[Recombination and expression of ORF1 and ORF2 gene of porcine circovirus type 2 and gene of pseudorabies virus].
ORF1 and ORF2 gene of porcine circovirus type 2 were cloned by PCR with the specific primers designed according to genome of PCV2 (AY035820). Following extraction and digestion, PCR products were subsequently inserted into universal transfer vector plECMV (deleted partial gE and gI of pseudorabies virus) to generate recombinant transfer plasmid pIEORF1-ORF2. The genomic DNA of PRV TK-/gE- /LacZ+ strain and pIEORF1-ORF2 were co-transfected into IBRS-2 cells with lipofectin, and recombinant virus TK- /gE- /gI- /ORF1-ORF2+ was selected by PCR with ORF1 gene and ORF2 gene primers respectively. The recombinant virus was analyzed with Southern blotting and Western blotting. The results indicated that ORF1 and ORF2 gene of PCV2 had been inserted into the genome of TK- /gE- /LacZ+ strain and the expressed ORF1-ORF2 fusion protein could react with PCV2 positive sera. Result of virus titers detection showed the insertion of ORF1 and ORF2 gene did not influence propagation of recombinant virus.
Ju
Chun-Mei
CM
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. juchunmei7625@163.com
Chen
Huan-Chun
HC
Fan
Hui-Ying
HY
Liu
Zheng-Fei
ZF
Cao
Sheng-Bo
SB
D000818:Animals; D002460:Cell Line; D017925:Circovirus / Q000145:classification* / Q000235:genetics*; D018014:Gene Transfer Techniques; D005814:Genes, Viral; D011558:Herpesvirus 1, Suid / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D013552:Swine
D011994:Recombinant Proteins