摘要
为研究绞股蓝皂苷对牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)的作用,采用体外细胞培养技术、细胞病变效应观察与CCK-8相结合的方法,首先确定绞股蓝皂苷和α-单月桂酸甘油酯的安全浓度,然后采用先加药后加病毒、先加病毒后加药,及药和病毒预先作用这3种加药方式,利用Western blot和定量PCR检测BVDV E2蛋白基因和5'-UTR表达量的变化,探究其在体外对病毒的抑制作用.结果表明,当绞股蓝皂苷和α-单月桂酸甘油酯的浓度分别为16 μmol·L-1和25 μg·mL-1时,细胞活性与空白组相比的差异无统计学意义(P＞0.05),将其确定为两种药物的最大安全浓度.与对照组相比,绞股蓝皂苷可极显著(P＜0.01)抑制BVDV E2蛋白基因和5'-UTR的表达,且其直接灭活作用优于α-单月桂酸甘油酯.此外,绞股蓝皂苷还显示出了显著(P＜0.05)的吸附阻断作用,但其复制阻断作用与单独病毒组无显著差异.
Abstract
To investigate the effect of gypenoside on bovine viral diarrhea virus(BVDV),the in vitro cell culture technique combined with cytopathic effect(CPE)observation and CCK-8 assay was employed.The safe concentra-tions of gypenoside and α-glycerol monolaurate were determined.Three different treatment protocols were used:pre-treatment with compound followed by viral infection,post-treatment with compound after viral infection,and pre-in-cubation of compound and virus prior to infection.Western blot and quantitative PCR were conducted to evaluate the changes of expression levels of BVDV E2 protein gene and 5'-UTR,to explore the inhibitory effect in vitro.The re-sults showed that the cell viability at the concentration of 16 μmol·L-1 for gypenoside and 25 μg·mL-1 for α-glycerol monolaurate was not significantly different from the control group(P＞0.05),indicating that these concentrations were the maximum safe concentrations of the two compounds.Compared to the control group,gypenoside exhibited significant(P＜0.01)inhibition on the expression levels of BVDV E2 protein gene and 5'-UTR,and its direct viru-cidal effect was superior to the α-glycerol monolaurate.Gypenoside also showed significant(P＜0.05)adsorption inhibition against BVDV,while no significant difference was found in replication inhibition when compared to the vi-rus control group.
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