Expression And Purificition Of Interleukin-18 In Eukaryote And Prokaryotic Cell

Interleukin-18(IL-18)is a new cytokine of the Interleukin's family. Interleukin-18 not only induces IFN-(, but also interacts with a lot of cytokine, which play an important role on the response to immunization. Interleukin-18's capital activity of biology as follows:1)Host defense;2) Antitumor immune;3)Proinflammation cytokine;4) Antiallergic reaction. So investigation with IL-18 not only has the significance of the foundation, but also the clinic. Now, the most investigation limited in the relationaship between IL-18 and diseases. The expression and purification of IL-18 in Escherichia coli has been explored; but nobody investigate the expression and purification of IL-18 in pichia pastoris. Our aims are to express and purify IL-18 in Escherichia coli and pichia pastoris, which would provide a opportunity to further study. IL-18 expresses and purify in Escherichia coli ER2566. We construct the Prokaryotic expression vector: pTYB1IL-18, which had been introduced into competence Escherichia coli ER2566. To get the best expressing effect, the optimum temperature and time of expressiong and concentration of IPTG induciong were grobed for. To achieve IL-18, the fusion protein secreted was purified by affinity chromatographic column of chitin beads. We constructed the Prokaryotic expression vector, and identified it by PCR, enzyme cutting and DNA sequence analysis. Then the fusion protein was assayed by 10% SDS-PAGE and Western blot. We expressed IL-18 in the Escherichia coli ER2566 successfully. When the condition are 30℃,pH7,IPTG0.5mM/L and 4hr, the IL-18 can achieve the optimum expression. The expression amount of protein is 22%. After the protein was purified, the amount reached to 90%. IL-18 expresses and Purify in pichia pastoris GS115. We constructed the eukaryote expression vector pPIC9IL-18 by recombinant PCR and asymmetry PCR. To take advantageous of purification, a inteinTag was added to the vector. The new vector was introduced into Pichia pastoris GS115 activated by PEG1000, and the target gene was integrated into the genome of the host. Then the recombinant Pichia pastoris strains were selected and the protein was expressd under the optimum inducing condition. We purified IL-18 by affinity chromatographic column of chitin beads. To achieve a high level of expression the IL-18' in Pichia pastoris, we selected the recombinant Pichia pastoris strains by G418. The eukaryote expression vector pPIC9KIL-18 was constructed succeedly. We utilized the recombinant PCR and asymmetry PCR to construct the eukaryote expression vector pPIC9IL-18. Moreover adding the intein Tag simplify the purification. By PCR, enzyme cutting and DNA sequence analysis; the recombinant vector has been confirmed correct. Then by extracting and linearizing by Sac I, the vector was introduced into pichia pastoris GS115 activated by PEG1000, which is more effective and convenient. Then the recombinant Pichia pastoris strains were selected, and were expressed under the optimum condition. The fusion protein was assayed by 10% SDS-PAGE and Western blot. The amount of expressed protein in the supernatant was 12%. After the protein was purified, the amount of IL-18 reached to 90%. Biologic activity: We cultured the lymphocyte extracted from the blood with the IL-18 and measured the expression of the biologic activity of IL-18 by MTT assays. We cultured the lymphocyte extracted from the blood with the IL-18 and measured the expression of the biologic activity of IL-18 by MTT assays. In a conclusion, the IL-18 cDNA was successfully cloned and expressed in E. coli ER2566 and pichia pastoris GS115. The soluble IL-18 protein was obtained by renaturing and affinity chromatographic column of chitin beads. The recombinant pichia pastoris GS115 successfully expressed the IL-18 initially. The IL-18 product expressed and purified has the biologic activity. The amount of expression product is highly in the E. coli ER2566, but the product was mixed with lots of other protein, most of which ar...