Constrction And Efficiency Of Multivalency Immune-Regulating DNA Vaccins Against Eimeria Tenella

The immunization dose of immune-regulative DNA vaccine pcDNA3.1-TA4-IL-2 was optimized.Four experimental groups i.e.200μg,100μg,50μg,and 25μg dose groups were designed and the challenged and unchallenged control groups were also designed.At the age of 14 days,experimental chickens were intramuscularly immunized with the corresponding immunization doses.A booster immunization was given by the same method as the first immunization 7 days later.The control groups were intramuscularly immunized with TE.At 28 days of age,the chickens were challenged with 5×10~4 sporulated oocysts of E.tenella JS except the unchallenged control group.Seven days post challenge,all the chickens were slaughtered.Body-weight gain,oocysts per gramfeces(OPG),lesion score, and ACI of each group were calculated.The results illustrated that ACIs of groups 25μg and 50μg were 196.45,182.67,respectively,which showed significantly effective protection against avian coccidiosis;ACI of group 100μg was 172.16,which showed effective protection against avian coccidiosis;ACI of group 200μg was 157,which showed ineffective protection against avian coccidiosis.The immunization route of DNA vaccine pcDNA3.1-TA4-IL-2 was optimized.Five experimental groups i.e.subcutaneous injection,oral administration,intravenous injection, intramuscular injection and intranasal administration groups were designed and the challenged and unchallenged control groups were also designed.At the age of 14 days, every experimental chicken was immunized with 25μg DNA vaccine by the corresponding immunization route.A booster immunization was given by the same method as the first immunization 7 days later.The control groups were immunized with TE.At 28 days of age, chickens were challenged with 5×10~4 sporulated oocysts of E.tenella JS except the unchallenged control group.Seven days post challenge,all the chickens were slaughtered. Body-weight gain,oocysts per gramfeces(OPG),lesion score,and ACI of each group were calculated.The results illustrated that ACIs of experimental groups were beyond 160, which showed effective protection against avian coccidiosis.ACIs of intravenous injection, subcutaneous injection and intranasal administration groups were 173.38,171.44,168.17, respectively,which showed effective protection against avian coccidiosis.ACI of oral administration was 180.04,which showed significantly effective protection against avian coccidiosis.Intramuscular injection induced a highest ACI of 193.97,which demonstrated intramuscular injection was the best immunization route of the five immunization routes.The primary immunization age and immunization times of DNA vaccine pcDNA3.1-TA4-IL-2 was optimized.Four experimental groups i.e.group immunized once at 7 days old,group immunized twice at 7 and 14 days old respectively,group immunized once at 14 days old,and group immunized twice at 14 and 21 days old respectively were designed and the challenged and unchallenged control groups were also designed. According to the corresponding primary immunization age and immunization times,every experimental chicken was intramuscularly immunized with 25μg DNA vaccine.The control groups were immunized with TE.At 28 days of age,chickens were challenged with 5×10~4 sporulated oocysts of E.tenella JS except the unchallenged control group.Seven days post challenge,all the chickens were slaughtered.Body-weight gain,oocysts per gramfeces(OPG),lesion score,and ACI of each group were calculated.The results illustrated that ACIs of experimental groups were beyond 160,which showed effective protection against avian coccidiosis.Younger primary immunization age groups showed a little higher ACI than older primary immunization age groups.There was no significant difference between the booster immunization groups and non-booster immunization groups.The stability of DNA vaccine pcDNA3.1-TA4-IL-2 was analysised.The DNA vaccines were stored at -20℃,4℃and room temperature for 1 month,3 months,and 6 months,respectively.With 25μg of the stored DNA vaccines,7-day-old chickens were intramuscularly immunized.A booster immunization was given by the same method as the first immunization 7 days later.The control groups were immunized with TE.At 28 days of age,chickens were challenged with 5×10~4 sporulated oocysts of E.tenella JS except the unchallenged control group.Seven days post challenge,all the chickens were slaughtered. Body-weight gain,oocysts per gramfeces(OPG),lesion score,and ACI of each group were calculated.The results illustrated that ACIs of experimental groups were beyond 160, which showed effective protection against avian coccidiosis.Preservation time and temperature had little effect on the immunizing efficacy of the vaccine in 6 months, indicating that the vaccine could be preserved for at least 6 months.The cross-protection of DNA vaccine pcDNA3.1-TA4-IL-2 was analysised.With 25μg of E.tenella DNA vaccine pcDNA3.1-TA4-IL-2,7-day-old chickens were intramuscularly immunized.A booster immunization was given by the same method as the first immunization 7 days later.The control groups were immunized with TE.At 28 days of age,chickens were challenged with sporulated oocysts of the corresponding Eimeria spp. JS except the unchallenged control group.Seven days post challenge,all the chickens were slaughtered.Body-weight gain,oocysts per gramfeces(OPG),lesion score,and ACI of each group were calculated.The results illustrated that the vaccine could provide partial cross-protection against the challenge with E.necatrix and E.acervulina,but not with E. maxima.T cell epitopes of MZ5-7 gene and SO7 gene were predicted with DNAStar software. Results showed that the T cell epitopes of MZ5-7 gene located at the segments of 115-435 and 547-897,the first segment was named ml encoding 107 amino acids and the second segment was named m2 encoding 117 amino acids.The T cell epitopes of SO7 gene located at the segments of 7-291 and 370-609,the first segment was named s1 encoding 95 amino acids and the second segment was named s2 encoding 80 amino acids.Several pairs of primers were designed and m1,m2,s1and s2 segments were amplified by PCR with pMD18-T-SO7 and pMD18-T-MZ5-7 as templates.PCR products were cloned into pMD18-T vector.Identification showed that m1,m2,s1and s2 segments were cloned into pMD18-T vector successfully.m1,m2,s1 and s2 were inserted into pVAX1 vector with chIFN-γand chIL-2 as gene adjuvant,generating recombinant plasmids pVAX1-m1-m2-s1-s2-IFN口pVAX1-m1-m2-s1-s2-IL2,pVAX1-m1-s1-IFN口pVAX1-m1-s1-IL2,pVAX1-m1-s2-IFN口pVAX1-m1-s2-IL2,pVAX1-m2-s1-IFN口pVAX1-m2-s1-IL2,pVAX1-m2-s2-IFN口pVAX1-m2-s2-IL2,pVAX1-m1-m2-s1-s2,pVAX1-m1-s1,pVAX1-m1-s2,pVAX1-m2-s1, pVAX1-m2-s2,pVAX1-m1,pVAX1-m2,pVAX1-s1 and pVAX1-s2.After identified by restriction enzyme digestion,these recombinant DNA plasmids were extracted and injected into the leg muscle of 7 days old chickens.A week later the injected tissue was sampled respectively to check whether these DNA plasmid expressed or not by RT-PCR and Western-blot.The results indicated that aim genes could be successfully transcribed and expressed in injected tissues.Immune protection experiment was carried out with the 19 constructed plasmids. Nineteen experimental groups i.e.pVAX1-m1-m2-s1-s2-IFN口pVAX1-m1-m2-s1-s2-IL2, pVAX1-m1-s1-IFN口pVAX1-m1-s1-IL2,pVAX1-m1-s2-IFN口pVAX1-m1-s2-IL2, pVAX1-m2-s1-IFN口pVAX1-m2-s1-IL2,pVAX1-m2-s2-IFN口pVAX1-m2-s2-IL2, pVAX1-m1-m2-s1-s2,pVAX1-m1-s1,pVAX1-m1-s2,pVAX1-m2-s1,pVAX1-m2-s2, pVAX1-m1,pVAX1-m2,pVAX1-s1 and pVAX1-s2 groups were designed.The empty vector,challenged and unchallenged control groups were also designed.At the age of 14 days,experimental chickens were intramuscularly immunized with 100μg of the corresponding DNA vaccines.A booster immunization was given by the same method as the first immunization 7 days later.The empty control group was intramuscularly immunized with 100μg pVAX1.The other two control groups were intramuscularly immunized with TE.At 28 days of age,the chickens were challenged with 5×10~4 sporulated oocysts of E.tenella JS except the unchallenged control group.Seven days post challenge, all the chickens were slaughtered.Body-weight gain,oocysts per gramfeces(OPG),lesion score,and ACI of each group were calculated.The results illustrated that all the DNA vaccine constructed could induce effective protection against E.tenella.The DNA vaccine with multi-stage antigen genes of E.tenella could induce better protection than the ones with single stage.The DNA vaccines with cytokines could induce better protection than the without cytokines.Immunization with pVAX1-m1-m2-s1-s2-IFN口induced the most effective protection against E.tenella.