mOX40Ig表达及其生物学活性初步探究.docVIP

mOX40Ig表达及其生物学活性初步探究 　 作者：刘艳菲, 林志娟, 冯永堂, 许传武, 史琳, 苗乃法 【摘要】 　　目的: 构建pcDNA3.1mOX40Ig真核表达载体, 转染CHO细胞进行稳定表达, 获得有生物学活性的mOX40Ig融合蛋白。方法: RTPCR法扩增获得hIgG1Fc段基因, 构建pcDNA3.1hIgG1Fc重组质粒并经测序证实。从本室保存的pIRES2EGFPmOX40重组质粒中PCR扩增mOX40胞外段, 将其插入pcDNA3.1hIgG1Fc重组质粒中构建pcDNA3.1mOX40Ig真核表达载体。脂质体法转染CHO细胞获得稳定表达, 用RTPCR与ELISA法检测其表达, 蛋白A亲和纯化后, SDSPAGE进行鉴定。3HTdR掺入法研究OX40信号对B细胞的体外促增殖作用。结果: 测序证实hIgG1Fc段、 mOX40胞外段及mOX40Ig基因序列正确, RTPCR与ELISA证实mOX40Ig的表达, SDSPAGE证实其为mOX40Ig融合蛋白, 3HTdR掺入法显示mOX40Ig在体外能有效地促进B细胞增殖。结论: 成功地构建pcDNA3.1mOX40Ig真核表达载体, 并获得有生物学活性的mOX40Ig融合蛋白的稳定表达, 为进一步开展OX40相关领域的横向应用研究奠定了基础。 【关键词】 OX40 mOX40 Ig CHO细胞 真核表达 　　[Abstract] AIM: To construct a recombinant eukaryotic expression vector containing pcDNA3.1mOX40Ig fusion gene and obtain mOX40Ig fusion protein with bioactivity by transfecting CHO cells. METHODS: The gene fragment encoding the human IgG1Fc was amplified by RTPCR and the eukaryotic expression vector pcDNA3.1hIgG1Fc was constructed. After sequencing, mOX40 extracellular gene was cloned from pIRES2EGFPOX40 by PCR and then inserted into the recombinant vector pcDNA3.1hIgG1Fc. The right recombinant was transfected into CHO cells with lipofectin Ragent and its expression was detected by RTPCR and sandwichELISA. After purified by protein A affinity column chromatography, the mOX40Ig fusion protein was identified by SDSPAGE and its effect on the proliferation of B cells in vitro was studied by 3HTdR method. RESULTS: The hIgG1Fc, mOX40 extracellular gene and mOX40Ig gene were consistent with DNA sequencing.The expression of mOX40Ig fusion protein in CHO cells was confirmed by RTPCR, sandwichELISA and SDSPAGE. 3HTdR analysis showed the mOX40Ig fusion protein stimulated the proliferation of B cells in vitro. CONCLUSION: A eukaryotic expression vector containing pcDNA3.1mOX40Ig has been constructed successfully and the stable expression of mOX40Ig fusion protein with bioactivity has been acquired, which lays a solid basis for