Expression Of Recombinant Human Interleukin 2 In Pichia Pastoris And Preliminary Studies On Pure IL-2

Interleukin-2(IL-2) is a kind of cytokine with a range of biological activity. It can promote the activated T cell'proliferating intoTd cells and Tc cells,and activate CTL,NK cells and LAK cells. It can induce killer cells to secrete cytokines such as IFN-γ,TNF-α,GM-CSF and so on. IL-2 can also stimulate NK cell selectively and activate B cells directly to promote their proliferation,differentiation and Ig secretion. IL-2 has been used in treatment of cancer and pain and immune adjuvant.IL-2 is used widely and it's need is very high,so this study will explore a series of processes from mass production to activity determination of rhL-2.1 Studies on expression conditions on rhIL-2 in PichiaRhIL-2 gene from E coli to Pichia by conversion was identifide by PCR,after that,when we induced IL-2 gene expressing in Pichia and got high level expressiong strains.We analyed the fermentation supernatants of different time by SDS-PAGE electrophoresis to make sure the high expression time. Then we cultured Pichia with different pH value to make sure the high expression pH. As a result,after electroporation,we got positive results with identification by PCR.That showed the target gene has been inserted into Pichia Pastoris.In later experiments we used the high level expressiong strains we got. The highest expression appers when the fourth day.Therefore, the expression can be drawn the most appropriate time for four days.And the best pH value was pH5.0.We selected a high expression strain,determined the optimal time and optimal pH value of the expression,to provide a good theoretical basis.2 Large-scale fermentation and purification process of rhIL-2 in Pichia After making sure the expression conditions of rhIL-2 in Pichia, we induced large scale fermentating of it in 80 L fermenter. As the fermentation conditions vary widely ,ultimately, we determined the expression of the number of days to three days. Later the fermertation supernatants went through the SP Sepharose XL cationic resin and SourceTM 30RPC hydrophobic interaction chromatography resin.After removal of methanol it was analyzed by AKTA explorer100. Experiment got the pure rhIL-2 and determined it's concentration with Bradford'method. The results showed that we got the target protein with high concentration after he fermertation supernatants goes through the SP Sepharose XL cationic resin and SourceTM 30RPC hydrophobic interaction chromatography resin.It was proved that we have got the protein with high concentration and good purity.The result was 0.191mg/ml after determinning it's concentration by Bradford method,then getting pure protein of rhIL-2.3 Determination of biological activity of rhIL-2It needs to find out that the pure rhIL-2 was active or not. There are a lot of methods to determine the biological activity of rhIL-2,such as establishing animal model,cell culture. In this study, using mouse splenopypens as research object, we measured the biological activity of rhIL-2 by MTT. The mouse splenopypens were culched with concentration rhIL-2 and standard IL-2 and the growth of the cells was measured by MTT to reflecte the activity of rhIL-2. In result, with the increasing concentration of the sample, the absorbance increased to 0.187 from 0.15, it indicated that rhIL-2 showed good biological activity. Compared with the standard,activity of rhIL-2 was lower,but the result was very good. My study started with importing rhIL-2 gene into Pichia,then exploring the fermentation conditions by small-scale fermentation.After that we culched Pichia in 80 L fermertor. The fermentation supernatants went through the cationic resin and hydrophobic interaction chromatography resin and we acquired the pure target protein.It's concentration was 0.191mg/ml.We adopted the MTT method to determine activity of cells.This method is simple andresults clear.Finally the target IL-2 had good activity.So this study established the experimental procedure from expression,separation and purification to determination of activity of rhIL-2.Details of each were explored and discussed and thin provide a good foundation in theory and practice for further production and scientific research.