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Signal regulatory protein alpha fragment-anti-FcRn single-chain antibody fusion protein as well as preparation and application thereof

A technology of signal regulation protein and fusion protein, which can be applied in the direction of peptide/protein composition, plasma life extension fusion, DNA/RNA fragment, etc. It can solve the problems of weak pH dependence, only days or even hours, and low affinity.

Active Publication Date: 2020-08-04
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, except for a small number of therapeutic antibody drugs that genetically engineer Fc to extend the half-life to a time comparable to that of natural antibodies (~21 days), the half-life of most antibody drugs and other fusion protein drugs is still only a few days or even a few hours. This is mainly due to the relatively low affinity of these drugs with FcRn or the weak pH dependence of their binding.

Method used

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  • Signal regulatory protein alpha fragment-anti-FcRn single-chain antibody fusion protein as well as preparation and application thereof
  • Signal regulatory protein alpha fragment-anti-FcRn single-chain antibody fusion protein as well as preparation and application thereof
  • Signal regulatory protein alpha fragment-anti-FcRn single-chain antibody fusion protein as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Construction of the expression vector of the fusion gene of the signal regulatory protein α fragment-anti-FcRn single-chain antibody fusion protein (SIRPα-F8)

[0073] 1.1 Experimental materials

[0074] 1.1.1 Reagent consumables

[0075] HEK293-6E cell line: Shanghai Cell Bank, Chinese Academy of Sciences

[0076] FreeStyle TM 293 Medium: Cat#12338018

[0077] Gibco opti-MEM Medium: Cat#31985-070

[0078] G418: 50mg / ml Solution, Invitrogen, Cat#10131-027

[0079] 293fectin (Invitrogen): Cat#12347

[0080] DNA Marker: DL100, DL500, D12000

[0081] HotStarTaq Plus Master Mix Kit (1000): QIAGEN, Cat#203645

[0082] Gel recovery kit: Qiagen, Cat#28606

[0083] Plasmid Mini Kit: Qiagen, Cat#12123

[0084] Endotoxin-free plasmid extraction kit: Qiagen, Cat#12165

[0085] Plus2 Pre-Stained Standard, Invitrogen, Cat#LC5925

[0086] HisTrap FF: GE Healthcare, Cat#11-0004-58

[0087] Protein-free blocking buffer: Pierce, Cat#37573

[0088] Amine Coupli...

Embodiment 2

[0127] Example 2 Expression and purification of fusion gene of signal regulatory protein α fragment-anti-FcRn single-chain antibody fusion protein (SIRPα-F8)

[0128] 2.1 Conversion:

[0129] The synthetic expression vector pTT5 was transformed into competent DH5a bacteria and cultured overnight by streaking on the agarose gel plate;

[0130] 2.2 A large number of plasmids were extracted for HEK293-6E cell transfection and protein expression.

[0131] Amplify the selected colony culture solution containing the correct gene sequence in LB medium with ampicillin at 37°C and 250rpm until the OD600 is about 1.5, and then amplify the bacterial solution to a ratio of 1:1000 to 1 Add the above LB medium, 37°C, 250rpm, and culture overnight for 12-16 hours;

[0132] Collect bacterial pellet by centrifugation, 20000g, 4°C, 15 minutes;

[0133] Resuspend the pellet harvested per 500ml of bacterial liquid with 20ml of buffer P1;

[0134] Add 20ml buffer P2, mix vigorously, an...

Embodiment 3

[0166] Example 3 Extended half-life functional identification of fusion protein

[0167] 3.1 Experimental materials and instruments

[0168] 3.1.1 Experimental materials

[0169] HEK293 / EGFP-HLA-A2 cell line: prepared by the method in Document 1 (Document 1: YS Qiu, Singlechain antibody fragments with pH dependent binding to FcRn enabled prolonged circulation of therapeutic peptide in vivo.Journal of Controlled Release 229(2016)37 -47.)

[0170] HEK293 / EGFP-hFcRn cell line: prepared by the method in Document 1

[0171] Hygromycin B: invitrogen, Cat#10687-010

[0172] DMEM medium: GIBCO, Cat#11995065

[0173] Penicillin-Streptomycin: GIBCO, Cat#15140122

[0174] Protein-free blocking buffer: Pierce, Cat#37573

[0175] PBS1: PBS, pH7.4, containing 0.05% Tween20

[0176] PBS2: PBS, pH6.0, containing 0.05% Tween20

[0177] Half-hole high adsorption 96-well plate: Corining, Cat#3690

[0178] Streptavidin: PROZYME, Cat#SA26

[0179] HRP linked anti-His antibody: Genscript,...

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Abstract

The invention relates to the technical field of biology, in particular to a signal regulatory protein alpha fragment-anti-FcRn single-chain antibody fusion protein as well as preparation and application thereof. The fusion protein contains a signal regulatory protein alpha fragment and an anti-FcRn single-chain antibody, wherein the signal regulatory protein alpha fragment is connected with the anti-FcRn single-chain antibody through a connecting peptide. According to the fusion protein disclosed by the invention, the binding specificity and pH dependence with hFcRn are well retained; the SIRPalpha-F8 monoclonal antibody prepared by the method is high in specificity and long in half-life period, can be used as a fusion protein carrier for development and research of long-acting macromolecular drugs, has a wide application prospect and huge economic and social values in development of long-acting polypeptides and protein drugs, and can be used for treating tumors by combining SIRP alpha-F8 with the monoclonal antibody targeting tumor cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a signal regulation protein α fragment-anti-FcRn single-chain antibody fusion protein and its preparation and application. Background technique [0002] Signal regulatory protein α (SIRPα) is a transmembrane protein widely expressed on the surface of myeloid cells such as macrophages and dendritic cells, and belongs to the immunoglobulin superfamily molecule. The extracellular region of SIRPα contains one IgV and two IgC immunoglobulin-like domains, and this diverse IgV-like domain can effectively bind to its ligand CD47. CD47 is widely expressed in tissue cells. By binding to SIRPα on the surface of macrophages, CD47 can generate a "don't eat me" signal and organize the phagocytosis of macrophages. Some existing anti-CD47 antibodies or SIRP-Ig fusion proteins contain Fc fragments in the protein structure. The Fc fragment has two main functions: one is that it can prolong the half-...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K38/17A61K47/68A61P35/00
CPCC07K14/70596A61K47/6849A61P35/00C07K2319/31A61K38/00
Inventor 徐宇虹吴凤岚邱阳生
Owner SHANGHAI JIAO TONG UNIV
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