Construction Of Coxsackievirus B5 Particle-like Vaccine And Preliminary Evaluation Of Its Immunogenicity And Protection

Objective:In this project,the Coxsackievirus B5 virus-like particle vaccine was constructed by the insect baculovirus system.Methods:1.The optimized synthetic CB5 P1（precursor protein）and 3CD（encoding viral protease）genes were inserted into the plasmid pOET5 to obtain the recombinant plasmid pOET5-P1-3CD.The insect cell-baculovirus expression system（flash BAC）expressed CB5 P1 and Recombinant baculovirus of the 3CD gene,infected with sf9 insect cells,expressing CB5 virus-like particles（VLPs）,using Western Blot to identify the expression of capsid protein,concentrated by polyethylene glycol precipitation method and released into cell culture The supernatant CB5 VLPs were purified by sucrose gradient centrifugation,and the morphology of VLPs was observed by transmission electron microscope.2.Six 6-week-old BALB/c female mice in each group,a total of 4 groups;CB5 VLPs vaccine is the experimental group,CB5 inactivated vaccine group is the positive control,wild-type baculovirus group is the negative control,PBS group is the blank control;Supplemented with complete/incomplete Freund’s adjuvant;each mouse was vaccinated intraperitoneally at a dose of 10 μg/time,and Week0,Week2,and Week4 were immunized once,and serum was collected before each immunization,respectively in Week6 and Week12 Blood was taken from the tail,and the serum specific IgG antibody levels of each group of mice were determined by ELISA.The neutralization antibody titer against CB5 in the mouse serum was detected by micro-neutralization experiment.3.Two 6-week-old ICR female mice in each group,a total of 4 groups;grouping and immune implantation route and dose are the same as in Method 2.Each mouse was subcutaneously transplanted at a dose of 10 μg / 0.1 ml,and immunized once at week 0,week 2 and week 4,respectively.Mate the female mice on Week3.After pregnancy at Week6～7 o’clock,take 10 1-day-old suckling mice as a group;each group was infected intracranially with 50 times the LD50 of CB5 mouse adapted strain,and continued to observe for 15 days and record The survival rate and clinical symptoms of suckling mice.Results:1.The anti-serum of immunized mice with inactivated CB5 whole virus was used as the primary antibody,and HRP-labeled goat anti-mouse IgG antibody was used as the secondary antibody.The protein expression of sf9 insect cells was verified by Western blot.The target bands appeared at 36 k Da and 28 k Da.They are VP1 and VP3,respectively.The morphology of the purified CB5 VLPs particles is similar to that of the inactivated CB5 virus particles under transmission electron microscopy,with a diameter of about 30 nm.2.ELISA results show that specific IgG antibodies can be detected in the serum of mice immunized with CB5 VLPs vaccine and CB5 inactivated vaccine,and the trend is the same: in the second week after booster immunization,IgG antibody levels increased significantly and reached a peak,The antibody continued to be at a relatively high level despite a slight decrease thereafter.Compared with the negative control group and the PBS group,the IgG antibody levels in the serum of mice immunized with CB5 VLPs vaccine and CB5 inactivated vaccine were significantly increased（p<0.05）.The results of micro-neutralization experiments showed that the average neutralizing antibody titer of CB5 VLPs vaccine mouse serum against CB5 was 1:160,and the average neutralizing antibody titer of CB5 mouse vaccine serum against CB5 was 1:128.Compared with the negative control group and the PBS group,the neutralization effect of CB5 VLPs vaccine and CB5 inactivated vaccine sera immunized mice was significantly enhanced（p<0.05）.3.The results of the mouse challenge protection experiment showed that the mice born from CB5 VLPs immunized mother mice were completely protected,and the mice born from CB5 inactivated virus immunized mother mice were partially protected.Their final survival rates were 100% and 80%,respectively.Conclusions:In this study,the insect baculovirus system was used to successfully construct and express CB5 VLPs as a candidate vaccine.The candidate vaccine can induce mice to produce specific IgG antibodies,and can confer protection of RD cells against CB5 virus in vitro and confer neonatal mice in vivo.Aiming at the effective protection of CB5 virus,it laid a theoretical and application basis for the development of CB5 vaccine.