Expression Of Porcine Circovirus Type 2 Capsid Protein Gene In Prokaryotic And Eukaryotic Systems

Porcine circovirus type 2(PCV2)is the major pathogen of post-weaning multi-systemic wasting syndrome(PMWS)in pigs,which has caused significant economic losses to the worldwide swine industry.PCV2 capsid protein is an important structural and functional protein encoded by the ORF2 of PCV2.In this work,we used three different expression systems including Escherichia coli,Pichia pastoris and Aspergillus.niger to express Cap protein.Our work provided alternative strategies to produce the proactive antigen Cap,which might facilitate developing PCV2 subunit vaccines.?Recombinant PCV2-Cap gene was expressed in E.coli expression system.According to the PCV2 genome sequence(DQ104422.1)from Gene Bank,Codon-usage optimized PCV2-Cap without nuclear localization signal was inserted into p ET-28a(+)expression vector and then transformed into E.coli expression strains BL21,recombinant protein was successfully expressed via extracellular secretion.Affinity chromatography can get a high degree of purification under 60% buffer B condition.And the purified Cap protein showed good immunogenicity based on SDS-PAGE and Western blot detection.?PCV2-Cap gene was recombinantly expressed via P.pastoris-based secretory expression system.Codon-usage optimized PCV2-Cap was inserted into p PICZ? A expression vector.Recombinant strains with the expression cassette integrated into the X-33 were selected by PCR.High-density fermentation in 5 L bioreactor revealed that the expression level of Cap protein in culture supernatant reached 0.53 mg/m L.Serum antibody tests demonstrated that Cap protein induced PCV2-Cap antibody with high specificity in piglets.PCV2-Cap protein without nuclear localization signal achieved secretory expression in P.pastoris,which has good immunogenicity.?PCV2-Cap gene was expressed in A.niger expression system.Codon-usage optimized PCV2-Cap were inserted into p MD18-pyr G-Tgla A general expression vector and integrated into the genome of A.niger host by homologous recombination.Results showed that the fusion protein of exogenous PCV2-Cap with endogenous glucoamlyse gene(gla A)was successfully expressed in A.niger via extracellular secretion.This is the first achievement of PCV2-Cap heterogeneous expression in filamentous fungi expression system.