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Application of peptide tag to purification of fusion polypeptide

A technology that fuses peptides and peptide tags, applied in the field of macromolecules, can solve the problems of reducing patient treatment compliance, making different antibodies less likely to be made into the same dosage form, etc., so as to reduce the volume and frequency of clinical administration and enhance the therapeutic effect , Excellent therapeutic effect

Active Publication Date: 2020-11-03
杭州皓阳生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, on the one hand, sequential injection of antibodies will reduce patients’ treatment compliance; on the other hand, due to the differences in the physicochemical properties of different antibodies, it is difficult or almost impossible to make different antibodies into the same dosage form

Method used

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  • Application of peptide tag to purification of fusion polypeptide
  • Application of peptide tag to purification of fusion polypeptide
  • Application of peptide tag to purification of fusion polypeptide

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Mouse immunization: choose 6-8 weeks old female BALB / c mice as experimental animals. For the initial immunization, 50 μg of human CD73 protein was mixed with complete Freund’s adjuvant to form an emulsion, and the mice were injected intraperitoneally at an injection volume of 0.5ml / mouse. Booster immunization was carried out every two weeks. A total of four booster immunizations were performed. One week after the last immunization, Collect and separate serum to detect antibody titer by ELISA method, refer to the instructions for the method. Select mouse cells with high titer to prepare hybridoma to prepare single splenocyte suspension. Collect logarithmically grown myeloma cells to prepare immune spleen cell suspension, mix myeloma cells and spleen cells in a certain proportion, wash with incomplete culture medium, centrifuge to discard the supernatant, and place the cell pellet and 1ml PEG-4000 respectively Preheat in a water bath at 40°C, then mix to form a reaction ...

Embodiment 2

[0051] The light chain variable region and the heavy chain variable region of the murine anti-CD73 antibody were amplified by PCR, and the complementarity-determining region sequence was obtained after excluding the framework region sequence; the three complementarity-determining region CDR-L1 amino acids of the light chain The sequence is shown in SEQ ID NO: 1; the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 2, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 3; the amino acid sequence of the three complementarity determining regions CDR-H1 of the heavy chain is shown in SEQ ID The amino acid sequence of NO:4, CDR-H2 is shown in SEQ ID NO:5, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO:6. The humanized form of anti-human CD73 antibody was prepared by referring to the preparation method of Molecule Immunol, and the humanized template that best matched the non-CDR region of the mouse anti-CD73 antibody was selected from the Germline database, ...

Embodiment 3

[0055] The heavy chain variable regions (SEQ ID NO: 8, SEQ ID NO: 9) of the humanized anti-human CD73 monoclonal antibody were respectively connected with the human antibody IgG1 heavy chain constant region (SEQ ID NO: 13) to obtain the corresponding Full-length heavy chain sequence. The light chain variable regions (SEQ ID NO: 11, SEQ ID NO: 12) of the humanized anti-human CD73 monoclonal antibody were connected with the constant region (SEQ ID NO: 14) of the human antibody Kappa light chain respectively, and corresponding The full-length sequence of the light chain of the humanized antibody was obtained by combining all the above-mentioned full-length sequences of the heavy chain and the full-length sequence of the light chain, which were ligated into the TL10-11 (vector backbone pEGFP-N1) vector by enzyme digestion. Anti-human CD73 antibody YS79b-85 (heavy chain amino acid sequence shown in SEQ ID NO: 8, light chain amino acid sequence shown in SEQ ID NO: 11, heavy chain co...

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Abstract

The invention provides an application of a peptide tag to purification of a fusion polypeptide. The peptide tag sequence is connected with the fusion polypeptide to screen and purify the fusion polypeptide, and the fusion polypeptide comprises a CTLA4 extracellular region, a fusion polypeptide of an anti-CD73 antibody and a pharmaceutical composition containing the protein.

Description

technical field [0001] The invention relates to the field of macromolecules, in particular to the use of a peptide tag in purifying fusion polypeptides. Background technique [0002] Peptide tags can be biotinylated on lysine residues by biotin ligase in vivo or in vitro to achieve protein biotinylation. However, avidin or streptavidin can specifically bind to biotin. It is based on these two reactions that peptide labeling technology can be applied to protein fixation, purification and imaging. Basically all proteins can be effectively and specifically labeled with the help of peptide tagging technology. [0003] Cytotoxic T lymphocyte-associated antigen 4 (CTLA4), also known as CD152, is a leukocyte differentiation antigen. The ligands of CTLA4 and CD28 are both B7 molecules, and the combination of CTLA4 and B7 molecules induces T cell anergy and participates in the negative regulation of immune responses. [0004] CD73 is an extracellular-5'-nucleotidase encoded by the...

Claims

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Application Information

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IPC IPC(8): C07K14/705C07K19/00
CPCC07K14/70521C07K16/40C07K2317/56C07K2317/565C07K2319/00C07K2319/02
Inventor 靳维维于聪陈滨
Owner 杭州皓阳生物技术有限公司
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