| ObjectiveA new type of interferon was discovered by two independent groups in 2003,thus IFN-λ family, which is composed of three members, including IFN-λ1( IL-29),IFN-λ2( IL-28A) and IFN-λ3( IL-28B). IFN-λs signal through the IFN-λ receptor complexes and activate JAK-STAT pathways to induce biological activities, including antivirus, antitumor and immune regulation. Compared to IFN-α, the activated time of STAT1/2 phosphorylation intracellular is significantly longer when IFN-λs act on liver cells. Further studies demonstrated the receptor of IFN-λ distribute in blood and nervous systems significantly less than IFN-α, so that treatment of IFN-λ will decrease the side effect in blood and nervous system. So IFN-λ may be an even better choice for those patients who take no response to IFN-α or cannot stand the serious side effects caused by IFN-α.We cloned the mouse IFN-λ3 gene and transfected it into BHK cells, then we research the bioactivity of the IFN-λ3 on the cell line A549. In this paper, the antiviral effect of m IFN-λ against HBV was determined in a hydrodynamic injection(HI)mouse model. This work will help to reveal the mechanism of antiviral effect of IFN-λin mouse model and develop novel antiviral drugs for HBV infection.1、The plasmid of mouse IFN-λ3 was gotten from the company, and specific primers were designed. The total DNA sequences of IFN-λ3 were amplified by PCR using the specific primers. And then the PCR products were cloned into p MD18-T vectors.2 、 The p MD18-T-m IFN-λ3 vector and p IRES2-EGFP were digested by restrict endonucleases SalⅠand Bam HⅠ, then m IFN-λ3 were combined with p IRES2-EGFP to get the expression vector p IRES2-EGFP-m IFN-λ3.3. The expression vector pIRES2-EGFP-IFN-λ3 was transfected into BHK cells by Lipofectamine 2000 to set up cell line BHK-IFN-λ3 and injected into the tail vein of mice in a volume of 0.9% Na Cl equivalent to 10% of the mouse body weigh with different amounts. The supernatant of BHK-m IFN- λ 3 and serum of the mouse were collected to detect the expression of m IFN-λ3.4. The biological activity of IFN-λ3 secreted by expressing cell line BHK-IFN-λ was detected, such as anti-viral and anti-tumor.5、10mg p AAV-HBV1.2 was injected into the tail vein of mice in a volume of 0.9%Na Cl equivalent to 10% of the mouse body weigh together with 20 mg pm IFN-a4、20mg pm IFN-λ3 、 20 mg pm IFN-a4 and 20 mg pm IFN-λ3 separately. The mice injected only with p AAV-HBV1.2 plasmid was used as negative control and the mice injected only with pm IFN-a4 was used as positive control.6、Many indicators in mouse serum samples were collected at 1, 4, 7, 10, 15, 20, 25,30, 40, 50 and 60 days post injection(dpi) and determined by commercial enzyme-linked immunosorbent assay(ELISA), including the amount of m IFN-a4,m IFN-λ3, HBs Ag and HBe Ag.7、Liver tissue was taken from the mice receiving HI at 1, 10, 20, 30 and 60 dpi and embedded in paraffin. The liver sections were also stained with hematoxylin to track the inflammation of the liver.8、Serum HBV DNA level was detected by real-time quantitative PCR by specific commercial kits. Meanwhile, total DNA was extracted from collected liver tissue at indicated time points. Then HBV DNA copies were detected by using real-time quantitative PCR.9、Total RNA was extracted from collected liver tissue at indicated time points by commercial kits. RNA was reverse-transcribed and the product was used for analyzing the copy number of mouse IRF3, IRF5, IRF7, ISG15, USP18, OAS and MX1 c DNA by using real-time quantitative PCR.Results1、The expression vector p IRES2-EGFP-m IFN-λ3 was successfully constructed,identified by PCR, restriction enzyme digestion and sequencing, the results consistent with the known one. And pm IFN-λ3 successfully expressed m IFN-λ3.2、 The plasmid of pIRES2-EGFP-m IFN-λ3 transfected into BHK cell expressed m IFN-λ3 efficiently. The concentration of supernatant was up to 1750 pg/ml at point of 24 hours after transfection, and the high concentration maintained for about 72 hours.3、 Mouse IFN-λ3 expressed by the cell, transiently transfected with the plasmid of p IRES2-EGFP-m IFN-λ3, has biological activity of anti-proliferation on the cell line A549 and antivirus against EMCV.4、Compared to the control group injected with p AAV-HBV 1.2 alone, the serum HBs Ag level of the mice co-injected with pm IFN-λ3 significantly decline within15 dpi, and HBe Ag decline within 20 dpi, and HBV DNA level significantly decline within 30 days. On the other hand, the serum HBs Ag and HBe Ag and HBV DNA level in the mice co-injected with pm IFN-a and pm IFN-λ significantly decline compared to the control group injected with p AAV-HBV 1.2 alone within dpi 30 days, yet the amount of HBV DNA in the liver show no significance.4、Different degrees of inflammation were observed during the period of different points in this HBV replication mouse model. The inflammation in group injected with p AAV-HBV 1.2 alone and co-injected with p AAV-HBV 1.2 and pm IFN-λ3were more remarkable than the other two groups,indicating that m IFN-α4 alone or m IFN-α4 combined with m IFN-λ3 can inhibit inflammatory cells of infiltration to the inflammation.5、Compared to the group injected with p AAV-HBV 1.2 alone, the expression of IRF3,IRF5, IRF7, ISG15, USP18, OAS and MX1 of the group co-injected with p AAV HBV-1.2 and pm IFN-λ3 were up-regulated slightly, but have no significance between them. However, these indicators of the group co-injected with pm IFN-a4 and pm IFN-λ3 were up-regulated at different points compared to the group injected with p AAV-HBV-1.2 alone at different degrees, showing that m IFN-α4combined with m IFN-λ3 can activate JAK-STAT pathway and a large number of interferon-stimulating genes. These effectors block viral transcription, degrade viral RNA and inhibit the process of HBV replication.Conclusion1、 The expression vector p IRES2-EGFP-m IFN-λ3 was successfully constructed.And pm IFN-λ3 efficiently expressed m IFN-λ3 in BHK cell and the liver of mouse.2、 Mouse IFN-λ3 expressed by the cell, transiently transfected with the plasmid of p IRES2-EGFP-m IFN-λ3, has biological activity of anti-proliferation on the cell line A549 and antivirus against EMCV.3、m IFN-λ3 alone only suppress significantly the level of serum HBs Ag, HBeAg and HBV DNA at the early stage, within 15 dpi, in the mouse model based on hydrodynamic injection.4、m IFN-α combined with m IFN-λ can activate JAK-STAT pathway and lead to the activation of a large number of interferon-stimulating genes. These effectors block viral transcription, degrade viral RNA and inhibit the process of HBV replication. |
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