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Biological film for promoting gene editing T cell activation and amplification, preparation method and application

A gene editing and biofilm technology, which is applied in the fields of bioengineering and biomedicine, can solve the problems of not being able to change the function of T cells, avoid cleaning steps and contamination opportunities, solve expensive problems, and simplify the culture procedures and steps.

Pending Publication Date: 2018-03-13
CHENGDU MEDGENCELL CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the absence of CD3 antibody signaling, stimulation of CD137 molecules did not alter T cell function, suggesting that CD137 is only a co-stimulatory molecule

Method used

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  • Biological film for promoting gene editing T cell activation and amplification, preparation method and application
  • Biological film for promoting gene editing T cell activation and amplification, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Preparation and purification of recombinant biofilm with rCD137L membrane protein

[0026] Step 1. Use the CRISPR-CAS9 gene editing method to insert the CD137L gene into the K562 cell genome at a fixed point. The specific steps are:

[0027] 1) Synthesize the full sequence of the CD137L gene with 6 His tag gene sequences, the sequence is shown in SEQ ID NO:1 below;

[0028] 2) Design, prepare and construct 3 gRNAs according to the target gene sequence.

[0029] 3) The constructed gRNA was constructed and transfected into K-562 cells, and the editing efficiency of gRNA in cells was detected.

[0030] 4) Compare and analyze the editing efficiency of the three gRNAs in cells, select the gRNA with the highest editing efficiency and design the corresponding ssDNA.

[0031] 5) Co-transfect the prepared gRNA and ssDNA with the highest editing efficiency into K-562 host cells.

[0032] 6) The transfected cells were identified by PCR amplification and Sanger sequen...

Embodiment 2

[0038] Example 2: Activation and proliferation of gene-edited T cells by recombinant biofilm with rCD137L protein

[0039] The recombinant biofilm with rCD137L membrane protein obtained in Example 1 was used to activate and expand gene-edited T cells.

[0040] Human peripheral blood mononuclear cells are now used as processed samples, and CRISPR-Cas9 technology is used to knock out the human PD-1 gene to construct PD-1 gene knockout T cells, and the gene-edited activated T cells are efficiently expanded and cultured in vitro. Specific steps are as follows:

[0041] Step 1: Isolation of peripheral blood mononuclear cells from blood

[0042] Directly extract 80-100ml of venous blood from the donor, and add anticoagulant-heparin;

[0043] Centrifuge the collected blood sample at 2000rpm for 10min at room temperature, and carefully absorb the upper plasma layer for later use; restore the blood sample to its original volume and mix well; slowly add the diluted blood to Ficoll, an...

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PUM

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Abstract

The invention provides a preparation method of a recombination biological film for promoting gene editing T cell activation and amplification. The recombination biological film is prepared through 1),leading a modified CD137L gene sequence to a K562 cell unit through a gene recombination method to obtain a steady transfer strain cell of CD137L protein stably expressed and modified at the K562 cell surface, wherein the cell is named as rCD137L-K562 cell; 2), regularly culturing the rCD137L-K562 cell in step 1), and breaking the cell through the cell lysis buffer; carrying out a differential speed centrifugal method to obtain the biological film containing the modified CD137L protein; gathering, extracting and purifying the biological film containing the modified CD137L protein by an affinity chromatography and a magnetic bead separating method. The preparation method can improve the irritation of the gene editing T cell, and is free from any bad and side effects.

Description

technical field [0001] The invention belongs to the field of biomedicine, especially the field of bioengineering technology, and specifically relates to a method for preparing a biofilm containing modified CD137L protein and includes the aspects of the recombinant biofilm activating gene-edited T lymphocytes and promoting the proliferation of gene-edited T lymphocytes Applications. The gene-edited T lymphocytes cultured with the recombinant biofilm can be applied to clinical cellular immunotherapy, and can also be used for basic medical research, clinical application research and biotechnology research, especially for immune system research and tumor killing effect research. Background technique [0002] T lymphocytes are one of the most important immune cells in the human body. They are differentiated from lymphatic stem cells in the thymus and migrate to the peripheral blood after maturation; they can be divided into three subgroups according to their functions: helper T c...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N5/10C12N5/0783C07K14/705
CPCC07K14/70578C12N5/0636C12N2501/599
Inventor 邓涛喻堃徐国锋卢铀
Owner CHENGDU MEDGENCELL CO LTD
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