Objective Indentifying effective adjuvants,immunity dose and procedure for the mycoplasma pneumoniae(MP)subunit vaccine in BALB/c mice.Methods Groups(n=16)of female 6-week-old BALB/c mice were immunized with vaccine alone(5μg and 20μg dose),Alum-adjuvant vaccine,PolyI:C-adjuvant vaccine,CpG-adjuvant vaccine,Alum-PolyI:Cadjuvants vaccine,Alum-CpG-adjuvant vaccine,PolyI:C-CpG-adjuvantsvaccine,Alum-PolyI:C-CpG-adjuvants vaccine by int-ramuscular injection in the hind legs four times at a 2-week interval.All animals bled via the venous sinus of eye on the day before each immunization.The serum was isolated to determine antibody titers using an ELISA.Spleens were aseptically removed from mices after the final immunization.Thesplenical cells were prepared,and then stimulated with 10μg mycoplasma pneumoniae fusion protein(MP559)for 72h.ELISA kit were used to detect supernatant from splenical cell cultured of IL-1,IL-4,IFN-γ.The differences were analyzed statistically,to determine the optimized immunization scheme.Results Two weeks after the third immunization,the antibody levels in the 20μg dose with Alum-PolyI:C-adjuvants group was the highest,and the antibody titers remain stable after the third immunization.Titers in animals vaccinated with 20μg MMP559 with Alum-PolyI:C-adjuvants were significantly higher or higher than other groups for the four immunization.For cellular immunity,in Alum-PolyI:C-20μg MP559 group,the level of IFN-γ was the highest,whereas IL-2 and IL-4 levels in Alum-PolyI:C-20μg MP559 group was less than Alum-PolyI:C-CpG-20 μg MP559.Conclusion The results prove that the best immunization schemes was vaccinated with 20μg MP559 mixed with Alum-PolyI:C-adjuvants three times at a 2-week interval.This study for the next step of mycoplasma pneumoniae subunit vaccine clinical research laid the foundation.