Transcription Analysis And Preliminary Identification Of Proteins Function Of Porcine Circovirus-like Virus P1

Porcine circovirus-like virus P1(P1 virus)was isolated from the sera of suspectable cases of clinical postweaning multisystemic wasting syndrome(PMWS).The genomes of P1 virus were the smallest among animal viruses,with a total length of 648 nucledtides.The nucleotide sequences of P1 virus shared high homology with the open reading frame 2(ORF2)of porcine circovirus type 2(PCV2).Previous studies showed that P1 virus caused the apoptosis in PK-15 cells and clinical symptoms similar to PMWS in pigs.The main contents of the paper are as follows:1.The identification of P1 ORF2 and ORF3To identify the ORF2 and ORF3 of the three main open reading frames of P1 virus on the transcriptiol and protein level,total RNA was extracted by Trizol from PK-15 cells transfected with the P1 molecular cloning(pSK-P1),purified and reverse transcribed into cDNA by RT-PCR.And then the cDNA was amplified by PCR and sequenced.The 5'-and 3'-ends of ORF2 and ORF3 were amplified by the rapid-amplification of cDNA ends(RACE).Simultaneously,B-cell epitopes of PI ORF2 and ORF3 were predicted according to their amino acid sequences,and then the corresponding epitope peptides were synthetized with stepwise solid-phase synthesis method and coupled to keyhole limpet hemocyanin(KLH).Polyclonal antibodies were prepared by the New Zealand White Rabbits immuned the synthetic peptides of ORF2 and ORF3,respectively.Titers of the polyclonal antibodies were detected by conventional indirect ELISA.Reactivity between PK-15 cells transfected with pSK-P1 and the two polyclonal antibodies were detected by immunohistochemistry.The results showed that the target genes fragments were amplified by RT-PCR with the specific primers of ORF2 and ORF3,which shared 100%sequence homology with P1 ORF2 and ORF3,respectively.The 5'-and 3'-ends of ORF2 were at nt 648 and nt 481,and that of ORF3 were at nt 364 and nt 70,respectively.The amino acid sequences of the synthetized epitope peptides were(ORF2)CLSRLPQSERPPGRW and(ORF3)CVYPKVRERRVLKMP,respectively.Polyclonal antibodies prepared with KLH-conjugated peptides of ORF2 or ORF3 developed specific chromogenic reaction with P1 virus in immunohistochemistry,respectively.The titers of the two polyclonal antibodies were higher than 1:512000.In conclusion,the presence of P1 ORF2 and ORF3 wsa determined on the transcription and protein level with transcriptional analysis and immunohistochemistry,respectively.2.Expression and analysis of three main proteins of PI virus in insect cellsTo identify whether the proteins encoded by three main open reading frames(ORFs)of P1 virus can self-assemble into virus-like particles(VLPs)in vitro,the genes of P1 ORF1,ORF2 and ORF3 were amplified by PCR,cloned into the pFastBacTM1 vectors and then transfected into Sf9 cells by liposome method,respectively.Three recombinant baculoviruses(rBac-ORF1,rBac-ORF2 and rBac-ORF3)were obtained.The expressions of three recombinant baculoviruses were identified on transcription and protein level by RT-PCR and Western blot,respectively.And then the three recombinant proteins were detected by transmission electron microscope.The results showed that the Sf9 cells presented cytopathy after transfected the recombinant plasmids;the corresponding genes fragments were amplified from the total RNA extrac;ted from inoculation cells after removal genomic DNA by RT-PCR;specific reactions between the expression of rBac-ORF1 and the anti-P1 VP1 monoclonal antibodies and polyclonal antibodies were observed in Western blot.And the inclusion bodies nearly elliptic formed by the three proteins in Sf9 cells were observed under electron microscope.In conclusion,the ORF1,ORF2 and ORF3 genes of PI virus were successfully expressed in Sf9 cells with the baculovirus expression vector system,and the recombinant proteins could not self-assemble into VLPs.The study may lay a foundation for further exploring other functional properties of the proteins of P1 virus.