LbCas12a蛋白的原核表达及活性检测

doi:10.16431/j.cnki.1671-7236.2022.05.003

摘要/Abstract

摘要： 【目的】获得具有体外切割活性的来自毛螺旋菌属(Lachnospiraceae) ND2006的LbCas12a蛋白，以期为LbCas12a的应用研究提供重要的生物学工具。【方法】根据pMBP-LbCas12a质粒(Addgene，113431)的基因序列合成LbCas12a基因，并将其与pET-28a(+)线性化载体进行同源重组，构建重组质粒pET28a-LbCas12a，经测序和NheⅠ、SalⅠ双酶切鉴定后将阳性重组质粒转入大肠杆菌BL21(DE3)感受态细胞中进行IPTG诱导表达。通过12% SDS-PAGE凝胶电泳检测确定IPTG诱导的最佳浓度及温度，鉴定重组蛋白的表达形式，通过Ni-NTA树脂亲和层析的方法进行纯化、超滤法浓缩，BCA法检测蛋白浓度。将浓缩后的LbCas12a与猪细小病毒(PPV)靶标DNA、CRISPR RNA (crRNA)及偶联荧光基团的非特异性单链DNA(ssDNA)探针FQ共孵育，设置1个无LbCas12a蛋白的对照组、4个不同蛋白浓度(125、250、500、1 000 nmol/L)的试验组，检测不同组别ssDNA探针的荧光强度，检测测试位点PPV活性。【结果】测序和NheⅠ、SalⅠ双酶切鉴定结果表明，重组质粒pET28a-LbCas12a构建成功且无移码突变。12% SDS-PAGE凝胶电泳检测结果表明，IPTG诱导表达的最佳浓度为0.5 mmol/L，最佳温度为37 ℃，表达形式主要为可溶性表达。蛋白浓度检测结果表明，浓缩后的蛋白浓度为485 ng/μL，质量为143 ku。活性检测结果表明，125、250、500、1 000 nmol/L LbCas12a蛋白组的荧光强度均极显著高于对照组(P<0.01)。【结论】本研究成功表达出高活性的LbCas12a蛋白，且LbCas12a蛋白具有体外切割ssDNA的反式活性，为后续基于CRISPR-LbCas12a系统的分子检测技术奠定了基础。

关键词: LbCas12a蛋白; 原核表达; 蛋白纯化; 反式活性

Abstract: 【Objective】 The purpose of this study was to obtain LbCas12a protein from Lachnospiraceae ND2006 with in vitro cleavage activity,in order to provide an important biological tool for the application of LbCas12a.【Method】 LbCas12a gene was synthesized according to the gene sequence of pMBP-LbCas12a plasmid (Addgene,113431),and homologous recombination was performed with pET-28a(+) linearized vector.The recombinant plasmid pET28a-LbCas12a was constructed.After sequencing and double enzyme digestion,the positive recombinant plasmid was transferred into the BL21(DE3) for IPTG induced expression.The optimal concentration and temperature induced by IPTG were detected by 12% SDS-PAGE,and the expression form was identified.The protein concentration was detected by Ni-NTA resin affinity chromatography and ultrafiltration concentration,and the protein concentration was detected by BCA method.The concentrated LbCas12a was co-incubated with Porcine parvovirus (PPV) target DNA,CRISPR RNA (crRNA) and ssDNA reporter probe FQ,a control group without LbCas12a protein and four experimental groups with different protein concentration gradients (125,250,500,1 000 nmol/L) were set up.The fluorescence intensity of ssDNA probes in different groups was detected by automatic microplate reader,and the activity of PPV at the test site was detected.【Results】 Sequencing and NheⅠ and SalⅠ double enzyme digestion results showed that the recombinant plasmid pET28a-LbCas12a was successfully constructed.The results of 12% SDS-PAGE showed that the optimum induction concentration of IPTG was 0.5 mmol/L,the optimum induction temperature was 37 ℃,and the expression form was mainly soluble expression.The results of protein concentration detection showed that the concentration of protein was 485 ng/μL and the weight was 143 ku.The activity test results showed that the fluorescence intensity of 125,250,500,1 000 nmol/L LbCas12a protein was extremely significantly higher than that of control group (P<0.01).【Conclusion】 In this study,LbCas12a protein with high activity was successfully expressed,and LbCas12a protein had trans-cleavage activity of ssDNA in vitro,which laid a foundation for the subsequent molecular detection technology based on CRISPR-LbCas12a system.

Key words: LbCas12a protein; prokaryotic expression; protein purification; trans-activity

中图分类号:

S852.65

刘茹, 李小龙, 张晓倩, 田小欢, 余梅, 赵书红, 曹建华. LbCas12a蛋白的原核表达及活性检测[J]. 中国畜牧兽医, 2022, 49(5): 1621-1629.

LIU Ru, LI Xiaolong, ZHANG Xiaoqian, TIAN Xiaohuan, YU Mei, ZHAO Shuhong, CAO Jianhua. Prokaryotic Expression and Activity Identification of LbCas12a Protein[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1621-1629.

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