Gene Cloning And Expression Of IFN-λ And Its Effects On Liver Injury And Hepatoma Cell Growth

IFN-λ, a new class of interferon, was founded in2003. It has three members: IFN-λ1、IFN-λ2、IFN-λ3. Through the receptor complex IL-28Rα and IL-10Rβ, it exerts its biological function.IFN-λ has three functional genes in human, the sequence of IFN-λ2and IFN-λ3is of highsimilarity and so is their non-coding flanking sequences. They share part of the cis-acting elementsand have the same mode of regulation with IFN-λ1. However, mice only have two functionalgenes, IFN-λ2and IFN-λ3, which are highly conserved with that in human genome sequence.After binding to the member receptor complex, IFN-λ activated the JAK-STAT signalingpathway. Activated STAT heterodimer combined the interferon-stimulated response element(ISRE) to regulate the expression of interferon-stimulated genes (ISGs). While activated STAThomodimer combined the IFN-γ activation sequence(GAS) to regulate IFN-γ expression.The mediated signaling pathway of IFN-λ was similar with type I interferon, so the biologicalfunction of IFN-λ and IFN-α is overlap. Because of the anti-virus function, IFN-λ can inhibit thereplication of HBV, HCV and other virus in the cells. It also has anti-proliferation and anti-tumoractivity. IFN-λ participated in immune regulation mediated Th1/Th2response, regulated theactivity of immune cells, and relate to the occurrence of allergic inflammatory diseases.This study we used prokaryotic technology to express and purify abundant IFN-λ protein andresearched the role of IFN-λ to murine hepatocellar injury induced by Con A and discussed theinfluence to the growth of hepatoma cell.Methods:1. We used cloning technology to clone IFN-λ cDNA and insert into the prokaryoticexpression pET-44vector, and then constructed pET-44-IFN-λ vector.2. Transformed the pET-44-IFN-λ vector into E.coli BL21(DE3) stain, and induced it expressthe targeted protein containing6×His tag. Then we analyzed its solubility, expressed and purifiedthe IFN-λ protein.3. Con A-induced liver injury model was estabilished. ICR mice were randomly divided intofour groups：①normal control group (NS)；②ConA+pcDNA3.1(+) group (Con A+CP)；③ConA+pcDNA3.1-IFN-λ3group (Con A+IFN-λ3)；④ConA group. Through High-PressureFluid Dynamics inject pcDNA3.1(+) and pcDNA3.1-IFN-λ3plasmid into mice. Detected the pathological changes by HE staining, tested relatinonal cytokines by real-time PCR, and thenevaluating the effect of IFN-λ to the Con A-induced liver injury.4. Detected the expression of IFN-λ receptor in HepG2cells. Using different concentrations (0,10ng/mL,50ng/mL,100ng/ml,500ng/ml,1000ng/ml) to treat HepG2cells24h or48h, test theinfluence of hepatoma cell.Results:1. IFN-λ was amplified by PCR, and insert into prokaryotic expression vector pET-44,successfully construct the pET-44-IFN-λ3prokaryotic expression plasmid.2. E. coli BL21(DE3) stain transformed with pET-44-IFN-λ3was induced to express proteinfor24h under1mM IPTG, at20oC. A purity of about93%IFN-λ3protein was purified by usingNi-NTA Agarose.3. Compared with NS group, the lobular architecture of liver tissue had different degree ofdamage and the cells were edema in ConA+CP, ConA+IFN-λ3and ConA groups, while thedamage was more serious and some cells were necrosis or apoptosis in ConA+IFN-λ3group. Theexpression of IFN-λ and TNF-α in the last three groups of mice liver tissue were significantlydifferent compared with NS group, and ConA+IFN-λ3group was the highest; At the same time,the expression of IL-4and IL-12in the last three groups has significant difference from NSgroup，but there was no statistically significant in ConA+IFN-λ3group and ConA+CP, Con Agroups.4. HepG2cells can express IFN-λ receptor IL-28Rα and IL-10Rβ. Use differentconcentrations (0,10ng/mL,50ng/mL,100ng/mL,500ng/ml,1000ng/ml) of IFN-λ to treatHepG2cells. We found it can affect the proliferation of HepG2cells without a dose or timedependent.. While different concentration of IFN-λ could affect cell cycle, made it arrest G1phasewithout dose or time dependent.Conclution:1. We successfully constructed prokaryotic expression vector pET-44-IFN-λ3;2. We induced IFN-λ3expression in prokaryotic cells and purified IFN-λ3proteinsuccessfully;3. IFN-λ3participated in the mice liver cell injury process induced by Con A. It aggravatedthe cell damage and made the cell necrosis or apoptosis. IFN-λ3stimulated inflammation may beinvolved in Th1response mediated the activation of T cells and macrophages and increased theexpression of IFN-γ and TNF-α.4. IFN-λ1could inhibit the proliferation of HepG2cells without dose or time dependent, itmight regulate the cell cycle enabling cell cycle arrest in G1phase.