【目的】目前国内外西尼罗病毒(West Nile virus,WNV)的核酸检测中缺乏安全、稳定的RNA标准参考样品,对检测结果的精确度和可信度造成一定的影响。本研究旨在研制一种安全稳定的WNV核酸检测质控品,为西尼罗热监测提供技术支持。【方法】...【目的】目前国内外西尼罗病毒(West Nile virus,WNV)的核酸检测中缺乏安全、稳定的RNA标准参考样品,对检测结果的精确度和可信度造成一定的影响。本研究旨在研制一种安全稳定的WNV核酸检测质控品,为西尼罗热监测提供技术支持。【方法】将WNV核酸检测靶基因、MS2噬菌体外壳蛋白CP及成熟酶蛋白A基因序列插入到表达载体得到重组质粒pET-CPA-WN,经转化、表达、纯化后,制备WNV装甲RNA质控品,并对其进行实时荧光定量PCR和数字PCR定量、均匀性及稳定性分析,评估其作为标准物质的可能性。【结果】PCR检测、双酶切和基因测序结果均显示已成功构建重组质粒pET-CPA-WN,表达纯化后得到了大小均一、直径为23~28 nm的病毒样颗粒。核酸酶消化后实时荧光定量PCR检测结果显示,颗粒溶液中几乎无核酸残余且形成了包封靶基因的装甲RNA;其定植结果为8.80×10~9拷贝/mL。稳定性试验结果表明,该装甲RNA可在37℃稳定保持20 d,随机抽取10个样本进行均匀性检验证明其均匀性良好。【结论】本研究基于MS2噬菌体制备的装甲RNA拷贝数高,均匀性和稳定性良好,可为WNV分子检测提供安全、稳定的参考样品。展开更多
Extractable glycolipids of mycobacteria,such as lipooligosaccharides(LOSs),play crucial roles in responding to environmental stress and modulating the host immune response.Although the biosynthesis of LOS is likely re...Extractable glycolipids of mycobacteria,such as lipooligosaccharides(LOSs),play crucial roles in responding to environmental stress and modulating the host immune response.Although the biosynthesis of LOS is likely regulated at multiple levels to ensure proper composition of the cell wall,the key regulators remain unknown.In this study,we investigated B11,a conserved mycobacterial small RNA(sRNA),and found that it post-transcriptionally regulates LOS synthesis in Mycobacterium marinum.Through a combination of RNA-seq and mass spectrometry screening,we identified specific genes within the LOS synthesis locus that are directly regulated by B11.We confirmed in vivo sRNA-mRNA interactions using MS2-tagged RNA affinity purification,and found that B11 utilizes the cytosine-rich loop of its Rho-independent transcriptional terminator to interact with guanine tracks adjacent to the ribosome binding sites of its target genes,thereby impeding translation and promoting mRNA degradation.Moreover,deletion of B11 altered the colony morphology associated with LOS composition.These comprehensive functional studies of the mycobacterial sRNA B11 reveal sRNA-based regulation of LOS synthesis,providing new insights into the regulatory mechanisms controlling the biosynthesis of the complex mycobacterial cell wall.展开更多
文摘【目的】目前国内外西尼罗病毒(West Nile virus,WNV)的核酸检测中缺乏安全、稳定的RNA标准参考样品,对检测结果的精确度和可信度造成一定的影响。本研究旨在研制一种安全稳定的WNV核酸检测质控品,为西尼罗热监测提供技术支持。【方法】将WNV核酸检测靶基因、MS2噬菌体外壳蛋白CP及成熟酶蛋白A基因序列插入到表达载体得到重组质粒pET-CPA-WN,经转化、表达、纯化后,制备WNV装甲RNA质控品,并对其进行实时荧光定量PCR和数字PCR定量、均匀性及稳定性分析,评估其作为标准物质的可能性。【结果】PCR检测、双酶切和基因测序结果均显示已成功构建重组质粒pET-CPA-WN,表达纯化后得到了大小均一、直径为23~28 nm的病毒样颗粒。核酸酶消化后实时荧光定量PCR检测结果显示,颗粒溶液中几乎无核酸残余且形成了包封靶基因的装甲RNA;其定植结果为8.80×10~9拷贝/mL。稳定性试验结果表明,该装甲RNA可在37℃稳定保持20 d,随机抽取10个样本进行均匀性检验证明其均匀性良好。【结论】本研究基于MS2噬菌体制备的装甲RNA拷贝数高,均匀性和稳定性良好,可为WNV分子检测提供安全、稳定的参考样品。
基金supported by the Natural Science Foundation of China(81991532 and 32170179 to C.W.and 82272376 to Q.G.)National Key R&D Program of China(2022YFE0111800 to C.W.)the Shanghai Municipal Science and Technology Major Project(ZD2021CY001).
文摘Extractable glycolipids of mycobacteria,such as lipooligosaccharides(LOSs),play crucial roles in responding to environmental stress and modulating the host immune response.Although the biosynthesis of LOS is likely regulated at multiple levels to ensure proper composition of the cell wall,the key regulators remain unknown.In this study,we investigated B11,a conserved mycobacterial small RNA(sRNA),and found that it post-transcriptionally regulates LOS synthesis in Mycobacterium marinum.Through a combination of RNA-seq and mass spectrometry screening,we identified specific genes within the LOS synthesis locus that are directly regulated by B11.We confirmed in vivo sRNA-mRNA interactions using MS2-tagged RNA affinity purification,and found that B11 utilizes the cytosine-rich loop of its Rho-independent transcriptional terminator to interact with guanine tracks adjacent to the ribosome binding sites of its target genes,thereby impeding translation and promoting mRNA degradation.Moreover,deletion of B11 altered the colony morphology associated with LOS composition.These comprehensive functional studies of the mycobacterial sRNA B11 reveal sRNA-based regulation of LOS synthesis,providing new insights into the regulatory mechanisms controlling the biosynthesis of the complex mycobacterial cell wall.
