Construction, expression and biological assessment of BPI23-Fcgamma1 recombinant protein prokaryotic expression vector.
- Author:
Yunqing AN
1
,
2
;
Yuanzhi GUAN
;
Yan KE
;
Guizhen YANG
Author Information
- Publication Type:Journal Article
- MeSH: Antimicrobial Cationic Peptides; Blood Bactericidal Activity; drug effects; Blood Proteins; biosynthesis; genetics; pharmacology; Carrier Proteins; biosynthesis; genetics; pharmacology; Cell Adhesion Molecules; Escherichia coli; genetics; metabolism; Genetic Vectors; HL-60 Cells; Humans; Membrane Proteins; Recombinant Fusion Proteins; biosynthesis; genetics; pharmacology
- From: Chinese Medical Sciences Journal 2002;17(3):140-147
- CountryChina
- Language:English
-
Abstract:
OBJECTIVETo construct pBV-BPI600-Fcgamma1(700) recombinant expression vector, to transform it into Escherichia coli DH5alpha, and to induce the expression of BPI23-Fcgamma1 anti-bacterial recombinant protein.
METHODSGenes coding for BPI23 and Fcgamma1 were amplified by RT-PCR from mRNA extracted from HL-60 cell and normal human leukocytes; recombinant cloning vector and recombinant expression vector were then constructed. pBV-BPI600-Fcgamma1(700) recombinant expression vector was transformed into the competent Escherichia coli DH5alpha and BPI23-Fcgamma1 recombinant protein was expressed by a temperature-induced method.
RESULTS(1) Expected amplified products BPI600hp and Fcgamma1(700bp) were obtained by RT-PCR method. (2) pUC18-BPI180, pUC18-BPI420 and pUC18-Fcgamma1(700) recombinant cloning vector were successfully constructed, and sequences were identical with the reported ones. 3) pBV-BPI600-Fcgamma1(700) recombinant expression vector was successfully constructed, and the enzyme digestion analysis showed an expected result. (4) The expression level of BPI23-Fcgamma1 recombinant protein accounted for 20% of total bacterial proteins. (5) The renatured BPI23-Fcgamma1 recombinant protein showed bacteriocidal activity and biological function of complement fixation, and opsonization.
CONCLUSIONpBV-BPI600-Fcgamma1(700) recombinant expression vector was successfully constructed, and BPI23-Fcgamma1 recombinant protein with double biological activity of BPI and IgGFc was expresed in Escherichia coli.
