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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。
This study designed and constructed a human tumor necrosis factor receptor II extracellular domain and human adiponectin fusion gene sTNFRII-gAD, and the corresponding fusion protein in mammalian cells BHK-21S serum-free culture system The expression was achieved and the fusion protein was preliminarily identified. Firstly, the extracellular domain of human TNF receptor type II gene was amplified by RT-PCR from the total RNA of peripheral blood lymphocytes of human and fused with the adiponectin gene fragment and cloned into pAAV2neo expression vector. Constructed into pAAV2neo-sTNFRII-gAD. Subsequently, BHK-21S cells were transfected with pAAV2neo-sTNFRII-gAD to obtain G418 resistant cells BHK-21S / pAAV2neo-sTNFRII-gAD; then, the original serum-containing culture medium was replaced by a serum-free chemically defined culture medium, Finally, the supernatant of BHK-21S / pAAV2neo-sTNFRII-gAD serum-free suspension culture was collected and the sTNFRII-gAD fusion protein was identified and analyzed. Restriction endonuclease digestion and sequencing results showed that the constructed pAAV2neo-sTNFRII-gAD plasmid was correctly constructed and the sequence of sTNFRII-gAD was as expected. Anti-human tumor necrosis factor receptor II and anti-adiponectin globulin monoclonal antibody The BHK-21S cells transiently transfected with pAAV2neo-sTNFRII-gAD were detected by immunofluorescence and the expression of sTNFRII-gAD fusion protein was detected by Western blot in the supernatant of BHK-21S cells stably transfected with pAAV2neo-sTNFRII-gAD. And exists as a multimer above monomers, trimers and trimers. The results of activity assay showed that sTNFRII-gAD fusion protein significantly inhibited the activity of TNFα in killing L929 cells. Therefore, this study provides a good foundation for the next large-scale preparation of sTNFRII-gAD fusion protein for in vitro and in vivo studies.