可溶性肿瘤坏死因子受体II-脂联素球部融合蛋白的真核表达及生物学活性检测  

Eukaryotic expression and bioactivity determination of the fusion protein sTNFRII-gAD consisting of soluble tumor necrosis factor receptor II and globular domain of adiponectin

作　　者：陈素云[1,2] 何秋山[3] 董小岩[2,4] 吴小兵[2] 高基民[1,3] 

机构地区：[1]温州医学院浙江省医学遗传学重点实验室,温州325035 [2]中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室,北京100052 [3]南方医科大学生物技术学院,广州510515 [4]北京五加和分子医学研究有限公司,北京100176

出　　处：《生物工程学报》2010年第2期207-215,共9页Chinese Journal of Biotechnology

基　　金：国家新药创新重大专项(No.2009ZX09103-649);浙江省重大科技专项(No.2008C14082);浙江省自然科学基金(No.R2080407);浙江省卫生高层次创新人才培养工程(No.Y2008);病毒基因工程国家重点实验室开放课题(No.2008-S-0008)资助~~

摘　　要：本研究设计和构建了一种人肿瘤坏死因子受体II胞外区与人脂联素球部的融合基因sTNFRII-gAD,且相应的融合蛋白在哺乳动物细胞BHK-21S的无血清培养体系中实现了表达,并对该融合蛋白进行了初步鉴定。首先,用RT-PCR方法从人的外周血淋巴细胞总RNA中扩增人肿瘤坏死因子II型受体胞外区基因片段,与脂联素球部基因片段融合,克隆至pAAV2neo表达载体中,构建成pAAV2neo-sTNFRII-gAD。随后,用pAAV2neo-sTNFRII-gAD转染BHK-21S细胞获得G418抗性细胞BHK-21S/pAAV2neo-sTNFRII-gAD;然后,将原来含有血清的培养液换成无血清的化学成分限定的培养液,细胞从贴壁培养方式转换成悬浮培养方式;最后,收集BHK-21S/pAAV2neo-sTNFRII-gAD无血清悬浮培养24h后的培养上清,进行sTNFRII-gAD融合蛋白的鉴定分析。酶切鉴定和测序结果显示,所构建的pAAV2neo-sTNFRII-gAD质粒结构正确,sTNFRII-gAD序列与预期一致;分别用抗人肿瘤坏死因子受体II和抗人脂联素球部的单克隆抗体检测pAAV2neo-sTNFRII-gAD瞬时转染的BHK-21S细胞,免疫荧光呈现阳性;免疫印迹分析在pAAV2neo-sTNFRII-gAD稳定转染的BHK-21S细胞上清中检测到sTNFRII-gAD融合蛋白的表达,并以单体、三聚体和三聚体以上的多聚体形式存在。活性测定结果表明,sTNFRII-gAD融合蛋白具有显著抑制TNFα杀伤L929细胞的活性。因此,本研究为下一步大量制备sTNFRII-gAD融合蛋白用于体内外功能研究提供了良好基础。In order to get soluble TNF receptor （sTNFR） Ⅱ with good neutralizing activity against TNFα, we constructed the fusion gene sTNFRⅡ-gAD, which encoded human sTNFR Ⅱ and the globular domain of adiponectin （gAD）, and then expressed it in mammalian cells and analyzed its anti-TNFα activity. First, sTNFRⅡ cDNA was obtained by RT-PCR from the total RNA of human peripheral blood lymphocytes, and fused in frame with gAD gene. Then, the fusion gene sTNFRⅡ-gAD was cloned into the expression vector pAAV2neo to result in the plasmid pAAV2neo-sTNFRⅡ-gAD. By immunofluorescent staining with monoclonal antibody either against TNFRⅡ or against adiponectin, we demonstrated that the pAAV2neo-sTNFRⅡ-gAD-transiently-transfected BHK-21S cells were positive. To obtain G418-resistant BHK-21S/pAAV2neo-sTNFRⅡ-gAD cells, we cultured the transfected BHK-21S cells above in 10% FBS containing DMEM media with 800 μg/mL G418 for 15 days, and changed the serum-containing culture media to a serum-free chemically defined media so as to change the cells culturing style from adhesion to suspension. 24 hours later, we harvested the supernatant of the culture for sTNFRⅡ-gAD fusion protein characterization and anti-TNFα activity analysis. With monoclonal antibody either against TNFRⅡ or against adiponectin, the Western blotting analysis showed that the sTNFRⅡ-gAD fusion protein was expressed and existed as monomer, trimer and multimer forms in the supernatant. The bioactivity assay demonstrated that the sTNFRⅡ-gAD fusion protein had the ability to neutralize TNFα so as to inhibit the cytotoxicity of TNFα on L929 cells. Put together, this study has laid the groundwork for large-scale preparation of sTNFRⅡ-gAD fusion protein.

关 键 词：肿瘤坏死因子α拮抗剂 可溶性肿瘤坏死因子受体Ⅱ 脂联素 融合蛋白 真核表达 

分 类 号：R363[医药卫生—病理学]