Secreted protein with chemotactic activity, its coding sequence and medical use
A protein and encoding technology, applied in the application field of the protein, polynucleotide and its pharmaceutical composition, can solve the problems of large investment and heavy workload, and achieve the effect of broad application prospects
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Embodiment 1
[0064] Example 1, construction of pcDNA3.1-FAM3D-myc-his6 fusion protein expression plasmid
[0065] The pcDNA3.1-FAM3D-myc-his6 plasmid was constructed to express the FAM3D-myc-his6 fusion protein.
[0066] 1. Method:
[0067] The FAM3D coding sequence (SEQ ID NO: 2 was inserted into the pcDNA3.1-myc-his6 (Invitrogen Company) expression vector to construct the pcDNA3.1-FAM3D-myc-his6 expression plasmid. After the correctness of the plasmid was verified by sequencing, plasmid amplification was carried out , Axygen plasmid extraction kit was used to extract plasmids for cell transfection.
[0068] 2. Results:
[0069] The sequence of the coding region was correct by DNA sequencing.
Embodiment 2
[0070] Example 2, plasmid transfection cells to obtain FAM3D expression supernatant
[0071] 1. Method:
[0072] HEK293T cells adjusted to 6×10 5 Cells / 2ml concentration in 6-well plate at 37°C 5% CO 2 Culture for 24 hours for transfection, pCDNA3.1-FAM3D-myc-his6 DNA 2μg, vigofect (Vigofect Biotechnology Co., Ltd.) 2μl, the mixture was slowly dropped into the prepared cells, and pcDNA3.1-myc -his6 empty vector transfected cells as control, after 6 hours, the serum-free medium HEKG was changed, cultured at 37°C for 48 hours, and the supernatant after transfection was harvested.
[0073] Use Western Blotting to check the expression of the target protein:
[0074] After 12.5% SDS-PAGE electrophoresis, the protein was transferred to the PVDF membrane with 1XCaps electrotransfer solution, after adding Anti-c-myc antibody (MBL company) to act, then adding IRDyeTM 800-labeled anti-mouse IgG (LICOR Bioscience), using Odyssey Imaging System detection system direct detection.
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Embodiment 3
[0078] Embodiment 3, the separation of human peripheral blood mononuclear cells:
[0079] 1. Method:
[0080] Concentrated white blood cells were provided by Beijing Blood Center, and human peripheral blood mononuclear cells were obtained using lymphocyte separation medium (Shanghai Huajing Bio-Technology Co., Ltd.). Culture with RPMI 1640 (Life Technologies, Inc.) containing 10% heat-inactivated fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin.
[0081] 2. Results:
[0082] Human peripheral blood mononuclear cells were obtained and cultured in vitro for future use.
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