| Interferon is a very important cytokine with high antiviral activity. It plays a keyrole in innate immune responses, as well as defense against viral infection. Interferoncan be classified into three types: type I, type II and type III. Among them, type Iinterferon received most of the attention. Many IFN-α preparations have been used inclinical treatment. Especially in the treatment of hepatitis C virus (HCV) infection,Type I interferon has its unshakable status.Hepatitis C is now a global epidemic, especially in China. Hepatitis C virus cancause chronic liver inflammation and may result in liver fibrosis and hepatic necrosis.Some will develop into hepatic cirrhosis, or even turn into liver cancer. In the comingyears, mortality caused by hepatitis C virus infection is expected to continue toincrease. It has become a serious social and public health safety problem. Currenttreatment for chronic hepatitis C includes recombinant polyethylene glycol (PEG)IFN-α plus ribavirin. Although this treatment can produce durable antiviral responsein more than half of the individuals, it’s also accompanied by some of the obviousside effects, due to the broad tissue distribution of INF-α receptor.Type III Interferon, which was discovered in2002-2003, has similar functioncompared to Type I interferon. They both can induce cells into antiviral state. Unliketype I Interferon, target cells of type III Interferon are relatively limited, includingmainly epithelial cells and hepatocytes. It has been demonstrated that type IIIInterferon can be a potential drug to replace type I Interferon in the treatment of viralinfection and tumors. Since its discovery, with its characteristics of having antiviralactivity and fewer side effects, type III Interferon has been proposed as a replacementof IFN-α in the treatment of hepatitis C. However, although type III interferon hassimilar antiviral activity to IFN-α, its efficacy of anti-hepatitis C virus is not as goodas IFN-α. On the other hand, natural type III interferon is more difficult to express and purify in vitro using gene engineering techniques. This is an important factor limitingthe use of type III interferon.Given the background about hepatitis C treatment and interferons, this workanalyzed the sequence and structural characteristics of type III Interferon familymembers at both gene and protein levels; and using biological modeling techniques,we designed a series of new genes encoding novel interferon analogues; then usinggenetic engineering technology, we expressed these artificial genes in Escherichiacoli, and by optimizing refolding conditions, we obtained refolded correspondingrecombinant proteins. Using protein purification technologies including HighPerformance Liquid Chromatography (HPLC), Affinity Chromatography and IonExchange Chromatography, we obtained novel recombinant human type III interferonanalogues with high yield and purity. Finally, we chose HepG2cell line to measurethe antiviral activity of those recombinant proteins. We chose two genes related tocellular antiviral responses-Mx (myxovirus resistance) and OAS(2’,5’-oligoadenylate synthetase), as reporters, and used real-time fluorescentquantitative RT-PCR technique to measure the expression of Mx and OAS andevaluate the antiviral activity of the novel interferon analogues. In summary, througha series of studies including in vitro expression, re-folding, purification, and activityassay, we obtained three new recombinant human interferon analogues with antiviralactivity but are easier to express and produce than natural type III IFNs. The resultsform a foundation to develop new generation of IFN pharmaceuticals with highefficacy, low cost, and most importantly, fewer side effects. |
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