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Preparation method and application of double-targeting anti-tumor recombinant protein based on antibody and macropinocytosis

A recombinant protein, macropinocytosis technology, applied in antitumor drugs, targeting specific cell fusion, peptide/protein components, etc., to achieve the effect of strong tissue penetration, significant therapeutic effect, and good application prospects

Active Publication Date: 2018-06-08
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation and application of a class of anti-tumor drugs based on the double-targeting effect of antibodies and macropinocytosis described in the present invention has not been reported at home and abroad so far

Method used

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  • Preparation method and application of double-targeting anti-tumor recombinant protein based on antibody and macropinocytosis
  • Preparation method and application of double-targeting anti-tumor recombinant protein based on antibody and macropinocytosis
  • Preparation method and application of double-targeting anti-tumor recombinant protein based on antibody and macropinocytosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] "Example 1" construction of recombinant expression vector pPIC9K-fv-ldp-3d

[0041] The complete structure of the recombinant protein Fv-LDP-3D is scFv-connecting peptide (G 4 S) 2 -LDP-connecting peptide (G 4S) 2 -3D, whose entire gene expression sequence was optimized and synthesized by GenScript based on the preferred codons of Pichia pastoris. The gene sequences of the experimental control recombinant proteins Fv-LDP and LDP-3D were constructed by designing primers using molecular biology techniques. Invitrogen TM Synthesized by the company, the expression vector is pPIC9K purchased from Invitrogen, and the Escherichia coli competent DH5α is a product of Genstar.

[0042] P1: 5'-TCTG TACGTA ATGGCCCAGGTCCAGCTTC-3' (the underline is the SnaB I restriction site)

[0043] P2: 5'-ATAAGAAT GCGGCCGC TTAGTGATGGTGATGG-3' (the underline is the Not I restriction site)

[0044] P3: 5'-TCTG TACGTA GCTCCAGCTTTCTCTG-3' (the underline is the SnaB I restriction site)

...

Embodiment 2

[0050] "Example 2" Expression and purification of recombinant protein in Pichia pastoris

[0051] The pPIC9K-fv-ldp-3d and the experimental control expression vectors pPIC9K-ldp-3d and pPIC9K-fv-ldp were transformed into Escherichia coli DH5α respectively, single clone strains were selected, and positive vector strains were confirmed by PCR identification and sequencing identification. The expression plasmid was extracted, linearized by Sal I enzyme digestion, and electrotransformed into Pichia pastoris GS115 competent cells after gel recovery. Pick a single colony for colony PCR identification, pick multiple His + The positive colonies were induced to express. After the expression conditions were optimized, the expression yield of the recombinant protein was detected, and the strain with higher expression was selected as the expression strain. The strain is named GS115-FL3, and was sent to the General Microorganism Center of China Microbiological Culture Collection Managemen...

Embodiment 3

[0053] "Example 3" Recombinant protein and EGFR protein and tumor cell affinity activity analysis

[0054] 1. ELISA to detect the affinity activity of recombinant protein to EGFR protein and pancreatic cancer cells

[0055] EGFR protein powder was dissolved in PBS, diluted to 5 μg / ml, 50 μl / well was added to a 96-well plate, and coated overnight at 4°C. Rinse and wash with PBS for 3 times, discard the liquid in the well for later use; pancreatic cancer cells BxPC-3, MIA PaCa-2, AsPC-1 and PANC-1 were treated with 1×10 4 Cells / well density were seeded in a 96-well plate, cultured at 37°C for 24 hours, rinsed twice with PBS, added 50 μl / well of pre-cooled 0.05% glutaraldehyde at 4°C, fixed the cells at 4°C for 20 minutes, and fixed the cells Rinse with PBS 3 times, dry the residual liquid and set aside. The 96-well plate coated with EGFR and the cells fixed was blocked with 5% skimmed milk solution at 200 μl / well at room temperature for 2 hours; washed with PBST buffer (contai...

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Abstract

The invention relates to a double-targeting recombinant protein based on an antibody and targeting macropinocytosis and an application of a coupling chemotherapeutic drug composition in the preparation of anti-tumor drugs. A research shows that a double-targeting action of an anti-EGFR single-chain antibody scFv and a macropinocytosis recombinant protein mediated by LDP and albumin D III is utilized to realize the coupling of chemotherapeutic drugs, and chemotherapeutic drugs such as lidamycin and the like can be conveyed targetedly to tumor cells, so that a more effective anti-tumor effect isexerted. The invention discloses a preparation method and application of the anti-tumor drugs based on the double-targeting action the antibody and the macropinocytosis, and no relevant reports havebeen found at home and abroad so far.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering medicinal proteins, and relates to the preparation and application of a class of antibody-based macropinocytosis dual-targeting anti-tumor recombinant proteins. Background technique: [0002] The Ras protein encoded by the proto-oncogene Ras gene has GTPase activity, and the signaling pathway mediated by Ras molecules plays an important role in regulating cell proliferation and differentiation. The mutation frequency of K-Ras gene in colorectal cancer, lung cancer and pancreatic cancer is as high as 30%-90%. The mutation of K-Ras gene is directly related to the poor prognosis of patients and the low overall survival rate. At present, there is no anti-tumor drug targeting K-Ras in clinical use. The study found that compared with K-Ras wild-type pancreatic cancer cells, tumor cells with K-Ras mutations can rapidly take up extracellular albumin through macropinocytosis, and macropinocytosis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/81A61K38/16A61K47/68A61P35/00
CPCA61K38/00C07K14/00C07K2319/33C12N15/815
Inventor 王晓飞盛唯瑾甄永苏李良王阳阳张胜华李毅刘秀均
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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