高致病性猪繁殖与呼吸综合征病毒株JXA1反向遗传平台的构建

Construction of reverse genetic platform of highly pathogenic porcine reproductive and respiratory syndrome virus strain JXA1

【目的】为高致病性猪繁殖与呼吸综合征病毒(Highly pathogenic porcine reproductive and respiratory syndrome virus,HP-PRRSV)的结构功能和致病机理的研究奠定基础。【方法】运用反向遗传技术将HP-PRRSV JXA1株的全基因组分段克隆至改造过的低拷贝载体pOKq上,并在病毒基因组两端分别添加CMV启动子和BGH终止信号肽以及在病毒全基因组第510位核苷酸突变引入FseI酶切位点,作为遗传标记位点。采取基于DNA-launched途径进行病毒拯救,并对拯救的病毒进行生物学特性分析。【结果】构建的PRRSV JXA1毒株的全长cDNA克隆具有感染性;成功拯救了病毒,命名为rJXA1;成功引入了拯救病毒的遗传标记;拯救病毒与亲本病毒的生长曲线相似,二者达到最高滴度的时间均为感染后72 h。【结论】成功构建了JXA1株反向遗传平台,为进一步研究HP-PRRSV的致病机理、基因功能以及新型疫苗研发奠定了基础。

【Objective】To provide a basis for studying the structural function and pathogenic mechanism of highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV).【Method】Fragments of the whole genome of HP-PRRSV JXA1 strain were cloned into the modified low copy vector pOKq with reverse genetics technique.The CMV promoter and BGH termination signal peptide were added into the terminals of the viral genome.The FseI restriction site,as a genetic marker locus,was introduced at the 510th nucleotide of the whole genome of the virus by mutation.DNA-launched approach was used for viral rescue and the biological properties of rescued viruses were analyzed.【Result】The full-length cDNA clone of the constructed PRRSV JXA1 strain was infectious.The virus was successfully rescued and named rJXA1.The genetic marker was successfully introduced into the rescued virus.The rescued virus and parental virus had similar growth curves with reaching the maximum titer at 72 h after infection.【Conclusion】The reverse genetic platform of the JXA1 strain has been successfully constructed,which will lay a foundation for further research on the pathogenesis,gene function and vaccine development of PRRSV.