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Anti-CD127 antibody, cell line secreting the antibody, preparation method and application thereof

A technology of CD127 and cell lines, which is applied in the fields of biomedicine and immunochemistry, can solve problems such as difficult determination, and achieve the effect of good affinity

Active Publication Date: 2021-08-03
SHANDONG ANALYSIS & TEST CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the inventors found that the immunogens of current anti-CD127 monoclonal antibodies are generally recombinant proteins or recombinant extracellular proteins, which makes it difficult to determine the epitope or functional domain of the specific site of CD127 in the prepared monoclonal antibody

Method used

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  • Anti-CD127 antibody, cell line secreting the antibody, preparation method and application thereof
  • Anti-CD127 antibody, cell line secreting the antibody, preparation method and application thereof
  • Anti-CD127 antibody, cell line secreting the antibody, preparation method and application thereof

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preparation example Construction

[0045] In yet another specific embodiment of the present invention, the preparation method includes:

[0046] naive CD4 + T(naive CD4 + T) cells to immunize mice, and then use synthetic OVA-coupled polypeptides as antigens to immunize mice to obtain immune splenocytes; fusion of the splenocytes with myeloma cells to obtain hybridoma cells; culturing the hybridoma cells (in vitro culture can be used) or in vivo culture), collected and purified.

[0047] In yet another specific embodiment of the present invention, the initial CD4 + T cells were obtained by aseptically sorting naive CD4 cells from spleen cells of OT-II mice + T cells. Further, the initial CD4 + T cells are CD44 low CD62L + CD25 - CD4 + CD8 - B220 - CD3 + initial CD4 + T cells.

[0048] In the present invention, initial CD4 + T cells can specifically recognize the polypeptide of OVA protein 323-339, so more polypeptide-OVA antigens are presented. Thereby more GC B cells can be obtained. And fu...

Embodiment 1

[0068] Example 1 Preparation of anti-hCD127 monoclonal antibody 7E7

[0069] 1.1 Using OT-II mice to construct a model for enhancing mouse immunity

[0070] (1) Aseptically sort initial CD4 from spleen cells of OT-II CD45.1 mice + T cells. Dissect the spleen of the mouse in a sterile ultra-clean bench, prepare suspended single cells by grinding, and perform fluorescent labeling on the proteins on the cell surface, and mark the following Markers: CD3-AF700, B220-APC-cy7, CD8-BV605, CD4 - PE-cy7, CD25-PE, CD44-FITC, CD62L-BV421, 7-AAD. Using FACS III sorting marker CD44 low CD62L + CD25 - CD4 + CD8 - B220 - CD3 + initial CD4 + T cells.

[0071] (2) C57BL / B6 CD45.2 mice were immunized with tail vein cells (marked as D-(-1) day). With PBS as the negative control, 100 μl / rat was injected into the tail vein of the control group and the experimental group.

[0072] (3) C57BL / B6 CD45.2 mice were immunized with antigen (marked as D-0 day).

[0073] Inject OVA-CFA intr...

Embodiment 2

[0102] Example 2 Application of anti-hCD127 monoclonal antibody 7E7

[0103] 2.1 Monoclonal antibody 7E7 recognizes the amino acid sequence of hCD127 protein

[0104] The method of Western Blot was used to identify the recognition of monoclonal antibody 7E7 to the amino acid of hCD127 protein expressed in eukaryotic cells.

[0105] (1) Preparation of protein samples. Passage K562 cells, 293T cells and Hela cells in 6-well plates. After 24 hours, when the cell density reaches 90%, the cells are directly lysed with loading buffer and incubated in a metal bath at 100°C for 10 minutes.

[0106](2) Western Blot identification of monoclonal antibody 7E7 to recognize the amino acid sequence of hCD127 full-length protein. ①Run denatured gel SDS-PAGE: 5% upper gel, 10% lower gel, take 40μL of protein and load, voltage 60-80V. ②Transfer film. NC film is used, and film transfer parameters are set: constant voltage 100V, time 1.5h. ③ closed. Add Odyssey blocking buffer and incubate ...

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Abstract

The invention provides an anti-CD127 antibody, a cell line secreting the antibody, a preparation method and application thereof, and belongs to the technical fields of biomedicine and immunochemistry. The antibody or the antigen-binding fragment of the antibody specifically binds to CD127 and specifically recognizes the epitope sequence of CD127. The anti-hCD127 monoclonal antibody prepared by the present invention and the monoclonal antibody hybridoma cell line CD127-7E7 secreting the monoclonal antibody can specifically recognize human CD127 and have good affinity, which can not only be used for qualitative or quantitative CD127 protein Detection; at the same time, it can also determine the epitope or functional domain of CD127 protein, so it has good practical application value.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and immunochemistry, and specifically relates to an anti-CD127 antibody, a cell line secreting the antibody, a preparation method and application thereof. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] CD127 is an interleukin-7 (IL-7) receptor α chain (IL-7Rα), which plays a role in different stages of the T lymphocyte life cycle. (1) It plays an important role in the development of T lymphocytes in the thymus. The differentiation of thymocytes depends on the expression of IL-7, and there are only two stages in the entire T lymphocyte life cycle that lack the expression of CD127. (2) It i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N5/20C12N15/13A61K39/395A61P37/00G01N33/68G01N33/577
CPCA61K2039/505A61P37/00C07K16/2866C07K2317/76C07K2317/92G01N33/577G01N33/68
Inventor 余迪田文静张宇张浩
Owner SHANDONG ANALYSIS & TEST CENT