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Neutralizing monoclonal antibody resisting to tetanus toxin and application thereof

A monoclonal antibody and anti-tetanus technology, which is applied in the field of genetic engineering and immunoglobulin, can solve problems such as monoclonal antibodies that have not yet appeared, and achieve the effect of prolonging the death time

Active Publication Date: 2016-05-04
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Invention patents CN102690789B, CN101220096B, CN1305904C, and CN100391975C have all disclosed anti-tetanus toxin monoclonal antibodies, but no monoclonal antibody with excellent neutralizing activity and protective effect has yet appeared, so that there is no monoclonal antibody Anti-development to the stage of commercialization and industrialization

Method used

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  • Neutralizing monoclonal antibody resisting to tetanus toxin and application thereof
  • Neutralizing monoclonal antibody resisting to tetanus toxin and application thereof
  • Neutralizing monoclonal antibody resisting to tetanus toxin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Preparation of anti-tetanus neutralizing monoclonal antibody

[0050] 1. Cloning of mouse monoclonal antibody gene

[0051] 1.1 Preparation of mouse monoclonal antibody

[0052] Using recombinant tetanus TeNT-Hc protein (CN200910135972.0) as immunogen, four strains of high-affinity and high-specificity mouse-derived anti-tetanus monoclonal antibodies TY1, TY5, TY8 and TY11 were obtained by conventional monoclonal antibody preparation methods Hybridoma cell line, the subtype of antibody molecule secreted is IgG1, and the light chain is Kappa subtype.

[0053] 1.2 Cloning of mAb variable region gene

[0054] The monoclonal antibody is composed of a variable region and a constant region. Because the gene variation of the variable region of the mouse monoclonal antibody is large, and the gene sequence of the constant region is very conservative, the 5'-RACE method is used for cloning. A primer sequence was added to the 5' end, and PCR amplification was perform...

Embodiment 2

[0133] Embodiment 2: ELISA method detects the binding activity of monoclonal antibody and TeNT-Hc, TT protein

[0134] Recombinant TeNT-Hc protein or tetanus toxoid TT diluted to 2 μg / ml, 100ul / well coated with enzyme-linked plate, overnight at 4°C, blocked with blocking solution (20mMPB, 0.15M NaCl, 5% milk powder) at 37°C for 1h the next day After washing the plate, add the sample and incubate at 37°C for 1h to wash the plate, add 1:1000 diluted HRP-labeled goat anti-human Fc (Sigma, A0170), wash the plate after incubating at 37°C for 1h, develop color with TMB, and measure OD with a microplate reader 450 . The experimental results show that the four monoclonal antibodies all have good specificity and can specifically bind to TeNT-Hc and TT proteins, but have no binding activity to other proteins, see Figure 4 .

Embodiment 3

[0135] Example 3: Protective activity of monoclonal antibody in mice

[0136] Using BALB / c mice, female, 18-20g / mouse, 10 / group, mix different concentrations of TY1, TY5, TY8 and TY11 monoclonal antibodies and mixtures with 2 times the lethal dose of tetanus toxin, 37°C After incubation for 1 hour, it was injected into the abdominal cavity of mice, and the incidence and survival of mice were observed for 10 consecutive days. from Figure 5 It can be seen from the experimental results that monoclonal antibodies can significantly prolong the death time of mice and have partial protective activity. The toxin protection rates of TY1, TY5, TY8, and TY11 monoclonal antibodies to mice with double lethal doses are 50%, 30%, respectively. %, 40%, and 40%. from Image 6 It can be seen from the experimental results that when the four antibodies are used in combination with a dose of 50 μg or more, they can completely protect the mice from the toxin challenge of less than twice the let...

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Abstract

The invention discloses a neutralizing monoclonal antibody resisting to tetanus toxin. Hybridoma cells are obtained from tetanus toxin heavy chain C fragment immune mice, light chain and heavy chain variable region genes of the antibody are taken, human-mouse chimeric complete antibody expression vectors are built, CHO cell transfection is carried out, purification is carried out, and then the antibody is obtained. The antibody has the activity of specific binding to tetanus toxin, the monoclonal antibody can partially protect mice against attack of tetanus toxin, and four antibodies can completely protect mice against a twofold lethal dose of tetanus toxin in combination.

Description

technical field [0001] The invention relates to an anti-tetanus monoclonal antibody polypeptide and the application of the gene and polypeptide in the preparation of tetanus therapeutic medicine, belonging to the technical fields of genetic engineering and immunoglobulin. Background technique [0002] Tetanus is an infectious bacterial disease caused by Clostridium tetani. Under anaerobic conditions (such as contaminated necrotizing wounds), this ubiquitous bacillus can produce a very toxic neurotoxin - tetanospasm toxin. Tetanospasm toxin blocks inhibitory neurotransmitters in the central nervous system, causing the muscle rigidity and spasms that are typical of tetanus. Tetanus can occur at any age, and even with modern intensive care conditions, the case-fatality rate is very high. All open injuries have the possibility of tetanus. The vast majority of tetanus cases are related to childbirth, mainly in developing countries, mainly in newborns and pregnant women with un...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/13C12N15/85C12N5/10A61K39/395A61P31/04
CPCA61K2039/505C07K16/1282
Inventor 陈薇于蕊徐俊杰侯利华于长明李建民房婷刘树玲宋小红付玲
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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