摘要
目的构建人补体受体2(CR2)-Fc融合蛋白基因表达载体,在中国仓鼠卵巢细胞(CHO)中表达人CR2-Fc蛋白。方法将人CR2片段和IgG1 Fc片段进行连接,克隆入PCI-neo表达载体。脂质体法将测序正确的重组真核表达载体转染CHO K1细胞进行蛋白表达,采用SDS-PAGE、Western blot法对蛋白表达进行检测和鉴定。结果利用双酶切及基因测序结果证实CR2-Fc基因序列正确,重组真核表达载体构建成功;SDS-PAGE检测纯化产物的相对分子质量(M r)与预期结果一致,Western blot法在同样位置检出条带。结论成功构建了CR2-Fc重组真核表达载体,获得了有活性的融合蛋白。
Objective To construct a eukaryotic expression vector containing human complement receptor 2 （CFI2）-Fc and express the CR2-Fc fusion protein in Chinese hamster ovary （CHO） ceils. Methods The extracellular domain of human CF2 and IgG1 Fc were respectively amplified, ligated and inserted into the eukaryotic expression vector PCI-neo. After verified by restriction enzyme digestion and sequencing, the recombinant plasmid was transfected into CHO K1 cells. The ones with stable expression of the fusion protein were obtained by means of G415 selection. The expression of the CR2-Fc fusion protein was detected and cor^nTed by SDS-PAGE and Western blotting, Results FP_,striction enzyme digestion and sequencing demonstrated that the recombinant plasmid was valid. SDS-PAGE showed that relative molecular mass （Mr） of the purified product was consistent with the expected value. Western blotting further proved the single band at the same position. Coactasion We constructed the eukaryotic expression vector of CR2-Fc/PCI-neo successfully. The obtained fusion protein was active and can be used for the further study of the role in HIV control.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2014年第1期1-3,7,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81072501)
