Construction And Identification Of Recombinant Adenoviruses Containing Specific Antigen Epitopes Of The Cap Protein From Porcine Circovirus 2

Porcine Circovirus type 2(PCV2) is a key pathogen causing postweaning muLtisystemic wasting syndrome(PMWS) and porcine circovirus-associated disease(PCVAD).PCV2 ORF2 contains two main specific antigen epitope area coding Cap protein and having excellent antigen characteristics,which can distingwish PCV1 and PCV2.The study amplified PCV2 ORF2 framgent contained two main specific antigen epitopes with the help the PCR technology and cloned and constructed.As a resuLt,it was expressed in adenoviruses and identified the protein.According to the sequence of PCV2 gene from Genebank,a pair of specific primers was designed to amplify the ORF2 gene, Kpnâ… and Hindâ…¢enzyme digestion sites were added in the prime1 and primer2 seprately.Amplified ORF2 gene fragment from the affected PK-15 cell by PCV2,found an about 276bp strip in agarose gel electrophoresis,reclaimed and cloned it to pShuttle-CMV of adenoviruses serotype 5 vector. It was identified to be true by sequencing and BLAST software,enzyme digestion by Kpn I and Hindâ…¢and PCR. A Recombinant plasmid was constructed successfuLly and named it rpShuttle-CMV-276.First of all,adenovirus backbone plasmid pAdeasy-1 was transformed into copetent E.coli BJ5183 by chemical method, BJ5183/p strain was selected with ampicillin.Second-ly, BJ5183/p competent cells were prepared by chemical technique and the recombinant linearization and dephosphorylated digestion by Pmeâ… shuttle plasmid carrying interesting gene ORF2 was transformed into them. Two plasmids were homologous recombinated in it.A recombination plasmid named rPAD-276 was successfuLly constructed after identified by PCR,sequence and Pacâ… digestion.Purified alkaline lysis rPAD-276 plasmid and digested by Pacâ… ,then transfored them into the HEK-293A cell and builded up recombinant adenoviruses.It was certified expression of PCV2 specific Cap protein gene in HEK-293A cell and had good antigen characteristic through PCR,IPMA,immunofluorescence and mouse experiment identification. The study builded up recombinant adenoviruses contained specific antigen epitope of PCV2 Cap protein epitope and expressed successfuLly.It laid strongly the foundation for the further study the recombinant adenoviruses vaccine against PCV2,monoclonal antibody and detection dipstick for PCV2.