Cloning and Expression of Therapeutic Recombinant Interleukin 29
In this study, we have successfully cloned the E. coli BL21 (DE3) strain that harbors gene encoding for recombinant human interleukin 29 (rh-IL29). Recombinant human IL-29 protein has been highly expressed in LBkan with induction of IPTG 1mM, at 37 oC, 200 rpm, and 4 h of induction time. The protein was purified by FPLC purification process including re-folding, buffer exchange, cation exchange chromatography and gel filtration, resulting the purified protein of 95 % purity, 1 mg/ml total protein concentration, pH at 3.5 – 4.5, molecular weight of 18-20 kDa, no residual host cell DNA and peptide contamination, endotoxin free. The biological activity of rh-IL29 was determined in an antiviral assay using human Hep-2C cells challenged with encephalomyocarditis virus (EMCV). The obtained effective dose 50 (ED50) of the product is at 2.86 ng/mL.
Keywords: Interleukin 29, Cloning and Expression.
1. Introduction
Hepatitis B and C are global health problems in regards to major cause of serious liver diseases. There are approximately 2 billion people having hepatitis B virus (HBV) infection and 130-170 million hepatitis C virus (HCV) carriers worldwide accounting for 2-3 % of the world population (WHO, 2012). Vietnam has high rates of HBV and HCV infection with about 1 to 3 % of Vietnamese population [3]. In which, approximately 80 % of patients failed to clear and become chronic carriers, and 10-20 % of them may progress to liver cirrhosis and hepatocellular carcinoma (HCC) [5,7]. Since 1991, interferons were approved in the United States by the Food and Drug Administration (FDA) as antiviral treatments for disease caused by hepatitis viruses. These products including interferon-2b (Intron A), interferon-2a (Roferon), consensus interferon (Infergen) and PEG-IFNα2a/b combined with ribavirin (RBV) were utilized in the treatment of chronic HBV and HCV infection. However, the current ‘standard’ therapy, a combination of Type I interferon (IFN) or PEG-IFN-α2a/b with ribavirin (RBV), does not produce sustained virologic response (SVR) in all patients treated. This treatment also associated with undesirable hematological and neurological side effects, resulting in reduced compliance and fewer patients completing the treatment.
Moreover, in 2009, genetic study correlated single nucleotide polymorphisms (SNP) on a linkage disequilibrium block in the vicinity of IL-28B (Type III IFN) has been implicated the association between the SNP in proximity to the IL-28B gene affect the SVR of patients treated with PEG-IFN-α/RBV [2]. In addition,interleukin 29 (IL-29) (also known as IFN lambda 1) which belongs to Type III IFN, has amino acid sequence similar to that of IL-28 [8]. IL-28 and IL-29 share common amino acid level with Type I IFN and bind to two receptors on the cell surface including IFNLR1 and IL10R2 [4,1]. These new kind of cytokines were classified based on type receptors which they bound to, resemble genomic structure of the IL-10 family and have some common characteristics with Type 1 IFN such as inhibition of cell proliferation, antiviral and in vitro antitumor [6,10,1]. Furthermore, several studies have confirmed that IL-29 lead to address the problem of the non-response in chronic HCV infected patients [9]. Recently, clinical trials of ZymoGenetics, Inc. (Seattle, WA) in collaboration with Britol-Myers Squibb Company have demonstrated that PEG-IL-29 and ribavirin in treatment-naïve patients with chronic HCV infection appeared a significant safety and efficacy than PEG-IFN-α2a.
Along with the development of recombinant biotechnology, the production of recombinant interferon is considered as effective antiviral therapy of chronic hepatitis B and C and has opened up tremendous promise for valuable scientific researches and practices. However, in Vietnam, few studies on recombinant human IL-29 (rhIL-29) production have been conducted. Therefore, this work would initially research on production of rhIL-29 protein which aims to produce raw materials for PEG-IL29 using in treatment of chronic hepatitis C as well as for further studies.
(Nguồn tin: )