Recombinant fusion protein capable of simultaneously blocking HER2 and VEGFR signal paths
A technology of protein and composition, applied in the direction of drug combination, peptide/protein composition, recombinant DNA technology, etc.
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Embodiment 1
[0067] Example 1 Construction of Anti-HER2-VEGFR Bifunctional Fusion Protein Expression Vector
[0068] 1. Amino acid sequence and corresponding nucleic acid sequence of anti-HER2-VEGFR bifunctional fusion protein
[0069] The amino acid sequence of the anti-HER2 light chain (the sequence is derived from WO1992022653) is shown in SEQ ID NO: 1, and the corresponding nucleotide coding sequence optimized according to the codon preference of mammalian cells is shown in SEQ ID NO: 2.
[0070] Anti-HER2 heavy chain-VEGFR1D2-VEGFR2D3 (amino acid sequence consisting of an anti-HER2 antibody heavy chain, a Ser(Gly4Ser)3 linker sequence, the D2 domain of VEGFR1 and the D3 domain of VEGFR2, wherein the anti-HER2 heavy chain sequence is derived from The amino acid sequence of WO1992022653, VEGFR1D2-VEGFR2D3 sequence derived from US7070959) is shown in SEQ ID NO: 3, and the corresponding nucleotide coding sequence optimized according to the codon bias of mammalian cells is shown in SEQ ID ...
Embodiment 2
[0079] Example 2 Expression of recombinant anti-HER2-VEGFR bifunctional fusion protein
[0080] 1. Preparation of HEK293-T cell factories
[0081] HEK293-T cells with good growth status and viability (ratio of living cells, which can also be written as "living rate") over 95% were divided into 1.8×10 7 Each inoculum was inoculated in a ten-layer cell factory (NUNC company), cultured with DMEM medium (purchased from Corning company) containing 10% fetal bovine serum (purchased from Gibco company), and placed in the cell factory at 37°C after repeated inversion and mixing , 5% CO2 incubator for 48 hours, the cells are completely attached to the wall, and the density reaches 80%, which can be used for transient transfection.
[0082] 2. Cell Transient Transfection and Collection of Cell Culture Supernatant
[0083] After filtering the X0GC2-anti-HER2-VEGFR1D2-VEGFR2D3 plasmid with a 0.22 μm filter membrane, absorb 1330 μg of the plasmid and add it to 66 ml of serum-free DMEM me...
Embodiment 3
[0084] Example 3 Purification of recombinant anti-HER2-VEGFR bifunctional fusion protein
[0085] The purification process of the recombinant bifunctional fusion protein is as follows: figure 2 shown.
[0086] 1. Pretreatment of cell expression fermentation broth
[0087] The harvested cell culture supernatant was centrifuged at 7000rpm for 20min to remove the precipitate. After the supernatant of the cell fermentation broth was filtered through a 0.2 μm filter membrane, it was concentrated by 10K membrane bag ultrafiltration and replaced with 20 mM PB buffer, adding 150 mM sodium chloride, pH 7.4. Filter through a 0.2 μm filter to remove precipitates before applying chromatographic column purification. The operation of this step was carried out at 4°C.
[0088] 2. rProtein A affinity chromatography purification
[0089] AKTA explorer 100 protein purification system (GE Healthcare) and affinity chromatography column rProtein A Sepharose Fast Flow (16mm I.D., 15ml, GE Hea...
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