Objective: To optimize the expression conditions of SARS-CoV-2 papain-like protease (papain-like protease, PLpro) in Escherichia coli (E.coli) and prepare highly specific polyclonal antibody against rat PLpro. Methods: The optimal expression conditions of PLpro in E.coli were determined by optimizing induction time, induction temperature and IPTG concentration. After PLpro being isolated and purified by HisTrapTM affinity chromatography column, the rat was immunized with PLpro as antigen to prepare anti-PLpro polyclonal antibody. The titer, antigen specificity and sensitivity of polyclonal antibody were determined by enzyme-linked immunosorbent assay (ELISA) and Western Blot experiments. Results: The optimum induction temperature of PLpro was 20 ℃, the induction time was 10 h, and the concentration of IPTG was 0.2 mmol/L. The titer of anti-PLpro polyclonal antibody reached to 1∶256 000 with good antigen specificity, and the detection limit of anti-PLpro polyclonal antibody was 12.5 ng with good sensitivity. Conclusion: The expression conditions of PLpro in E.coli was successfully optimized and highly specific polyclonal antibody against rat PLpro successfully prepared, which laid a foundation for the study of the immunological function of PLpro in COVID-19.
LIU Zhicheng
,
YAN Gang’an
,
YAN Haohao
,
LIU Xiaoli
,
LIU Xiaoping
,
CHEN Yunyu
. Optimization of prokaryotic expression condition of SARS-CoV-2 papain-like protease and preparation and identification of polyclonal antibody[J]. Journal of Baotou Medical College, 2024
, 40(1)
: 85
-90
.
DOI: 10.16833/j.cnki.jbmc.2024.01.015
[1] Cooper DM, Afghani B, Byington CL, et al. SARS-CoV-2 vaccine testing and trials in the pediatric population: biologic, ethical, research, and implementation challenges[J]. Pediatr Res, 2021, 90(5): 1-5.
[2] Wang X, Zhang XX, He JJ. Challenges to the system of reserve medical supplies for public health emergencies: reflections on the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic in China[J]. Biosci Trends, 2020, 14(1): 3-8.
[3] Romano CM, Chebabo A, Levi JE. Past, present, and future of COVID-19: a review[J]. Braz J Med Biol Res, 2020, 53(9): e10475.
[4] Mohammadi M, Shayestehpour M, Mirzaei H. The impact of spike mutated variants of SARS-CoV2[Alpha, Beta, Gamma, Delta, and Lambda] on the efficacy of subunit recombinant vaccines[J]. Braz J Infect Dis, 2021, 25(4): 101606.
[5] Li QQ, Wu JJ, Nie JH, et al. The impact of mutations in SARS-CoV-2 spike on viral infectivity and antigenicity[J]. Cell, 2020, 182(5): 1284-1294.
[6] Wu F, Zhao S, Yu B, et al. A new coronavirus associated with human respiratory disease in China[J]. Nature, 2020, 579(7798): 265-269.
[7] Yang HT, Rao ZH. Structural biology of SARS-CoV-2 and implications for therapeutic development[J]. Nat Rev Microbiol, 2021, 19(11): 685-700.
[8] Shamsi A, Mohammad T, Anwar S, et al. Potential drug targets of SARS-CoV-2: from genomics to therapeutics[J]. Int J Biol Macromol, 2021, 177: 1-9.
[9] Arya R, Kumari S, Pandey B, et al. Structural insights into SARS-CoV-2 proteins[J]. J Mol Biol, 2021, 433(2): 166725.
[10] Gordon DE, Jang GM, Bouhaddou M, et al. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing[J]. Nature, 2020, 583(7816): 459-468.
[11] Maiti BK. Can papain-like protease inhibitors halt SARS-CoV-2 replication[J]. ACS Pharmacol Transl Sci, 2020, 3(5): 1017-1019.
[12] Mehdipor AR, Tasche G, Geurink PP, et al. Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity[J]. Nature, 2020, 587(7835): 657-662.
[13] 闫干干, 李淼, 戚海燕, 等. 新型冠状病毒木瓜样蛋白酶在大肠杆菌中的可溶表达与酶活性测定[J]. 中国现代应用药学, 2022, 39(1): 5-11.
[14] 陈云雨, 付正豪, 闫干干, 等. 基于密码子优化策略的新型冠状病毒主蛋白酶在大肠杆菌中的表达条件优化与活性鉴定[J]. 生物工程学报, 2021, 37(4): 1334-1345.
[15] 陈云雨, 牛夏忆, 李淼, 等. 大肠杆菌Fts Z蛋白原核表达及多克隆抗体的制备与鉴定[J]. 生物工程学报, 2019, 35(6): 1117-1125.
[16] 闫干干, 戚海燕, 闫浩浩, 等. 大鼠抗新冠病毒主蛋白酶多克隆抗体的制备与鉴定[J]. 细胞与分子免疫学杂志, 2021, 37(11): 1032-1037.
[17] 陈云雨, 牛夏忆, 林媛, 等. 大肠杆菌丝状热敏蛋白Z相互作用蛋白A(Ec-ZipA)原核表达?纯化与大鼠多克隆抗体的制备[J]. 细胞与分子免疫学杂志, 2018, 34(10): 942-948.
[18] Klemm T, Ebert G, Calleja DJ, et al. Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2[J]. EMBO J, 2020, 39(18): e106275.
[19] Lindner HA, Lytvyn V, Qi H, et al. Selectivity in ISG15 and ubiquitin recognition by the SARS coronavirus papain-like protease[J]. Arch Biochem Biophys, 2007, 466(1): 8-14.
[20] 杨宇东, 孙莉, 陈忠斌. SARS冠状病毒PLpro蛋白酶的结构与功能[J]. 中国生物化学与分子生物学报, 2010, 26(1): 15-21.
[21] 杜倩倩, 张智慧, 廉政, 等. 新型冠状病毒PLpro蛋白酶结构与功能的生物信息学分析[J]. 病毒学报, 2021, 37(1): 43-51.
[22] 彭彦皓, 肖树, 陈丽, 等. 鼠源胰岛素降解酶的原核表达及活性检测[J]. 生命科学研究, 2021, 25(4): 283-292.
[23] 乔圣泰, 王曼琦, 徐慧妮. 番茄SlTpx原核表达蛋白的体外功能分析[J]. 中国生物工程杂志, 2021, 41(8): 25-32.
[24] 葛晨晨, 尚超, 张翠玲, 等. 新型冠状病毒PLpro的克隆、表达及其对抗病毒天然免疫反应的调节作用[J]. 中国病原生物学杂志, 2021, 16(10): 1129-1133,1138.
