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

A monoclonal antibody and antigen polypeptide technology, applied in the field of cell engineering, can solve the problem of no immunogen antigen polypeptide, etc., and achieve the effect of strong specificity, single biological activity and high uniformity

Inactive Publication Date: 2014-01-08
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 peptides

[0030] Taking the TRPC6 protein as the research object, look for a sequence on the TRPC6 molecule, which should not be too long, about 15 amino acids, must be in a conserved region, and will not change between different species; and determine whether it is an intracellular or extracellular segment (TRPC6 molecule Complicated, with 6 transmembrane structures); compare the homology between species (protein blast in NCBI); at the same time, it is necessary to consider whether other proteins also have this sequence, and it is best for the antibody used for identification and diagnosis to have this sequence in the same Other molecules in the family do not have specificity, otherwise they are prone to cross-reaction and cannot be used as in vitro diagnostic reagents.

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

[0032] The selected seque...

Embodiment 2

[0034] Example 2 Preparation of monoclonal antibody against TRPC6

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

[0036] According to the antigenic polypeptide designed in Example 1, the polypeptide is coupled with KHL, and the healthy BALB / c mice are immunized, and the age of the mice is 6-12 weeks, male or female. In order to avoid poor reaction of mice or death during immunization, 3-4 mice can be immunized at the same time. Multi-point immunization was used. After the second and third immunizations, the titer of mouse serum was determined by indirect ELISA method, and the one with the highest immune titer was selected for booster immunization to prepare for 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 selected to be cultured in RPMI-1640 medium with 10% feta...

Embodiment 3

[0051] Example 3 Application of Monoclonal Antibody Detection in TRPC6 Molecular Overexpression Tissues

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

[0053] The monoclonal antibody was labeled with horseradish peroxidase to construct a double-antibody sandwich method. The labeling method is sodium periodate method, marked

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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 relates to the technical field of cell engineering, in particular to a TRPC6 antigen polypeptide and an anti-TRPC6 monoclonal antibody. Background technique [0002] TRPC6 (transient receptor potential cation channel 6) protein is one of the important podocyte membrane proteins, and its gene mutation product can lead to massive proteinuria, thereby causing focal segmental glomerulosclerosis (FSGS) and progressive renal failure. Studies have found that in human non-hereditary proteinuric kidney diseases such as FSGS, minimal change nephropathy and membranous nephropathy, the expression of TRPC6 is significantly increased, and it is distributed in groups, with segmental intensive. FSGS is a common glomerular disease with an incidence of about 2 per million in the population, and its clinical features are massive proteinuria, nephrotic syndrome and progressive renal insufficiency. FSGS accounts for about 7-15% of children with steroid-resis...

Claims

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Application Information

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Patent Type & Authority Patents(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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