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

Abstract

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

CLC Number:

S852.659.1

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.

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[1]	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.
[2]	ZHANG Hao-hao，CHEN Long-xin，CAO Yang，CHEN Ying，GUAN Gui-quan，MA Run-lin. Construction of Bacterial Artificial Chromosome (BAC) Library of Ovine Babesia motasi [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(8): 1324-1329.
[3]	ZHANG Hao-hao，CHEN Long-xin，CAO Yang，CHEN Ying，GUAN Gui-quan，MA Run-lin. Construction of Bacterial Artificial Chromosome (BAC) Library of Ovine Babesia motasi [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(8): 1324-1329.

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

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