[Preparation of recombinant IL-1 fusion protein as the substrate for HCV NS3 serine protease].
OBJECTIVE
To prepare recombinant IL-1 fusion protein as the substrate for the HCV NS3 serine protease.


METHODS
NS5A-B gene fragment (2,412-2,427aa) synthesized by PCR was subcloned into prokaryotic expression vector pBVIL1 to fuse with IL-1 gene and the recombinant vector pBVIL1/NS5A-B was transformed into E.coli strain HB101. The fused protein was induced to express at 42degreesCelsius and purified by two-step column chromatography. The proteolysis of the purified IL-1 fusion protein catalyzed by NS3 serine protease was analyzed with SDS-PAGE, Western blot and ELISA.


RESULTS
NS5A-B fragment gene was correctly subcloned into pBVIL1 vector and the fusion protein was expressed as inclusion body in transformed HB101 cells. The recombinant fusion protein can be cleaved into smaller fragments by NS3 protease.


CONCLUSIONS
The recombinant fusion protein can be cleaved by NS3 serine protease successfully and specifically, suggesting that it can be used as a surrogate substrate of NS3 serine protease in searching for inhibitors of this protease.
Hu
Wei
W
Department of Vaccine Engineering, Beijing Institute of Basic Medical Sciences, Beijing 100850, China. sdhuwei@hotmail.com
Liu
Wen
W
Song
Xiao-guo
XG
Zhu
Cui-xia
CX
Ling
Shi-gan
SG
D000595:Amino Acid Sequence; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics; D016174:Hepacivirus / Q000378:metabolism*; D007375:Interleukin-1 / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
D007375:Interleukin-1; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D012697:Serine Endopeptidases