| H9N2 subtype avian influenza inactivated vaccine has been widely used in China since 1998,but the endemic epidemic of H9N2 avian influenza suggests that the vaccine currently used can not provide satisfactory protective efficacy.Therefore,it is very important to develop a more effective vaccine for pre-control of H9N2 avian influenza.Virus-like particles(VLPs)are hollow protein particles formed by the self-assembly of the structural proteins of one or more viruses,which do not contain the genetic material of the virus,but retain the structure and immunogenicity of natural virions,so it is a safe form of vaccine.Therefore,in order to cope with the epidemic of G57 H9N2subtype avian influenza in China in recent years,we constructed VLPs displaying the HA protein of G57 H9N2 subtype avian influenza virus by using insect cell baculovirus expression system,using M1 protein of avian influenza virus and Gag protein of mouse leukemia virus as skeletons,by comparing their Immunogenicity and immune protection efficacys,in order to provide technical support for the development of H9N2subtype avian influenza vaccine.Firstly,the G57 genotype H9N2 subtype avian influenza virus strain preserved in the laboratory was selected,and the recombinant baculoviruses expressing HA,M1 and gag were constructed by using the insect cell-baculovirus expression system,which were named r BV-HA,r BV-M1 and r BV-gag respectively.Then,r BV-M1 or r BV-gag were co-infected with r BV-HA by MOI=5,respectively,and the cell suspension was collected 96 hours later.Purified virus-like particles VLP-M1 and VLP-gag,were obtained by sucrose density gradient centrifugation and identified by hemagglutination activity test,Western blot and transmission electron microscope.The results showed that the hemagglutination titers of purified VLP-M1 and VLP-gag were 2~7 and 2~8,respectively.The target proteins HA and M1 of VLP-M1 detected by Western blot are about 63KD and 27KD,and the target proteins HA and gag of VLP-gag are about 63KD and 55KD,and their sizes were consistent with the expected values.The shape of particles similar to that of wild-type avian influenza virus was observed under transmission electron microscope,and the diameters of the two virus-like particles were obviously different:the diameter of VLP-M1 was about 100nm and the diameter of VLP-gag was about 150nm.This shows that VLP-M1 and VLP-gag have been correctly constructed in this study,and the diameter of VLP-gag is significantly larger than that of VLP-M1.In order to further compare the immunogenicity and immune protective efficacy of the two groups,the constructed VLP-M1 and VLP-gag were injected intramuscularly to 10-day-old broilers with three different doses of 40?g,30?g and 15?g,respectively.Through the immune challenge test,we compared the immunogenicity and immune protective efficacy of VLP-M1 and VLP-gag by humoral immunity,cellular immunity,in vivo carrying virus and in vitro detoxification.The results showed that the titer of HI antibody in 40?g VLP-gag group was the highest and the activation ability of lymphocytes was the strongest.There was no significant difference among 40?g VLP-M1 group,30?g VLP-gag group and commercial vaccine group.And the HI antibody titer in the 30?g VLP-gag group was significantly higher than that in the 30?g VLP-M1 group(p<0.05)on the 7,14,28,and 35 days after immunization.After 7-28 days of immunization,the 40?g VLP-gag group was significantly higher than the 40?g VLP-M1 group(p<0.001)and there was no significant difference between the VLP-M1group and the VLP-gag group in the 15?g dose group(p>0.05).After 21 days of immunization,the ability of lymphocyte activation in VLP-gag group was significantly higher than that in VLP-M1 group(p<0.0001).The results showed that the level of humoral and cellular immunity induced by VLP-gag was significantly higher than that of VLP-M1 at the same immune dose.In addition,the results of challenge test showed that 40?g VLPs group,30?g VLPs group and 15?g VLP-gag group had shorter detoxification time in vitro and lower viral load in vivo than 15?g VLP-M1 group and commercial vaccine group,indicating that 40?g VLPs,30?g VLPs group and 15?g VLP-gag group could better prevent virus replication,and the ability of VLP-gag group to prevent virus replication was better than that of VLP-M1 group at the same immune dose.In summary,two virus-like particles VLP-M1 and VLP-gag containing HA and M1proteins of H9N2 subtype avian influenza virus,HA of H9N2 subtype avian influenza virus and gag protein of mouse leukemia virus were correctly prepared in this study.And both VLP-M1 and VLP-gag had good immunogenicity.When the immune dose was the same,VLP-gag had better immune protection efficacy than VLP-M1.The results of this study provide new technical support for the research and development of a new vaccine against H9N2 subtype avian influenza and the effective prevention and control of the disease. |
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