| Interferon is one kind of cytokine with high activity and many biological functions secreted by monocyte. Since 1957, the study of interferon has never been interrupted. The fact that interferon plays a important role in anti-viral, anti-tumor and immune regulation has been well known all over the world, and it has been utilized for the benefit of mankind. Interferon α-2b was one of the earliest discovered subtype of interferon family, and was also one of the most widely used interferon. As type I interferon, the IFN-α-2b has the optimal function of anti-viral, anti-tumor and immune regulation among all of the subtypes of interferon. The expression of interferon preferinactive inclusion body, although the renaturation rate has increased with the development of science and technology, most of the protein can not be used efficiently.rhIFN with a lot of biological activities plays an important role in clinical applications especially in HBV, HCV and other viral diseases, and cancer such as HCL,Melanoma,NHL,KS. As biological agent, rhIFN has common problems : side effects; short half-life; easy to induce the production of neutralizing antibodies and so on. Therefore, research on structure modification for it is continuing.Pegylated interferon as a derived product of interferon is composite product obtained by using the click chemistry theory and various reactions of organic chemistry to couple the free radicals of interferon with non-toxic and non-irritating PEG.The coupled interferon has all of the advantages of interferon, and the molecular weight and the half-life are increased. Although biological properties of interferon decreased by the addition of the PEG, the side effects and neutralizing antibodies decreased.The study aims at solving the current situation that the expression of the IFNα-2b in E. coli is inclusive. Genetic engineering was used to reform it and add chaperone TrxA, Smt3 etc. It is able to express soluble and use simple purification technology and SUMO protease to purify. The single conjugation polyethylene glycol mPEG-IFNα-2b was extracted and purified by using the simple purification steps.And then the HPLC and activity tests was used in vitro to determine the purity and biological activity.Experimental content I :We used a multi-chaperone expression vector to express the fusion protein IFN α-2b in Escherichia coli and then researched the purification process of the protein;The bacterial culture, plasmid extraction, plasmid digested and connected, bacteria induced expression, protein purification technology were used to clone the artificially designed and synthesized Smt3-IFNα-2b into self-constructed P32-TrxA vector,and then the recombinant plasmid P32-TrxA- Smt3-IFN α-2b was transformed into Escherichia coli BL21(DE3). The bacteria was induced expression by IPTG,then analyzed by SDS-PAGE.The expressed fusion protein was soluble by using metal chelate chromatography, protease cleavage of SUMO, anion exchange chromatography and gel chromatography to purify the IFNα-2b. And a new purification process of soluble expression of IFNα-2b was established.Experimental content II: the feature that the PEG point modify the IFN was used to get the single polyethylene glycol-modified pegylated interferon, and to research purification process of PEG-IFNα-2b; Through the property of IFNα-2b involved in the experiment and the property of polyethylene glycol reported, we choose the mPEG-SPA20000 to couple by using biology statistical software to design the 5 factors4 levels orthogonal experiment and to obtain the best reaction condition of the interferon and polyethylene glycol coupling.Anion exchange chromatography, affinity chromatography, and gel filtration chromatography were used to extract and purify single polyethylene glycol conjugated interferon mPEG-IFNα-2b with good biological activity, and to establish the purify technology of mPEG-IFNα-2b.Experimental content III : the characteristics of anti-virus, anti-tumor of interferon and pegylated interferon were applied to research the biological activity of human amniotic cells and various tumor cells in vitro;By reagent Lowry and BSA, the concentrations of the two proteins were determined. Using HPLC, the purity of the two interferon was determined. The RPMI1640 with 10% FBS and 10% DMEM was used to dialysis to filter and do the cytopathic inhibition assay according to People’s Republic of China Pharmacopoeia(2015) to determine the antiviral activity of the two interferon. Cell experiments in vitro was applied to detect the restrain and kill effect of different tumor cells of the two interferon. |
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