In Vitro Expression And Immunogenicity Analysis Of Porcine Circovirus Type 2 Cap Gene

Objective:This project was designed to express the codon-optimized porcine Cap protein gene with yeast Pichia pastoris expression system,and mice were used to demostrate the immunogenicity function of Cap protein.Methods:According to the codon usage preference of pichia pastoris,the recombinant plasmid Cap was constructed.The recombinant Plasmid pwPICZalpha-Cap was transformed into pichia pastoris by electroporation.The proteins was expressed with methanol and then purified with ProteinIsoTM Ni-NTA resin.The purification fractions were analyzed using SDS-PAGE and Western blot with Rabbit anti-6×His polyclona antibody.Cap protein toxicity was detection by acute and chronic toxicity test.Cap groups(40 p,g,10 ?g and 2.5 ?g),positive control group and negative control group were set up in this research.The humoral immune-realated and cellular immune-related indexes were measured at 14 d(Booster injections with the same dose),28 d(challenged with PCV2),42 d,49 d and 56 d after immunization.In addition,Cap protein was mixed with ISA206 and GEL01 adjuvant respectively,and then specific antibody was detected at 14 d,28 d,56 d,70 d,84 d and 98 d after immunization.Result:The results showed it had specific bands in 28.5 kD analysed recombinant protein Cap by SDS-PAGE and Western blot.Acute toxicity and chronic toxicity test results showed that injection Cap protein in mice of indicators compared witn PBS control group,no significant differences(P>0.05).The results of immunoprotection test showed that each group has different levels of specific antibody and neutralizing antibody produced,of which 40 ?g group work well.40 ?g group specific antibody and neutralizing antibody levels were significantly higher than the negative control group(P<0.05),40 ?g group specific antibody and neutralizing antibody leveals compared with the positive control group,no significant differences(P>0.05).The results of challenge test showed that PCV2 copies of immunized groups was significantly lower than non-immunized group in lymph nodes(P<0.05).Adjuvant combined test results showed that the specific antibody expression level of adjuvant mixed group was significantly higher than 40 ?g Cap protein group(P<0.05).Conclusion:We preliminary expressed the immune protective effect of porcine circovirus type 2 Cap protein,which laid a methodological foundation for the development of PCV2 subunit vaccine.