论文部分内容阅读
目的克隆并构建含小鼠4-1BB胞外段和人IgG Fc融合基因的真核表达质粒,表达具有生物学活性的4-1BB-Fc融合蛋白,初步探讨其体外生物学效应。方法借助RT-PCR技术,从小鼠脾脏总RNA中扩增4-1BB全长编码基因,并导入克隆载体pGEM-T Easy。经测序证实,用PCR扩增其膜外区cDNA,酶切后与hIgG1 Fc基因一起装入真核表达载体pcDNA3．1中。应用脂质体将重组子pcDNA3．1-4-1BB-Fc转染COS-7及CHO细胞,经G418筛选,获得稳定表达4-1BB-Fc融合蛋白的CHO细胞株。双抗体夹心FLISA检测融合蛋白表达,经蛋白A亲和层析纯化,借助免疫印迹进行鉴定。应用FACS检测融合蛋白与DC细胞系DC2．4表面4-1BBL结合情况。采用MTT及CFSE(羧基荧光素乙酰乙酸)标记法检测4-1BB-Fc融合蛋白对同种异基因小鼠T细胞增殖的抑制作用。结果经测序证实,所克隆和构建的小鼠4-1BB-Fc cDNA阅读框及连接部位序列正确;ELISA与免疫印迹证实4-1BB- Fc蛋白的表达及其对T细胞增殖具有明显抑制作用。结论成功构建4-1BB-Fc融合基因并获稳定表达,可望用于探讨4-1BB参与移植排斥的作用及其机制,并为研究4-1BB-Fc的其他生物学作用奠定初步基础。
Objective To clone and construct a eukaryotic expression plasmid containing mouse 4-1BB extracellular domain and human IgG Fc fusion gene and express the 4-1BB-Fc fusion protein with biological activity to investigate its in vitro biological effects. Methods The full-length 4-1BB gene was amplified from total RNA of spleen of mice by RT-PCR and introduced into the cloning vector pGEM-T Easy. After sequencing confirmed, PCR amplification of its outer membrane cDNA, digested with hIgG1 Fc gene into the eukaryotic expression vector pcDNA3.1. The recombinant pcDNA3.1-4-1BB-Fc was transfected into COS-7 and CHO cells by lipofectamine and then screened by G418 to obtain a CHO cell line stably expressing 4-1BB-Fc fusion protein. The fusion protein was detected by double antibody sandwich ELISA and purified by protein A affinity chromatography and identified by immunoblot. The binding of fusion protein to 4-1BBL on the surface of DC2.4 DCs was detected by FACS. MTT and CFSE (carboxyfluorescein acetoacetate) labeling method was used to detect the inhibitory effect of 4-1BB-Fc fusion protein on T cell proliferation in allogeneic mice. Results The sequence of the 4-1BB-Fc cDNA was cloned and constructed correctly. The sequence of 4-1BB-Fc cDNA was confirmed by ELISA and western blot. The results showed that the 4-1BB-Fc protein had significant inhibitory effect on the proliferation of T cells. Conclusion The 4-1BB-Fc fusion gene was successfully constructed and stably expressed, which is expected to be used to investigate the role of 4-1BB in graft rejection and its mechanism, and to lay a preliminary foundation for the further biological study of 4-1BB-Fc.