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CD127 resisting antibody, cell strain for secreting antibody and preparation method and application of antibody

A CD127, cell line technology, 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: 2020-03-20
SHANDONG ANALYSIS & TEST CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the inventors have 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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  • CD127 resisting antibody, cell strain for secreting antibody and preparation method and application of antibody
  • CD127 resisting antibody, cell strain for secreting antibody and preparation method and application of antibody
  • CD127 resisting antibody, cell strain for secreting antibody and preparation method and application of antibody

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preparation example Construction

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

[0046] Initial CD4 + T(naive CD4 + T) Cells immunize mice, and then immunize mice with synthetic OVA-conjugated polypeptides as antigens to obtain immune spleen cells; fuse the spleen cells with myeloma cells to obtain hybridoma cells; culture the hybridoma cells (in vitro culture can be used Or in vivo culture), collected and purified.

[0047] In another specific embodiment of the present invention, the initial CD4 + The method for obtaining T cells is: aseptically sort the initial CD4 from the 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, the initial CD4 + T cells can specifically recognize the polypeptides of OVA protein 323-339, so more polypeptide-OVA antigens are presented. Thus more GC B cells can be obtained. And further differentiate...

Embodiment 1

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

[0069] 1.1 Use OT-II mice to build a model to enhance mouse immunity

[0070] (1) Aseptically sort the initial CD4 from the spleen cells of OT-II CD45.1 mice + T cells. The mice were dissected in a sterile clean bench and their spleens were taken, and single cells were prepared by grinding and fluorescently labeled with the protein on the cell surface. Markers were marked as follows: 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 a negative control, the control group and the experimental group were injected with 100 μl per mouse through the tail vein.

[0072] (3) Antigen immunize C57BL / B6 CD45.2 mice (marked as D-0 day).

[0073] Intraperitoneal injection of OVA...

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

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

[0106] (2) Western Blot identified the recognition of the amino acid sequence of the full-length hCD127 protein by monoclonal antibody 7E7. ①SDS-PAGE with denaturing gel: 5% for the upper gel and 10% for the lower gel. Take 40μL of protein and load the sample with a voltage of 60-80V. ② Transfer film. Use NC film, transfer film parameter setting: constant voltage 100V, time 1.5h. ③Closed. Add Odyss...

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Abstract

The invention provides a CD127 resisting antibody, a cell strain for secreting the antibody and a preparation method and application of the antibody, and belongs to the technical fields of biopharmaceutics and immunochemistry. The antibody or an antigen binding fragment of the antibody is specially bound with CD127 and can specially recognize epitope sequences of CD127. The prepared hCD127 resisting monoclonal antibody and the monoclonal antibody hybridoma cell strain CD127-7E7 for secreting the monoclonal antibody can specifically recognize human CD127, have favorable affinity, can be used for qualitative or quantitative detection of CD127 protein, and besides, can also be used for judging the epitope or functional domain of the CD127 protein, so that the monoclonal antibody and the cellstrain have favorable practical application value.

Description

Technical field [0001] The present invention belongs to the technical field of biomedicine and immunochemistry, and specifically relates to an anti-CD127 antibody, a cell line secreting the antibody, and a preparation method and application thereof. Background technique [0002] Disclosure of the background information is only intended to increase the understanding of the overall background of the present invention, and is not necessarily regarded as an acknowledgement or any form of suggestion that the information constitutes the prior art known to those of ordinary skill in the art. [0003] CD127 is the interleukin-7 (IL-7) receptor alpha chain (IL-7Rα), which plays a role in different stages of the life cycle of T lymphocytes. (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. There are only two stages in the life cycle of T lymphocytes that lack the expression of CD127. (2) It ...

Claims

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Application Information

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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
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