SENP1 protein inhibitor and use thereof
An inhibitor and protein technology, applied in the field of SENP1 protein inhibitor and its application, can solve the problem that there is no target for the treatment of malignant hematological tumors
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Embodiment 1
[0083] Example 1: Detection of SENP1 expression in CML.
[0084] The inventors performed magnetic bead sorting on the bone marrow cells of 8 CML patients and donors, and obtained CD34 + Cells, the expression of SENP1 mRNA was detected by real-time quantitative PCR, and it was found that the expression of SENP1 in CML patients was higher than that in donors ( figure 1 -A). Take two cases of CML CD34 + The cells were sorted for stem and progenitor cells to obtain myeloid progenitor cells (CMP), granulomonocytic progenitor cells (GMP), erythroid megakaryocytic progenitor cells (MEP) and hematopoietic stem cells (HSC), followed by PCR detected, and found that the SENP1 expression level of the HSC fraction was the highest ( figure 1 -B). The proteins of two samples were taken for western blot analysis, and it was found that the protein of SENP1 was in CML CD34 + cells than donor CD34 + The expression of cells should be high ( figure 1 -C).
Embodiment 2
[0085] Example 2: Construction and identification of shRNA (short hairpin RNA, small hairpin RNA) interference vector that reduces SENP1 gene expression.
[0086] Purchase 1 set (including 5 pieces) of SENP1 interference vector set (SHCLNG, NM-014554, including TRCN0000004395, TRCN0000004396, TRCN0000004397, TRCN0000004398, TRCN0000004399) from sigma company, and use lipofectin2000 transfection reagent to transfer engineering cell line to extract total HEK293 cells, RNA, and then use RT-qPCR detection to obtain the carrier with the highest interference efficiency. The specific process is: using the reverse-transcribed cDNA as a template, qPCR (quantitative PCR) to detect the expression of the SENP1 gene, using β-actin as an internal reference; The empty vector of the vector is used as a control, and 2-ΔΔCT is the expression of SENP1 after interference. The small hairpin sequence in the vector with the highest interference efficiency is: 5'CCGGGCGCCAGAUUGAAGAACAGAACUCGAGUUCUGUU...
Embodiment 3
[0088] Example 3: Preparation of lentivirus containing human SENP1-shRNA and the inhibitory effect of SENP1-GFP-shRNA on K562 cells.
[0089] After the successful construction of the shRNA interference vector that reduces the expression of the SENP1 gene, the lentivirus was prepared and purified. First inoculate 293T cells 4-4.5×10 6in a 10cm petri dish. The next day, the cells were confluent to 80-85%, and the lentiviral plasmid SENP1-GFP-shRNA was transfected. The lentiviral plasmid was transfected into 293T cells by the calcium phosphate transfection method, and the specific steps were: pMD2G: 3 μg, pSAX2: 9 μg (the vector was purchased from Addgene), and the target plasmid (interference vector): 12 μg. The three plasmids were mixed well. Add 63 μl of 2.0M CaCl 2 , fill up to 500μl with 0.1×TE, and mix well. Slowly drop the above liquid into 500μl 2×HBS, and mix well while adding. The liquid was in a turbid state after mixing, and stood at room temperature for 25-30mi...
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