Production Of PCV2 Cap Protein In Silkworm Larvae For Immunity Research Of Virus-like Particles

Porcine circovirus(PCV)belongs to the Circoviridae family and the Circoviridae genus.It is the smallest single-stranded circular DNA virus with a genome size of about 1.7 kb.According to its different serotype,porcine circovirus virus can be divided into three types,PCV1,PCV2 and PCV3.PCV2 is strongly pathogenic to pigs.After infection,PCV2 causes postweaning multisystemic wasting syndrome(PMWS)and other associated diseases.PCV2 mainly consists of four open reading frames(ORF1,ORF2,ORF3 and ORF4).ORF1 encodes Rep protein,which is involved in virus replication.ORF2 encodes capsid protein named Cap.ORF3 encodes a protein inducing apoptosis of host cells.ORF4 encodes a protein that inhabites caspase activity as well as regulation of CD4 and CD8 T lymphocytes.The Cap protein has a size of about 30 k Da and contains a signal peptide of about 4 k Da and a core of about 26 k Da that contains major antigens and epitopes of PCV2.With suitable conditions,Cap protein can be self-assembled into virus-like particles(VLPs).Therefore,the VLPs of PCV2 have a good application prospect in the development of vaccine and the establishment of stable serum detection.Expression systems for obtaining recombinant protein include prokaryotic expression system,yeast expression system and other eukaryotic expression systems(insect cells,silkworm,etc.).Prokaryotic expression system and baculovirus-silkworm larva expression system are the two expression systems commonly used in the application of protein expression research.However,the recombinant protein expressed by prokaryotes is easy to form inclusion bodies as well as lacks post-processing,while eukaryotic expression system has safe and efficient genes that can accommodate multiple or large fragments,and does not infect humans or other mammals.Moreover,as a eukaryotic expression system,it can modify the target protein after translation and enhance the activity of the protein.In this study,the sequence for expression of PCV2Cap in E.coli BL21(DE3)strain and insect cells was optimized through bioinformatics analysis,of which Cap protein was highly expressed by the baculovirus Multi Bac expression system.The optimized Cap gene was cloned into the prokaryotic expression vector p ET30 a and the eukaryotic transfer vector p FBDM,and 6×His label was introduced respectively.Enhanced green fluorescent protein(e GFP)was used as the eukaryotic expression fluorescence reporter gene.The recombinant strain BL21(DE3)-p ET30a-PCV2Cap was obtained by transformation,screening and identification.The expression of recombinant strain was induced at different temperatures(16,25 and 37 ℃)and concentrations of IPTG(0.4,0.6,0.8 and 1.0 m M),and the expression conditions were optimized,which proved that the best expression conditions were obtained at 37 ℃ and 0.8m M IPTG concentration.The recombinant transfer vector p F-PCV2Cape GFP was introduced into Ac Multi Bac and Bm Multi Bac vectors through Tn7 transposition using the characteristics of the transfer vector p FBDM containing Tn7 transposition to obtain the recombinant strain of E.coli r Sw106-Ac-p F-PCV2Cap-e GFP and E.coli r Sw106-Bmp F-PCV2Cap-e GFP.Due to the deletion of asd gene,the recombinant strains E.coli r Sw106-Ac-p F-PCV2Cap-e GFP and E.coli r Sw106-Bm-p F-PCV2Cap-e GFP directly infected the insect cells Sf9 and Bm N without liposome transfection under the mediation of infection protein.The recombinant virus Ac MNPV-PCV2Cap-e GFP and Bm NPV-PCV2Cap-e GFP were confirmed by observation of green fluorescence after 72 h infected by insect cells.The supernatant of the medium was collected and centrifuged to obtain the recombinant virus.Western-blot identification proved that the Cap protein with a size of about 25.7 k Da was obtained,meaning the recombinant virus was successfully constructed.The Cap protein was stably expressed in insect cells.The recombinant virus Bm NPV-PCV2Cap-e GFP was injected into silkworm larvae at the fifth instar.Green fluorescence was observed in the silkworm body 4 d later.Western-blot analysis was performed on silkworm skin and hemolymph.The target band of 25.7 k Da was obtained,which proved that Cap protein was successfully expressed in silkworm larvae.After denaturation of the prokaryotic expressed inclusion body proteins,Cap proteins were purified and renatured by Ni column affinity chromatography.The epidermis of silkworm larvae was collected,processed,purified,and later self-assembled virus-like particles(VLPs)which were obtained under suitable conditions.Polyclonal antibodies against PCV2Cap were obtained by immunizing mice with VLPs and confirmed by western-blot and enzyme linked immunosorbent assay(ELISA).In conclusion,through codon optimization of PCV2Cap gene,Cap gene was obtained through gene synthesis.Prokaryotic expression system and baculovirus-silkworm larva expression system were constructed respectively,with which expression conditions and purification conditions were optimized.As a result,the expected VLPs was finally formed,while mice were successfully immunized as well as polyclonal antibodies were obtained.