CD137L融合蛋白真核表达载体的构建与表达  

Construction and Expression of Eukaryotic Expression Vector of CD137L Fusion Protein

作　　者：徐胜 王淑珍[1] XU Sheng;WANG Shu-zhen(School of Life Science and Technology,China Pharmaceutical University,Nanjing 211198,China)

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京211198

出　　处：《药物生物技术》2021年第3期233-238,共6页Pharmaceutical Biotechnology

摘　　要：真核表达系统因具有完整的翻译后修饰、表达蛋白免疫原性较小而成为制药行业生产蛋白药物的新方向。该研究目的是构建CD137L融合基因真核表达载体,瞬时转染HEK293F细胞实现高效表达并进行蛋白纯化与活性分析。以该实验室保存的含有CD137L融合基因的质粒p ET11a-FP为模板,采用PCR技术在目的基因N端添加长腹水蚤荧光素酶信号肽和Kozak基因序列、C端添加6×His纯化标签,使用XbaⅠ、EcoRⅠ限制性内切酶酶切并插入pc DNA3.1(+)载体,并对其进行双酶切和测序鉴定,通过转染试剂PEI将构建成功的pc DNA3.1-FP真核表达质粒瞬时转染进入HEK293F细胞,对转染复合物比例及表达时间等参数进行优化。SDSPAGE电泳和Western印迹检测结果表明每毫升细胞转染1.5μg质粒和7.5μg转染试剂、表达4 d时融合蛋白表达水平最高。镍柱亲和纯化结果显示,每升培养基上清可以纯化获得1 mg目的蛋白,功能活性分析表明CD137L融合蛋白能够有效激活人PBMC细胞增殖和抑制人HUVEC细胞的生长。综上所述,该研究成功构建了CD137L融合基因的真核表达载体并在HEK293F细胞中实现了分泌表达,为该融合蛋白的进一步稳定表达、大规模放大和成药性研究奠定了基础。The eukaryotic expression system has become a new direction in the pharmaceutical industry for the production of protein drugs because of its complete post-translational modification and less immunogenicity.The purpose of the present study was to construct and to express the eukaryotic expression vector of CD137 L fusion gene(FP)by transient transfection of human embryonic kidney 293 F(HEK293 F)cell line,and to test the functional activity of the CD137 L fusion protein after purification by Ni-affinity chromatography in small scale level.The plasmid p ET11 a-FP containing the CD137 L fusion gene was used as template which was stored in the authors laboratory.The DNA sequences coding Gaussia luciferase signal peptide and Kozak sequence were added at the N-terminal and 6×His flag were added at the C-terminal of the fusion gene by PCR technology and ligated with pc DNA3.1(+)vector.The successfully constructed expression plasmid pc DNA3.1-FP,which was verified by Xba I、EcoR I restriction endonuclease digestion and DNA sequencing,was transient transfected into HEK293 F cells by using polyetherimide(PEI).Expression condition of FP such as the ratio of transfection complex and the number of expression days were optimized.The best expression level of FP protein was achieved when DNA and PEI concentrations were 1.5μg/m L and 7.5μg/m L,separately,and expressed for 4 days as revealed by SDS-PAGE and Western-blotting.About 0.1 mg of FP protein was purified from 1 L media by Ni-affinity chromatography.Furthermore,the FP could promote the proliferation of human peripheral blood mononuclear cells(PBMC)and inhibit the proliferation of human umbilical vein endothelial cells(HUVEC)as desired and analyzed by cell counting kit-8(CCK-8).Above all,the present study successfully expressed and purified CD137 L fusion protein in HEK293 F cells,which laid a foundation for future stable expression,large-scale amplification and drug gability research of the CD137 L fusion protein.

关 键 词：CD137L 融合蛋白 真核表达 瞬时转染 HEK293F 蛋白纯化 信号肽 

分 类 号：Q789[生物学—分子生物学]