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MIP3 alpha-FGFR1-PD1/Fc fusion protein and nucleic acid molecule and application thereof

One-FGFR1-PD1, FGFR1 technology, applied in the field of anti-tumor drugs, can solve the problems of complex in vitro operation, high cost, and difficult standardization, and achieve the effect of shrinking tumor volume, inhibiting growth, and reducing tumor quality

Active Publication Date: 2019-07-16
THE FIRST AFFILIATED HOSPITAL OF HAINAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, DC vaccines prepared by commonly used methods such as DC loaded with tumor cell antigens or polypeptides, cultured in vitro, transformed with total RNA of tumor cells, or genetically modified with specific tumor antigens have been used in clinical applications, but this "workshop" DC vaccine preparation method exists. In vitro operations are complex, costly, and difficult to standardize, etc.

Method used

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  • MIP3 alpha-FGFR1-PD1/Fc fusion protein and nucleic acid molecule and application thereof
  • MIP3 alpha-FGFR1-PD1/Fc fusion protein and nucleic acid molecule and application thereof
  • MIP3 alpha-FGFR1-PD1/Fc fusion protein and nucleic acid molecule and application thereof

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preparation example Construction

[0047] The present invention also provides a method for preparing the nucleic acid molecule described in the above technical solution, which includes the following steps:

[0048] 1) Mix primer 1 to primer 84, and use primer 1 to primer 84 as templates and primers to perform the first round of PCR amplification to obtain mixed amplification products;

[0049] The nucleotide sequences of primer 1 to primer 84 are shown in SEQ ID NO. 3 to SEQ ID NO. 86 in sequence;

[0050] 2) Using the mixed amplification product as a template, primer 1 as an upstream primer, and primer 84 as a downstream primer, perform the second round of PCR amplification to obtain the nucleic acid molecule of the MIP3α-FGFR1-PD1 / Fc fusion protein.

[0051] In the present invention, the nucleotide sequences of primer 1 to primer 84 are preferably shown in SEQ ID NO. 3 to SEQ ID NO. 86.

[0052] In the present invention, it is preferable to obtain primer 1 to primer 84 by artificial synthesis, and to mix the obtained p...

Embodiment 1M

[0073] Example 1 Construction of nucleic acid molecules of MIP3α-FGFR1-PD1 / Fc fusion protein

[0074] 1) Mix equal amounts of primer 1 to primer 84 to obtain a primer mixture with a concentration of 20 mM for primer 1 to primer 84, and primer 1 to primer 84 in the primer mixture are primers and templates for each other. Perform the first round of PCR amplification under the following conditions Increase to obtain mixed amplification products.

[0075] The PCR amplification system used in the first round of PCR amplification is 50 μL: 33 μL ddH 2 O, 5μL of 10×PCRBuffer, 1μL of dNTP with a concentration of 2.5mM each, 10μL of a mixture of primers 1 to primer 84 with a concentration of each primer of 20mM, and 1μL of Pfu high-fidelity DNA polymerase with a concentration of 2.5U / L;

[0076] The PCR reaction program adopted in the first round of PCR amplification is: 95°C for 5 min; 94°C for 30s, 55°C for 30s, 72°C for 30s, 25 cycles; 72°C for 5 minutes.

[0077] 2) Using the mixed amplifi...

Embodiment 2

[0096] Example 2 Construction, screening and identification of target plasmid

[0097] 1) LB medium preparation

[0098] Liquid culture medium: tryptone 10g, yeast extract 5g, NaCl 10g, add 950ml deionized water to dissolve, adjust to pH 7.2 with 5mol / L NaOH, finally add water to 1000ml, and autoclave 10 pounds for 20min after filling.

[0099] Solid medium: 1.5g agar powder, dissolved in 100ml LB liquid medium, autoclaved 10 pounds for 20min.

[0100] 2) Enzyme digestion of the target gene fragment and plasmid

[0101] Double enzyme digestion of pcDNA3.1(+) with AflII and XbaI. The digestion reaction system is: pcDNA3.1(+) 12μL, 10buffer 2μL, AflII1μl, XbaI 1μL and ddH 2 O 4μL, 20μL in total.

[0102] Place the above system in a 37°C water bath for 6 hours, and then inactivate the restriction enzymes at 65°C for 15 minutes. The digested products were purified by gel recovery using the gel recovery kit of Jiangsu Forte Biotechnology Co., Ltd. After identification by 10g / L agarose electr...

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Abstract

The invention provides MIP3 alpha-FGFR1-PD1 / Fc fusion protein and a nucleic acid molecule and application thereof, and relates to the technical field of antitumor medicines. The MIP3 alpha-FGFR1-PD1 / Fc fusion protein is obtained through coding a nucleic acid molecule containing PD1, FGFR1 and a mouse antibody Fc segment. The MIP3 alpha-FGFR1-PD1 / Fc fusion protein has significant tumor vessel targeting effects, and is in high-concentration enrichment in tumor tissue or tumor new vessels. The fusion protein contains PD1 protein, and is combined with PDL1 in tumor microenvironment; through competitive combination, the combination of PD1 on the surfaces of T cells and ligands PDL1 and PDL2 on the surfaces of tumor cells is reduced through competitive binding, and further continuous activationof a PD1 signal channel is blocked, so that the effect of the fusion protein for eliminating immunosuppression can be fully exerted, and efficient tumor immunity treatment effects are provided.

Description

Technical field [0001] The invention relates to the field of anti-tumor drugs, in particular to a nucleic acid molecule of MIP3α-FGFR1-PD1 / Fc fusion protein, and a preparation method and application thereof. Background technique [0002] Macrophage inflammatory protein 3α (MIP3α) is currently the most important specific chemokine found in DC. CC Chemokine Receptor 6 (CCR6) is the only receptor for MIP3α. Studies have shown that MIP3α can recruit pathogen-infected tissues or tumor areas by attracting immature DCs expressing CCR6, so that the recruited DCs can be activated and exert an antigen presentation effect. It can be seen that if we try to increase the level of MIP3α in tumor tissues, a large number of DCs can be recruited in the local tumor tissues, which will greatly enhance the ability of these DCs to process and present antigens. Therefore, in order to enable DCs recruited by MIP3α chemotaxis in vivo to target tumor cells more effectively and improve their biological a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/10C12N15/85C12N5/10A61K38/19A61K47/62A61P35/00
CPCC07K14/71C07K14/70521C07K14/47C12N15/85A61P35/00C07K2319/30A61K38/00
Inventor 郑少江张晓钿赵焕阁林岷格刘思汝吴新来
Owner THE FIRST AFFILIATED HOSPITAL OF HAINAN MEDICAL UNIV
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