Expression And Fermentation Of The Fusion Protein Of Human Serum Album/Interleukin-2

Human interleukin-2(IL-2) is produced by T cells in vivo, it is a kind of glycoprotein which is about 15 kDa. It can enhance immunity cases through promoting the reproduction of T cells in human body. The half-life of native IL-2 is about 6.9 min. In order to prolong the half-life, the plasmid pPIH which includes the encoding gene of IL2-HSA was constructed. According to the research, exchanging dissoving oxygen and methanol concentration were tested, a result was showed that the most expressing level of P. pastoris KM71/pPIH is 44 mg/L, there is no more potential in this transformant.Then the recombinant plasmid pPIH was transformed into three different kinds of hosts,they were P. pastoris GS115, P. pastoris KM71 and P. pastoris SMD1168. After the orthogonal test, it can be showed that the best expressing host was P. pastoris GS115. The concerntration of IL2-HSA is 314 mg/L. Western blot showed that the fusion protein contained both IL-2 and HSA. The molecular weight of the product was 81 kDa, which was reached the expected. After desalting and freeze-drying,the bioactivity of the crude protein was checked by CTLL-2/MTT method and the result showed that the fusion protein IL2-HSA can stimulate the division of CTLL-2 cell.The IL-2 bioactivity of the crude protein was 1.05×106 IU/mg.As the encoding sequence of the nature human interleukin2 and the preference codon of the Pichia pastoris were analyzed, the mutant human IL-2 gene which had changed cysteine-125 into alanine was designed and composed. The recombinant transformant P. pastoris GS115/pPHIm was constructed. The expressing level after orthogonal test is 143 mg/L. After desalting and freeze-drying,the bioactivity of the crude protein was checked by CTLL-2/MTT method.The IL-2 bioactivity of the crude protein was 1.51×106 IU/mg.