Abstract: To estabish LwaCas13a molecular detection platform,LwaCas13a was expressed in E.coli and its collateral RNase activity was determined.The recombinant plasmid pET His6-TwinStrep-SUMOLwaCas13a was transformed into Rosetta(DE3) competent cells to induce expression.The results of SDSPAGE electrophoresis and Western blot analysis showed that the soluble LwaCas13a recombinant protein was successfully expressed. Optimization of inducing temperature and time showed that maximal production of LwaCas13a was obtained at 15 h after induction at 16℃ .The expressed LwaCas13a recombinant protein was purified by nickel column affinity chromatography to obtain a single target band with ideal purification effect.In addition,the collateral RNase assay was also established with in vitro synthesized CRISPR RNA and target RNA.The assay could be performed within 30 min at constant 37℃ ,with the detection limit of 31.2 nmol/L.To sum up,the LwaCas13a was successfully expressed in E.coli Rosetta (DE3).Purified LwaCas13a exhibited ideal collateral RNase activity,and the collateral RNase assay for LwaCas13a was optimized and established.

Key words: CRISPR/Cas13a, Prokaryotic expression, Ni affinity chromatography, Collateral RNase activity, Nucleic acid detection

摘要： 为建立LwaCas13a连带剪切酶活性的检测方法，在大肠杆菌中表达LwaCas13a重组蛋白，并对其连带剪切酶活性进行鉴定。将重组质粒pET His6-TwinStrep-SUMO-LwaCas13a转化至Rosetta(DE3)感受态细胞诱导表达，SDS-PAGE电泳和Western blot分析结果显示，成功表达了可溶性LwaCas13a重组蛋白。对诱导温度和时间进行优化，结果显示，在16 ℃条件下诱导培养15 h时，LwaCas13a重组蛋白的可溶性表达量最高。通过镍柱亲和层析法对表达的LwaCas13a重组蛋白进行纯化，得到单一的目的条带，纯化效果较为理想。此外，通过体外转录合成了1对CRISPR RNA及靶标RNA，建立LwaCas13a连带剪切酶活性的检测方法，可在37 ℃条件下30 min内完成检测，对靶标RNA的检出限为31.2 nmol/L。综上，成功利用大肠杆菌Rosetta(DE3)菌株可溶性表达了LwaCas13a重组蛋白，且纯化后的LwaCas13a重组蛋白具有良好的连带剪切酶活性，建立了LwaCas13a连带剪切酶活性的检测方法。

关键词: CRISPR/Cas13a蛋白, 原核表达, 镍柱亲和层析, 连带剪切酶活性, 核酸检测