[Fusion expression of the ORF5 gene of porcine reproductive and respiratory syndrome virus in insect cells].
A 0.75kb fragment containing the complete cDNA of glutathione-S-transferase (GST) and a modified thrombin cleavage sites were amplified and cloned into pFastBacl, the donor plasmid of Bac-to-Bac baculovirus expression systems, downstream from the polyhedrin promoter (pPolh), resulting in the GST fusion transposition plasmid pFGST. After transposition and transfection, SDS-PAGE analysis showed that the developed GST fusion expression system can highly express GST. The signal sequences of the major structural protein gene ORF5 of porcine reproductive and respiratory syndrome virus (PRRSV) strain YA was removed by PCR and the truncated ORF5 gene was inserted into pFGST and fused with GST to generate the transposition plasmid expressing GST-ORF5 fusion protein. The recombinant baculovirus rvBacGST53 was obtained by transposition and transfection. GST-ORF5 fusion protein was identified by SDS-PAGE and Western blot. The fusion protein is 45kD and is specific to the monoclonal antibody against the E protein of PRRSV. The fusion protein was directly injected into mice and the anti-sera can react with the PRRSV-infected MARC-145 cells by indirect immunofluorescent assay (IFA), indicating that the fusion protein have better immunogenicity.
Fang
Liurong
L
Laboratory of Animal Virology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
Xiao
Shaobo
S
Niu
Chuanshuang
C
Zhang
Hui
H
Chen
Huanchun
H
D000818:Animals; D019084:Fluorescent Antibody Technique, Indirect; D005982:Glutathione Transferase / Q000235:genetics*; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D018411:Spodoptera; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C114789:glycoprotein 5, PRRSV; D005982:Glutathione Transferase