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A monoclonal antibody cell line that can stably secrete anti-conotoxin and its application

A technology of monoclonal antibody and conotoxin, which is applied in the field of cell engineering, can solve the problems of rare ω-CTXMVIIA immunological detection method, no detection, and inability to obtain antibodies by direct immunization of animals

Active Publication Date: 2021-01-29
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since ω-CTX MVIIA is a small molecule polypeptide, which is antigenic but not immunogenic, the corresponding antibody cannot be obtained by direct immunization of animals, so the immunological detection method of ω-CTX MVIIA is rarely seen
There are only immunoassays for ω-CTX MVIIA based on polyclonal antibodies, single-chain antibodies, and monoclonal antibodies (IgM), but no immunoassays for ω-CTX MVIIA based on monoclonal antibodies (IgG)

Method used

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  • A monoclonal antibody cell line that can stably secrete anti-conotoxin and its application
  • A monoclonal antibody cell line that can stably secrete anti-conotoxin and its application
  • A monoclonal antibody cell line that can stably secrete anti-conotoxin and its application

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Embodiment 1

[0027] Embodiment 1 Preparation and identification of monoclonal antibody of the present invention

[0028] 1. Antigen carrier construction

[0029] 1) Obtaining the protein sequence and gene sequence of ω-CTX MVIIA The sequence of the functional mature peptide region in ω-CTX MVIIA found on the NCBI website is: CKGKGAKCSR LMYDCCTGSC RSGKC. According to the amino acid sequence of the mature peptide region of conotoxin and the codon preference of Escherichia coli, the DNA sequence of the toxin is deduced, and enzyme cleavage sites are directly added to the two ends of the sequence. After chemical synthesis, it can be directly inserted into the vector middle. The final sequence is as follows:

[0030] Positive strand: 5'- GA TCC TGC AAA GGT AAA GGT GCG AAA TGC TCT CGT CTG ATGTAC GACTGC TGC ACC GGT TCT TGC CGT TCT GGT AAA TGC TGA C-3'

[0031] Negative strand 5'- TC GAG TCA GCA TTT ACC AGA ACG GCA AGA ACC GGT GCA GCAGTC GTACAT CAG ACG AGA GCA TTT CGC ACC TTT ACC TTT GCA ...

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Abstract

The invention belongs to the field of antibody engineering, in particular to a monoclonal antibody cell strain capable of stably secreting conotoxin (ω-CTX MVIIA) and its application. The hybridoma cell line 2E5 has been preserved in the China General Microorganism Culture Collection Management Center on January 31, 2018, and the preservation number is: CGMCC NO.15295. In the present invention, the prokaryotic expression and purification of GST-ω-CTX MVIIA recombinant antigen is used as the immune antigen, and the TRX-ω-CTX MVIIA recombinant antigen is used as the detection antigen to coat the microtiter plate. A positive hybridoma cell line was screened by iELISA, and the monoclonal antibody secreted by the hybridoma cell line laid the foundation for the establishment of an ω‑CTX MVIIA immunoassay method.

Description

technical field [0001] The invention belongs to the field of cell engineering, in particular to a hybridoma cell line (IgG) capable of stably secreting monoclonal antibody against conotoxin (ω-CTX MVIIA) and its application. Background technique [0002] Conotoxin (CTX) is a biologically active small molecule polypeptide neurotoxin secreted by cone snails for predation and defense. It generally contains 10-30 amino acids, and most of them are rich in disulfide bonds. , targeting ion channels and receptors in the nervous system, people and animals generally suffer from paralysis, convulsions, convulsions and even death after being bitten. ω-conotoxin MVIIA (ω-CTX MVIIA) contains 25 amino acid residues, is a specific and reversible N-type voltage-sensitive calcium ion channel blocker, and can be used as an analgesic to treat severe and chronic pain . It is a research hotspot in the fields of neurobiology, drug pharmacology, peptide chemistry, molecular biology, etc., so its ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/577
CPCC07K16/18G01N33/577G01N2333/43504
Inventor 王荣智钟燕芳杨航王俊程汪世华凌素美
Owner FUJIAN AGRI & FORESTRY UNIV