| This study carried out the preparation process optimization of PCV2 Cap protein antigen,Mhp antigen,HPS serotypes 4 and 5 antigens,and determined the minimum usage of various antigens in the triple inactivated vaccine.Further,carried out the safety and effectiveness evaluation of the vaccine and completed the antibodies comparative study with similar products for the duration of immunity.The preparation process of PCV2 Cap protein was determined as follows:initial cell density:3.0×10~6?4.0×10~6 cells/m L,MOI:1.0?5.0,27?,35?50r/min,DO:30%?50%and incubation time:96?120 hours.Using BEI with a final concentration of 1%to inactivate at 37°C for 24 hours.The inactivated virus solution was clarified by a 3?6?m filter plate,and the clarified solution was concentrated 3 to4 times in a 50KD membrane package,and then purified via a chromatography column,the recovery rate was more than 85%and the purity was higher than 90%,respectively.After measuring the protein concentration,it was used for vaccine preparation.The preparation process of Mhp antigen was determined as follows:the inoculation amount was 8%?10%.The static cultivation process required 4?5 days with 37?,and to harvest when the p H value drops to about 6.8.The fermentation cultivation process required 3?4 days with 37?,50r/min,DO:10%?20%,and to harvest when the p H value drops to about 6.8.Using BEI with a final concentration of1%to inactivate at 37°C for 24 hours.The inactivated bacterial solution was concentrated 10 times with a 50KD ultrafiltration membrane,centrifuged at 4?10000r/min for 60 minutes,collected the sediment bacteria,and resuspended to 1/100original volume with PBS,sonicated for 5 minutes.After measuring the protein concentration,it was used for vaccine preparation.The preparation process of HPS 4 and HPS 5 antigen were determined as follows:1%the seed inoculation,more than 20%oxygen,37?,p H 7.0 under ventilation flow rate and stirring speed control,the HPS 4 needs to feed 4 times with5%culture volume at 8,10,12,and 14 hours,and finally,the bacterial liquid was harvested at 16 hours.The HPS 5 needs to feed 4 times with 5%culture volume at 6,8,10,and 12 hours,and finally,the bacterial liquid was harvested at 14 hours.The bacterial liquid was inactivated for 24 hours with the 0.3%formaldehyde solution.The inactivated bacteria solution was concentrated 10 times by a 0.1?m hollow fiber column,then centrifuged at 4?6000r/min for 20 minutes,then the sediment was resuspended with PBS(0.01mol/L,p H 7.2)for vaccine preparation.The vaccine preparation standard was determined as follows:Cap protein50?g/Dose,Mhp antigen 100?g/dose,HPS 4 antigen 4.0×10~9CFU/dose,and HPS 5antigen 2.0×10~9CFU/dose.The safety test showed that the spirit,feeding,aspiration,feces,and behavior were normal,and there was no significant difference between the control group and the immunized group,no local or systemic adverse reactions were observed.The ELISA antibody and MAT antibodies all turned positive.The protective efficacy against PCV2 or HPS 4 or HPS 5 challenge was above 80%,and the lung lesion rate was reduced by more than 60%against the Mhp challenge.There was no difference in the immune antibody duration from related products on the market.The vaccine in this study is the first report.It can prevent and control three major diseases that affect the pig industry at the same time with one shot,which will supplement the shortage of this product in China. |
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