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PFTM3GW recombinant vector as well as preparation method and application thereof

A technology of recombinant vector and recombinant lentivirus, which is applied in the field of genetic engineering, can solve the problems of insufficient infection efficiency, low TCR sensitivity, complex and cumbersome preparation methods, etc., and achieve the effect of enhancing the ability to recognize tumor antigens

Inactive Publication Date: 2018-06-01
HENAN HUALONG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CN107502596A provides a kind of T cell receptor that contains specific recognition NY-ESO-1 and expresses said TCR and cytokine at the same time, constructed the recombinant expression vector pGreen puro-TCR (NY-ESO-1), packaged And purify lentivirus, lentiviral vector to infect T lymphocytes, but the TCR prepared by the above prior art has low sensitivity, weak signal transduction, insufficient infection efficiency, complicated and cumbersome preparation method
[0008] In summary, the current T cell receptor (TCR) has low sensitivity to recognize tumor antigens and weak signal transduction. Therefore, a recombinant vector was developed to express a large number of NY-ESO-1 specific TCRs, Has broad application prospects and huge market value

Method used

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  • PFTM3GW recombinant vector as well as preparation method and application thereof
  • PFTM3GW recombinant vector as well as preparation method and application thereof
  • PFTM3GW recombinant vector as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1 Construction of recombinant vector

[0051] In order to facilitate the construction of the pFTM3GW recombinant vector, the restriction sites BamHI and XbaI required for vector construction were added when synthesizing NY-ESO-1; the sequence contains α chain protein, furin, SGSG flexible amino acids, and connecting peptide P2A and T cell receptor β-catenin, synthesized by Shanghai Sangon Bioengineering Co., Ltd., and cloned into the pUCK vector. The specific double enzyme digestion reaction system is as follows:

[0052] The double enzyme digestion reaction system is as follows:

[0053]

[0054] The above is a reaction system, five reaction systems were done in total, that is, repeated five times, the reaction conditions are as follows: 1 hour at 30°C, 1 hour at 37°C, the enzyme digestion and identification results of pUCK-NY-ESO-1 plasmid are as follows: figure 1 shown by figure 1 It can be seen that NY-ESO-1 was digested successfully with a length of...

Embodiment 2

[0062] Embodiment 2 packaging virus

[0063] (1) Virus packaging

[0064] 1. Inoculate 293T cells in T175 cell culture dishes, and the cell density grows to about 70% about 24 hours after plating;

[0065] 2. 40 minutes before transfection, absorb the cell culture medium and add 20mL of fresh culture medium without antibiotics and serum. Note: It is best to use fresh culture medium with carefully adjusted pH value for transfection. The culture medium can be subpackaged and frozen after preparation, and then thawed when used;

[0066] 3. Take the recombinant vector plasmid DNA, add them to 1.8mL calcium chloride solution, mix well and let stand at room temperature for 5min;

[0067] 4. Add DNA-calcium chloride solution to 1.8mL BBS solution, mix well and incubate at room temperature for 25min, no visible precipitation will occur at this time;

[0068] 5. Evenly add the DNA-calcium chloride-BBS mixture to the entire T175 cell culture dish, and culture it in a 37°C cell cultur...

Embodiment 3

[0076] Example 3 Verification of target gene transcription and expression at the gene level in cells

[0077] The recombinant lentivirus pFTM3GW-NY-ESO-1 and the control virus pFTM3GW-GFP were used to infect SUPT-1 cells and T cells, and RT-PCR was used to detect gene transcription and expression. The experimental steps were as follows:

[0078] 1. Add the following reaction mixture to the PCR tube on ice:

[0079]

[0080] Mix gently and centrifuge for 3-5 seconds in an instant centrifuge to keep the solution at the bottom of the tube;

[0081] Optional: If the RNA template has a high GC content or contains secondary structures, it can be incubated at 65°C for 5 minutes and then cooled on ice.

[0082] 2. Place the tube on ice and add the following ingredients in the specified order:

[0083]

[0084] Mix gently and centrifuge for 3-5 seconds in an instant centrifuge to keep the solution at the bottom of the tube;

[0085] 3. Perform reverse transcription on a PCR ma...

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Abstract

The invention provides a pFTM3GW recombinant vector as well as a preparation method and application thereof. The pFTM3GW recombinant vector comprises a specific T cell receptor and a lentiviral vectorpFTM3GW, wherein the specific T cell receptor is used for identifying an antigen NY-ESO-1; the specific T cell receptor used for identifying the antigen NY-ESO-1 sequentially comprises an alpha chain, furin, SGSG flexible amino acid, a linker peptide and a beta chain. In the pFTM3GW recombinant vector, the T cell receptor comprising the alpha chain, the furin, the SGSG flexible amino acid, the linker peptide and the beta chain is inserted into the pFTM3GW vector, and all the components have synergistic interaction, so that the alpha chain and the beta chain of the T cell receptor are promotedto form a stable structure and achieve high-efficiency expression, the signal transduction is promoted, the transfection efficiency and a targeted effect are improved, an immunotherapy effect is promoted, and the pFTM3GW recombinant vector is wide in application prospect and market value.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a pFTM3GW recombinant vector and its preparation method and application. Background technique [0002] Today, adoptive cellular immunotherapy has been recognized as the fourth effective method for treating tumors after surgery, chemotherapy, and radiotherapy. Among them, T-cell-based adoptive cell therapy mediates the elimination of tumor cells by enhancing the body's immunity. It is a specific and non-toxic new cancer therapy that has received high attention in recent years. The reason why tumors can proliferate and metastasize in vivo is because they lack molecules that can activate T cells, such as MHC antigens, and cannot be recognized by T cells, so they cannot activate T cell-mediated active immunity. Therefore, in order to induce a strong anti-tumor immune response, the expression and presentation of tumor-associated antigens and immune effects mediated by c...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/62C12N7/01C12N5/10C12N9/50C07K19/00A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K2319/00C07K2319/33C12N5/0636C12N7/00C12N9/50C12N15/86C12N2510/00C12N2740/15021C12N2740/15043C12N2740/15052
Inventor 韦丹范宇清连杰秦志华马亚锋皇甫晶晶
Owner HENAN HUALONG BIOLOGICAL TECH
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