Construction And Immune Effects Of Recombinant PCV2 ORF2 And IL-18 Gene Of PRV

Porcine circovirus(Porcine circovirus,PCV)was first found in 1974 in pig kidney cell line,which is the smallest known genome,non-enveloped,covalently closed,and single negative-stranded DNA virus so far.Porcine circovirus disease(Porcine circovirus-associated disease,PCV AD)was first discovered in Canada in the early 1990s.Since then,this disease was reported in the United States,France,Japan,Korea and other countries.PCV is one of the important pathogens caused serious harm to swine industry in the world.Except for the primary infection and even death caused by PCV2 in pigs,the immune suppression and/or secondary infection of other bacteria and virus were also occurred,which all could aggravate the epidemic situation and cause huge economic losses to the swine industry worldwide.In this study,we isolated 17 PCV2 virus strains from pig farms in Henan Province,including two complete genome sequence of 1768bp,and others were 1767bp.The homology between their mutual relationships and evolutionary analysis of gene sequences homology is 94.9%to 99.9%.Compared to other PCV2 genomic sequence isolated from home and abroad,most of the evolutionary relationships of Henan isolates have closer genetic relationships.Swine pseudorabies virus(Porcine pseudorabies,PRV)is a linear double-stranded DNA genome,formed by the unique long segment(Unique Long,UL),short sequences(Unique Short,US),US both sides of the terminal repeat(TR)and the internal repeat sequence(IR).Pseudorabies virus genome contains at least 70 genes encoding 70-100 proteins.In this study,the nucleocapsid protein of PCV2 ORF2 gene got at the isolated ZZ-1(Genbank NO.KC753772)strain and porcine IL-18 gene were constructed to attenuated vaccine pseudorabies virus strain HB98,to get the recombinant virus,in order to explore the development of recombinant PCV2 vaccine for prevention of PCV2.In this study,according to the PRV gG gene sequence published in GenBank we designed a pair of specific primers,to amplify a sequence containing the PRV gG gene,partial pK gene and partial gD gene from the FA strain of pseudorabies virus,the amplified sequence length of 3.1 kb.The 3.1 kb fragment of plasmid DNA digested with SphI and KpnI and subcloned into the vector pUC19,getting the recombinant plasmid PUP.Using pcDNA3.1(?)vector as the template,primers were designed to obtain about 0.3 kb of the SV40 Poly(A)fragment and cloned into the PUP which digested with BamHI and Pstl?,building recombinant plasmid PUPS.And eliminating the Hind ? restriction sites of the recombinant plasmid PUPS and eliminating the BamHI and Pst? restriction sites of pEGFP-nl vector.According to the pEGFP-n1 vector gene sequences to design a pair of primers to amplify the target gene of EGFP(including GFP,CMV,MCS,SV40 Poly(A)),the destination cloned into pMD18-T vector.After digested with Pst? of PUPS,dephosphorylated and then connected with EGFP treated by Pst? digestion.After amplified by PCR,restriction enzyme digestion,sequencing confirmed that the transfer vector was successfully constructed and named PG.Plasmid pIRES-IL18 and vector PG were digested with Bgl? respectively,then recycling.After dephosphorylated with PG fragment,the IL-18 gene was connected to PG constructing a recombinant plasmid PG18.Plasmid psy538-ORF2 and PG were digested with BamHI respectively,then recycling.After dephosphorylated with PG fragment,the ORF2 gene was connected to PG constructing a recombinant plasmid PGO.Plasmid pIRES-IL18 and PGO were digested with Bgl? respectively,then the IL18 gene was connected to PGO constructing a recombinant plasmid PGO18.After amplified by PCR,restriction enzyme digestion and sequencing,we proved that the three transfer vectors were successfully constructed with the correct orientation.The PGO 18 recombinant plasmid was transfected to ST cells which treated with attenuated vaccine strain PRV HB98.And PGO 18 was able to see a lot of green fluorescence by fluorescence microscope.After three times of plaque purification of PGO 18 recombinant plasmid,and detected by PCR,Western-blotting,lymphocyte proliferation assays,transcriptome detection,IPMA detection and genetic stability analysis,the virus was confirmed to be the recombinant virus.The purified recombinant virus PGO 18 and PGO which purified by Chao anjun of our laboratory were tested immunity of mice,and DMEM control group also seted.The detection of PCV2 and PRV antibodies,and MTT lymphocyte proliferation test,T lymphocyte subsets and viruses challenge tests were done,and results showed that the recombinant virus could significantly stimulate mice to produce antibodies,and stimulated the proliferation of lymphocyte,T lymphocyte subsets number,and have very good protection effect to the virulent attack.