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

发布时间：2018-05-23 22:21

本文选题：重组粘粒 + 重组火鸡疱疹病毒　；参考：《中国预防兽医学报》2017年11期

【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。
[Abstract]:In order to construct a recombinant turkey herpesvirus (rHVT) expressing both AIVH HA protein and IBDVV VP2 protein of avian influenza virus (AIV), five recombinant mucks were constructed from the whole genome of continuously covering turkey herpesvirus (HVT) constructed in our laboratory. On the basis of Fos, Fos, Fos, Fos D and p Fos E). The HA gene and VP2 gene were sequenced and cloned into p CAGGs vector to construct the recombinant plasmid pCA-HA-2A-VP2. Using Gateway LR cloning technique, the expression cassette of HA-2A-VP2 in the recombinant plasmid Pec-HA-2A-VP2-POLYA) was inserted between HVT053 and HVT054 gene, the non-essential region of the HVT fragment in the p Fos C clay. The recombinant clay p Fos C-5354-HA-VP2was constructed. Five recombinant mucosomes, p Fos Agna and p Fos C-5354-HA-VP2, were co-transfected into chicken embryonic fibroblasts (CEF) to prepare recombinant turkey herpesvirus (rHVT-5354-HA-VP2P) expressing HA protein and VP2 protein. After the recombinant virus was transmitted to CEF for 20 generations, the results of indirect immunofluorescence and western blot analysis showed that HA and VP2 genes could be stably expressed in the recombinant virus, and the growth kinetics showed that HA and VP2 genes could be expressed correctly in the recombinant virus. The proliferation efficiency of recombinant virus in CEF was not significantly different from that of wild-type virus HVT. The preparation of the recombinant virus laid a foundation for the development of AIV-IBDV- Marek virus triple vaccine.
【作者单位】：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;
【基金】：“十二五”农村领域国家科技计划(2015BAD12B03)
【分类号】：S852.65

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