Anti-rat CD3 recombinant immunotoxin as well as preparation method and application thereof
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A technology of immunotoxin and toxin, which is applied in the biological field and can solve problems such as different half-lives
Inactive Publication Date: 2019-01-25
GUANGDONG PHARMA UNIV
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Zhirui Wang et al. reported recombinant anti-human and anti-monkey CD3 T cell immunotoxins. This type of immunotoxin contains DT390, which is covalently coupled to its C-terminus through (G4S)3, and then fused with the single-chain antibody CD3+ScFv, but uses the same The latter is different from the connection method between the anti-CD3 Fv region and DT390, the affinity of the recombinant toxin is increased by more than 30 times, and the half-life in peripheral blood and lymphatic circulation is also different
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Embodiment 1
[0047] (1) Expression vector construction of mouse anti-CD3 recombinant immunotoxin:
[0049] Design and optimize the codon nucleotide sequence of mouse anti-CD3 recombinant immunotoxin, see SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; introduce KpnI restriction site and EK recognition at the 5' end respectively Site-(Asp) 4 Lys coding sequence and design start codon ATG, wherein EK recognition site-(Asp) 4 Lys coding sequence in order to excise the His-Tag of the final expressed fusion protein; design 6 histidine codons CGTCGTCGTCGTCGTCGT, stop codon TAATGA and introduce XhoI restriction site at the 3′ end, so that each construct is easy to protein Purification and detection, while removing the stop codon at the 3' end of the coding sequence.
[0050] The nucleotide sequence was synthesized by the company, then amplified by PCR technology, and m...
Embodiment 2
[0059] In vitro cell model analysis of biological characteristics of mouse anti-CD3 recombinant immunotoxin and its effect on mouse CD3 phenotype knockout
[0060] 1. In vitro cell model analysis of mouse anti-CD3 recombinant immunotoxin binding to mouse CD3 and phenotype knockout
[0061] Analysis of murine anti-CD3 recombinant immunotoxin binding and killing of murine peripheral blood mononuclear cells in vitro using healthy mouse peripheral blood cells.
[0062] (1) Prepare enough mouse peripheral blood lymphocytes and dilute them with cell culture medium to 2.0×10 6 cells / mL, 500 μL per well was added to a 24-well plate.
[0063] (2) Dilute the purified mouse anti-CD3 recombinant immunotoxin with lymphocyte culture medium to 1.0×10 -6 M, 1.0×10 -7 M, 1.0×10 -8 M and 1.0×10 -9 M.
[0064] (3) Add 50 μL of diluted mouse anti-CD3 recombinant immunotoxin or negative control into corresponding wells, and incubate in a 37° C. incubator for 48 hours.
[0065] (4) Collect c...
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Abstract
The invention discloses anti-rat CD3 recombinant immunotoxin as well as a preparation method and application thereof and belongs to the technical field of biology. A genetic engineering technology anda genetic engineering strategy are adopted, and a combination manner of a single-chain antibody and DT390 is changed to improve the affinity; codon series are optimized to obtain three types of recombinant immunotoxin which take a DT structure as a toxic center, and a prokaryotic expression system is constructed; anti-rat single-chain antibody coupled immunotoxin of a targeting T cell receptor CD3epsilon is obtained and T lymphocytes of a peripheral immune organ are eliminated; a transplanting immune model is established by utilizing the immunotoxin, and treatment researches including immunetolerance, autoimmune diseases and the like are carried out. The anti-rat CD3 recombinant immunotoxin proves the effectiveness of eliminating rat peripheral blood and lymph node T cells by the targeting anti-rat CD3epsilon recombinant immunotoxin. The anti-rat CD3 recombinant immunotoxin has application meanings in the aspects of researches of eliminating the rat T lymphocytes, treating the autoimmune diseases, transplanting the organs, treating immune tolerance and treating tumors, and possibly has a potential of pre-clinical pharmaceutical researches and conversion subsequently.
Description
technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to a fusion protein for selectively destroying cells with a specific marker, which is a chimeric recombinant formed by combining a cell selective ligand and a toxin molecule through gene fusion And its preparation method and application. Background technique [0002] Immunotoxin (IT) can be used as a targeted therapy drug, and it is usually a chimeric protein composed of a cell-selective ligand and a toxin molecule through chemical linkage or gene fusion. The mechanism of action was proposed by German immunologist Ehrilich in the early 20th century. Commonly used targeting carriers include monoclonal antibodies such as CD3, CDS, CD22, IL-2 (interleukin 2), IL-3 (interleukin 3), GM-CSF (granulocyte colony-stimulating factor), and short peptide hormones, etc. ; Toxins such as ricin, Pseudomonas Exotoxin (PE), diphtheria toxin (diphtheriatoxin, DT) and so on. The composition ...
Claims
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