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。
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 DH10 Bcells 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 gCrecovered virus was generated by replacement of mini-F sequences with gCgene 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.Fivestrains of mini-F recombinant HVT was obtained.One of the five recombinants（HVTmini-F/ΔgC）was selected for isolation of DNA to transfer into E.coli DH10 Bcells 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 BACHVT-G.The recombinant HVT was rescued successfully from BACHVT-G,named HVTBAC-ΔgC.And the gCrecovered virus（HVTBAC-gC-R）was successfully obtained through homologous recombination.The HVTBAC-gC-R and HVT FC126 showed no significant difference for growth kinetics or immunity.However,the propagation ability of HVTBAC-ΔgC was significantly decreased compared to HVT and the immunity against MD of HVTBAC-ΔgC was also decreased.This study successfully constructed a novel HVT bacterial artificial chromosome,which was an infectious clone of the wh
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2016年第10期2071-2080,共10页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
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)
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
herpesvirus of turkey
bacterial artificial chromosome
glycoprotein C
growth kinetics
immune efficiency
