一种新型火鸡疱疹病毒细菌人工染色体的构建与鉴定

doi:10.11843/j.issn.0366-6964.2016.10.016

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (10): 2071-2080.doi: 10.11843/j.issn.0366-6964.2016.10.016

一种新型火鸡疱疹病毒细菌人工染色体的构建与鉴定

王志胜^1，3，刘芳^1，2，许梦微^1，2，乔永峰^1，3，侯继波^1，3，王继春^1，3*

（1.江苏省农业科学院/ 国家兽用生物制品工程技术研究中心，南京 210014；2.南京农业大学动物医学院，南京 210095；3.江苏省动物重要疫病与人兽共患病防控协同创新中心，扬州 225009）

收稿日期:2016-04-11 出版日期:2016-10-23 发布日期:2016-10-24
通讯作者: 王继春，研究员，E-mail：jcwang@263.net，Tel：025-84392068
作者简介:王志胜(1982-)，男，山东济南人，助理研究员，博士，主要从事畜禽病毒活载体疫苗的研究，E-mail：zhisheng.wang@163.com
基金资助:
公益性行业(农业)科研专项(201303046)；江苏省农业科技自主创新基金项目［CX(12)3061］；江苏省自然科学基金项目(BK20131334)

Construction and Identification of a Novel Bacterial Artificial Chromosome of Herpesvirus of Turkey

WANG Zhi-sheng^1,3，LIU Fang^1，2，XU Meng-wei^1，2，QIAO Yong-feng^1,3，HOU Ji-bo^1,3，WANG Ji-chun^1,3*

（1.National Research Center of Engineering and Technology for Veterinary Biologicals，Jiangsu Academy of Agricultural Sciences，Nanjing 210014，China；2.College of Veterinary Medicine，Nanjing Agricultural University，Nanjing 210095，China；3.Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses，Yangzhou 225009，China）

Received:2016-04-11 Online:2016-10-23 Published:2016-10-24

摘要/Abstract

摘要：

拟构建一种新型火鸡疱疹病毒（HVT）全基因组细菌人工染色体（BAC），通过同源重组方法将mini-F载体插入HVT基因组的糖蛋白C（glycoprotein C，gC）基因的等位位点得到mini-F重组HVT，提取重组病毒的DNA转入大肠杆菌DH10B细胞，再转入GS1783细胞获得BAC，拯救病毒获得mini-F重组病毒和gC恢复病毒后，与亲本病毒(HVT FC126)比较生长动力学和对鸡马立克病的免疫效力。结果：获得数个BAC阳性克隆，取其中一个克隆（BAC^HVT-G）成功拯救出HVT mini-F重组病毒（HVT^BAC-ΔgC），并成功获得了gC恢复毒株(HVT^BAC-gC-R)。生长特性和免疫效力试验表明HVT^BAC-gC-R与HVT FC126无显著差异，而HVT^BAC-ΔgC增殖能力和免疫效力均明显下降。成功构建了HVT全基因组的感染性BAC；HVT的gC基因是一个非必需基因，但对病毒的增殖能力和免疫效力有重要影响。

关键词: 火鸡疱疹病毒, 细菌人工染色体, 糖蛋白C, 生长动力学, 免疫效力

Abstract:

This study was conducted to construct a novel bacterial artificial chromosome (BAC) of herpevirus of turkey (HVT) as a technical platform for generation of recombinant HVT live vectored vaccine.The mini-F sequences were inserted into the genome of HVT in lieu of glycoprotein C (gC) gene through homologous recombination.The mini-F recombinant HVT were selected by GFP and gpt labeling.Then the DNA of mini-F recombinant HVT was transferred into E.coli DH10B cells to construct the HVT BAC.Following identification of correct construction of HVT whole genome BAC through RFLP method，HVT BAC DNA was transferred into E.coli GS1783 cells for further study after checking again.Then the HVT BAC was transfected into chicken embryo fibroblasts (CEF) for rescuing of virus.And also the gC recovered virus was generated by replacement of mini-F sequences with gC gene through homologous recombination again.Finally，the growth characteristics and protective efficacy against Marek’s disease of the gC recovered HVT，mini-F recombinant HVT and the parental virus (HVT FC126) were investigated.Five strains of mini-F recombinant HVT was obtained.One of the five recombinants (HVT^mini-F/ΔgC) was selected for isolation of DNA to transfer into E.coli DH10B cells to construct HVT BAC which was identified successfully through RFLP.Following up，DNA of HVT BAC was transferred into E.coli GS1783 cells successfully to obtain several positive clones and one of these clones was selected and named BAC^HVT-G.The recombinant HVT was rescued successfully from BAC^HVT-G，named HVT^BAC-ΔgC.And the gC recovered virus(HVT^BAC-gC-R) was successfully obtained through homologous recombination.The HVT^BAC-gC-R and HVT FC126 showed no significant difference for growth kinetics or immunity.However，the propagation ability of HVT^BAC-ΔgC was significantly decreased compared to HVT and the immunity against MD of HVT^BAC-ΔgC was also decreased.This study successfully constructed a novel HVT bacterial artificial chromosome，which was an infectious clone of the whole genome of HVT.The gC gene of HVT is a non-essential gene，but plays an important role for propagation of virus.The deletion of gC gene will reduce the immunity of HVT against Marek’s disease.

Key words: herpesvirus of turkey, bacterial artificial chromosome, glycoprotein C, growth kinetics, immune efficiency

中图分类号:

S852.659.1

王志胜，刘芳，许梦微，乔永峰，侯继波，王继春. 一种新型火鸡疱疹病毒细菌人工染色体的构建与鉴定[J]. 畜牧兽医学报, 2016, 47(10): 2071-2080.

WANG Zhi-sheng，LIU Fang，XU Meng-wei，QIAO Yong-feng，HOU Ji-bo，WANG Ji-chun. Construction and Identification of a Novel Bacterial Artificial Chromosome of Herpesvirus of Turkey[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(10): 2071-2080.

参考文献

［1］MELCHIOR F J，FREDRICKSON T，LUGINBUHL R.Neutralization studies with Marek’s disease virus and turkey herpesvirus［J］.Appl Microbiol，1973，26(6)：925-933.
［2］RAHAUS M，AUGUSTINSKI K，CASTELLS M，et al.Application of a new bivalent Marek’s disease vaccine does not interfere with infectious bronchitis or Newcastle disease vaccinations and proves efficacious［J］.Avian Dis，2013，57(2 Suppl)：498-502.
［3］AFONSO C，TULMAN E，LU Z，et al.The genome of turkey herpesvirus［J］.J Virol，2001，75(2)：971-978.
［4］SONDERMEIJER P，CLAESSENS J，JENNISKENS P，et al.Avian herpesvirus as a live viral vector for the expression of heterologous antigens［J］.Vaccine，1993，11(3)：349-358.
［5］TSUKAMOTO K，SAITO S，SAEKI S，et al.Complete，long-lasting protection against lethal infectious bursal disease virus challenge by a single vaccination with an avian herpesvirus vector expressing VP2 antigens［J］.J Virol，2002，76(11)：5637-5645.
［6］DARTEIL R，BUBLOT M，LAPLACE E，et al.Herpesvirus of turkey recombinant viruses expressing infectious bursal disease virus (IBDV) VP2 immunogen induce protection against an IBDV virulent challenge in chickens［J］.Virology，1995，211(2)：481-490.
［7］ESAKI M，GODOY A，ROSENBERGER J，et al.Protection and antibody response caused by turkey herpesvirus vector Newcastle disease vaccine［J］.Avian Dis，2013，57(4)，750-755.
［8］PALYA V，KISS I，TATÁR-KIS T，et al.Advancement in vaccination against Newcastle disease：recombinant HVT NDV provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions［J］.Avian Dis，2012，56(2)：282-287.
［9］RAUW F，PALYA V，GARDIN Y，et al.Efficacy of rHVT-AI vector vaccine in broilers with passive immunity against challenge with two antigenically divergent Egyptian clade 2.2.1 HPAI H5N1 strains［J］.Avian Dis，2012，56(4 Suppl)：913-922.
［10］PALYA V，TATÁR-KIS T，MATÓ T，et al.Onset and long-term duration of immunity provided by a single vaccination with a turkey herpesvirus vector ND vaccine in commercial layers［J］.Vet Immunol Immunopathol，2014，158(1-2)：105-115.
［11］LIU S，SUN W，CHU J，et al.Construction of recombinant HVT expressing PmpD，and immunological evaluation against chlamydia psittaci and Marek’s disease virus［J］.PLoS One，2015，10(4)：e0124992.
［12］PANTIN-JACKWOOD M，KAPCZYNSKI D，DEJESUS E，et al.Efficacy of a recombinant turkey herpesvirus h5 vaccine against challenge With H5N1 clades 1.1.2 and 2.3.2.1 highly pathogenic avian influenza viruses in domestic ducks (Anas platyrhynchos domesticus)［J］.Avian Dis，2016，60(1)：22-32.
［13］LIU J，CHEN P，JIANG Y，et al.A duck enteritis virus-vectored bivalent live vaccine provides fast and complete protection against H5N1 avian influenza virus in ducks［J］.J Virol，2011，85(21)：10989-10998.
［14］CHEN Y，GUO W，XU Z，et al.A novel recombinant pseudorabies virus expressing parvovirus VP2 gene：Immunogenicity and protective efficacy in swine［J］.Virol J，2011，8：307.
［15］兰德松，石星明，王云峰，等. 火鸡疱疹病毒细菌人工染色体的构建［J］.微生物学报，2008，48（6）：811-817.
LAN D S，SHI X M，WANG Y F，et al.Reconstituting turkey herpesvirus with bacterial artificial chromosome clones［J］.Acta Microbiologica Sinica，2008，48(6)：811-817.（in Chinese）
［16］WARDEN C，TANG Q，ZHU H.Herpesvirus BACs：past，present，and future［J］.J Biomed Biotechnol，2011：124595.
［17］TISCHER B，SMITH G，OSTERRIEDER N.En passant mutagenesis：a two step markerless red recombination system［J］.Methods Mol Biol，2010，634：421-430.
［18］OSTERRIEDER N，SCHUMACHER D，TRAPP S，et al.Establishment and use of infectious bacterial artificial chromosome (BAC) DNA clones of animal herpesviruses［J］.Berl Munch Tierarztl Wochenschr，2003，116：373-380.
［19］MESSERLE M，CRNKOVIC I，HAMMERSCHMIDT W，et al.Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome［J］.Proc Natl Acad Sci U S A，1997，94：14759-14763.
［20］OSTERRIEDER N.Construction and characterization of an equine herpesvirus 1 glycoprotein C negative mutant［J］.Virus Res，1999，59：165-77.
［21］WANG J，OSTERRIEDER N.Generation of an infectious clone of duck enteritis virus and generation of a vectored DEV expressing hemagglutinin of H5N1 avian influenza virus［J］.Virus Res，2011，159(1)：23-31.
［22］TISCHER B，VON EINEM J，KAUFER B，et al.Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli［J］.Biotechniques，2006，40：191-197.
［23］REDDY S，SUN A，KHAN O，et al.Cloning of a very virulent plus，686 strain of Marek’s disease virus as a bacterial artificial chromosome［J］.Avian Dis，2013，57(2 Suppl)：469-473.
［24］WANG J，GE A，XU M，et al.Construction of a recombinant duck enteritis virus (DEV) expressing hemagglutinin of H5N1 avian influenza virus based on an infectious clone of DEV vaccine strain and evaluation of its efficacy in ducks and chickens［J］.Virol J，2015，12：126.
［25］MA C，ZHANG Z，ZHAO P，et al.Comparative transcriptional activity of five promoters in BAC-cloned MDV for the expression of the hemagglutinin gene of H9N2 avian influenza virus［J］.J Virol Methods，2014，206：119-127.
［26］BAIGENT S，PETHERBRIDGE L，SMITH L，et al.Herpesvirus of turkey reconstituted from bacterial artificial chromosome clones induces protection against Marek’s disease［J］.J Gen Virol，2006，87(Pt 4)：769-776.
［27］HALL R，MEERS J，FOWLER E，et al.Identification of non-essential loci within the Meleagrid herpesvirus 1 genome［J］.Virol J，2015，12：130.
［28］RAUW F，GARDIN Y，PALYA V，et al.The combination of attenuated Newcastle disease (ND) vaccine with rHVT-ND vaccine at 1 day old is more protective against ND virus challenge than when combined with inactivated ND vaccine［J］.Avian Pathol，2014，43(1)：26-36.
［29］PRANDINI F，SIMON B，JUNG A，et al.Comparison of infectious bursal disease live vaccines and a HVT-IBD vector vaccine and their effects on the immune system of commercial layer pullets［J］.Avian Pathol，2016，45(1)：114-125.
［30］KAPCZYNSKI D，ESAKI M，DORSEY K，et al.Vaccine protection of chickens against antigenically diverse H5 highly pathogenic avian influenza isolates with a live HVT vector vaccine expressing the influenza hemagglutinin gene derived from a clade 2.2 avian influenza virus［J］.Vaccine，2015，33(9)：1197-1205.
［31］ADAMIAK B，TRYBALA E，MARDBERG K，et al.Human antibodies to herpes simplex virus type 1 glycoprotein C are neutralizing and target the heparan sulfate-binding domain［J］.Virology，2010，400：197-206.
［32］JAROSINSKI K，OSTERRIEDER N.Further analysis of Marek’s disease virus horizontal transmission confirms that U(L)44 (gC) and U(L)13 protein kinase activity are essential，while U(S)2 is nonessential［J］.J Virol，2010，84：7911-7916.

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一种新型火鸡疱疹病毒细菌人工染色体的构建与鉴定

Construction and Identification of a Novel Bacterial Artificial Chromosome of Herpesvirus of Turkey

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