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Preparation method of B7-H4 protein and application of B7-H4 protein in preparation of drugs for resisting excessive immune response or cytokine storm

A B7-H4, 1.B7-H4 technology, applied in the field of B7-H4 protein preparation, can solve problems such as troublesome sequelae, and achieve the effect of inhibiting the secretion of cytokines by CD8+ T cells

Pending Publication Date: 2022-02-25
上海奢旭企业管理中心(有限合伙)
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sequelae (necrosis of the femoral head) caused by high-dose corticosteroid shock therapy is a very troublesome problem

Method used

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  • Preparation method of B7-H4 protein and application of B7-H4 protein in preparation of drugs for resisting excessive immune response or cytokine storm
  • Preparation method of B7-H4 protein and application of B7-H4 protein in preparation of drugs for resisting excessive immune response or cytokine storm
  • Preparation method of B7-H4 protein and application of B7-H4 protein in preparation of drugs for resisting excessive immune response or cytokine storm

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Experimental program
Comparison scheme
Effect test

Embodiment 1B7-H4

[0039] The preparation of embodiment 1B7-H4 protein

[0040] S1.B7-H4 protein expression identification:

[0041] 1. Construction of B7-H4 gene and plasmid construction

[0042] Using PET-30a(+) as the prokaryotic expression vector (the plasmid has been verified to be correct by full-length sequencing, with his tag), the B7-H4 gene recombinant plasmid PET-30a(+)-B7-H4 was rebuilt (Table 1).

[0043] Table 1

[0044]

[0045] 2. Obtain the cDNA fragment of B7-H4 gene

[0046] (1) Design of specific amplification primers: the puc57-B7-H4 gene recombinant plasmid was used as the amplification template, and the two restriction endonuclease sites of NdeI and HindIII were selected as the amplification fragments to be ligated into PET-30a (+) The entry point of the vector. When designing primers, the restriction site sequence, protected base sequence, plasmid coding direction, GC content, etc. should be considered, and the codon usage preference of E.coli should also be conside...

Embodiment 2

[0144] Example 2 Inhibitory Effect of B7-H4 Protein on Mouse CD8+ T Cells

[0145] (1) Extraction of mouse spleen T cells: Take five 6-8-week-old female SPF grade C57BL / 6 mice, kill them by decapitation, and take spleens. After grinding, filter the cell suspension through a 70um filter and transfer to a centrifuge. Tube, centrifuge at 300g at 4°C for 5 minutes, discard the supernatant; add 5ml red blood cell lysate, mix well, incubate at room temperature for 5 minutes, add 20ml PBS, centrifuge at 300g at 4°C for 5 minutes, discard the supernatant; add 20ml PBS, Filter through a 40um filter, centrifuge at 300g at 4°C for 5 minutes after resuspension, discard the supernatant; resuspend the cells in 10ml PBS and count, centrifuge at 300g at 4°C for 5 minutes, discard the supernatant;

[0146] (2) Isolation, culture and identification of mouse spleen CD8+ T cells: with 10 7 cells as a unit, every 10 7Add 90ul MACS buffer and 10ul CD8+ Microbead to each cell, and incubate at 2-8°...

Embodiment 3

[0148] Example 3 Inhibitory effect of B7-H4 protein on human solid tumor infiltrating T cells

[0149] (1) Isolation, culture and enrichment of solid tumor infiltrating T cells (TIL): In a sterile environment, remove impurities such as necrosis and blood clots on the solid tumor tissue, and cut them into 1-8mm 3 Left and right, put it in a 24-well culture plate, add a medium containing a large dose of IL-2 cytokines and incubate for 2-4 weeks, collect the corresponding TILs for amplification and freeze them; at the same time, place the cell suspension in a T25 culture bottle for further development Routine culture, amplified to a certain number and then sorted by human CD8+ magnetic beads (MACS).

[0150] (2) Identification of CD8+ T cells in TILs: The above sorted T cells were centrifuged with PBS, then incubated with human CD8 flow cytometry antibody for 15 minutes, washed by PBS centrifugation twice, and CD8+ clusters in TILs were detected on the machine. See Figure 7.

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Abstract

The invention provides a preparation method of B7-H4 protein and application of the B7-H4 protein in preparation of drugs for resisting excessive immune response or cytokine storm, and belongs to the technical field of bioengineering. The preparation method of the B7-H4 protein comprises the following steps: S1, identification of the expression of the B7-H4 protein; S2, expression and purification of the B7-H4 protein; and S3, B7-H4 protein verification. The B7-H4 protein prepared by the invention has a very good inhibition effect on excessive immune response and cytokine storm, and tests prove that the B7-H4 protein inhibits CD8+T cells of mice and human from secreting cytokines.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a preparation method of B7-H4 protein and its application in the preparation of anti-excessive immune response or anti-cytokine storm medicine. Background technique [0002] Many serious infections, such as the new coronavirus, SARS, HIV, etc., will cause the appearance of cytokine storm (cytokinestorm), which will lead to the deterioration of the disease and become a critically ill patient. This is caused by an excessive immune response, and the patient will suffer from complications such as hypotension, coagulation disorders, and multiple organ failure, which will eventually lead to the death of the patient. Similarly, it is used to prevent acute graft-versus-host disease (GvHD) in leukemia patients after bone marrow transplantation, and the above-mentioned excessive immune response may also occur, including organ transplantation, such as liver transplantation, heart-lun...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61P37/06C12N15/70C12N15/66C12N15/62C07K14/705
CPCA61K38/1774A61P37/06C12N15/70C07K14/70532C07K2319/21
Inventor 童茵
Owner 上海奢旭企业管理中心(有限合伙)
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