Anti-human CD133 monoclonal antibody and preparation method and applications thereof

A monoclonal antibody, hybridoma cell line technology, applied in the direction of antibodies, biochemical equipment and methods, anti-animal/human immunoglobulin, etc., can solve problems such as affecting the expression level of CD133

Inactive Publication Date: 2014-12-31
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently used commercial anti-CD133 antibodies (293C3, AC133, AC141, Miltenyi Biotec) have certain limitations in application (mainly targeting the glycosylation site of the molecule, so changes in glycosylation levels directly affect CD133 detected expression level)

Method used

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  • Anti-human CD133 monoclonal antibody and preparation method and applications thereof
  • Anti-human CD133 monoclonal antibody and preparation method and applications thereof
  • Anti-human CD133 monoclonal antibody and preparation method and applications thereof

Examples

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Embodiment 1

[0039] Embodiment one : Preparation of anti-human CD133 monoclonal antibody

[0040] This example describes the preparation of the anti-human CD133 monoclonal antibody 14D7 of the present invention.

[0041] (1) Establishment of transgenic cells L929 / CD133 and L929 / mock

[0042] (a) Cloning of human CD133 gene:

[0043] Using AC133-2 as a template, two pairs of primers (Table 1) were used for segmented PCR amplification. The reaction conditions were denaturation at 94°C for 60 s, annealing at 58°C for 60 s, and extension at 72°C for 1-3 min. After a total of 35 cycles, Then extended at 72°C for 10 min to obtain two fragments, A and B. After the PCR product was purified by the kit, 133A (SEQ ID NO:1) and 133B (SEQ ID NO:4) were used as primers, and the reaction conditions were denaturation at 94°C for 60s, annealing at 58°C for 60s, and extension at 72°C for 3 min15s, a total of 33 After three cycles, it was extended at 72°C for 10 min to obtain the full-length CD133-1 g...

Embodiment 2

[0062] Example 2: In vitro biological effects of monoclonal antibody 14D7 on SHI-1 cells

[0063] 1) Inhibitory effect of monoclonal antibody 14D7 on the proliferation of SHI-1 cells in vitro: SHI-1 cells were treated with anti-CD133 monoclonal antibody 14D7 at different concentrations of 0.5 μg / ml and 5 μg / ml according to the above method. Inhibit the proliferation of SHI-1 cells within a certain range (compared with mouse IgG group respectively, p Figure 9 ).

[0064] The above experimental results showed that monoclonal antibody 14D7 inhibited the proliferation of SHI-1 cells.

[0065] 2) Monoclonal antibody 14D7 blocks the cycle of SHI-1 cells: This experiment analyzed the cycle distribution of SHI-1 cells under the action of 14D7. The result is as Figure 10 Shown: In the mouse IgG control group, 56% of the cultured cells are in the S phase, and about 40% of the cells are in the G1 / G2 phase, indicating that most cells are in active DNA synthesis and cell division phase....

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Abstract

The invention discloses an anti-human CD133 monoclonal antibody and a preparation method and applications thereof, and particularly relates to a monoclonal antibody. The monoclonal antibody is secreted by a hybridoma cell strain which is the hybridoma cell strain 14D7 secreting the mouse anti-human CD133 molecular monoclonal antibody and which is preserved in the China General Microbiological Culture Collection Center (CGMCC) (address: Building 3, No. 1 Precincts, Beichen West Road, Chaoyang district, Beijing) on May 9, 2012 and has the preservation number of CGMCCNo.6095. The anti-human CD133 monoclonal antibody 14D7 can be used as a biological test index in tumor cell tests; and the experiments in vitro show that the anti-human CD133 monoclonal antibody has proliferation inhibition effect on tumor cells.

Description

technical field [0001] The invention relates to a monoclonal antibody, in particular to an anti-human CD133 monoclonal antibody and a preparation method thereof, as well as the application of the monoclonal antibody in the in vitro proliferation of human monocytic leukemia cell line SHI-1. Background technique [0002] Many receptor-ligand interactions are known to be involved in the induction, establishment and regulation of antigen-specific immune responses. In order to effectively activate a T cell response, at least two signals are usually required. Among them, in addition to the T cell antigen receptor (TCR) recognizing the MHC-antigen complex on the antigen-presenting cell (APC) to provide the first signal, the antigen-specific signal, it is also necessary to obtain the costimulatory molecule expressed by the T cell and APC. Non-antigen-specific, non-MHC-restricted secondary signal generated after action. The second signal is co-stimulatory signal or co-stimulatory s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C12N5/09C07K16/28G01N33/577A61K39/395A61P35/02C12R1/91
Inventor 张学光陈旭勤居颂光
Owner SUZHOU UNIV
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