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Antitetanus toxin neutralizing antibody as well as preparation method and application thereof

An antibody and antigen technology, applied in the field of cellular immunology, can solve the problems of unresolved industrial production of antibodies and low titer of neutralizing toxins, and achieve the effect of controllable standards and low cost

Active Publication Date: 2018-10-02
JINAN UNIVERSITY +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although these reported antibodies can bind to tetanus toxin, the titer of neutralizing toxin is not high, and the problem of industrial production of antibody has not yet been solved

Method used

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  • Antitetanus toxin neutralizing antibody as well as preparation method and application thereof
  • Antitetanus toxin neutralizing antibody as well as preparation method and application thereof
  • Antitetanus toxin neutralizing antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Mononuclear Cell Separation and Plasma Cell Sorting

[0069] (1) Collect 15mL blood samples from the blood of healthy volunteers injected with 1500IU tetanus toxoid vaccine on the 0th, 14th and 21st days respectively, put them in anticoagulant tubes containing heparin, separate them with Ficoll, and absorb mononuclear cells ( PBMC) layer suspension was washed 3 times with PBS, and the supernatant was discarded by suction;

[0070](2) Filter with a 40 μm filter membrane, count the cells, and use the BD FACSria flow cytometer to sort out the target cell population from the PBMC, and select a single cell with a good shape and place it in a 96-well PCR (Polymerase Chain Reaction, polymerase chain reaction) Plate (20 μL single-cell lysate per well), so that each well contains one B cell, and store in a -80°C refrigerator for later use.

Embodiment 2

[0071] Example 2 Isolation of Antibody Variable Region Genes from Single B Cells Using RT-PCR

[0072] (1) RT-PCR: Add 0.5 μM constant region primers of heavy and light chains of different subtypes to a 96-well plate containing a single B cell (primers are designed at specific sites by conventional methods) and Superscript III reverse transcriptase , incubate at 37°C for 1 hour, and carry out PCR amplification according to the following conditions: 95°C for 15 min; 95°C for 1 min, 55°C for 1 min, 72°C for 1 min, 30 cycles; 72°C for 10 min; 4°C for 5 min; the obtained product cDNA was stored at -20 Store at ℃;

[0073] (2) PCR: 5 μL of reverse transcription product, HotStarTaq Plus enzyme, dNTPs, and 0.5 μM specific primers for the variable regions of heavy and light chains of different subtypes in a 50 μL system (primers were designed at specific sites by conventional methods) ), carry out PCR amplification according to the following conditions: pre-denaturation at 94°C for 5...

Embodiment 3

[0075] Embodiment 3 constructs the expression vector of recombinant antibody

[0076] The PCR product of the variable region gene of the antibody identified as positive by gel electrophoresis and whose heavy chain and light chain can be paired is connected to the pcDNA3.3 vector by TA cloning method to construct a fully human neutralized anti-tetanus toxin The expression vector of the antibody, and then transform the expression vector into DH5α competent bacteria, culture on a plate containing ampicillin at 37°C overnight, pick 10 single colonies and perform PCR with specific primers, the reaction conditions are: 94°C pre-denaturation for 3 minutes; Denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 100s, 28 cycles; extension at 72°C for 5min. Take 5 μL of PCR products and detect them by 1% agarose gel electrophoresis.

[0077] The results showed that transformants containing antibody heavy chain and light chain genes were identified among the posi...

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Abstract

The invention provides an antitetanus toxin neutralizing antibody as well as a preparation method and an application of the antitetanus toxin neutralizing antibody, the amino acid sequence of CDR in alight chain variable region of the antibody is shown in SEQ ID NO.1-3; the amino acid sequence of CDR in a heavy chain variable region of the antibody is shown in SEQ ID NO.4-6. The neutralizing antibody has no immunogenicity, is good in specificity, high in affinity, is clear in ingredients, has no potential risks like pathogen contamination, and is widely suitable for various crowds; the preparation method is simple and efficient, and is suitable for standardized production.

Description

technical field [0001] The invention belongs to the field of cellular immunology, and relates to a fully human anti-tetanus toxin neutralizing antibody; in addition, the invention also relates to a preparation method and application of the neutralizing antibody. Background technique [0002] Tetanus is an acute and fatal zoonotic neurological disease caused by infection with Clostridium tetani. Clostridium tetani widely exists in the intestines and soil of humans and animals, with a carrier rate of up to 25%. Wounds of different types and sizes can become portals for the invasion of Clostridium tetani. When a person is infected, during the incubation period Clostridium tetani multiplies in large numbers at the wound under anaerobic conditions and releases toxins, and the tetanus toxin invades the central nervous system by destroying human nerve cells, resulting in convulsions, rigidity of limbs, and horns. Symptoms such as opisthotonus eventually lead to death from suffocat...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/13C12N15/70C12N1/21G01N33/577G01N33/569A61K39/395A61P31/04
CPCA61K2039/505A61P31/04C07K16/1282C07K2317/24C07K2317/565C07K2317/76C12N15/70G01N33/56911G01N33/577G01N2800/28
Inventor 廖化新郑伟宏王月明袁晓辉
Owner JINAN UNIVERSITY
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