Long-acting interleukin 15 fusion protein and preparation method and application thereof
A fusion protein and interleukin technology, applied in the biological field, can solve problems such as increasing the molecular weight of fused or modified protein drugs, reducing the yield and clinical efficacy of fused protein drugs
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Embodiment 1
[0083] Example 1 Design and preparation of novel long-acting interleukin-15 fusion protein
[0084] The schematic diagram of the designed novel long-acting interleukin-15 fusion protein is shown in figure 1 . Will encode IL15Rα-GGGGS-sFc (SEQ ID NO:5), IL15Rα-(GGGGS) 3-sFc (SEQ ID NO:6), IL15Rα-Fc (SEQ ID NO:7), IL15(N72D) (sequence is SEQ ID NO:4) The gene was synthesized by Nanjing GenScript (Nanjing, Genescript) Company. And the gene was cloned into the eukaryotic expression vector PTT (purchased from Thermo Fisher Company). After the successful construction of the plasmid, it was transiently co-transfected into Expi293 cells (purchased from Thermo Fisher) for expression (where IL15Rα-(GGGGS)3-sFc was co-transformed with IL15(N72D) to obtain FL115-2a, IL15Rα-GGGGS-sFc and IL15 (N72D) was co-transformed to obtain FL115-2b, IL15Rα-Fc and IL15 (N72D) were co-transformed to obtain the Fc fusion protein of IL-15), and purified by protein G resin (purchased from GE Healthcare)...
Embodiment 2
[0085] Embodiment 2 Novel long-acting interleukin-15 fusion protein physicochemical properties
[0086] The new long-acting interleukin-15 fusion proteins (FL115-2a and FL115-2b) were expressed and purified according to the method in Example 1. At the same time, the Fc fusion protein of IL-15 was designed and constructed in the same way, and the unified expression system was used to express and purify the Fc fusion protein to obtain related proteins. Purification by protein G resin (see: Ying Tianlei et al., JOURNAL OF BIOLOGICAL CHEM ISTRY, 2012, 287(23): 19399-19408 for specific transformation, expression and purification methods), and the yield of the new long-acting interleukin-15 fusion protein is as high as 10mg / L, compared with the yield of IL-15 Fc fusion protein 1mg / L, it has obvious advantages, and theoretically has a significant advantage in industrial production costs. At the same time, we took the above-mentioned protein with an equal volume of 100ul for high-pe...
Embodiment 3
[0087] Example 3 Evaluation of biological activity of novel long-acting interleukin-15 fusion protein in CTLL-2 cell line
[0088] To evaluate the biological activity of the novel long-acting interleukin-15 fusion protein, a proliferation assay on the CTLL-2 cell line was used (Santos-Savio A. et al., BiotecnAplic. 2000, 17: 221-4). According to the procedure described below, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (purchased from Sigma) for mitochondrial Staining (see: Mossman T.J. Immunol. Methods. 1983, 65(1-2): 55-63 for the specific method), the biological activity can be measured by stimulating the proliferation of these cells. In a 96-well culture plate (purchased from Costar, USA), in a volume of 50 μL supplemented with 10% fetal bovine serum (FBS) (purchased from Gibco) and 50 μg / mL gentamicin (purchased from Sigma Company) In RPMI medium (purchased from Gibco), serial dilutions of FL115-2a, FL115-2b, positive control (IL15&IL15Rα) an...
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