The expression of porcine circovirus type 2 ORF2 gene in insect cells and its character.
- Author:
Hui-Ying FAN
1
;
Huan-Chun CHEN
;
Tie-Zhu TONG
;
Chun-Mei JU
;
Jian-Qiang LU
;
Hong-Liang HUANG
Author Information
1. Laboratory of Animal Virology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. hzauvet@public.wh.hb.cn
- Publication Type:Journal Article
- MeSH:
Animals;
Baculoviridae;
genetics;
metabolism;
Circovirus;
genetics;
metabolism;
Escherichia coli;
genetics;
metabolism;
Insecta;
cytology;
metabolism;
Open Reading Frames;
genetics;
Recombinant Proteins;
genetics;
metabolism;
Swine;
Viral Proteins;
genetics;
metabolism;
Virion
- From:
Chinese Journal of Biotechnology
2005;21(6):975-978
- CountryChina
- Language:Chinese
-
Abstract:
To produce the recombinant baculovirus transfer plasmid pFast-ORF2, the ORF2 gene of Porcine Circovirus type 2 (PCV2) was subcloned into baculovirus transfer vector (pFastBac(TM1) ) using Bac-to-Bac baculovirus expression system. E. coli DH10Bac (Gibco BRL) containing baculovirus shutter vector (bacmid) and helper vector was transformed with recombinant plasmid pFast-ORF2. Within E. coli DH10Bac, the ORF2 gene was transposed into the bacmid. The colonies of E. coli containing recombinant bacmid (Bac. ORF2) were collected by blue/white selection. The Bac. ORF2 was transfected into sf9 cells to yield AcNPV carrying the PCV2 ORF2 gene, referred to as Ac. ORF2. Expression of the ORF2 gene of PCV2 was confirmed by indirect immunofluorescent assay (IIFA), SDS-PAGE and Western-blotting. The expressed ORF2 gene product had a molecular mass of 28kD and could be recognized by the positive serum of PCV2. The results indicated the ORF2 gene was properly expressed in sf9 cell. It was noteworthy that many self-assembled virus-like particles (VLPs) were found in purified and phosphotungstic acid (PTA) stained PCV2 ORF2 protein by electron microscope. The particles were of similar morphology to the PCV2 virion and some self-assembled virus-like particles had darkly stained centers that made them appear to be empty capsids. Both PCV2 particles and self-assembled particles were approximately 17 nm in diameter.
