|
|
|
| Expression of Porcine Circovirus Subtype 2 d Capsid Protein in Baculovirus/insect Cell Expression System |
| 2, 2, 2, 2 |
|
|
|
|
Abstract In order to express capsid protein of porcine circovirus type 2 (PCV2) in insect cells, nuclear-localization-signal (NLS) free capsid protein gene of PCV2 Shandong strain was amplified from clinical samples, then, the gene was inserted into baculovirus transfer vector pFastBac-1, the resulted plasmid was transferred to competent Escherichia coli DH10-Bac cells, the recombinant shuttle Bacmid was transfected into sf9 insect cells and cytopathic effect was observed. Capsid protein expression was identified by PCR, indirect immunofluorescence test, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The results showed that a 614 bp-length fragment was amplified from suspected samples, the amplified capsid protein gene of PCV2 Shandong strain shared highest homology with PCV2d strains at nucleotide level (99.8%) and amino acid level(100%), and belong to PCV2d subtypes revealed by evolutionary tree. Comparing with vaccine strains (PCV2a or PCV2d) widely used in China, this PCV2d strain showed a different antigenic domain at 160~180 aa. pFastBac1-PCV2d- Cap and Bacmid-PCV2d-Cap were successfully constructed and identified by PCR, restriction enzyme digestion and sequencing. The recombinated baculovirus induced cytopathogenic effect in sf9 insect cells, and successfully expressed a 24 kD protein, which could react with PCV2 antiserum and His-tag monoclonal antibody. The good immunogenicity of expressed capsid protein provides possible application in the development of porcine circovirus virus type 2d subunit vaccine and ELISA kit. This is the first report about PCV2d capsid protein expressed in baculovirus/insect cell expression system.
|
|
Received: 25 March 2016
Published: 22 July 2016
|
|
|
|
|
|
|
