双黄连水煎液对猪繁殖与呼吸综合征病毒的体外作用效果研究

Effect of Shuanghuanglian decoction on porcine reproductive and respiratory syndrome virus in vitro

【目的】通过建立的荧光定量PCR方法对猪繁殖与呼吸综合征病毒载量的检测,探究双黄连水煎液在体外对猪繁殖与呼吸综合征病毒增殖的影响。【方法】建立非洲绿猴胚胎肾细胞与猪繁殖与呼吸综合征病毒共感染模型,并应用CCK8法筛选双黄连水煎液的最大安全质量浓度。根据DNA序列数据库公布的猪繁殖与呼吸综合征病毒-N蛋白参考序列,设计特异性引物,构建猪繁殖与呼吸综合征病毒N基因质粒,建立检测猪繁殖与呼吸综合征病毒N基因的荧光定量PCR方法,研究最大安全质量浓度下的双黄连水煎液对猪繁殖与呼吸综合征病毒载量的影响。【结果】双黄连水煎液对非洲绿猴胚胎肾细胞的最大安全质量浓度为6.25 mg/mL,在此安全质量浓度下对猪繁殖与呼吸综合征病毒具有良好的阻断吸附和抑制复制作用,作用效果呈时间和剂量依赖性,而对猪繁殖与呼吸综合征病毒的直接杀灭作用效果不显著。【结论】成功建立了检测猪繁殖与呼吸综合征病毒载量的荧光定量PCR方法,通过此方法检测到双黄连水煎液在6.25 mg/mL时具有抗猪繁殖与呼吸综合征病毒作用。

【Objective】The effect of Shuanghuanglian decoction on proliferation of porcine reproductive and respiratory syndrome virus(PRRSV) in vitro was investigated by fluorescence quantitative PCR method for viral load detection of PRRSV.【Methods】The co-infection model of Marc-145 cells and PRRSV was established, and the maximum safe concentration of Shuanghuanglian extract was screened by CCK8 method. According to the PRRSV-N protein reference sequence published in GenBank, specific primers were designed to construct PRRSV-N gene plasmid. A fluorescence quantitative PCR method for detecting the PRRSV-N gene was established to study the effect of Shuanghuanglian decoction at the maximum safe concentration on the viral load of PRRSV. 【Results】The maximum safe concentration of extract of Shuanghuanglian on Marc-145 cells was 6.25 mg/mL. At this safe concentration, it had a good effect on blocking adsorption and inhibiting replication of PRRSV, and the effect is time and dose-dependent, while the direct killing effect on PRRSV was not significant. 【Conclusion】In this study, a fluorescence quantitative PCR for PRRSV was established, which was showed that Shuanghuanglian decoction had an anti-porcine reproductive and respiratory syndrome virus effect at 6.25 mg/mL.