LwaCas13a蛋白的表达纯化与活性分析

doi:10.16431/j.cnki.1671-7236.2023.07.034

摘要/Abstract

摘要： 【目的】获得高纯度、高浓度、优良反式切割活性的韦德纤毛菌（Leptotrichia wadei）的Cas13a （LwaCas13a）蛋白，为研发基于CRISPR-LwaCas13a系统的动物病毒RNA检测新方法提供重要的生物学工具。【方法】通过PCR、双酶切与连接反应构建pET15b-SUMO-LwaCas13a重组质粒，并进行原核诱导表达与条件优化；发酵100 mL大肠杆菌BL21（DE3）菌诱导表达LwaCas13a蛋白后进行纯化，纯化后利用超滤法浓缩蛋白，使用BCA法检测蛋白浓度。将LwaCas13a蛋白、CRISPR RNA （CrRNA）、猪流行性腹泻病毒（PEDV）靶标RNA及荧光标记单链RNA （ssRNA）探针共孵育，利用全波长荧光分析仪检测CRISPR-LwaCas13a反式切割活性，与商业化Cas13a蛋白进行活性对比，并优化LwaCas13a与CrRNA最佳结合比例。【结果】成功构建重组质粒pET15b-SUMO-LwaCas13a；LwaCas13a重组蛋白的最佳诱导表达条件为0.4 mmol/L IPTG、18 ℃诱导16 h；经过纯化最终获得95%纯度、7.5 mg/mL浓度的LwaCas13a蛋白。荧光检测结果显示，LwaCas13a蛋白具有明显的反式切割活性，为商业化Cas13a蛋白活性的1.25倍，且LwaCas13a蛋白与CrRNA的最佳结合比例为1：2。【结论】试验成功表达纯化出高纯度、高浓度的LwaCas13a重组蛋白，其反式切割活性优于商业化Cas13a蛋白，并明确了该蛋白与CrRNA的最佳配比，为利用LwaCas13a进行动物病毒RNA检测新技术的研发提供了依据。

关键词: LwaCas13a蛋白; 原核表达; SUMO酶切; 反式切割活性

Abstract: 【Objective】 The production collection of high-purity, high-concentration LwaCas13a protein capable of excellent trans-cleavage activity from Leptotrichia wadei was undertaken in vitro, which could provide an important biological tool for the development of new methods in animal virus RNA detection based on CRISPR-LwaCas13a system.【Method】 The recombinant plasmid pET15b-SUMO-LwaCas13a was constructed by PCR, double digestion as well as ligand reaction, and prokaryotic expression and condition optimization were performed.Ferment 100 mL of Escherichia coli BL21(DE3) was used to obtain the expression product and assess the purification procedure, the induced expression and purified products were concentrated by ultrafiltration, and the protein concentration was detected by BCA.The purified LwaCas13a protein was co-incubated with CRISPR RNA (CrRNA), target RNA from Porcine epidemic diarrhea virus (PEDV) and fluorescent-labeled single stranded RNA (ssRNA).A full-wavelength fluorescence analyzer was used to verify the trans-cleavage activity of CRISPR-LwaCas13a, which was also compared with commercial Cas13a protein.The optimal binding ratio between LwaCas13a and CrRNA was optimized for further research.【Result】 The recombinant plasmid pET15b-SUMO-LwaCas13a was successfully constructed.The optimal IPTG concentration of LwaCas13a recombinant protein was 0.4 mmol/L, and the optimum induction temperature and time was 18 ℃ for 16 h.After a series of purifications, LwaCas13a protein was produced at a purity of 95% and concentration of 7.5 mg/mL.The purified LwaCas13a protein was verified to have readily detectable trans-cleavage activity of ssRNA probes, and found its trans-cleavage activity was better than that of commercial Cas13a protein by about 1.25 times at the same concentration.The optimal binding ratio between LwaCas13a protein and CrRNA was 1:2.【Conclusion】 In this study, LwaCas13a protein at high purity and concentration was successfully obtained, and the purified LwaCas13a had excellent trans-cleavage activity than commercial Cas13a in vitro with the optimal binding ratio between LwaCas13a and CrRNA, which provided a basis for the development and use of CRISPR-LwaCas13a for animal virus RNA detection.

Key words: LwaCas13a protein; prokaryotic expression; SUMO enzyme digestion; trans-cleavage activity

中图分类号:

S852.65

豆玲, 李泽轩, 贾訸潇, 张彩云, 左富江, 李婷, 谢元江, 段晓雷. LwaCas13a蛋白的表达纯化与活性分析[J]. 中国畜牧兽医, 2023, 50(7): 2941-2950.

DOU Ling, LI Zexuan, JIA Hexiao, ZHANG Caiyun, ZUO Fujiang, LI Ting, XIE Yuanjiang, DUAN Xiaolei. Expression, Purification and Activity Analysis of LwaCas13a Protein[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2941-2950.

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