|
Abstract Porcine reproductive and respiratory syndrome virus(PRRSV) is a major pathogen of economic losses in pigs(Sus domesticus), causing severe reproductive failure in pregnant sows and boars, and respiratory problems in young pigs. This study was aimed to detect the expression of the recombinant co-expressing GP3 and GP5(N-glycosylation protein) and M(membrane protein) fusion protein of PRRSV in PK-15 cells (Porcine kidney cells), and to detect their immunogenicity. The eukaryotic recombinant plasmid pcDNA3.1-ORF3-ORF5-ORF6 was transfected into PK-15 cells by lipofectamineTM 2000 reagent, the stable expressive cells were selected (72 h) by G418 (μg/mL). We designed three pairs of primers based on the published sequences of PRRSV, the ORF3 and ORF5 and ORF6 was transfected into PK-15 cells, and then detected by RT-PCR, the GP3-GP5-M fusion protein was detected by IFA (indirect immunofluorescence assay). The immunogenicity of GP3-GP5-M was detected by Western blot and ELISA (enzyme linked immunosorbent assay). S/P (the ratio of sample/postive control) reached 1.80 after the second immunization, and lasted for at least 1 monthes. The results showed that the fusion protein GP3-GP5 (N- glycosylation protein)-M (membrane protein) could express in mammalian cells. Moreover, the fusion protein showed good immunogenicity. The recombinant plasmid pcDNA3.1-ORF3-ORF5-ORF6 may be an attractive candidate vaccine for the prevention and control of PRRS.
|
|
Received: 06 February 2012
|
|
|
|
|
|
