表达IFN-α的重组猪繁殖与呼吸综合病毒的构建及生物学特性分析

Construction of a Recombinant Porcine Reproductive and Respiratory Syndrome Virus Expressing IFN-αand Analysis of Its Biological Characteristics

猪繁殖与呼吸综合征(PRRS)是危害我国生猪业的重要疫病之一,其病原是猪繁殖与呼吸综合征病毒(PRRSV),是一种免疫抑制性病毒。干扰素(IFN)是一类具有免疫调节功能和抗病毒作用的细胞因子。IFN-α除更直接的抗病毒作用外,还可以调节宿主的先天性和适应性免疫。论文构建表达IFN-α的重组PRRSV,分析重组病毒的生物学特性以及IFN-α的生物学活性。通过反向遗传操作方法将IFN-α插入到PRRSV ORF1b和ORF2a之间,重组质粒转染细胞后可以拯救出重组病毒(rGXAM-P-IFN-α)。插入到PRRSV基因组中的IFN-α可遗传稳定9代。重组病毒生长特性分析可发现rGXAM-P-IFN-α复制能力显著低于亲本病毒。rGXAM-P-IFN-α感染猪肺泡巨噬细胞(PAM)可显著上调抗病毒基因(PKR,ISG15和ISG54)mRNA表达水平,为进一步研发新型PRRSV疫苗提供参考。

Porcine reproductive and respiratory syndrome(PRRS)is one of the major diseases that threaten the pig industry in China.Interferon(IFN)is a kind of cytokines with immunomodulatory function and antiviral effect.In addition to its more direct antiviral effects,IFN-αcan also regulate host innate and adaptive immunities.In order to investigate the increased antiviral activity of exogenous IFN-α,we constructed a recombinant plasmid(pGXAM-P-IFN-α)expressing porcine IFN-αusing an infectious PRRSV cDNA clone as a vector.The recombinant virus could be stably propagated in Marc-145 cells for 3 generations.The biological characteristics of the successfully rescued recombinant virus were analyzed and the effect of the inserted foreign genes on the expression of antiviral genes in alveolar macrophages(PAMs)was detected.The results showed that the IFN-αin the successfully rescued virus could stably exist in 9 generations,and the replication level of the recombinant virus was lower than that of the parent virus.In addition,the mRNA expression levels of antiviral genes(PKR,ISG15 and ISG54)were upregulated in PAMs infected with rGXAM-P-IFN-α.This study provides a reference for the further development of novel PRRSV vaccines.