Construction Of A Recombinant Pseudorabies Virus Expressing ORF2 Gene Of Porcine Circovirus Type 2 And Porcine Interleukin-2 Gene

Porcine circovirus(PCV) is a small animal virus with a single-stranded circular DNA genome, which is icosahedral and nonenveloped. PCV contains PCV1 and PCV2. Porcine circovirus-associated disease(PCVAD) was first described in the early 1990 s and subsequently, in the USA, France, Japan, Korea and other countries. PCV2 could cause progressive weight lose and high wasting,in the other hand the virus proliferates in the immune system of piglet,and cause disorder of immune function.The virus has caused great economic lose in the pig industry. It can be said that the virus has been prevalent in China at present.In this study we have expressed the major capsid protein of PCV2 and IL-2 gene of swine,used Swine Pseudorabies Vaccine HB98 strain to the parental strain,Construction of a recombinant pseudorabiesvirus co-expressing ORF2 gene of PCV2 and porcine IL-2 gene.According to the published gene sequences of PRV complete sequence in GenBank, a pair of specific primers was designed and systhesized, and PRV gene was amplified, then cloned and sequenced. The plasmid containing the SphI 15 kb fragment of genomic DNA of pseudorabies virus(PRV)Min-A strain was digested with SphI and KpnI. About 2.8kb DNA fragment containing the complete gG gene pK and gD partial gene were recovered and cloned into the same sites of modified vector pUC19,which resulted in the recombinant plasmid PUPD. 0.3kb SV40 Poly(A)DNA fragments were amplified with pCDNA3.1 by PCR and were cloned into PUPD, which resulted in the recombinant plasmid PUPDS.Plasmids PUPDS and pEGFP –n1 were reformed by digestion, fill-linking and the connection methods. The Hind? site of plasmid PUPDS was eliminated to reform PUPDS which was named as PH. The BamHI and PstI sites of plasmid pEGFP –n1 were eliminated to reform pEGFP –n1 which was named as pEGFP-P. A pair of primers were designed according pEGFP-n1 gene sequence, PstI restriction sites were introduced at downstream 5'end in the primers, EGFP gene(including GFP, CMV, MCS, SV40 Poly(A)) was amplified by PCR with plasmid pEGFP-P as template and cloned into pMD18-T vector to generate the recombinant plasmid. Digestion and PCR fragment proved the inserted fragment was EGFP gene, and recombinant plasmid was named as pMD-EGFP. Both the recombinant plasmid vector pMD-EGFP and PH were digested with PstI, and then recoveried. EGFP was cloned into plasmid PH after phosphorlation of plasmid PH. PCR amplification, restriction enzyme digestion, sequencing proved that the success of the transfer vector, and named as PG.The plasmid 538-ORF2 containing the 702 bp fragment of genomic DNA of ORF2 and vector PG were digested with BamHI and recoveried. After vector PG digested with BamHI was phosphorlated, ORF2 gene was linked into vector PG to construct the recombinant plasmid PGO. Trough amplifing by PCR, restriction enzyme digestion, sequencing proved that the success of the transfer vector. IL-2 was amplified by PCR with plasmid pIRES-pIL2 as template, and cloned into PGO, which resulted in the recombinant plasmid PGOIL2. The transfer plasmid was co-transfeeted into ST cells with the genome of HB98 constructing the recombinant pseudorabies virus. The recombinant pseudorabies virus was identified by fluorescence assay and PCR test.