A biological probe for detecting the degradation activity of ubiquitin-proteasome system
A biological probe and proteasome technology, applied in the biological field, can solve the problem of inability to detect ubiquitin phosphorylation, and achieve the effects of high sensitivity, convenient use and enhanced fluorescence
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Embodiment 1
[0044] Construction of Bioluminescent Probe Ub-cpEGFP
[0045] The amino acid sequence of the designed biological fluorescent probe Ub-cpEGFP is shown in SEQ ID NO.1, and its coding sequence is shown in SEQ ID NO.2. The base sequence was entrusted to the company to synthesize, and then the pDNA3.1 / Ub-cpEGFP plasmid was obtained by molecular cloning between the EcoRI and XhoI multiple cloning sites of the pDNA3.1 plasmid. Sequencing confirmed the correctness of the inserted sequence.
[0046] Excitation and emission spectra of fluorescent probes such as figure 1 shown.
Embodiment 2
[0048] Inhibition of ubiquitin-dependent proteasome activity by proteasome inhibitor MG132 detected by flow cytometry and immunoblotting
[0049] a. Transfection of PCDNA3.1 / Ub-cpEGFP plasmid in HEK293 cells: 400,000 HEK293 cells were planted in a 6-well cell culture plate, and after 24 hours, lipo3000 liposomes were used for transfection to make the cells express Ub- cpEGFP.
[0050] b. 24 hours after transfection, add 10 μM MG132 (proteasome inhibitor), 0, 1, 2, 3, 4, 5 hours after the addition, trypsinize, 1000 rpm, and centrifuge for 5 minutes to collect the cells .
[0051] c. Resuspend the received cells with phosphate buffer, then use a flow cytometer to detect the fluorescence intensity of cpEGFP, calculate the average fluorescence intensity of the cells, and analyze the inhibitory effect of MG132 on the proteasome.
[0052] d. After the flow cytometric detection, the cells were recovered, and the cells were lysed by a cell lysis kit, detected by SDS gel electrophore...
Embodiment 3
[0055] Probes for inhibition of ubiquitin-dependent proteasome degradation by proteasome inhibitor MG132 or coexpressed ubiquitin kinase PINK1 by fluorescence microscopy
[0056] a. Transfect PCDNA3.1 / Ub-cpEGFP or PCDNA3.1 / Ub-EGFP alone in HEK293 cells, or co-transfect two plasmids, PCDNA3.1 / Ub-cpEGFP and PCDNA3.1 / sPINK1, or Co-transfect two plasmids, PCDNA3.1 / Ub-EGFP and PCDNA3.1 / sPINK1: Plant HEK293 cells in a 24-well cell culture plate with a round cover glass at the bottom at a density of 30,000 / well, and use lipid method of transfection.
[0057] b. After 24 hours of transfection, add 10 μM MG132 to the cells transfected with PCDNA3.1 / Ub-cpEGFP or PCDNA3.1 / Ub-EGFP alone for 5 hours. All cells were fixed with freshly prepared 4% paraformaldehyde.
[0058] c. Confocal microscopy was used to observe the expression of green fluorescence.
[0059] The result is as image 3 As shown, the two probes were transfected alone, weak green fluorescence could be seen, and the MG132...
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