Identification of a Novel Insertion Site HVT-005/006 for the Generation of Recombinant Turkey Herpesvirus Vector.
Identification of a Novel Insertion Site HVT-005/006 for the Generation of Recombinant Turkey Herpesvirus Vector.
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DOI：
10.3389/fmicb.2022.886873
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发表时间：
2022
影响因子：
5.2
通讯作者：
中科院分区：
文献类型：
--
作者：
火鸡疱疹病毒（HVT）已被广泛用作鸡马立克氏病（MD）的成功活病毒疫苗超过五十年。越来越多地，HVT也被用作针对多种禽类病原体的高效重组疫苗载体。涉及病毒基因组克隆的常规重组或重组工程技术以及最近的基因编辑方法已用于产生基于重组HVT的疫苗。在这项研究中，我们使用了NHEJ依赖性CRISPR/Cas9方法来插入mCherry盒，以筛选HVT基因组并确定插入外源基因的新的潜在位点。在HVT基因组的独特长（UL）区域中鉴定了一个新的基因间位点HVT-005/006，并且发现mCherry在插入该位点时稳定表达。为了确认该位点是否适合插入其他外源基因，将H9 N2病毒的血凝素（HA）插入该位点，并拯救重组HVT-005/006-HA。HVT-005/006重组菌生长良好，能稳定表达HA蛋白，表明HVT-005/006是一个很有前途的外源基因插入位点。
Turkey herpesvirus (HVT) has been widely used as a successful live virus vaccine against Marek's disease (MD) in chickens for more than five decades. Increasingly, HVT is also used as a highly effective recombinant vaccine vector against multiple avian pathogens. Conventional recombination, or recombineering, techniques that involve the cloning of viral genomes and, more recently, gene editing methods have been used for the generation of recombinant HVT-based vaccines. In this study, we used NHEJ-dependent CRISPR/Cas9-based approaches to insert the mCherry cassette for the screening of the HVT genome and identifying new potential sites for the insertion of foreign genes. A novel intergenic site HVT-005/006 in the unique long (UL) region of the HVT genome was identified, and mCherry was found to be stably expressed when inserted at this site. To confirm whether this site was suitable for the insertion of other exogenous genes, haemagglutinin (HA) of the H9N2 virus was inserted into this site, and a recombinant HVT-005/006-HA was rescued. The recombinant HVT-HA can grow well and express HA protein stably, which demonstrated that HVT-005/006 is a promising site for the insertion of foreign genes.
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