UNcle高通量蛋白稳定系统分析3种反转录酶  

Application of an all-in-one UNcle stability platform for the thermostability analysis of reverse transcriptases

作　　者：武瑶 安琳 Wu Yao;An Lin(State Key Laboratory of Plant Genomics,Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101)

机构地区：[1]中国科学院微生物研究所,北京100101

出　　处：《分析仪器》2022年第2期64-72,共9页Analytical Instrumentation

基　　金：中科院仪器设备功能开发技术创新项目(292019000090)。

摘　　要：目的:反转录酶的热稳定性是重要的质控参数,利用UNcle高通量蛋白稳定分析仪多种检测功能对反转录酶热稳定性进行表征。方法:比较qRT-PCR方法及UNcle系统对3种反转录酶的热稳定检测,并利用UNcle荧光光谱分析核苷类反转录酶抑制剂去羟肌苷对反转录酶的影响。结果:利用高通量蛋白稳定性分析系统UNcle进行反转录酶熔解温度Tm、聚集起始温度Tagg及蛋白均一度分析。和qRT-PCR检测结果相比,3种反转录酶的热稳定性顺序一致,但各自检测的Tm数值差距较大。比较这两种方法的前处理,UNcle不需要外源添加染料,即没有引入外来变量直接检测,可以更加准确反映反转录酶本身的Tm值。结论:提供了一种新的快速准确表征反转录酶热稳定性的方法,为反转录酶的研发质控提供重要检测依据,并为UNcle在其他蛋白的热稳定分析提供借鉴参考意义。Objective Reverse transcriptase is an enzyme that synthesizes complementary DNA(cDNA)using RNA as template.The thermal stability of reverse transcriptase is an important quality control parameter.Using the principle of Nano Differential Scanning Fluorimetry(nano DSF),The high-throughput protein stability detecting system-UNcle has a variety of detection functions,which can be used to analyze the thermal stability of proteins such as Reverse transcriptase and verify the interaction between biological molecules.Methods In this study,the thermal stability detection results of reverse transcriptase by qRT-PCR and UNcle system were compared,and the effect of nucleoside reverse transcriptase inhibitor dehydroxyinosine on reverse transcriptase was analyzed by UNcle fluorescence spectrum.Results The UNcle system was used to analyze the melting temperature Tm,aggregation initiation temperature Tagg and protein size of the three commercial reverse transcriptases.Compared with the detection results obtained by the qRT-PCR method,the thermostability sequence of the three reverse transcriptases was the same,but the Tm values were quite different.After comparing the pre-treatment conditions of these two methods,it was found that UNcle did not require additional dyes,that is,it did not introduce foreign variables for direct detection.And it had stronger accuracy in reflecting the Tm value of reverse transcriptase.Conclusion This study provides a new method to quickly and accurately characterize the thermal stability of reverse transcriptase.It is a reference for UNcle’s thermal stability analysis of other proteins.

关 键 词：反转录酶 蛋白热稳定性 核苷类逆转录酶抑制剂 去羟肌苷 

分 类 号：Q55[生物学—生物化学]