Fusion protein comprising cfms extracellular fragment and its preparation method and application
A technology of fusion proteins and extracellular fragments, applied in the field of biomedical engineering, can solve problems such as differences in expression and different intracellular signaling pathways
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Embodiment 1
[0049] Construction of expression plasmids:
[0050] The coding sequence of the extracellular region of cFms was obtained by gene synthesis method according to GenBank (BC047521.1).
[0051] The Fc fragment (684bp) is obtained by PCR amplification using the lymph node cDNA (BD) as a template and primer 1 and primer 2:
[0052] Primer 1: 5'-ctatctcacacatcgacaattcgaagacaaaactcacacatgcccac-3'
[0053] Primer 2: 5'-aagggaatctagagcggccgctcatttacccggagacaggggag-3'
[0054] YY-001 consists of the 1st, 2nd and 3rd immunoglobulin-like regions (D1, D2, D3) of the extracellular region of cFms (see figure 2 ) fused with human immunoglobulin Fc. Between the extracellular domain D3 and Fc, a connecting peptide is added. The obtained DNA sequence of YY-001 is shown in SEQ ID NO.19. Among them, the PCR fragment (831bp) of D1 to D3 is obtained by PCR amplification with primer 3 and primer 4:
[0055] Primer 3: 5'-ccgctcgagatcccagtgatagagcccagt-3'
[0056] Primer 4: 5'-gcataccggttaccacc...
Embodiment 2
[0073] Transfection of host cells and production of fusion proteins:
[0074] A plurality of fusion proteins in the present invention are expressed in CHO-K1, CHO-S and DG44 cells, secreted into culture fluid, and purified by Staphylococcus A protein affinity precipitation method.
[0075] Transient expression of YY-001 to YY-007 fusion protein in the present invention, the recombinant plasmid was purified with a DNA purification kit (Qiagen Company), and then transfected into CHO-K1 (ATCC#CCL61) cells with Liposome 2000 (Invitrogen Company) . Supernatants were collected after 3 days of culture in serum-free medium OPTI-MII. After the concentration of the purified fusion protein was determined by ELISA, it was verified by SDS-PAGE and western blot analysis.
[0076] The fusion protein of YY-001 to YY-007 in the present invention is stably expressed, and the purified plasmid is transfected into CHO-S cells or DG44 cells (Invitrogen Company) by electroporation. After 48 hours...
Embodiment 3
[0078] In vitro binding experiment of fusion protein with IL-34 and M-CSF:
[0079] The present invention uses a highly sensitive and specific IL-34 and M-CSF detection kit (R&D Systems Company) to determine the affinity of each fusion protein to IL-34 and M-CSF. Fusion proteins (YY-001 to YY-007) of different concentrations (0 to 100nM) were incubated with 250pM human IL-34 or with 50pM human M-CSF (R&D Systems) overnight at room temperature, and then treated with IL-34 or The M-CSF Detection Kit detects free IL-34 and free M-CSF that are not bound by the fusion protein. Table 1 shows the affinity obtained after nonlinear fitting of the experimental results with the software program Prism5 (GraphPad Company).
[0080] Table 1
[0081]
[0082] Under the experimental conditions, the fusion proteins YY-001 to YY-007 all exhibit different degrees of affinity to IL-34 or M-CSF. Among them, the affinity of YY-001 to IL-34 and M-CSF is about 0.101-0.149nM / 0.099-0.169nM respec...
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