葫芦多糖及其硫酸化修饰物抗新城疫病毒活性

Anti-Newcastle disease virus activity of Lagenaria siceraria(Molina)standl polysaccharide and sulfated modifications

为了探究硫酸化修饰对葫芦多糖(LSP)抗新城疫病毒(NDV)活性的影响,本试验利用Sephadex G-100方法纯化得到LSP,通过氯磺酸-吡啶法得到硫酸化修饰的sLSP_(1.3),用苯酚硫酸法对LSP及sLSP_(1.3)进行多糖含量、蛋白含量及硫酸根取代度测定,经红外光谱测定LSP及sLSP_(1.3)的官能团变化,结果显示,氯磺酸-吡啶法能够对LSP进行硫酸化修饰。利用MTT法测定LSP及sLSP_(1.3)对鸡胚成纤维细胞(CEF)的安全质量浓度,结果LSP及sLSP_(1.3)的安全质量浓度分别为50,10 mg/L,LSP经硫酸化修饰后对CEF的安全质量浓度下降。在安全质量浓度范围内以3种不同的加药方式(先添加LSP后接种病毒、先接种病毒后添加LSP、LSP和病毒混合后同时加入)检测LSP与sLSP_(1.3)体外对NDV活性的影响,结果显示,LSP经硫酸化修饰后其抗NDV活性明显增强,其中sLSP_(1.3)在5 mg/L时抗NDV感染能力和杀灭NDV的作用显著高于40 mg/L时(P<0.05)。检测LSP与sLSP_(1.3)对新城疫模型鸡血清HI抗体效价及攻毒后的死亡率、保护率和痊愈率,结果显示,攻毒后各治疗组及病毒对照组抗体都逐渐升高,sLSP_(1.3)组效价显著高于病毒对照组(P<0.05),但LSP和sLSP_(1.3)之间无显著性差异(P>0.05)。攻毒后病毒对照组死亡率高达86.7%,而sLSP_(1.3)组死亡率降低为66.7%(P<0.05);治疗组中sLSP_(1.3)组的保护率显著高于LSP组,痊愈率达16.7%,保护率达33.3%。上述结果表明,LSP经硫酸化修饰后抗NDV活性显著增强且LSP使用的有效质量浓度显著降低。

In order to explore the effect of sulfation modification on the activity of Lagenaria siceraria(Molina)standl.Polysaccharide(LSP)against Newcastle disease virus(NDV),in this experiment,LSP was purified by the SephadexG-100 method,and the sulfated modified sLSP_(1.3) was obtained by the chlorosulfonic acid-pyridine method.The polysaccharide content,protein content and sulfate substitution degree of LSP and sLSP_(1.3) were tested by Phenol sulphuric acid method.The functional group changes of LSP and sLSP_(1.3) were determined by infrared spectroscopy.The results showed that LSP can be sulfated modified by chlorosulfonic acid-pyridine method and The safe concentrations of LSP and sLSP_(1.3) on chicken embryo fibroblasts(CEF)were determined by MTT assay.The safe concentrations of LSP and sLSP_(1.3) respectively were 50,10 mg/L,and after LSP was modified with sulfuration,the safe concentration of CEF decreased,The safe and the effects of LSP and sLSP_(1.3) on NDV in vitro were detected by three different dosing methods(adding LSP first and inoculating virus,adding virus first and inoculating LSP,and adding LSP and virus at the same time)within the safe concentration range.The results also showed that the antiNDV activity of LSP was significantly enhanced after sulfur modification,and the anti-NDV effect and NDV killing effect of sLSP_(1.3)at 5mg/L were significantly higher than the LSP at 40 mg/L(P<0.05).LSP and sLSP_(1.3),there was no significant difference(P>0.05).Poison attack after virus mortality rate control group is 86.7%,sLSP_(1.3)group had a significantly lower mortality rate was 66.7%(P<0.05);sLSP_(1.3)group protection in treatment group is significantly higher than the LSP group,the cure rate was 16.7%,and the protection rate was 33.3%.The above results indicated that the anti-NDV activity of LSP was significantly enhanced and the effective concentration of LSP used was significantly decreased after the sulfur modification,the significance and innovation of this to study the effect of sulfuration modification of LSP and 3different administration methods on the anti NDV activity.