Membrane-like system containing FGFRs (Fibroblast Growth Factor Receptor), phospholipid bilayer and membrane scaffold protein (MSP) and preparation method thereof
A phospholipid bilayer, skeleton protein technology, applied in the biological field, to achieve the effect of uniform size and controllable size
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Embodiment 1
[0056] S.O.C medium, Tris, imidazole, and antibiotics were purchased from Shanghai Sangong, other salts were purchased from Sinopharm, and concentrated tubes were purchased from Millipore. The FPLC instrument used is GE's The system, the heparin affinity chromatography column used, the Ni-sepharose chromatography column and the molecular exclusion chromatography column Hiload 16 / 60Superdex75pg were all purchased from GE Company. Insect cell culture medium (Sf-900TM II SFM) was purchased from gibco. The BCA kit was purchased from Thermo, and the FGFR tyrosine phosphorylated antibody was purchased from R&D. Cloning and positive screening of embodiment 1.FGFR1c
[0057] Using the synthesized FGFR1c as a template, PCR amplification is performed, and the sequence of the FGFR1c is shown in SEQ ID NO:1. The sequences of the two primers used are:
[0058] P1: 5'-CCCGGATCCATGTGGAGCTGGAAGTGCCTCCTC-3' (SEQ ID NO: 12),
[0059] P2: 5'-CGCAAGCTTCAAGCGGCGTTTGAGTCCGCCATTG-3' (SEQ ID NO...
Embodiment 2
[0074] Example 2. FGFR1c virus packaging
[0075] First place the 2×10 6 Put one sf9 cell into a T25 culture flask, add 5ml of insect cell culture medium without antibody, and let it stand for more than 30 minutes. On the other hand, use liposome transfection reagent for transfection, put 12uL of transfection reagent and 2ug of bacmid (isolated in Example 1) into 100uL of serum-free and non-resistant medium respectively, and incubate for 30 minutes Then put the two mediums containing 2ug bacmid into the medium containing 12ul transfection reagent, incubate for 30 minutes, and drop the mixture into the pre-prepared T25 culture flask. After culturing for 4 days, the supernatant virus was collected, which was the first-generation virus P1, and 1 mL of P1 was added to a mixture containing 1×10 7 In the T75 culture flask (15-20ml insect cell culture medium) of 1 cell, cultivate for 4 days, collect the supernatant virus and be the second generation virus P2, add 1mL P2 into contai...
Embodiment 3
[0076] Example 3. Expression of FGFR1c protein
[0077] Add 500ml of S.O.C medium to a sterile Erlenmeyer flask with a capacity of 2L, and add sf9 cells to make the cell concentration reach 0.5×10 6 cells / mL, cultivated for about 20 h, until the cell concentration reached 1.0×10 6 Each bottle was added with 5ml FGFR1c P3 generation virus, continued to culture for 36-48h, collected cells, and analyzed the expression of the collected cells by SDS-PAGE gel electrophoresis.
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