Recombinant bifunctional fusion protein and its preparation method and application

A technology of fusion protein and protein, applied in the field of biomedicine

Active Publication Date: 2019-08-09
HUABO BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bifunctional fusion protein drugs that can simultaneously bind to TGF-β1 and VEGF and simultaneously block the biological activities induced by these two targets have not been reported so far

Method used

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  • Recombinant bifunctional fusion protein and its preparation method and application
  • Recombinant bifunctional fusion protein and its preparation method and application
  • Recombinant bifunctional fusion protein and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0166] Construction of TβRII-D2-Fc expression vector

[0167] The coding sequence of the TβRII extramembrane region gene consists of 483 nucleotides, as shown in positions 70-552 in SEQ ID NO.:2. The VEGFR1-D2 gene coding sequence consists of 300 nucleotides, as shown in positions 553-852 in SEQ ID NO.: 2, including 279 nucleotides of the D2 coding sequence, 15 nucleotides of the upstream flanking sequence, The downstream flanking sequence is 6 nucleotides. 69 signal peptide coding sequences from TβRII (ie, positions 1-69 in SEQ ID NO.: 2) are added to the 5' end of the outer region of TβRII, forming 852 nucleotides.

[0168] A "HindIII" gene cloning site and an "EcoRI" gene cloning site were added to the amino terminus of the 852 nucleotides to form a gene fragment containing 873 nucleotides.

[0169] The synthetic product (synthesized by Nanjing GenScript Biotechnology Co., Ltd.) was digested with HindIII / EcoRI and cloned into the pHB-Fc plasmid vector to form the pHB-TbRI...

Embodiment 2

[0174] Expression of TβRII-D2-Fc, D2-TβRII-Fc, TβRII-Fc-D2

[0175] The host cells used for protein expression were CHO-K1 cells (Cat#CCL-61) purchased from ATCC Company. After a series of domestication steps, the cells are domesticated into CHO-K1 cells that can be cultured in suspension in serum-free medium (EX-CELLTM 302).

[0176] Using the cells, the plasmids pHB-TβRII-D2-Fc, pHB-D2-TβRII-Fc, and pHB-TβRII-Fc-D2 were respectively transferred into the cells by electroporation. The specific method is: collect the cells in the logarithmic growth phase under sterile conditions, centrifuge (1200rpm x 5min), resuspend in complete medium, and adjust the cell density to 1x 10 7 cells / ml. Transfer 350ul of the cell suspension to a 0.4cm electroporation cup, and pulse once under the set electroporation conditions (voltage range 200 to 350V, generally 260V, time about 20ms). Add 10-30ug of plasmid DNA to the electroporation cup containing the cells, mix gently, put the electropor...

Embodiment 3

[0181] TβRII-D2-Fc and target (VEGF and TGF-β1) binding activity detection

[0182] Enzyme-linked immunosorbent assay (ELISA) method was used to determine the binding properties of the fusion protein and the target (VEGF and TGF-β1). Specific steps are as follows:

[0183] TGF-β1 (Cat: 10804-HNAH, Sino biological Inc.) and VEGF-165 (Cat: 11066-HNAH, Sino biological Inc.) were coated with CBS (Sigma-Aldrich Co., Product code: 1001329288 C3041-100CAP) Inc.) were diluted to 500ng / ml, and 100ul was added to the ELISA plate (Nunc TM , Cat: 442404), 50ng per well. Place the coated plates in a 4°C refrigerator overnight. During the detection, the coated plate was washed once with 0.05% PBS-T, and then blocked with 3% skimmed milk at room temperature for 1 hour. 2-fold serially diluted TbRII-D2-Fc protein (100 nM, 50 nM, 25 nM, up to 0.0244 nM) was added to the coated plate, 100 ul per well. After incubating at room temperature for one hour, the sample was discarded, washed 5 tim...

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Abstract

The present invention relates to recombinant bifunctional fusion protein and its preparation method and application. Specifically, the present invention discloses a fusion protein, which has element A including the extramembrane region of TGF-β receptor, element B and immunoglobulin element C of the second extramembrane region D2 of VEGFR1, and the three are connected in series. The protein can simultaneously bind to two ligands of VEGF and TGF-β1, and has good binding activity, thereby inhibiting the biological activity of the ligand. The invention also provides the effect of the recombinant bifunctional fusion protein in disease treatment.

Description

technical field [0001] The invention relates to the field of biomedicine. More specifically, the present invention relates to a recombinant bifunctional fusion protein and its preparation method and application. Background technique [0002] Diseases such as tumors and liver fibrosis take tens of millions of lives and cause hundreds of millions of dollars in economic losses every year. The combination of TGF-β and VEGFR receptors on the cell membrane surface and the corresponding ligands is an important reason for the occurrence and development of such diseases, so the development of corresponding protein ligand drugs has a good application prospect. [0003] Currently known protein drugs include growth factors, hormone proteins, enzyme proteins, cytokines, interferons, erythropoietin, and fusion proteins. Except for fusion proteins, other protein drugs are homogeneous proteins, that is, they contain only one protein component. However, existing fusion protein drugs (such...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10C12P21/02A61K38/17A61K47/68A61P35/00A61P1/16
CPCA61K38/00A61P1/16A61P35/00C07K14/495C07K14/71C07K2319/00C07K2319/02
Inventor 田文志
Owner HUABO BIOPHARM
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