Construction of eukaryotic expression plasmid containing HCV NS3 segment and protein expression in human HL-7702 hepatocytes.
OBJECTIVE
To construct the eukaryotic expression plasmid containing HCV NS3 segment and to analyze the expression of NS3 protein in normal human hepatocyte HL-7702.


METHODS
We amplified HCV NS3 fragment from plasmid pBRTM/HCV 1-3011 containing the whole length of HCV genome, recombined it with expression vector pcDNA3.1(-) to form the eukaryotic expression vector pcDNA3.1(-)/NS3, and transfected human HL-7702 hepatocytes with the recombined plasmid by cationic polymers. The expressed HCV NS3 protein was detected and analyzed by immunohistochemical method and Western blot.


RESULTS
The amplified NS3 fragments had correct molecule weight and sequence. The successfully constructed eukaryotic expression plasmids were transfected to HL-7702 cells. The expressed NS3 proteins had correct molecular weight 70000.


CONCLUSIONS
Eukaryotic expression vector pcDNA3.1 (-)/NS3 containing NS3 segment of HCV can be constructed, the sequence of NS3 fragments is consistent with the template. Normal human HL-7702 hepatocytes can efficiently express specific HCV NS3 protein in vitro.
Microbiology and Immunology Department, Medical College, Jinan University, Guangzhou 510632, Guangdong Province, China. ljw6610@163.com
Jun-Wu
JW
Li
Ke
K
Li
Jing
J
Jiang
Xiao-Liang
XL
Xu
Ze-Qi
ZQ
Huang
D015153:Blotting, Western; D002460:Cell Line; D003062:Codon; D053938:DNA Fragmentation; D004279:DNA, Viral / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics*; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D003062:Codon; D004279:DNA, Viral; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases