A kind of rtn4b polypeptide, its monoclonal antibody, hybridoma cell line producing monoclonal antibody and their preparation and application

A hybridoma cell line, monoclonal antibody technology, applied in the biological field

Inactive Publication Date: 2018-06-12
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Secondly, reports in recent years have further found that RTN4B is likely to be related to vascular remodeling and even angiogenesis

Method used

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  • A kind of rtn4b polypeptide, its monoclonal antibody, hybridoma cell line producing monoclonal antibody and their preparation and application
  • A kind of rtn4b polypeptide, its monoclonal antibody, hybridoma cell line producing monoclonal antibody and their preparation and application
  • A kind of rtn4b polypeptide, its monoclonal antibody, hybridoma cell line producing monoclonal antibody and their preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 Obtaining of hybridoma cell lines and preparation of monoclonal antibodies

[0083] 1 Materials and Instruments

[0084] 1.1 Cell lines, tissue samples and animals

[0085] a. Myeloma cells (SP2 / 0)

[0086] b. BALB / C mice

[0087] 1.2 Main reagents

[0088] a. Freund's complete adjuvant, Freund's incomplete adjuvant, HAT, HT, ABTS, PEG (molecular weight 4000)

[0089] b. HRP-labeled rabbit anti-mouse IgG

[0090] c. RPMI 1640 medium: Hyclone company

[0091] d. Nitrocellulose membrane (NC): Hybond company

[0092] e. Calf serum: Hangzhou Sijiqing Bioengineering Materials Co., Ltd.

[0093] f. Mouse Monoclonal Antibody Subclass Typing Kit

[0094] g. Coating buffer: add 0.795g of sodium carbonate and 1.465g of sodium bicarbonate per 500ml of deionized water, the pH value is 9.6

[0095] h. Substrate buffer: add 9.2g of disodium hydrogen phosphate dodecahydrate and 2.55g of citric acid per 500ml of deionized water, the pH value is 5.0

[0096] i. ABTS ch...

Embodiment 2

[0164] Example 2 Preparation of Recombinant Monoclonal Antibody

[0165] Obtaining the heavy chain variable region and light chain variable region sequences:

[0166] 1. Culture and collect hybridoma cells,

[0167] 2. Conventional methods to extract total RNA from hybridoma cells

[0168] 3. Reverse transcription of RNA to cDNA

[0169] 4. Using the prepared cDNA as a template, obtain the variable region sequences of the heavy chain and light chain by conventional PCR amplification

[0170] 5. After testing, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 3, the coding sequence of the heavy chain variable region is SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is SEQ ID NO: 4, The coding sequence of the light chain variable region is SEQ ID NO:6.

[0171] 6. After obtaining the heavy and light chain variable region coding sequences by sequencing, the heavy and light chain constant region sequences of the known mouse ...

Embodiment 3

[0176] Example 3 Mass production of monoclonal antibodies and their functional studies

[0177] Apply this strain of hybridoma cells to 1×10 6 The amount of cells / mouse was injected intraperitoneally into 8-10 week old BALB / C female mice sensitized with liquid paraffin in advance, and the ascites was collected 10-14 days later, and RTN4B protein (prepared with reference ZL00111791.2) was injected. Coated, positive ascites was detected by ELISA (the results are shown in Table 1), and the ascites was used for Western blot to prove that it can recognize RTN4B protein, and this ascites was used for immunohistochemistry to prove that it could recognize natural RTN4B protein, the result Such as figure 1 shown. The monoclonal antibody secreted by this strain of hybridoma laid the foundation for further research on the new gene RTN4B.

[0178] Table 1 ELISA results (OD450 value)

[0179] Antigen concentration

100ng / well

10ng / well

1ng / well

0.1ng / well

...

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Abstract

The invention relates to an RTN4B polypeptide, its monoclonal antibody, a hybridoma cell line producing the monoclonal antibody, and their preparation and application. The RTN4B polypeptide of the present invention contains the amino acid sequence shown in SEQ ID NO:1. The invention further discloses the monoclonal antibody prepared by using the RTN4B polypeptide, the hybridoma cell line producing the monoclonal antibody and their related applications in tumor treatment or prevention.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an RTN4B-related polypeptide, its monoclonal antibody, a hybridoma cell line producing the monoclonal antibody, and their preparation and application. Background technique [0002] RTN4B belongs to the reticolon family (RTNs), named for its specific endoplasmic reticulum location, and has a conserved RHD structural box (reticulon-homology domain). This family contains four genes, RTN1, RTN2, RTN3 and RTN4, among which there are many research reports on RTN4. The RTN4 (also known as Nogo / ASY / RTN-X) gene has multiple alternative splices, mainly encoding RTN4A (GenBank accession number AF148537), RTN4B (GenBank accession number AF148538) and RTN4C (GenBank accession number AF087901) three proteins, Other splices are more specifically expressed in testis. [0003] RTN4B is widely expressed in normal tissues, and studies have found that RTN4B is closely related to the occurrence and dev...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K14/47C12N15/12C07K16/18C12N5/16C12N5/20C12N5/24C12N5/28A61K39/395A61P35/00G01N33/577G01N33/68
CPCA61K38/00C07K14/47C07K16/22A61K2039/505C07K2317/55C07K2317/76A61P1/16A61P35/00C07K7/08C07K16/18C07K2317/54C12N5/16G01N33/57496
Inventor 余龙季国庆吴燕华韩丁丁唐丽莎
Owner FUDAN UNIV
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