In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjc14FABP and Sjc26GST genes and identify their expression in vitro, Sj 14 and Sj26 genes were ob- tained by RT-PCR with total RNA ...In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjc14FABP and Sjc26GST genes and identify their expression in vitro, Sj 14 and Sj26 genes were ob- tained by RT-PCR with total RNA of Schistosoma japonicum adult worms as the template and cloned into eukaryotic expression plasmid pVAC to construct recombinant plasmids pVAC-Sjl4 and pVAC-Sj26. Then a 23 amino-acid signal peptide of human interleukin-2 （IL-2） upstream Sj 14 or Sj26 gene and a membrane-anchored sequence containing 32 amino-acids of carboxyi-terminal of human placental alkaline phosphatase （PLAP） downstream were amplified by PCR as the template of plasmid pVAC-Sj14 or pVAC-Sj26 only to get two gene fragments including Sj14 gene and Sj26 gene. The two modified genes were altogether cloned into a eukaryotic co-expression plasmid plRES, resulting in another new recombinant plasmid plRES-Sj26-Sj14. The expression of Sj 14 and Sj26 genes was detected by RT-PCR and indirect immunofluorescent assays （IFA） when the plasmid plRES-Sj26-Sj 14 was transfected into eukaryotic Hela cells. Restriction enzyme analysis, PCR and sequencing results revealed that the recombinant plasmids pVAC-Sj14, pVAC-Sj26 and plRES-Sj26-Sj 14 were successfully constructed and the expression of modified Sj 14 and Sj26 genes could be detected by RT-PCR and IFA. A bivalent membrane-anchored DNA vaccine encoding Sj 14 and Sj26 genes was acquired and expressed proteins were proved to be mostly anchored in cellular membranes.展开更多
Quantifying the microRNAs(miRNAs)levels in living cells,while essential for the study of fundamental biology and medical diagnostics,has barely been achieved due to insufficient probe delivery and unquantifiable signa...Quantifying the microRNAs(miRNAs)levels in living cells,while essential for the study of fundamental biology and medical diagnostics,has barely been achieved due to insufficient probe delivery and unquantifiable signals.We report a cell-anchored and self-calibrated DNA nanoplatform,a cholesterol-headed DNA nanowire that is capable of efficiently delivering to various cells and simultaneously detecting two target miRNAs.One miRNA target can be utilized as an endogenous control against cell-to-cell variations.Moreover,the photocleavable linkers inserted in the nanostructures allow us to precisely regulate the probe structure and fluorescence signaling at the desired time and location in vivo.As a second control,the maximum fluorescence can be elicited by UV light,which further facilitates the normalization of the absolute fluorescence signal.With two introduced internal controls,the maximum fluorescence and endogenous control gene,this approach displays excellent stability and self-calibration performance,effectively avoiding the interference from operating conditions and cellto-cell variations,such as the laser powers and intracellular probe concentrations.Importantly,this design is capable of unifying the output signal intensity between in vitro test and cell imaging,making the in vitro linear calibration curve appropriate for the quantification of miRNA expression in living cells.展开更多
基金This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30471603).
文摘In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjc14FABP and Sjc26GST genes and identify their expression in vitro, Sj 14 and Sj26 genes were ob- tained by RT-PCR with total RNA of Schistosoma japonicum adult worms as the template and cloned into eukaryotic expression plasmid pVAC to construct recombinant plasmids pVAC-Sjl4 and pVAC-Sj26. Then a 23 amino-acid signal peptide of human interleukin-2 （IL-2） upstream Sj 14 or Sj26 gene and a membrane-anchored sequence containing 32 amino-acids of carboxyi-terminal of human placental alkaline phosphatase （PLAP） downstream were amplified by PCR as the template of plasmid pVAC-Sj14 or pVAC-Sj26 only to get two gene fragments including Sj14 gene and Sj26 gene. The two modified genes were altogether cloned into a eukaryotic co-expression plasmid plRES, resulting in another new recombinant plasmid plRES-Sj26-Sj14. The expression of Sj 14 and Sj26 genes was detected by RT-PCR and indirect immunofluorescent assays （IFA） when the plasmid plRES-Sj26-Sj 14 was transfected into eukaryotic Hela cells. Restriction enzyme analysis, PCR and sequencing results revealed that the recombinant plasmids pVAC-Sj14, pVAC-Sj26 and plRES-Sj26-Sj 14 were successfully constructed and the expression of modified Sj 14 and Sj26 genes could be detected by RT-PCR and IFA. A bivalent membrane-anchored DNA vaccine encoding Sj 14 and Sj26 genes was acquired and expressed proteins were proved to be mostly anchored in cellular membranes.
基金This work was financially supported by the Project Fund for Shangdong Key R&D Program(no.2019GSF108169)the National Natural Science Foundation of China(no.21605091)+3 种基金the Taishan Scholar Program of Shandong Province of China(no.ts20110829)the State Key Laboratory of Analytical Chemistry for Life Science(no.SKLACLS2008)the Natural Science Foundation of Shandong Province of China(no.ZR2021YQ11)the Open Project of Chemistry Department of Qingdao University of Science and Technology(no.QUSTHX201926).
文摘Quantifying the microRNAs(miRNAs)levels in living cells,while essential for the study of fundamental biology and medical diagnostics,has barely been achieved due to insufficient probe delivery and unquantifiable signals.We report a cell-anchored and self-calibrated DNA nanoplatform,a cholesterol-headed DNA nanowire that is capable of efficiently delivering to various cells and simultaneously detecting two target miRNAs.One miRNA target can be utilized as an endogenous control against cell-to-cell variations.Moreover,the photocleavable linkers inserted in the nanostructures allow us to precisely regulate the probe structure and fluorescence signaling at the desired time and location in vivo.As a second control,the maximum fluorescence can be elicited by UV light,which further facilitates the normalization of the absolute fluorescence signal.With two introduced internal controls,the maximum fluorescence and endogenous control gene,this approach displays excellent stability and self-calibration performance,effectively avoiding the interference from operating conditions and cellto-cell variations,such as the laser powers and intracellular probe concentrations.Importantly,this design is capable of unifying the output signal intensity between in vitro test and cell imaging,making the in vitro linear calibration curve appropriate for the quantification of miRNA expression in living cells.
