P-VP8~* IgY在轮状病毒乳鼠模型中的药效学评价

Evaluation of pharmacodynamics of P-VP8~* IgY in rotavirus suckling mouse disease model

目的探讨P-VP8*IgY在轮状病毒(RV)乳鼠模型中的抗病毒作用。方法利用ZTR-68株RV灌胃攻毒6日龄乳鼠建立RV乳鼠模型。实验分为A组(对照组)、B组(感染组)、C组(预防组)、D组(低剂量实验组)、E组(中剂量实验组)、F组(高剂量实验组),C组灌胃攻毒前连续2 d早晚各给予P-VP8*IgY 20 mg·kg-1,D、E、F组灌胃攻毒后3 d早晚各给予P-VP8*IgY3.3,10,30 mg·kg-1。观察乳鼠症状和腹泻情况,以酶联免疫吸附实验检测粪便RV抗原,实时荧光定量聚合酶链反应检测小肠组织RV基因拷贝数,以蛋白质印迹法检测RV蛋白的表达水平,以苏木精-伊红染色法观察小肠组织病理改变,以免疫组化法检测小肠组织RV表达。结果 6日龄乳鼠灌胃攻毒1×107PFU ZTR-68株RV后出现黄色稀水样便,可成功制备乳鼠RV感染模型。治疗96 h后,E和F组腹泻评分分别为1.75±0.49,1.50±0.27,与B组(3.37±0.18)比较,差异有统计学意义(P<0.05);E和F组RV抗原量分别为(60.0±26.2),(40.0±18.5)EU·m L-1,与B组(130.0±41.4)EU·m L-1相比,差异有统计学意义(P<0.05);E和F组小肠组织中RV基因拷贝数和RV-VP6蛋白表达显著减少(P<0.05),小肠组织病理显示绒毛空泡样改变减轻,免疫组化显示RV抗原表达量显著降低。C和D组与B组比较差异无统计学意义(P>0.05)。结论 P-VP8*IgY可中和RV感染乳鼠中肠道及小肠组织中RV抗原,保护小肠绒毛,从而缓解腹泻症状,起到抗RV的作用。

Objective To explore the anti-virus effect of P-VP8*IgYin rotavirus suckling mouse disease model.Methods 6-day-old suckling mice were inoculumed with RV ZTR-68 strain to establish rotavirus suckling mouse disease model.The experiment animals were divided into six groups:group A(control group),group B(infection group),group C(prevention treatment group),group D(low dose experimental group),group E(medium dose experimental group)and group F(high dose experimental group).Group C was given P-VP8*IgY 20mg·kg-1in the morning and evening for 2 consecutive days before inoculation.D,E and F groups were given P-VP8*IgY 3.3,10,30mg·kg-1in the morning and evening at 3 d after inoculation.The symptoms and diarrhea were observed after inoculation,virus-antigen of stoolsample was tested by enzyme-linked immunosorbent assay.RV DNA gene copies of small intestine were tested by Real-time fluorescence quantitative polymerase chain reaction and the RV protein expression level were tested by Western Blot.Pathological changes were observed by hematoxylin-eosin staining(HE)and small intestine RVexpression were tested by immunohistochemical(IHC).Results 6-day-old suckling mice presented yellow dilute water stool after inoculation with a dose of Human RV of 1×107PFU,which indicated that the model of RV infection in suckling mouse was successful.after 96 h treatment,the diarrhea score in group E and group F were 1.75±0.49 and1.50±0.27,compared with group B(3.37±0.18),there was a significant decrease(P<0.05),stool sample RVantigens in group E and group F were(60.0±26.2)and(40.0±18.5)EU·m L-1,compared with group B(130.0±41.4)EU·m L-1,there was significant difference(P<0.05);the amount of RV DNA gene copies and the expression of RV-VP6 protein in small intestine had significant reduction(P<0.05),chorionic vacuolar changes were alleviated by HE stain.IHC showed a significant decrease in the expression of RV antigen.While there were no significant differences between group C,group D and group B.Conclusion P-VP8*IgY antibodies could protect the intestinal villi and alleviate diarrhea through neutralizing the RV antigen from fecal and small intestinal tissues in the suckling mouse disease model,playing the role of anti-rotavirus.