火鸡疱疹病毒细菌人工染色体的构建

Reconstituting turkey herpesvirus with bacterial artificial chromosome clones

摘要:

火鸡疱疹病毒（HVT）为一种a疱疹病毒，因其与马立克氏病病毒（MDV）抗原相关性而被广泛用作预防马立克氏病（MD）的活疫苗。【目的】本研究的目的是构建HVT全基因组感染性细菌人工染色体（BAC）。【方法】利用Eco-gpt (黄嘌呤鸟嘌呤磷酸核糖转移酶) 基因和BAC载体pBeloBAC11的基本功能序列，构建重组病毒转移载体pGAB-gpt-BAC11。通过将pGAB-gpt-BAC11与HVT感染细胞总DNA共转染原代鸡胚成纤维细胞（CEF），待出现病毒噬斑后，利用霉酚酸（MPA）阻断核酸代谢途径，经过筛选获得纯化的重组病毒purified-rHVT。提取purified-rHVT感染细胞总DNA电转化大肠杆菌DH10B感受态细胞，在氯霉素抗性平板上筛选阳性克隆，并用酶切和PCR方法对其进行鉴定。随机选取BAC克隆提取BAC DNA转染次代CEF，完成HVT重组病毒的拯救。【结果】经过6轮筛选后获得纯化的重组病毒，并筛选到25个BAC分子克隆化病毒。其中BAC6、BAC8和BAC10再次启动病毒感染，产生与野生型HVT感染CEF相似的病毒噬斑形态，说明已经获得拯救出的HVT重组病毒。【结论】本研究构建了HVT全基因组感染性细菌人工染色体，建立了HVT反向遗传操作技术平台。

Abstract:

Herpesvirus of turkey (HVT) is an alpherpesvirus and widely used as a live vaccine against Marek’s disease (MD) because of its antigenic relationship with Marek’s disease virus (MDV). [Objective] The aim of this study was to construct Herpesvirus of turkey Fc126 strain as an infectious bacterial artificial chromosome (BAC). [Methods] Using the selection marker Eco-gpt (Xanthine-guanine phosphoribosyl transferase)(1.3 kb) and BAC vector pBeloBAC11(7.4 kb), we constructed the transfer plasmid pGAB-gpt-BAC11. Then, the transfer plasmid and HVT-infected cells’ total DNA were cotransfected into primary chicken embryo fibroblasts (CEFs). After six rounds of selection in medium containing mycophenolic acid, xanthine and hypoxanthine, we obtained purified recombinant viruses. Genomic DNA was extracted and electroporated into Escherichia coli DH10B competent cells. BAC clones were identified by restriction enzyme digestion and PCR analysis, and then tested for infectivity after transfection into CEFs using calcium phosphate. [Results] We obtained 25 BAC clones, and reconstituted recombinant viruses by transfection of HVT-BAC6 DNA, HVT-BAC8 DNA and HVT-BAC10 DNA into CEFs respectively. [Conclusion] In this study, we cloned the complete genome of HVT Fc126 strain as an infectious bacterial artificial chromosome.