表达禽流感病毒HA蛋白及传染性法氏囊病毒VP2蛋白的重组火鸡疱疹病毒的构建  

Construction of recombinant turkey herpesvirus co-expressing HA protein of avian influenza virus and VP2 protein of infectious bursa disease virus

作　　者：唐猛[1] 陈普成[1] 高立[1] 柳金雄[1] 潘俊慧 王笑梅[1] 姜永萍[1] 陈化兰[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2017年第11期865-869,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金："十二五"农村领域国家科技计划(2015BAD12B03)

摘　　要：为构建同时表达禽流感病毒(AIV)HA蛋白及传染性法氏囊病毒(IBDV)VP2蛋白的重组火鸡疱疹病毒(r HVT),本研究以本实验室构建的连续覆盖火鸡疱疹病毒(HVT)全基因组的5个重组粘粒(p Fos A、p Fos B、p Fos C、p Fos D和p Fos E)为基础,利用猪捷申病毒2A蛋白的编码序列,将HA基因和VP2基因串联,并克隆于p CAGGs载体中,构建重组质粒p CA-HA-2A-VP2。利用Gateway LR克隆技术,将重组质粒中的HA-2A-VP2的表达盒(Pec-HA-2A-VP2-POLYA)插入到p Fos C粘粒中HVT片段的复制非必需区HVT053与HVT054基因之间,构建重组粘粒p Fos C-5354-HA-VP2。将p Fos A、p Fos B、p Fos D、p Fos E和p Fos C-5354-HA-VP2 5个重组粘粒共转染鸡胚成纤维细胞(CEF),制备表达HA蛋白及VP2蛋白的重组火鸡疱疹病毒(r HVT-5354-HA-VP2)。将重组病毒在CEF中连续传至20代后,通过PCR、间接免疫荧光和western blot鉴定分析,结果显示HA与VP2基因能够在重组病毒中稳定存在并正确表达;生长动力学结果表明,重组病毒在CEF中的增殖效率与野生型病毒HVT无显著差异。该重组病毒的制备为研制AIV-IBDV-马立克病毒三联苗奠定了基础。To construct a recombinant turkey herpesvirus co-expressing the HA protein of AIV and the VP2 protein of IBDV, five recombinant cosmids, pFosA, pFosB, pFosC, pFosD and pFosE, covering the whole genome of turkey herpesvirus（HVT） developed in our laboratory, were used in this study. The HA and VP2 genes were connected by 2A sequence of porcine teschovirus （PTV） and cloned into the plasmid pCAGGs to generate the recombinant plasmid pCA-HA-2A-VP2. By using Gateway LR cloning technology, recombinant cosmids pFosC-5354-HA-VP2 was constructed by inserting the expression cassette （Pec-HA-2A-VP2-POLYA） of the recombinant plasmid pCA-HA-2A-VP2 into the non-essential region between HVT053 and HVT054 of turkey herpesvirus. The five recombinant cosmids （pFosA, pFosB, pFosD, pFosE and pFoC-5354-HA-VP2） were then co-transfected into the chicken embryo fibroblast （CEF） monolayer cells to rescue the recombinant HVT （rHVT-5354-HA-VP2）. After 20 generations＇ subculture, HA and VP2 genes or proteins of the recombinant virus were confirmed by PCR, immuno- fluorescence assay （IFA） and western blot. The results indicated that HA and VP2 genes were stable in the recombinant virus and expressed correctly. The growth kinetics of the recombinant virus demonstrated that HA and VP2 insertion in the HVT genome had no effect on the replication of the recombinant virus. This recombinant virus provided a potential vaccine candidate against AIV, IBDV and MDV.

关 键 词：重组粘粒 重组火鸡疱疹病毒 三联苗 

分 类 号：S852.65[农业科学—基础兽医学]