Construction, expression and biological assessment of BPI23-Fcgamma1 recombinant protein prokaryotic expression vector.
OBJECTIVE
To 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.


METHODS
Genes 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.


CONCLUSIONS
pBV-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.
An
Yunqing
Y
Department of Microbiology and Immunology, Capital University of Medical Sciences, Beijing 100054. anyunq@cpums.edu.cn
Guan
Yuanzhi
Y
Ke
Yan
Y
Yang
Guizhen
G
D023181:Antimicrobial Cationic Peptides; D001770:Blood Bactericidal Activity / Q000187:drug effects; D001798:Blood Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D015815:Cell Adhesion Molecules; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D018922:HL-60 Cells; D006801:Humans; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology
D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; C106607:FCGBP protein, human; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C021623:bactericidal permeability increasing protein