摘要
目的构建scFv-RBP4融合蛋白。方法采用德泰生物最新开发的密码子优化软件MaxCodonTM Optimization Program(V13)对提供的scFv-RBP4蛋白氨基酸序列进行优化,设计全长拼接引物,通过双酶切法将scFvRBP4基因插入到表达载体proEM中,并通过酶切法和测序确认最终表达载体的准确性,最终转到DH5a克隆菌株中,通过质粒大抽试剂盒提取转染级质粒,之后将质粒通过转染试剂转染到哺乳动物细胞HEK293T中进行瞬时表达,再通过Ni-IDA亲和层析纯化scFv-RBP4蛋白。结果酶切和测序结果显示,scFv-RBP4蛋白构建正确,目标蛋白纯度>90%。结论构建的scFv-RBP4融合蛋白可以在HEK293T细胞内获得稳定表达,为后期融合蛋白的提取及功能研究打下基础。
Objective Construction of fusion protein of single chain variable fragment and RBP4. Methods MaxC- odonTM Optimization Program （V13） was used to optimize the amino acid sequence of scFv-RBP4 protein,and the full- length splice primers were designed by Detai Bio. The scFv-RBP4 gene was inserted into the expression vector proEM by double digestion, and the accuracy of the final expression vector was confirmed by restriction enzyme digestion and sequencing. The plasmid was transfected into DHSa clone strain and the plasmid was extracted from themammalian cell HEK293T by transfection reagent. The plasmid was purified by Ni-IDA affinity chromatography. The scFv-RBP4 was purified by Ni-IDA affinity chromatography. Results The results of digestion and sequencing showed that the scFv- RBP4 protein was constructed correctly and the purity of the target protein was〉 90%. Conclusion The scFv-RBP4 fusion protein can be expressed stably in HEK293T cells, which will be the foundation for the extraction and functional study of late fusion proteins.
出处
《中国实验诊断学》
2017年第6期1082-1085,共4页
Chinese Journal of Laboratory Diagnosis
基金
国家自然科学基金面上项目(81571455)
