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mip3α-fgfr1-pd1/fc fusion protein and its nucleic acid molecule and application

A -FGFR1-PD1, FGFR1 technology, applied in the field of anti-tumor drugs, can solve the problems of difficult specification, complicated in vitro operation, high cost, and achieve the effect of inhibiting growth, reducing tumor volume and reducing tumor mass

Active Publication Date: 2021-03-23
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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  • mip3α-fgfr1-pd1/fc fusion protein and its nucleic acid molecule and application

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preparation example Construction

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

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

[0049] The nucleotide sequences of the primers 1 to 84 are sequentially shown as SEQ ID NO.3 to SEQ ID NO.86;

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

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

[0052] In the present invention, the primers 1 to 84 are preferably artificially synthesized, and equal amounts of the obtained primers 1 to 84 are mi...

Embodiment 1M

[0073] Example 1 Construction of Nucleic Acid Molecules of MIP3α-FGFR1-PD1 / Fc Fusion Protein

[0074] 1) Mix primers 1 to 84 in equal amounts to obtain a primer mixture with a concentration of 20 mM for each of primers 1 to 84. Primers 1 to 84 in the primer mixture serve as primers and templates for each other, and perform the first round of PCR amplification according to the following conditions: 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. 5 μL of 10×PCRBuffer, 1 μL of dNTP with a concentration of 2.5 mM, 10 μL of a mixture of primers 1 to 84 with a concentration of 20 mM, and 1 μL of Pfu high-fidelity DNA polymerase with a concentration of 2.5 U / L;

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

[0077] 2) Using the mixed amplification pro...

Embodiment 2

[0096] Construction and screening, identification of embodiment 2 target plasmids

[0097] 1) Preparation of LB medium

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

[0099] Solid medium: 1.5g of agar powder, dissolved in 100ml of LB liquid medium, sterilized under 10 pounds of high pressure for 20min.

[0100] 2) Digestion of target gene fragments and plasmids

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

[0102] Put the above system in a water bath at 37°C for 6h, then inactivate the restriction enzyme at 65°C for 15min. The digested products were recovered and purified by gel recovery kits from Jiangsu Futai Biotechnology Co., Ltd. respectively. Aft...

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Abstract

The invention provides MIP3α-FGFR1-PD1 / Fc fusion protein and its nucleic acid molecule and application, and relates to the technical field of anti-tumor drugs. The MIP3α-FGFR1-PD1 / Fc fusion protein of the present invention is encoded by a nucleic acid molecule comprising PD1, FGFR1 and the Fc segment of a mouse antibody. The MIP3α-FGFR1-PD1 / Fc fusion protein of the present invention has a significant tumor blood vessel targeting effect, and is enriched at a high concentration in tumor tissue and or tumor neovascularization. The fusion protein of the present invention contains PD1 protein, which is compatible with the tumor microenvironment The combination of PDL1 in T cells reduces the binding of PD1 on the surface of T cells to the ligands PDL1 and PDL2 on the surface of tumor cells through competitive binding, thereby blocking the continuous activation of PD1 signaling pathways, thereby fully exerting the fusion protein’s role in eliminating immunosuppression and providing efficient Tumor immunotherapy.

Description

technical field [0001] The present invention relates to the field of antitumor drugs, in particular to nucleic acid molecules of MIP3α-FGFR1-PD1 / Fc fusion protein and its preparation method and application. Background technique [0002] Macrophage inflammatory protein 3α (MIP3α) is currently found to be the most important specific chemokine for 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, and activate the recruited DCs to play an antigen presentation role. It can be seen that if the level of MIP3α in tumor tissue is managed to increase, a large number of DCs can be recruited in the local tumor tissue, thereby greatly enhancing the ability of these DCs to process and present antigens. Therefore, in order to make DCs recruited by MIP3α in vivo to target tumor cells more effectively and improve their biological activity of killin...

Claims

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Application Information

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Patent Type & Authority Patents(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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