目的探究96孔板高通量筛选方法在TrxA-溶菌酶热稳定性改造中的可行性。方法通过易错聚合酶链式反应建立高质量突变体文库,待单克隆长出后利用96孔板进行微量表达,之后采用冷热循环裂解的方法破坏菌体,离心取上清后检测不同温度下的杀菌...目的探究96孔板高通量筛选方法在TrxA-溶菌酶热稳定性改造中的可行性。方法通过易错聚合酶链式反应建立高质量突变体文库,待单克隆长出后利用96孔板进行微量表达,之后采用冷热循环裂解的方法破坏菌体,离心取上清后检测不同温度下的杀菌活性,借此实现高通量筛选,最后通过摇瓶表达验证初筛结果。结果在终质量浓度20μg/mL条件下,突变体Y262M的完全失活温度从野生型的48℃提高到52℃,分子动力学模拟结果显示Y262M的均方根偏差(root mean square deviation,RMSD)及均方根波动(root mean square fluctuation,RMSF)值均低于野生型,疏水簇分析显示Y262M在野生型基础上将细胞壁结合域的2个单独疏水簇融合成一个较大的疏水簇,总面积增加了约25%。结论本研究采用96孔板高通量筛选方法成功筛选出热稳定性提高的突变体Y262M,证实了该方法在溶菌酶热稳定性改造中的可行性。此方法不需要深入了解目标蛋白质的空间结构,实验操作相对简单,且随机突变也使得改造结果具有多样性,同时进一步的结构分析也为了解溶菌酶结构与功能关系提供有益参考。展开更多
The implementation of multiple pathogen testing is essential for a rapid response to future outbreaks and for reducing disease transmission.This study introduces a 96-channel microfluidic chip,fabricated through a mol...The implementation of multiple pathogen testing is essential for a rapid response to future outbreaks and for reducing disease transmission.This study introduces a 96-channel microfluidic chip,fabricated through a molding process,which enables the batch detection of pathogens.It explores the rapid lysis and elution processes of pathogens within the microfluidic chips to ensure that nucleic acid extraction,elution,and amplification are completed entirely within the chip.This chip can extract nucleic acids from samples in just 10 min,achieving an extraction efficiency comparable to that of traditional in-tube methods.An oil phase is pre-loaded into the chip to effectively prevent aerosol contamination.This approach allows for the simultaneous detection of 21 common respiratory pathogens,with a detection limit of 10 copies per reaction.Furthermore,applications involving clinical samples demonstrate significant practicality.Compared to many traditional in-tube pathogen detection methods and molecular biology technologies that utilize microfluidic chips,this detection chip not only enables simultaneous detection of multiple pathogens but also demonstrates high sensitivity.展开更多
文摘目的探究96孔板高通量筛选方法在TrxA-溶菌酶热稳定性改造中的可行性。方法通过易错聚合酶链式反应建立高质量突变体文库,待单克隆长出后利用96孔板进行微量表达,之后采用冷热循环裂解的方法破坏菌体,离心取上清后检测不同温度下的杀菌活性,借此实现高通量筛选,最后通过摇瓶表达验证初筛结果。结果在终质量浓度20μg/mL条件下,突变体Y262M的完全失活温度从野生型的48℃提高到52℃,分子动力学模拟结果显示Y262M的均方根偏差(root mean square deviation,RMSD)及均方根波动(root mean square fluctuation,RMSF)值均低于野生型,疏水簇分析显示Y262M在野生型基础上将细胞壁结合域的2个单独疏水簇融合成一个较大的疏水簇,总面积增加了约25%。结论本研究采用96孔板高通量筛选方法成功筛选出热稳定性提高的突变体Y262M,证实了该方法在溶菌酶热稳定性改造中的可行性。此方法不需要深入了解目标蛋白质的空间结构,实验操作相对简单,且随机突变也使得改造结果具有多样性,同时进一步的结构分析也为了解溶菌酶结构与功能关系提供有益参考。
基金supported by grants from the National Key Research and Development Program of China(Nos.2023YFA0915200,2023YFA0915204)the Equipment Research and Development Projects of the Chinese Academy of Sciences(No.PTYQ2024YZ0010)+3 种基金the Science and Technology Commission of Shanghai Municipality Project(No.XTCX-KJ-2024-038)the Natural Science Foundation of Hebei Province of China(No.H2024206249)the Postdoctoral Fellowship Program of CPSF(No.GZC20232838)Science and Technology Commission of Shanghai Municipality(No.22S31901700).
文摘The implementation of multiple pathogen testing is essential for a rapid response to future outbreaks and for reducing disease transmission.This study introduces a 96-channel microfluidic chip,fabricated through a molding process,which enables the batch detection of pathogens.It explores the rapid lysis and elution processes of pathogens within the microfluidic chips to ensure that nucleic acid extraction,elution,and amplification are completed entirely within the chip.This chip can extract nucleic acids from samples in just 10 min,achieving an extraction efficiency comparable to that of traditional in-tube methods.An oil phase is pre-loaded into the chip to effectively prevent aerosol contamination.This approach allows for the simultaneous detection of 21 common respiratory pathogens,with a detection limit of 10 copies per reaction.Furthermore,applications involving clinical samples demonstrate significant practicality.Compared to many traditional in-tube pathogen detection methods and molecular biology technologies that utilize microfluidic chips,this detection chip not only enables simultaneous detection of multiple pathogens but also demonstrates high sensitivity.
