表达H9亚型禽流感病毒HA基因的重组火鸡疱疹病毒构建及应用
Construction and Application of Recombinant Herpesvirus of Turkey Expressing HA Gene of Subtype H9 Avian Influenza Virus
宋新宇 1张茜 1贡鑫 1戚亭 1陈雨 1车艳杰1
作者信息
- 1. 天津瑞普生物技术股份有限公司,天津 300308
- 折叠
摘要
为构建表达H9 亚型禽流感病毒HA基因的重组火鸡疱疹病毒(herpesvirus of turkey,HVT),以连续覆盖HVT全基因组的Fosmid文库为基础,利用Red/ET同源重组技术和Gateway LR克隆技术,将优化的携带Pec复合启动子的HA基因插入到HVT复制非必需区HVT053 与HVT054 位点处的 95 318～95 319 nt之间,获得了表达H9亚型禽流感病毒HA基因的重组HVT(rHVT-H9-HA株),并对构建的毒株进行鉴定.结果显示,成功构建了含有HA基因的入门质粒pEntr-HA,通过Gateway克隆的LR反应将含有HA基因的表达盒转移到黏粒pFosC DEST的第 53 和 54 号基因之间形成pFosC HA;提取 5 个黏粒后,转染次代CEF细胞获得拯救重组病毒rHVT-H9-HA株.连续传代和动物试验结果表明,rHVT-H9-HA株毒价高,遗传稳定,可以克服母源抗体干扰,免疫持续期长,能够提供针对H9亚型禽流感的保护;同时也实现了一针防两病,效果明显优于灭活疫苗.本研究为H9亚型禽流感病毒重组HVT活载体疫苗的研制提供了技术支撑.
Abstract
In order to construct the recombinant herpesvirus of turkey(HVT)expressing HA gene of H9 subtype avian influenza virus(AIV),based on a Fosmid library continuously covering the whole genome of HVT,the optimized HA gene carrying a Pec complex promoter was inserted between the loci of HVT053 and HVT054(95 318～95 319 nt)in HVT non-essential region for replication using Red/ET homologous recombination technology and Gateway LR cloning system,a recombinant HVT strain(rHVT-H9-HA)expressing HA gene was obtained and characterized.The results showed that the entry plasmid pEntr-HA containing HA gene was successfully constructed.pFosC HA was formed by transferring the expression cassette containing HA gene between No.53 and No.54 gene of the pFosC DEST cosmid by LR reaction;after extracting 5 cosmids,the rescued recombinant virus rHVT-H9-HA strain was obtained by transfecting secondary-generation CEF cells.Consecutive cell passages and animal tests showed that the rHVT-H9-HA strain was with high virus titer,genetic stability and anti-interference against maternal antibodies,and capable of fighting against H9 subtype AIV with long immunization duration;compared to inactivated vaccines,the effect of"one injection against two diseases"was better.A technical support was provided by the study to develop the recombinant HVT live-vector vaccine for H9 subtype AIV.
关键词
重组毒株rHVT-H9-HA/火鸡疱疹病毒/HA基因/Red/ET同源重组技术/Gateway/LR克隆技术Key words
recombinant rHVT-H9-HA strain/HVT/HA gene/Red/ET homologous recombination technology/Gateway LR cloning system引用本文复制引用
出版年
2024
国家科技期刊平台
NSTL
万方数据