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基因编辑与诊断工具酶LwCas13a的克隆表达、纯化与酶活性鉴定

A Cloning,Expression,Purification,and Enzyme Activity Identification of Gene Editing and Diagnostic Tool Enzyme LwCas13a
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摘要 目的开发可大量制备具有天然酶活性的基因编辑与诊断工具酶LwCas13a的技术,为研究基因编辑或开发基因诊断试剂提供稳定的酶。方法以LwCas13a蛋白基因序列为模板,采用PCR技术扩增目的基因LwCas13a,运用NotⅠ和BamHⅠ进行双酶切,将酶切产物与相同酶切获得的载体pC013用T4 DNA连接酶连接构建成含有融合表达His标签的原核表达载体pC013-Twinstrep-SUMO-huLwCas13a,选择经酶切和测序鉴定均正确的质粒转化到E.coli BL21感受态细胞中,用0.5 mmol·L^(-1)的异丙基-β-D-硫代丰乳糖苷在16℃条件下诱导16 h,表达LwCas13a酶,产物经SDS-PAGE电泳检测,获得可溶性表达条件后,进行大量蛋白诱导表达,使用Ni^(2+)亲和层析法,纯化LwCas13a蛋白。利用LwCas13a酶的“侧向切割活性”检测纯化获得的酶。结果PCR扩增获得3479 bp序列完全正确的LwCas13a基因,重组蛋白以融合蛋白形式可溶性高效表达,纯度达到98%。经比较优于商业化的LwCas13a酶。结论成功获得具有天然侧向剪切活性的LwCas13a酶,为开发使用LwCas13a酶的分子诊断试剂提供原材料奠定了基础。 Objective To obtain a gene editing and diagnostic tool enzyme LwCas13a with natural enzyme activity,and to provide a stable enzyme for the development of gene editing or diagnostic reagents.Methods The LwCas13a gene sequence was used as a template,the target gene LwCas13a sequence fragment was amplified using PCR method.NotⅠand BamHⅠwere used for double digestion,and the digested product was linked with the vector pC013 obtained from the same digestion using T4 DNA ligase to construct a prokaryotic expression vector pC013-Twinstrep SUMO-huLwCas13a that fused with His-tag.The plasmid,which was identified as correct by digestion and sequencing,was transformed into E.coli BL21 receptive cells,induced with 0.5 mmol·L^(-1) of IPTG at 16℃for 16 hours to obtain LwCas13a protein.The product was detected by SDS-PAGE electrophoresis to obtain soluble expression conditions.After obtaining a large amount of induced protein expression,Ni^(2+)affinity chromatography was used to purify LwCas13a protein.The characteristics of LwCas13a protein side branch cutting activity was used to detect the purified enzyme activity.Results The LwCas13a gene fragment with a completely correct 3479 bp sequence was obtained by PCR amplification,and the recombinant protein was highly expressed in the form of a fusion protein,with a purity of 98%.After comparison,the activity of LwCas13a enzyme was comparable to that of commercial LwCas13a enzyme.Conclusion The LwCas13a protein with natural lateral shear enzyme activity has been successfully obtained,laying a foundation for the development of molecular diagnostic reagents using LwCas13a protein as a raw material enzyme.
作者 李晓雨 倪江萍 朱玥 杨玉玛 薛依凡 张斯涵 妥蓉蓉 杨延辉 LI Xiaoyu;NI Jiangping;ZHU Yue;YANG Yuma;XUE Yifan;ZHANG Sihan;TUO Rongrong;YANG Yanhui(School of Basic Medical Sciences,Ningxia Medical University,Key Laboratory for Prevention and Treatment of Common Infectious Diseases in Ningxia,Yinchuan 750004,China)
出处 《宁夏医科大学学报》 2023年第12期1208-1213,共6页 Journal of Ningxia Medical University
基金 宁夏回族自治区重点研发计划项目(2021BEG03072) 宁夏医科大学校级科研项目(XM2020013)。
关键词 LwCas13a酶 原核表达 蛋白纯化 酶活性鉴定 enzyme LwCas13a prokaryotic expression protein purification enzyme activity identification
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