新冠病毒主蛋白酶特异性荧光底物ddRFP-M的制备与鉴定  

Preparation and characterization of a fluorogenic ddRFP-M biosensor as a specific SARS-CoV-2 main protease substrate

作　　者：张锐 闫浩浩 刘志成 刘晓丽 闫干干 刘晓平[1] 陈云雨 ZHANG Rui;YAN Haohao;LIU Zhicheng;LIU Xiaoli;YAN Gangan;LIU Xiaoping;CHEN Yunyu(Institute for Drug Screening and Evaluation,Wannan Medical College,Wuhu 241002,Anhui,China)

机构地区：[1]皖南医学院药物筛选与评价研究所,安徽芜湖241002

出　　处：《生物工程学报》2024年第2期496-506,共11页Chinese Journal of Biotechnology

基　　金：安徽省自然科学基金(1808085QH265);安徽省高等学校自然科学研究项目(KJ2021A0839);皖南医学院青年骨干人才资助项目(wyqnyx202104);安徽省研究生学术创新项目(2022xscx129)。

摘　　要：传统的新冠病毒主蛋白酶(main protease, Mpro)多肽底物具有制备成本高、稳定性差和合成工艺复杂等缺点,积极开发廉价稳定的新型底物具有重要意义。本研究基于二聚化红色荧光蛋白(dimerization-dependent red fluorescent protein, ddRFP)原理,以AVLQS为连接肽,利用基因工程技术制备Mpro特异性荧光底物ddRFP-M,用于Mpro抑制剂的药理活性评价。将连接肽基因插入到密码子优化的RFP-A_1与RFP-B_1基因之间,构建ddRFP-M基因,再将其克隆到pET-28a载体中构建重组质粒。将重组质粒转化至大肠杆菌Rosetta(DE3)感受态细胞中,以卡那霉素抗性法筛选重组子。重组子经低温诱导后,在大肠杆菌中进行荧光底物ddRFP-M的可溶表达,并以HisTrap~(TM)层析柱进行分离纯化。以荧光动力学检测法和电泳法测定ddRFP-M的底物特异性,并利用荧光底物ddRFP-M评价恩赛特韦和黄芩素的药理活性。结果显示,荧光底物ddRFP-M在大肠杆菌中呈可溶表达并成功进行了分离纯化,其具有良好的底物特异性、灵敏性和可靠性。新冠病毒Mpro特异性荧光底物ddRFP-M的制备,为新冠病毒Mpro抑制剂的药理活性评价奠定了基础。The conventional peptide substrates of SARS-CoV-2 main protease(Mpro)are frequently associated with high cost,unstable kinetics,and multistep synthesis.Hence,there is an urgent need to design affordable and stable Mpro substrates for pharmacological research.Herein,we designed a functional Mpro substrate based on a dimerization-dependent red fluorescent protein(ddRFP)for the evaluation of Mpro inhibitors in vitro.The codon-optimized DNA fragment encoding RFP-A1 domain,a polypeptide linker containing Mpro cleavage sequence(AVLQS),and the RFP-B1 domain was subcloned into the pET-28a vector.After transformation into Escherichia coli Rosetta(DE3)cells,the kanamycin resistant transformants were selected.Using a low temperature induction strategy,most of the target proteins(ddRFP-M)presented in the supernatant fractions were collected and purified by a HisTrapTM chelating column.Subsequently,the inhibition of Mpro by ensitrelvir and baicalein was assessed using ddRFP-M assay,and the biochemical properties of ddRFP-M substrate were analyzed.Our results showed that the fluorogenic substrate ddRFP-M was successfully prepared from E.coli cells,and this biosensor exhibited the expected specificity,sensitivity,and reliability.In conclusion,the production of the fluorogenic substrate ddRFP-M provides an expedient avenue for the assessment of Mpro inhibitors in vitro.

关 键 词：新冠病毒 主蛋白酶 荧光底物 二聚化红色荧光蛋白 恩赛特韦 黄芩素 

分 类 号：R914[医药卫生—药物化学]