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

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24 h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。

Abstract:

In order to get soluble TNF? receptor (sTNFR) II with good neutralizing activity against TNF?, 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.