋白抗血清的中和活性并鉴定VP3的线性B细胞表位。利用pGEX-4T-1表达载体，在BL21(DE3)宿主菌中原核表达DHAV-1 VP3基因，以切胶纯化出的蛋白质为抗原免疫兔制备多克隆抗体，通过鸡胚中和试验对多抗的中和效价进行检测；采用Karplus&Schulz、Emini、Jameson-Wolf和Parker方法分别对柔韧性、表面可及性、抗原性及亲水性进行分析，得到了4条候选线性B细胞表位，以制备的兔抗VP3多克隆抗体为一抗，通过间接ELISA方法对人工合成的B细胞表位进行鉴定，并进一步用临床鸭血清样品对鉴定的B细胞表位的抗体检测能力进行评估。结果显示，VP3在BL21(DE3)中以包涵体形式表达，大小约54 ku，Western blot分析表明重组蛋白质具有较好的反应原性。制备的兔抗VP3多克隆抗体的琼扩效价达到1∶16，并能中和DHAV-1，中和效价为1∶39；利用间接ELISA鉴定出GKRKPCRRPIHKPKNPPQEP（1—20 aa）、FNTGRYQMSWYPIADGEQSL（131—150 aa）和VNSSAPSNID（200—209 aa）为VP3的B细胞表位，抗体检测能力试验结果显示表位肽可检测临床DHAV-1鸭血清。本研究表明DHAV-1 VP3的抗血清具备一定的中和活性，1—20 aa、131—150 aa和200 —209 aa为VP3的B细胞表位且具有临床应用前景。

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.

中图分类号:

沈友林，汪铭书，程安春，贾仁勇，朱德康,陈舜,刘马峰，刘菲，杨乔,孙昆峰，陈孝跃. 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[J]. 畜牧兽医学报, 2016, 47(1): 141-148.

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.

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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

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

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