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Anti-REG gamma C terminal region monoclonal antibody and preparation and application thereof

A monoclonal antibody and regional technology, applied in the field of medicine, can solve the problems of different batches of REGγ antibodies, unfavorable long-term research and use, and large differences in stability, so as to improve objectivity and credibility, convenient and fast detection and Diagnosis, the effect of high sensitivity

Inactive Publication Date: 2020-05-29
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the REGγ antibodies on the market are all polyclonal antibodies, which have multiple subtypes and can recognize multiple epitopes. Compared with monoclonal antibodies, their specificity is poor, and it is easy to cause background in immunoassays. , such as heterogeneous bands in Western Blot or darker background staining in immunohistochemistry (IHC), etc.
Moreover, the batches of REGγ antibodies on the market are different, and the stability is quite different, which is not conducive to long-term research and use

Method used

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  • Anti-REG gamma C terminal region monoclonal antibody and preparation and application thereof
  • Anti-REG gamma C terminal region monoclonal antibody and preparation and application thereof
  • Anti-REG gamma C terminal region monoclonal antibody and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Bioinformatics analysis and comparison of amino acid sequence differences of REG family proteins

[0048] Using DNAMAN software to conduct bioinformatics analysis on the protein amino acid sequences of REG family members (REGα, REGβ, REGγ), and design REGγ is different from REGα, REGβ protein sequences and is the most suitable short peptide as an antigen, that is, the 13 amino acids at the C-terminus of REGγ Sequence (IKRPRSSNAETLY (SEQ ID NO: 1), as the target antigen peptide (such as figure 1 ).

[0049] Table 1 Amino acid sequences of REG family proteins

[0050]

Embodiment 2

[0051] Example 2 Preparation, purification and subtype identification of REGγ monoclonal antibody

[0052] The fused cells were selected through HAT selective culture, indirect Elisa screening and subcloning identification, and a cell line stably secreting REGγ antibody was obtained. The REGγ-specific antibody was obtained by expanding the fused cells, enriching the serum-free medium supernatant with proteinG, and concentrating. After identification by indirect ELISA method, the results showed that the REGγ-specific antibody was of IgG type (such as figure 2 A, 2B).

[0053] 1. Antigen preparation

[0054] The 13 amino acid sequence (IKRPRSSNAETLY (SEQ ID NO: 1)) located at the C-terminus of REGγ was used as the target antigen peptide, and coupled with hemocyanin (keyhole limpet hemocyanin, KLH). Then, the above-mentioned peptides were obtained by artificial synthesis as the final antigen.

[0055] 2. Immunization of mice

[0056] The synthesized above-mentioned peptides...

Embodiment 3

[0108] Embodiment 3: the indirect ELISA identification of the monoclonal antibody of the present invention

[0109] (1) Antigen coating: Add the synthesized monoclonal antibody against the C-terminal region of REGγ into a 96-well microtiter plate, and coat overnight at 4°C;

[0110] (2) Aspirate the coating solution, wash with PBST 3 times, 5 min each time;

[0111] (3) 200 μL of 3% bovine serum albumin (prepared in PBS) was added to each well, and blocked at 37° C. for 1 hour;

[0112] (4) repeat the washing method in step (2);

[0113] (5) The above-mentioned screened and cloned hybridoma cell culture supernatant was used as the primary antibody, added to the microtiter plate in an amount of 50 μL per well, and incubated in a 37° C. incubator for 1 h;

[0114] (6) repeat the washing method in step (2);

[0115] (7) Alkaline phosphatase-labeled goat anti-mouse Ig (diluted 1:4000) was used as the secondary antibody, added to the microtiter plate in an amount of 50 μL per we...

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Abstract

The invention discloses an anti-REG gamma C-terminal region monoclonal antibody and a preparation method and application thereof. The monoclonal antibody is secreted by a hybridoma cell Mc-IgG-REG gamma-C with the name of the hybridoma cell strain Mc-IgG-REG gamma-C, the preservation unit of China General Microbiological Culture Collection Center, the address of No.3, Yard 1, West Beichen Road, Chaoyang District, Beijing, postcode 100101, the preservation date of November 14, 2018, and the preservation number of CGMCC: No.16809. The monoclonal antibody has the characteristics of high potency,high sensitivity, high uniformity, high specificity and the like and can provide an effective tool for diagnosis and treatment of related diseases with high REG gamma expression.

Description

technical field [0001] The invention belongs to the field of medicine, and in particular relates to a monoclonal antibody against the C-terminal region of REGγ and its preparation method and application. Background technique [0002] REGγ, also known as 11Sγ, PA28γ, PSME3 or Ki (GeneID: 10197), is a member of the 11S proteasome activator family. It was first discovered in the serum of patients with systemic lupus erythematosus and named as Ki antigen, but its The relationship with patients with systemic lupus erythematosus is still not very clear. It was later discovered that the Ki antigen, like REGα and REGβ, belongs to the 11S proteasome activator family, and was named REGγ because it was discovered after REGα and REGβ. [0003] As a proteasome activating factor, REGγ binds to the α subunit of the 20S core particle, opens the 20S gate, and sends the substrate protein to the protease catalytic site of the β subunit of 20S, degrading the substrate protein. The REGγ-20S pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/574C07K16/18C12N5/20A61K39/395A61P35/00
CPCA61P35/00C07K16/18C07K2317/34C07K2317/73G01N33/57484G01N33/577G01N33/6893G01N2333/474G01N2800/06G01N2800/325
Inventor 李晓涛童璐夏艳阳黄婷妹李磊
Owner EAST CHINA NORMAL UNIV
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