Nucleic acid molecule SEPTIN1SR2 and application in preparing anti-leukemia medicament
A nucleic acid molecule and nucleotide technology, which is applied to the nucleotide molecule and its application in the preparation of drugs for treating leukemia, can solve the problems of low bone marrow probability, low immunity, huge manpower and material resources, etc.
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Embodiment 1
[0056] The expression situation in the organization of embodiment 1 Septin1
[0057] 1.1 Northern probe preparation
[0058] Clontech Multiple Tissue northen membrane was used to detect the distribution of septin1 gene in adult tissues, and the probe used was the cloned full-length open reading frame of septin1 (ie, positions 188 to 1291 in the nucleotide sequence shown in NCBI accession number NM_052838).
[0059] 1) Using specific primers of septin1 gene to amplify the PCR product from the constructed and sequenced plasmid. After the PCR product was purified with the QIAquick PCR purification kit, the concentration of the purified DNA was measured with a GeneQuant II UV spectrophotometer.
[0060] 2) After the DNA template was heat-denatured at 100°C for 2 minutes, [γ-32P]dCTP was incorporated with a random primer labeling kit using a random primer labeling method. After the labeling reaction, EDTA was added to a final concentration of 20mmol / L to stop the reaction, heat d...
Embodiment 2
[0068] Example 2 western-blot analysis of septin1 protein expression in 17 human cell lines
[0069] 1. Culture of 17 human cell lines
[0070] The selected 17 kinds of cells are all from the Cell Bank of the Chinese Academy of Sciences, and their tissue sources and culture conditions are as follows:
[0071] name
species
source of organization
Culture conditions
HEK293T
Humanity
embryonic kidney
90% DMEM high glucose complete medium, 10% fetal
Bovine serum, 37°C, 5% CO 2
Humanity
Same as HEK293T
U2OS
Humanity
90% 5A medium, 10% fetal bovine serum, 37
°C, 5% CO 2
PANC
Humanity
Same as HEK293T
SMMC772
1
Humanity
the liver
Same as HEK293T
MCF7
Humanity
breast
Same as HEK293T
H1299
Humanity
90% RPMI1640 medium, 10% fetal bovine...
Embodiment 3
[0082] Example 3 Localization of Septin1 in Jurkat cells
[0083] In the present invention, firstly, the cDNA of full-length septin1 is constructed into the Ds-Red-Cl fluorescent expression vector. The resulting RFP-septin1 recombinant was transfected into Jurkat cells, cultured for 48 hours, fixed, and observed under a confocal laser microscope. Specific steps are as follows:
[0084] (1) Collect the Jurkat cells cultured in suspension, disperse them fully, drop them on slides coated with poly-lysine, let them stand for 5 minutes, and then fix them in 4% paraformaldehyde at room temperature for 10 minutes (minutes).
[0085] (2) Wash three times with PBS, and punch holes in 0.2% Triton X-100 at room temperature for 15 minutes.
[0086] (3) Wash three times with PBS, block in TBS containing 10% horse serum and 1% BSA at room temperature for 1 hr (hour).
[0087] (4) Wash once with TTBS, add primary antibody (anti-septin1 antibody, 1:20) diluted in proportion with antibody d...
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