关键词: 新型冠状病毒(SARS-CoV-2), 非结构蛋白14(Nsp14), 原核表达, 蛋白质纯化, 核酸酶活性

Abstract: Objective To obtain the non-structure protein 14 (Nsp14) of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) with higher purity and enzymatic activity. Methods This study firstly analyzed the rare codons in the gene of nsp14 according to the codon usage bias of E. coli, followed by codon optimization. The optimized nucleotide fragment of nsp14 was cloned into four kinds of expressing vectors respectively. Comparative analysis of yield and solubility was performed among these expressed four fusion proteins. The best one was chosen for further optimization of expressing conditions. After the fusion protein was purified by glutathione affinity column, the fusion tag was removed by 3C protease, and then the protein was purified by glutathione affinity column and molecular sieve column for further analysis of enzymatic activity through urea polyacrylamide gel electrophoresis. Results Many rare codons were found in expression of SARS-CoV-2 nsp14 in E. coli, among which some rare codons were distributed in close range and tandem. The best recombinant plasmid for expressing the fusion protein was pGEX6P1-GST-OPTI-Nsp14, which gave an eptimal expression in 30 ℃ with high yield and solubility. After purification, a higher purity of Nsp14 with nuclease activity was obtained. Conclusions This study shows that the SARS-CoV-2 Nsp14 protein with nuclease activity is successfully prepared, which lays a foundation for the further research on the structure and function of SARS-CoV-2 Nsp14, and provides favorable conditions for screening antiviral drugs targeting at Nsp14 of SARS-CoV-2.

Key words: severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), non-structure protein 14(Nsp14), prokaryotic expression, protein purification, nuclease activity