Expression Of Escherichia Coli Derived HCV F Protein And Its Modulation Of Th1/Th2Cytokine Expression

In order to understand the role of HCV F protein in HCV chronic infection andhepatocellular carcinoma, this experiment was designed to adopt F protein to treatperipheral blood mononuclear cells, therefore ELISA was used to test IFN-γ andIL-5expression of PBMCs. We also analyzed the effect F protein puts on Th1/Th2cytokine expression patterns with the hope of understanding the function of thisprotein in hepatitis C diseases such as hepatocellular carcinoma. As well weobserved F protein expression in patient groups with different ages and stages bydetecting F antibodies in the serum. All we had done was to understand the what roleF protein played in HCV life cycle.Methods:(1)After extracting the recombinant plasmid, pEGFPC3-F, F genechimera was obtained by double enzyme cutting in BamH I and EcoR I. Then F genewas collected after electrophoresis and connected with pcoldⅡ plasmid, which cameto result in the target recombinant plasmid. The new recombinant pcoldⅡ-F wastransformed into Escherichia Coli BL21. Double enzyme cutting, as well as DNAsequencing, was used to identify the recombinant plasmid（2）After its transformationinto BL21, those bacterium were inducted by IPTG. Binding buffer was used todissolve bacterial precipitation,which was then sonicated on ice.F protein was elutedfrom Nickel column by elution buffer. The F protein solution was dialyzed in thetemperature of4℃.(3)After HCV1b-F protein was purified, it was then used asantigen to identify F antibodies in the serum of HCV patients. In the enzyme linkingimmunosorbent assay, Horseradish peroxidase labeled anti-human IgG antibody wasadopted in ELISA to identify F antibodies in40HCPs and20HCCPs.(4)PBMCs were extracted from HCV chronic patients and HCC patients. F protein was used asstimuli to PBMCs.72hours after that, cell supernatant was collected to identify thelevel of interleukin-5and interferon-γ. The ELISA results were analyzed by SPSSsoftware.Results:(1)The enzyme cutting assay results showed that F gene was correctlyinserted into pcold Ⅱplasmid. And the DNA sequencing also confirmed this.(2)SDS-PAGE electrophoresis showed that the expression of F protein came to havean approximate18kD mass.After F protein was renatured, the protein concentrationwas tested which came out to be0.94mg/ml after renaturation. Coomassie bluestaining showed the protein mass was18kD, which fits closely with our targetprotein. And the amino acid sequencing further confirmed that it was exactly Fprotein.(3)ELISA results indicated that F protein reacted with serum from22HCVchronic patients and12HCC patients.(4)After testing the cytokine level, it turnedout that the level of IL-5in F-Ab(+)CHP group was significantly higher than that inF-Ab(-)CHP group（P<0.05）, but level of interferon-γ in F-Ab(+)CHP group wassignificantly lower than that in F-Ab(-)CHP group.（P<0.05） As well, the level ofIL-5in F-Ab(+)HCC P group was significantly higher than that in F-Ab(-)HCCPgroup（P<0.05）,but the level of interferon-γ in F-Ab(+)CHP group was significantlylower than that in F-Ab(-) CHP group（P<0.05）.Conclusions:These results indicated that HCV1b F protein can up-regulate theexpression of IL-5but down-regulate the expression of interferon-γ. It was helpful toprovide clues for exploring the function of F protein in HCV chronicity.