真核表达重组小鼠IL-15/Fc融合蛋白的制备和活性鉴定

Preparation of eukaryotic and expressed mice IL-15/Fc fusion protein and confirmation of its bioactivity

目的制备真核表达小鼠白细胞介素15(IL-15)和IgGγ1Fc段融合蛋白,并探讨其对抗原特异性CD8+T细胞的作用。方法运用RT-PCR方法,从B6小鼠脾细胞中分离小鼠IL-15和IgGγ1绞链区-CH2-CH3Fc cDNA。在IL-153′端和Fc5′端引入BamHI酶切位点,将IL-15和Fc直接连接,构建小鼠IL-15/Fc融合蛋白基因,再连接到真核表达质粒载体pcDNA3.1(a+)上,转化CHO-S细胞表达、纯化后,研究其活性。结果融合蛋白486bp的编码由162个氨基酸组成,其中含48个信号肽氨基酸的小鼠IL-15前体蛋白和由681bp编码227个氨基酸的IgGγ1-CH2-CH3功能区构成;表达蛋白在IL-15信号肽引导下能高效分泌到培养液中,有二聚体和三聚体两种天然结构,单体分子量约50kDa,能特异性的结合IL-15R并抑制抗原诱导的CD8+T细胞的增殖反应。结论成功构建了小鼠IL-15/Fc融合蛋白真核表达载体,并在CHO-S细胞中得到高效表达。此融合蛋白具有较高的生物学活性,可有效地抑制抗原特异性CD8+T细胞的增殖反应。

OBJECTIVE Interleukin 15 is a powerful T cell growth factor which secreted by non T lymphocyte. Previous study has demonstrated that IL - 15 play a critic role in the growth, differentiation, development and homgentics of CD8^＋ T cell. It is also an important factor which facilitates the survival and maintenance of CD8^＋ memory T cell. Targeting IL - 15/IL - 15R system has been a hopeful therapeutic strategies in RA, multiple sclerosis autoimmune diseases. In current study, we prepared eukaryotic expressed mice interleuokine- 15（IL- 15） and mice IgCγ1 Fc fusion protein and investigated its function on antigen specific CD8^＋ T lymphocyte. METHODS We cloned mice pro - mature IL - 15 protein and IgGγ1 Fc（ hang - CH2 - CH3 region） eDNA with RT - PCR and liga- tied them by endonuclease and constructed IL - 15/Fc fusion protein eDNA. The IL - 15/Fc eDNA was then ligated into pcDNA3.1 （ a ＋ ） plasmid and expressed in CHO - S cell line. Function of IL - 15/Fc fusion protein was tested by inducing the proliferation and ex- pansion of antigen induced CD8^＋ T cell. RESULTS IL - 15/Fc fusion protein was composed of IL - 15 which had 162 amino acid including 48 signal peptide coden by 486 bp eDNA and IgGγ1 hang - CH2 - CH3 region which 227 amino acid eoden by 681 bp eDNA. The natural protein had two structures, dimer and trimer, which had a molecular weight of 100 kDa and 150 kDa. It could also bind to IL - 15R specifically and inhibit the proliferation and expansion of antigen specific CD8^＋ T cell. CONCLUSION Successfully, we prepared eukaryotic expressed mice IL - 15/Fc fusion protein with high bioactivity.