The Development And Safety Evaluation Of A DNA Vaccine Encoding TA4-IL-2 For Eimeria Tenella

Coccidiosis in Chicken is a protozoal disease caused by parasites of Eimeria.tenella in the intestine of chickens.Coccidiosis is globally distributed with a high rate of infection and harmfulness.Among them,Eimeria tenella was the most pathogenic and caused the most serious damage.The current prevention and treatment of coccidiosis mainly rely on drugs and a few live coccidiosis vaccines.However,the existing drug resistance and drug residue problems as well as the potential of virulence reversion and coccidiosis infection which makes the urgent need to develop of a new type coccidiosis vaccine.In this study,TA4 protein of E.tenella sporozoites was used as antigen molecule,pSMART2 b was used as eukaryotic expression vector,chicken IL-2 was in tandemed as an adjuvant.pSMART2b-TA4-IL-2 recombinant DNA vaccine was successfully constructed.The immune response and protection against E.tenella in chickens induced by the recombinant plasmid pSMART2b-TA4-IL-2 was determined.Finally,the vaccine was evaluated for safety.The construction,expression and identification of recombinant plasmid pSMART2b-TA4-IL-2 First,the EtTA4 and IL-2 genes were amplified by PCR,then the TA4-IL-2 gene was amplified by fusion PCR and ligated into the pMD18-T cloning vector.Finally,the TA4-IL-2 gene was ligated into the pSMART2 b vector.The recombinant plasmid was identified by PCR,double enzyme digestion and sequencing identification,then the recombinant plasmid pSMART2b-TA4-IL-2 was transfected into 293 T cells and analyzed by Western blot.pSMART2b-TA4 and pSMART2b-IL-2 was constructed as control group.The results showed that the lengths of amplified EtTA4 and IL-2 genes were 687 bp and 429 bp respectively.The recombinant plasmid pSMART2b-TA4-IL-2 was successfully constructed determined by PCR,double enzyme digestion and sequencing identification.Western blot results showed that there was a significant band around 76 KDa,indicated that the recombinant plasmid can be expressed in 293 T cells and recognized by chicken anti-E.tenella positive serum.Immune response induced by recombinant plasmid pSMART2b-TA4-IL-2 The plasmids were immunized by intramuscular immunization with the doses of 25 ?g per chicken,the chickens were immunized every other week after first immunization at 7 days.Antibody levels and cytokines levels of each group were measured by ELISA.The proliferation of T lymphocytes was test by MTT method.The results showed that the levels of IL-2 and IFN-? were significantly different between the pSMART2b-TA4-IL-2 group and the pSMART2b-IL-2 group(P<0.01),there was no significant difference between the pSMART2b-TA4-IL-2 and pSMART2b-TA4 control(P>0.05).At the level of antibody,there was no significant difference between pSMART2b-TA4-IL-2 group and pSMART2b-TA4 group(P>0.05).At the level of T lymphocyte proliferation,there was a significant differences between the pSMART2b-TA4-IL-2 group and pSMART2b-TA4 and pSMART2b-IL-2 group(P <0.05).The protective effect of recombinant plasmid against Eimeria tenella challenge After immunized by recombinant plasmids,the chickens were challenged with E.tenella spores oocysts.The survival rate,weight gain rate,cecal lesion score,and the number of oocysts were determined in each group,and the protective effect of the recombinant plasmid was evaluated by Anti-coccidial index(ACI).The results showed that the pSMART2b-TA4-IL-2 immunized group have better anti-coccidiosis protective effect than pSMART2b-TA4 and pSMART2b-IL-2 groups which ACI reached 186.62,showed a good anti-coccidial protection effect.The safety evaluation of recombinant plasmid pSMART2b-TA4-IL-2 The safety of the recombinant plasmid was evaluated in accordance with the Rules of Management on Agricultural Transgenic organision.The evaluation indexes included changes in the clinical response of the target animals under single and over-dose inoculation conditions,changes in hematological parameters,observation of major organ lesions,and determination of the residual time of the recombinant plasmid antigen genes in animals.The assessment of environmental safety mainly includes the detection of surrounding water and the target animal feces around the test sites.The results showed that the recombinant plasmids did not cause any pathological damage to the test animals either in the single dose or the overdose vaccination in the 4 months test period.The residual time of the antigen gene in the target animal was determined by PCR.The results showed that the presence of the TA4 gene could be detected within 2 months after the immunization,but after 2 months,TA4 gene can not be detected in each group,indicates that the antigen genes of the recombinant did not integrate into the host chromosome.The water around the test site and animal feces were detected by PCR,but no TA4 gene was detected in each group.In one word,the recombinant plasmid pSMART2b-TA4-IL-2 had no problems on biosafety to target animals and environment.