Prokaryotic expression and purification of the capsid protein of porcine getah virus and preparation of its polyclonal antibody.
- Author:
Yan JIANG
1
;
Dan-Ni HE
;
Xiao-Min ZHANG
;
Bin ZHOU
;
Pu-Yan CHEN
Author Information
1. Jiangsu Entry-exit Inspection and Quarantine Bureau, Nanjing 210001, China. jiangyan@jsciq.gov.cn
- Publication Type:Journal Article
- MeSH:
Alphavirus;
genetics;
immunology;
metabolism;
Alphavirus Infections;
immunology;
veterinary;
virology;
Animals;
Antibodies, Viral;
blood;
immunology;
Blotting, Western;
Capsid Proteins;
genetics;
immunology;
isolation & purification;
metabolism;
DNA Primers;
genetics;
Electrophoresis, Polyacrylamide Gel;
Escherichia coli;
genetics;
metabolism;
Gene Expression;
Genetic Vectors;
Humans;
Male;
Mice;
Mice, Inbred BALB C;
Plasmids;
genetics;
Polymerase Chain Reaction;
Recombinant Fusion Proteins;
Swine;
Swine Diseases;
immunology;
virology;
Zoonoses
- From:
Chinese Journal of Virology
2013;29(4):371-375
- CountryChina
- Language:Chinese
-
Abstract:
Based on a pair of specific primers, a 804-bp fragment was amplified from the plasmid pT-Cap containing Cap gene of Porcine Getah Virus(PGETV) and cloned into the prokaryotic expression vector pCold I which carried the His tag, this recombinant plasmid was then determined by enzyme digestion, PCR and DNA sequencing. This recombinant plasmid pCold-Cap was transformed into E. coli Rosetta 2, and PGETV Cap fusion protein was expressed through IPTG induction. The results showed that the Cap gene obtained efficient and soluble expression in Rosetta 2 induced by 0. Immol/L IPTG under 15"C for 24h, the expression quantity was 40. 2%. The product had a molecular mass about 32. 3kD as expected. The target protein was separated in gel slices and used to immunize Balb/c mice. The polyclonal antibody with high titer against Cap protein specifically analyzed by Western blot was obtained. The successful preparation of the polyclonal antibody laid the foundation for the further study on the detection and identification of PGETV.
