摘要
目的构建BP180NC16A-IgG1Fc融合蛋白的真核表达体系。方法①以逆转录PCR(reverse transcrip-tion-polymerase chain reaction,RT-PCR)方法从表皮组织提取总RNA中扩增编码人BP180NC16A的cDNA,与包含编码人免疫球蛋白IgG1Fc段恒定区基因的质粒pFUSE-hIgG1e4-Fc2融合,形成重组pFUSE-hIgG1e4-Fc2-BP180NC16A质粒。②重组质粒转染真核HEK293T细胞,48h后收集细胞培养上清。③通过免疫印迹的方法鉴定转染细胞上清中分泌的BP180NC16A-IgG1Fc融合蛋白。结果构建了pFUSE-hIgG1e4-Fc2-BP180NC16A真核表达质粒并在HEK293T细胞成功表达BP180NC16A-IgG1Fc融合蛋白。结论 BP180NC16A-IgG1Fc融合蛋白能够成功真核表达。
Objective To construct eukaryotic expression system of BP180NC16A-IgG1Fc fusion protein. Methods We cloned BP180NCI6A cDNA from total RNA extracted from the human epidelxnis by RT-PCR, then linked BPlg0NC16A cDNA with plasmid pFUSE-hIgGle4-Fc2 which encodes human IgG1Fc constant region, to construct recombinant plasmid pFUSE-hlgGle4-Fc2-BP180NC16A. Recombinant plasmids were then transfected in HEK 293T cells. Finally, we detected the BP180NC16A-IgG1Fc fusion protein in the supernatant by western blotting. Results HEK293T cells successfully expressed BP180NC16A-IgG1Fc fusion protein. Conclusion BP180NC16A-IgG1Fc fusion protein can be eukaryotically expressed.
出处
《中国皮肤性病学杂志》
CAS
北大核心
2012年第9期797-800,共4页
The Chinese Journal of Dermatovenereology
基金
高等学校博士学科点专项科研基金新教师基金课题(20090001120070)
国家自然科学基金青年科学基金项目(81000694)
