Expression Of The Mutant IL－2 In Pichia Pastoris,Purify And Analysion Bioactive

Interleukin-2(IL-2) was initially isolated as a T cell growth factor and had been shown to direct the expansion and differertiation of several hematopioetic cell types.Clinical studies using IL-2 in the treatment of AIDS have been encouraging,due to its critical role as a proliferative signal for several types of cancer,based on its stimulation of cytotoxic,antitimor cells .Today,human IL-2 is produced completely by genetically engineered method,and it has been proved that genetically engineered recombinant human IL-2 has almost the same function and clinical effect as wild IL-2. Objective: Nearly these years .although genetically engineered IL-2 has been focused all of us attention upon its treament of liver inflammation and several types of cancer ,there are still some toxic side effects on sick men,such as heating, shivering with cold ,nausea,feeling nauseated,fainting,expriencing exhausted and Capillary leakage syndrome when this drug was used with a large amount or for a long time .So it is necessary to reconstruct rhIL-2 for inprovement of its stability and bioactivity ,decreasing its toxic side effects,In order to obtain a high level express of human IL-2 cDNA with PCR based mutagenesis method. And also we had determined the new mutant variant human IL-2 cDNA structure and its protein products activity.This method can be used to study gene modification ,the structure or function of protein and the relationship between themselves .Methods:Mature human Interleukin-2 gene was amplified from IL-2cDNA by PCR methods,By using the technique of site-specific mutagenesis with a synthetic oligonucleotide primer ,we replaced the codon for cysteine-125 of human IL-2 with alanine ;the codon for leucine-18 with methionine;the codon for leucine-19 with serine;Restriction enzymine analysis was carried out and nucleotide sequences of the PCR products were determined by DNA sequence.Natural human IL-2 cDNA (NhIL-2 cDNA) and mutant variant human IL-2 cDNA(MvhIL-2) have been inserted downstream of promoter with pPIC9K expression vector ,The PCR product precisely engineered into an intermidiate vector pPIC9K which digested with SnaBI,EcoRI,ligated by T4 DNA ligase and phosphatase alkaline .In order to obtain a lot of pPIC9K-MvIL-2DNA,we transformed it into E.coli Top10F',expanding plasmids,then transferred into Pichia pastoris ,and expressed themutant protein(C125A/L18M/L19S)in Pichia pastoris.The biological activity of the purified protein(IL-2- C125A/L18M/L19S and wild IL-2) were determined with MTT assay using IL-2 dependent CTLL-2cells.Result:the mutant IL-2 was successfully constructed and expressed in Pichia pastoris .Linearing pPIC9K-MvIL-2DNA with Sall,we transformed it into eukaryotic expression system-pichia yeast.Transformants were fermented in a shaking incubator and fermentor,analyzing the supernatants for protein expressing by Coomassie-stained 15% SDS-PAGE,there are expression by MvhIL-2.It has immunocompetence by Western-blotting,we can get purified MvIL-2 by millipore filtration to concentration,Econo-PacS strongly alkali anion exchanger cartridge .Purified IL-2-C125A/L18M/L19S protein had stimulating activity similar to the wild type of IL-2 as assay by IL-2-dependent CTLL-2cells.However, the stability of IL-2-C125A/L18M/ L19S was superior than that of IL-2 at different temperatures . Pichia pastoris yeast cells integrating the plasmid pPIC9K MvhIL-2 had produced a high level of human IL-2 about 30% of the total yeast body protein .High level expression products of IL-2 with highly activity had been puried . Conclusion:The human IL-2cDNA can be reconstructed with PCR mutagenesis method to et a new human IL-2cDNA mutant .The activity of this MvhIL-2 expressed protein we have obtained was 5 times of the NhIL-2,So IL-2-C125A/L18M/L19S had an advantage over wild type of rhIL-2 in storage stability and activity as assayed by IL-2-dependent CTLL-2 cells.