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

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-A₁与RFP-B₁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。

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

Abstract:

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 HisTrap^TM 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.

Key words:SARS-CoV-2;main protease;fluorogenic substrate;dimerization-dependent red fluorescent protein;ensitrelvir;baicalein