中国兽医科学2025，Vol.55Issue(4)：449-455,7.DOI:10.16656/j.issn.1673-4696.2025.0070
含禽流感病毒M基因、H5和H7亚型HA基因的重组假病毒构建与鉴定
Construction and identification of recombinant pseudovirus containing avian influenza virus M gene,H5 and H7 subtype HA genes
摘要
Abstract
Generation of a pseudovirus containing the conserved regions of avian influenza virus(AIV)M gene and H5 and H7 subtype hemagglutinin(HA)genes aims to provide a safe,reliable,and stable positive control for multiple rapid molecular diagnostic methods for AIV.A recombinant shuttle plasmid,pLV-M57-IRES-ZsGreen1,was constructed by gene synthesis of a fused fragment containing the conserved regions of AIV M gene and H5 and H7 subtype HA genes.pLV-M57-IRES-ZsGreen1 was co-transfected with two backbone plasmid pMD2.G,psPAX2 into 293T cells,was detected by green fluorescence after 24h tranfec-tion.Then the pLV was detected by standard methods for the dected of M,H5,and H7 genes after 48 h.And quantified via digital PCR.PCR amplification and sequencing analysis showed that the fused fragment was correctly inserted into the shuttle plasmid.Green fluorescence was clearly observed at both 24 and 48 hours post-transfection.Specific amplification curves could be observed of the virus and the virus titer was 5.0 × 108 TU/mL,and all the three genes of M,H5 and H7 could be effectively detected.Digital PCR showed that titer of the recombinant pseudovirus was approximately 9.0 × 108 copies/μL.The study successfully developed a pseudovirus incorporating AIV M,H5 and H7 gene fragments,which exhibits effec-tive detection capabilities.This pseudovirus serves as a valuable positive control for molecular diag-nostic assays,ensuring comprehensive monitoring throughout the nucleic acid extraction and detection processes.关键词
禽流感病毒/重组假病毒/构建/鉴定Key words
avian influenza virus/recombinant pseudovirus/construction/identification分类
农业科技引用本文复制引用
潘俊慧,李超,王楷宬,王素春,周凯钰太,李涛,白婷,杨丁,隋金钰,魏世萌,祁倩..含禽流感病毒M基因、H5和H7亚型HA基因的重组假病毒构建与鉴定[J].中国兽医科学,2025,55(4):449-455,7.基金项目
"十四五"国家重点研发计划项目(2023YFD1800604) （2023YFD1800604）
