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

摘要:

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。

Abstract:

Abstract:［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-Aincubated 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．