Transient receptor potential channel-6 (TRPC6) antigen polypeptide and anti-TRPC6 monoclonal antibody

A technology of monoclonal antibodies and antigenic polypeptides, applied in the field of cell engineering, can solve the problems of no immunogenic antigenic polypeptides, etc., and achieve the effects of strong specificity, convenient manual processing and quality control, and uniform physical and chemical properties

Inactive Publication Date: 2012-09-12
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to establish a simple type B detection method for the diagnosis of FSGS, it is first necessary to screen the appropriate TRPC6 polypeptide sequence to obtain a good immunogen, but currently there is no antigenic polypeptide with good immunogenicity and high specificity

Method used

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  • Transient receptor potential channel-6 (TRPC6) antigen polypeptide and anti-TRPC6 monoclonal antibody
  • Transient receptor potential channel-6 (TRPC6) antigen polypeptide and anti-TRPC6 monoclonal antibody
  • Transient receptor potential channel-6 (TRPC6) antigen polypeptide and anti-TRPC6 monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 TRPC6 antigen polypeptide design

[0029] (1) Sequence screening of TRPC6 synthetic peptide

[0030] Taking the TRPC6 protein as the research object, look for a sequence on the TRPC6 molecule. It should not be too long, about 15 amino acids, and must be a conserved region, which does not change among different species; and determine whether it is the intracellular or extracellular segment (TRPC6 molecule Complex, with 6 transmembrane structures); Compare the homology between species (protein blast in NCBI); At the same time, consider whether other proteins also have this sequence. The antibody used for identification and diagnosis should preferably be in the same sequence. No other molecules in the family have specificity, otherwise they are prone to cross-reactions and cannot be used as in vitro diagnostic reagents.

[0031] (2) Analysis of antigenic epitope and hydrophilicity of TRPC6 synthetic peptide

[0032] The selected sequence is used as the synthetic peptid...

Embodiment 2

[0034] Example 2 Preparation of monoclonal antibodies against TRPC6

[0035] (1) Antigenic peptide synthesis and animal immunity

[0036] According to the antigen polypeptide designed in Example 1, the polypeptide is coupled with KHL to immunize healthy BALB / c mice, which are 6-12 weeks old, and are not limited to males or females. To avoid poor response of mice or death during immunization, 3 to 4 mice can be immunized at the same time. Multi-point immunization was used. After two or three immunizations, the indirect ELISA method was used to determine the serum titer of mice, and those with high immune titer were selected for booster immunization to prepare cell fusion.

[0037] (2) Preparation of myeloma cells and feeder cells

[0038] The mouse myeloma P3X63Ag8.653 cell line (purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) was cultured with RPMI-1640 medium containing 10% fetal bovine serum. The cells were semi-ad...

Embodiment 3

[0051] Example 3 Application of monoclonal antibodies in detection of TRPC6 molecular overexpression tissues

[0052] (1) Application of monoclonal antibodies in immunolabeling technology

[0053] Horseradish peroxidase was used to label monoclonal antibodies to construct a double antibody sandwich method. The labeling method is sodium periodate method.

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Abstract

The invention discloses a transient receptor potential channel-6 (TRPC6) antigen polypeptide and an anti-TRPC6 monoclonal antibody. The TRPC6 antigen polypeptide consists of 15 amino acids, and the specific sequence is shown as SEQ ID NO: 1. A mouse is immunized by using the protein molecule of the polypeptide as an antigen, a hybridoma cell strain H8 is screened and collected in CCTCC on March 7th, 2012, and the collection number is CCTCC-C201236. The cell strain can continually and stably secrete the anti-TRPC6 monoclonal antibody. The monoclonal antibody can specifically identify TRPC6 proteins expressed by various tissue cells, and can be used for establishing an enzyme-linked immunosorbent assay (ELISA) method to monitor diseases related with TRPC6 molecules, so that the monoclonal antibody can be used for preparing medicaments for diagnosing the diseases related with the TRPC6 molecules.

Description

Technical field [0001] The invention involves the field of cell engineering technology, which specifically involves a monoclonal antibody with a TRPC6 antigen peptide and anti -TRPC6. Background technique [0002] TRPC6 (instantaneous receptor electrical cationic channel 6) Protein is one of the important foot cell membrane proteins. Its genetic mutation products can cause a large amount of proteinuria, which causes the stove segmentation glomerular sclerosis (FSGS) and performed renal failure.Studies have found that among human non -genetic protein urinary renal diseases such as FSGS, micro lesion degenerative nephropathy, and membrane nephropathy, the TRPC6 expression increased significantly and distributed in groups, with segmented dense types.FSGS is a common glomerular disease. The incidence of crowd is about two -million -dollar. Its clinical characteristics are large amounts of proteinuria, nephrotic syndrome and cargiving.FSGS accounts for 7 ~ 15%of children with hormone ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N5/20C07K16/18G01N33/68G01N33/577
Inventor 张萃刘晓波李红枝黄超贤梅俪凡
Owner GUANGDONG PHARMA UNIV
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