Study On Gene Cloning, Expression In E.Coli And Protein Purification Of RhIL-15

So far, it has twelve years since human interleukin-15 has beendiscovered. With the development of study about the cytokine, moreand more biological funcation of hIL-15 has been understanded, whichindicates its wonderful prospect of clinical application.Human interleukin-15 is one of the multifunctional cytokine. AlthoughIL-2 and IL-15 are not closely linked and not share significantsequence homology, the organization of the two genes was found to bevery similar and characteristic of the helical cytokine family. Il-15shares many biological properties with IL-2. IL-15 may be a novelanabolic agent to increase skeletal muscle mass. IL-15 was originallydiscovered as a T cell stimulatory factor present in the culturesupernatants of a simian kidney epithelial line, CV-1/EBNA. Andersonet al. mapped the human IL-15 gene to chromosome 4 band 4g31.IL-15 can stimulate the proliferation of T lymphocytes, and acomplementary DNA clone encoding this new T cell growth factorwas isolated. The cytokine, designated as interleukin-15 (IL-15), isproduced by a wide variety of cells and tissues.In the present study, a series of researches correlated with expressionof genetically engineered E. coli transferred with IL-15 gene had beenobserved. Purification methods for the product were investigated andits activity, biochemical and physical characteristics were identified.After recovered, constructed E .coli were inoculated into ZYM-5052media and expressed under the control of temperature.Bacteria werecracked by ultrasonic vibrations and the amount of protein wasdetermined by SDS-PAGE electrophoresis combined with laminascanning. The protein was purified by three steps: prepation ofinclusion bodies;ion-exchange chromatography;gel filtration. Thepurity was measured by SDS-PAGE electrophoresis and laminascanning. Its biological activity was manifested by CTLL-2proliferation assay. The molecular weight of purified IL-15 wasestimated by reduced SDS-PAGE electrophoresis. Its homogeneitywas confirmed by UV scanning.Engineering bacteria were grown in ZYM-5052 media and thencultured at 42℃ for 7h. The quantity of recombinant IL-15represented 18.6% of total bacterial protein. the specific activity was1.1×107IU/mg, the yield was 42%. The purity reached to 95.3%detected by SDS-PAGE electrophoresis combined with laminascanning.After ion-exchange chromatography, there were two peaksand the second peak was the target protein of rhIL-15, A series ofbiophysical and biochemical features were also characterized and samewith the previous reports. The molecular weight was 12.9kDdetermined by denatured SDS-PAGE electrophoresis. The engineeredE .c oli expressed with high level of rhIL-15.Ideal purity ofIL -15 had been obtained through threes teps: prepationof inconlusion bodies;ion-exchange chromatography;gel filtration.The biophysical and biochemical features such as molecular weight,characteristic in UV scanning.These experienced results will make the technical process possible toexpand to large scale products and clinical usage.