重组猪肺表面活性蛋白A在体外可抑制PRRSV感染宿主细胞  

Recombinant porcine lung surfactant protein A inhibits porcine reproductive and respiratory syndrome virus infection into host cells in vitro

作　　者：张峰[1] 仲飞[1] 李秀锦[2] 王幸兴[1] 张考[1] 陈慧慧[1] 李振[1] 李文艳[1] 潘红丽[1] 韩冬梅[1] 

机构地区：[1]河北农业大学动物医学院基础兽医部,农业部动物疫病病原生物学华北科学观测实验站,保定071001 [2]燕山大学环境与化学工程学院生物工程系,秦皇岛066004

出　　处：《微生物学报》2012年第5期654-660,共7页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31140093);河北省自然科学基金(C2008000244)~~

摘　　要：【目的】研究重组猪肺表面活性蛋白A(SP-A)在体外对猪繁殖与呼吸综合征病毒(PRRSV)感染的抑制作用。【方法】采用PCR方法从含有猪SP-A基因的质粒中扩增SP-A基因,并将其插入到含有人CD5信号肽序列的真核表达载体pcDNA3.1A-CD5中,构建成SP-A基因的真核分泌型表达载体pcDNA-CD5-SPA/MH。将重组表达载体通过磷酸钙介导转染HEK293T细胞进行瞬时表达,通过Western blot方法鉴定表达产物,采用Ni-NTA琼脂糖凝胶亲和层析法从培养基中分离和纯化重组SP-A蛋白,通过ELISA方法检测SP-A蛋白与PRRSV的结合活性。将SP-A蛋白与PRRSV孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,感染72 h后测定病毒滴度,分析重组SP-A蛋白对PRRSV感染的抑制作用。【结果】结果表明构建的真核表达载体能够介导SP-A基因在HEK293T细胞中进行分泌表达;表达的重组猪SP-A蛋白能够与PRRSV进行剂量依赖性结合;用重组猪SP-A蛋白与PRRSV进行孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,结果显示SP-A处理的PRRSV感染细胞后的病变程度明显低于对照组。感染72 h后,SP-A处理组的PRRSV在MARC-145细胞和猪肺泡巨噬细胞的滴度明显低于SP-A非处理组。【结论】重组猪SP-A在体外对PRRSV的感染有明显的抑制作用,揭示SP-A具有抗PRRSV的活性。[Objective] To investigate the antiviral activity of porcine lung surfactant protein A（SP-A） to porcine reproductive and respiratory syndrome virus（PRRSV） in vitro.[Methods] The SP-A gene was amplified by PCR from the plasmid containing porcine SP-A gene,and subcloned into pcDNA3.1A-CD5 vector containing the human CD5 signal peptide to generate SP-A eukaryotic expression vector pcDNA-CD5-SPA/MH.The recombinant expression vector was transfected into HEK293T cells mediated with calcium phosphate.The expressed recombinant SP-A was identified by Western blot and purified from culture medium by Ni-NTA-Agarose beads.The binding activity of SP-A with PRRSV was identified by ELISA.The antiviral activity of SP-A to PRRSV was analyzed by viral titer reduction assays on MARC-145 cells and porcine alveolar macrophages（PAM）.[Results] The results showed that the eukaryotic expression vector of SP-A gene could mediate SP-A expression in HEK293T cells,the expressed SP-A could bind PRRSV in a dose dependent manner.The PRRSV incubated in advance with SP-A showed the lower infective activity compared with no-SP-A-incubated PRRSV on both MARC-145 cells and porcine alveolar macrophages.The SP-A-treated PRRSV titers in MARC-145 cells and PAM cells were significantly lower than that of SP-A-untreated PRRSV at 72 h post-infection.[Conclusion] Recombinant porcine SP-A significantly inhibit the infection of PRRSV to the host cells in vitro,which indicates that recombinant SP-A possesses anti-PRRSV activity.

关 键 词：猪肺表面活性蛋白A 抗病毒活性 猪繁殖与呼吸综合征病毒 真核表达 

分 类 号：S858.28[农业科学—临床兽医学]