[Construction and expression of plasmids containing different truncated core proteins of hepatitis C virus derived from tumor and non-tumor tissues].
OBJECTIVE
To study the role of different truncated core proteins (CORE) of hepatitis C virus (HCV) played in the pathogenesis of HCV persistent infection and hepatocellular carcinoma, and to construct seven different truncated prokaryotic expression plasmids of HCV CORE.


METHODS
The gene sequences of different truncated HCV genotype 1b CORE were amplified from plasmids containing CORE sequences derived from tumor and non-tumor tissues of a patient infected with HCV. Amino acid (aa) lengths of HCV BT (from tumor tissue, patient B) were: 1-172 aa, 1-126 aa, 1-58 aa, 59-126 aa, 127-172 aa; of BNT (non-tumor tissue, patient B) were: 1-172 aa and HCV C191 (HCV-J6): 1-172 aa. PCR products were cleaved with restriction enzymes BamH I and EcoR I and cloned into pGEX-4T-1. Positive clones were transformed into BL21 and glutathione S-transferase(GST)-CORE fusion proteins were expressed with isopropylthio-beta-D-galactoside (IPTG) induction, purified and verified by Western blot.


RESULTS
Different truncated GST-CORE fusion proteins were expressed with different quantities. Except the fragment of 59-126 aa, the longer the fragment, the less its expression. The levels of truncated expression of CORE of BT and BNT were higher than that of C191, even though they all contained 1-172 aa. Some of truncated CORE of HCV genotype 1b could form dimmers.


CONCLUSIONS
Successful construction of truncated GST-CORE expression plasmids lays a basis for future study of the function of different domains of CORE of different HCV strains; different expression levels of HCV COREs might be related to their different hydrophobicity, cytotoxicity and their functions in HCV structure and their roles played in the pathogenesis; the domain of 59-126 aa is responsible for the HCV genotype 1b CORE dimmer formation.
Yan
Xue-bing
XB
Institute of Infectious Diseases, the First Affiliated Hospital, College of Medical Sciences, Zhejiang University, Key Lab of Infectious Diseases, Hangzhou 310003, China.
Chen
Zhi
Z
Delphine
Boucreux
B
Nicole
Pavio
P
Christian
Brechot
B
D006528:Carcinoma, Hepatocellular / Q000821:virology; D003001:Cloning, Molecular; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics; D019698:Hepatitis C, Chronic / Q000821:virology*; D006801:Humans; D008113:Liver Neoplasms / Q000821:virology*; D010957:Plasmids / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D014758:Viral Core Proteins / Q000096:biosynthesis / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; C433031:hepatitis C protein F, Hepatitis C virus