Prokaryotic expression and purification of human immunodeficiency virus p24 antigen.
- Author:
Jun HOU
1
;
Pan-yong MAO
;
Shi-wen HONG
;
Yan HU
;
Jian-yang YANG
;
Hong-hui SHEN
;
Lei ZHU
Author Information
- Publication Type:Journal Article
- MeSH: Blotting, Western; Chromatography, Affinity; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Escherichia coli; genetics; Gene Expression; HIV Core Protein p24; genetics; isolation & purification; metabolism; Humans; Plasmids; genetics; Polymerase Chain Reaction; Recombinant Proteins; isolation & purification; metabolism
- From: Chinese Journal of Experimental and Clinical Virology 2005;19(1):28-31
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo express and purify the HIV p24 gene in E. coli cells, and identify p24 antigen activity.
METHODSThe full length gene fragment of HIV p24 was amplified by PCR and inserted into the pRSET vector in order to construct the pRSET-p24 recombined vector. After transforming into E. coil, the purified p24 protein was prepared by metal-ligand affinity chromatography (IMAC). The accuracy of inserted gene and activity, specificity of HIV p24 proteins were detected by two enzymes digestion technology, SDS-PAGE, Western Blot (WB) and ELISA.
RESULTSThe length of the HIV p24 gene fragment was 690 bp after digesting the recombinant plasmid T-p24 and pRSET-p24 with BamH I and Hind III. The expressed proteins had a single expected band of about 24 x 10(3) in SDS-PAGE. The specificity and activity of p24 protein were tested by WB and ELISA.
CONCLUSIONThe HIV p24 sequence from HIV-1 gene plasmid hasbeen expressed in E. coil. This protein possessed good specificity and activity.
