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目的建立人TACI-Fc基因转染细胞,获得该融合蛋白,研究其对多发性骨髓瘤细胞系XG6的生物学作用。方法利用RT-PCR方法从扁桃体细胞中克隆全长TACI cDNA片段,将该基因的胞外段sTACI与人IgG1Fc段连接到pcDNA真核表达载体上,使两者融合表达。脂质体法将重组质粒pcDNA-sTACI-Fc转入小鼠成纤维细胞系L929,ELISA法测定转染TACI-Fc基因的L929细胞培养上清中TACI2Fc的含量,培养上清经亲和层析柱纯化,获得纯化的融合蛋白TACI-Fc。然后应用台盼蓝染色活细胞计数和3H2TdR掺入法检测其对XG6细胞的生长和增殖的调节作用。结果经RT-PCR和ELISA方法检测表明,成功构建了稳定表达sTACI-Fc融合蛋白的基因转染细胞。基因转染细胞的培养上清经亲和层析柱纯化,得到TACI-Fc融合蛋白。体外检测其生物学活性结果表明,该融合蛋白可有效地阻断Blys对骨髓瘤细胞株XG6的促增殖作用。结论本实验成功地将TACI胞外段与人IgG1Fc段在真核细胞中进行融合表达,表达的蛋白质具有良好的生物学功能,这为进一步对TACI基因进行功能研究奠定了物质基础。
OBJECTIVE: To establish a human TACI-Fc gene-transfected cell line to obtain the fusion protein and study its biological effect on multiple myeloma cell line XG6. Methods The full-length TACI cDNA fragment was cloned from human tonsillar cells by RT-PCR. The extracellular domain of sTACI and human IgG1Fc were ligated into the eukaryotic expression vector pcDNA-pcDNA3. The recombinant plasmid pcDNA-sTACI-Fc was transfected into mouse fibroblast cell line L929 by liposome method. The content of TACI2Fc in the supernatant of L929 cells transfected with TACI-Fc gene was determined by ELISA. The culture supernatant was analyzed by affinity chromatography Column to obtain purified fusion protein TACI-Fc. Then, the viability of XG6 cells was measured by Trypan blue staining and 3H2TdR incorporation assay. Results The results of RT-PCR and ELISA showed that the transfected cells stably expressing sTACI-Fc fusion protein were successfully constructed. The culture supernatant of gene-transfected cells was purified by affinity chromatography to obtain TACI-Fc fusion protein. The results of in vitro bioassay showed that the fusion protein could effectively block the proliferation of myeloma cell line XG6 induced by Blys. Conclusion The experiment successfully fused the extracellular domain of TACI with the human IgG1 Fc fragment in eukaryotic cells. The expressed protein has good biological function, which lays the material foundation for further study on the function of TACI gene.