摘要
目的:利用原核系统对新型冠状病毒S蛋白的受体结合域(RBD)肽段进行克隆、表达和纯化,制备高纯度的RBD肽。方法:在RBD肽段N端加入肠激酶位点序列(DDDDK),对应的核酸序列参照原核系统表达偏好进行全基因合成,构建pET-DsbC-RBD融合表达载体,通过原核表达和亲和纯化获得DsbC-RBD融合蛋白,用肠激酶对融合蛋白进行酶切和二次亲和纯化,获得高纯度新冠病毒S蛋白RBD肽。结果:构建了pET-DsbC-RBD表达载体,融合蛋白DsbC-RBD在大肠杆菌中实现了高效表达,经亲和层析纯化,重组融合蛋白DsbC-RBD相对分子质量为58×103,纯度约90%。用肠激酶对融合蛋白进行酶切,酶切效率近100%,通过二次亲和纯化获得了RBD肽,相对分子质量为25×103,纯度92%以上。结论:利用原核表达系统获得可溶性DsbC-RBD融合蛋白,采用亲和纯化和肠激酶酶切联用的方法制备获得高纯度的新冠病毒S蛋白RBD肽,为后续抗体制备和抗病毒药物筛选奠定了基础。
Objective:To obtain high purify recombinant receptor binding domain(RBD) peptide of SARS-CoV-2 S protein by expression and purification in prokaryotic system.Methods:Enterokinase site sequence(DDDDK)was added to the N-terminal of the amino acid sequence of RBD peptide and the nucleotide sequence synthesized according to the principle of prokaryotic system expression was cloned into prokaryotic expression vector pET-DsbC.The constructed recombinant plasmid pET-DsbC-RBD was transformed to E.coli BL21(DE3) for expression under induction of IPTG. The DsbC-RBD fusion protein was expressed and purified by metal chelate affinity chromatography. After digesting by enterokinase, the high-purity RBD peptide would be obtain by secondary purification with affinity chromatography.Results:pET-DsbC-RBD was constructed successfully and recombinant DsbC-RBD fusion protein was expressed efficiently, which had a relative molecular mass of about 58×103 and purity of reached to90% according to the results of SDS-PAGE as correct. The fusion protein cleavage efficiency was close to 100%when digesting by enterokinase and RBD peptide was obtained by secondary purification with 25×103 and purity of more than 92%.Conclusion:The DsbC-RBD fusion protein was overexpressed solubly in prokaryotic system and high purity RBD peptide can be obtained by second affinity purification after enterokinase digestion, which should lay a foundation for subsequent antibody preparation and antiviral drug screening.
作者
侯江厚
张灵霞
黄国红
詹晓燕
杨奕梅
HOU Jiang-Hou;ZHANG Ling-Xia;HUANG Guo-Hong;ZHAN Xiao-Yan;YANG Yi-Mei(Kunming City Matermal and Child Health Hospital,Kunming 650013;Eighth Medical Center of General Hos-pital of PLA,Beijing 100091,China)
出处
《生物技术通讯》
CAS
2020年第3期292-296,共5页
Letters in Biotechnology
关键词
新型冠状病毒S蛋白
受体结合域(RBD)
原核表达
肠激酶
纯化
SARS-CoV-2 S protein
receptor binding domain(RBD)peptide
prokaryotic expression
enterki-nase
purification
