| Objective: In this study,anti-intestinal virus type 71 chicken egg yolk antibody(anti-EV71 IgY)was prepared,and it was carried out against enterovirus 71(EV71)in vitro and in vivo and against Coxsackievirus A16(CVA16)in vitro.The mechanism of the IgY against EV71 and CVA16 were investigated.In order to explore a new cross-reactive immunotherapy for hand-foot-and-mouth disease(HFMD)caused by EV71 and CVA16.Methods: EV71 strain was used as antigen,mixed with Freund's incomplete adjuvant at a ratio of 1:1 and beaten to make it emulsified evenly.BWEL-SPF hens were immunized by multiple injections,and IgY was collected and purified.The total protein concentration of IgY was detected by protein quantitative method.The purity of IgY was analyzed by polyacrylamide gel electrophoresis(SDS-PAGE).Indirect enzyme-linked immunosorbent assay(ELISA)was used to evaluate the titer and variation of specific IgY.The specificity of purified IgY was detected by western blot and bidirectional immune agar diffusion test.Cytopathic effect(CPE)of human rhabdomyosarcoma(RD)cells was observed to detect the dose-effect relationship,stability and time-effect of IgY antiviral activity in vitro.The effect of specific IgY against EV71 in African green monkey kidney cells(Vero)was detected by immunofluorescence.The protective effect of specific IgY on newborn BALB/C suckling mice infected with EV71 virus was detected by intragastric administration.Results: The concentration of IgY protein increased after immunization.The result of SDS-PAGE showed that the purified IgY has high purity and can be divided into IgY heavy chain(H)with molecular weight of 70 kDa and IgY light chain(L)with molecular weight of 30 kDa.We found that the levels of the IgY increased at the 7th day after immunization,reached the peak on the 7th week and maintained at a higher level for about 4 weeks by ELISA.The results of bidirectional immune agar diffusion test and western blot showed that the IgY had cross-binding ability with both EV71 and CVA16 strains,and can specifically identify the envelope proteins VP1 and VP3 of EV71 and CVA16.The results of neutralization assay of IgY in vitro showed that the infectivity of EV71 and CVA16 strains was cross-blocked by the IgY in a dose-dependent manner,and the difference is statistically significant.Specific IgY had strong inhibitory effect on EV71 virus in the early stage of infection,but had no obvious inhibitory effect in the late stage of infection.The antiviral activity of IgY was still stable after 48 h at 4 ?,room temperature and 37 ?,and the inhibition rate of 1.6 ?g/mL at 60 ? can still reach 100 %.Repeated freezing and thawing for 5 times had no effect on the antiviral activity of IgY.Specific IgY had strong inhibitory activity on enteroviruses EV71 and CVA16,but has no obvious inhibitory effect on other enteroviruses.Immunofluorescence showed that specific IgY at 3.72 ?g/mL could inhibit strain in Vero cells almost 100 %.The survival rate of newborn mice in the IgY group reached 100 %,compared with the control group,there were no emaciation,listlessness,abnormal hair growth and any other symptom.Conclusion:(1)The specific IgY extracted after immunization has high purity and antiviral specificity;(2)Specific IgY can recognize the envelope proteins VP1 and VP3 of EV71 and CVA16,and can effectively inhibit the activitythe infectivity of EV71 and CVA16 strains in vitro was cross-blocked by the IgY in with a dose-dependent manner.(3)Specific IgY had significantly better stability in early stage of EV71 infection than late treatment,and the stability of specific IgY is better.IgY has good stability on early infection.(4)Specific IgY can provide protection to suckling mice attacked by EV71 after intragastric administration. |
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