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Tetanus toxoid monoclonal antibody and preparation method and application thereof

A monoclonal antibody and anti-tetanus technology, applied in the field of biological immunology, can solve the problems of limited application of tetanus human blood immunoglobulin, high price, and difficult source of human blood

Active Publication Date: 2012-05-16
SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of tetanus human blood immunoglobulin is greatly limited due to the difficulty of human blood sources, high price, complicated industrial production, and susceptibility to virus contamination.

Method used

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  • Tetanus toxoid monoclonal antibody and preparation method and application thereof
  • Tetanus toxoid monoclonal antibody and preparation method and application thereof
  • Tetanus toxoid monoclonal antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1, preparation of anti-tetanus toxoid mouse monoclonal antibody

[0076] 1. Preparation of anti-tetanus toxoid monoclonal antibody hybridoma cell line

[0077] 1. Immunogen

[0078] Tetanus toxoid was purchased from the Production Department of Shanghai Institute of Biological Products.

[0079] 2. Immunization of Balb / c mice

[0080] Balb / c mice were purchased from Shanghai Slack Co., Ltd. All Balb / c mice used for immunization were 3-week-old, female, standardized disease-free, healthy purebred mice, which met the US FDA standards.

[0081] 3. Balb / c mouse immunization method

[0082] The first immunization: Mix 50 μg (250 μl) of antigen with 250 μl of aluminum adjuvant, prepare a 500 μl solution, and inject it into Balb / c mice at multiple points and paws subcutaneously. The second immunization: three weeks after the first immunization, the second immunization is carried out in the same way and with the same dose. The third and fourth immunizations: t...

Embodiment 2

[0098] Example 2, Cloning and Identification of Anti-tetanus Toxoid Monoclonal Antibody Variable Region Coding Sequence

[0099] Using the cDNA of the anti-tetanus toxoid hybridoma cell line (6D12) screened in Example 1 as a template, PCR technology was used to clone the anti-tetanus toxoid mouse monoclonal antibody variable region gene; after sequencing, no mutation and no termination were selected. The sequence of the codon, using 5'RACE technology to clone the functional V L and V H Gene.

[0100] 1. Cloning of the variable region coding sequence of anti-tetanus toxoid murine monoclonal antibody

[0101] (1) Extracting total RNA of anti-tetanus toxoid monoclonal antibody from hybridoma cell lines

[0102] Extracted with FAST1000 kit of Shanghai Feijie Biological Company.

[0103] (1) Take 4×10 5 Hybridoma cells, 1000rpm×3min, discard the supernatant. Wash once with PBS. Resuspend the cells in 100 μl PBS and place in a centrifuge tube.

[0104] (2) Add 1ml of RB1 s...

Embodiment 3

[0173] Example 3, Construction of anti-tetanus toxoid human-mouse chimeric monoclonal antibody eukaryotic expression vector

[0174] light chain V L gene and human Ig C L The genes were spliced ​​to form a light chain chimeric gene; a BamHI restriction endonuclease site was introduced at the 5' end of the light chain, and an EcoRI restriction endonuclease site was introduced at the 3' end of the light chain. Similarly, construct the heavy chain chimeric gene. The above light chain / heavy chain genes were respectively inserted into the monoclonal restriction site of pcDNA3.1(+ / -) expression vector (purchased from Invitrogen) to construct the expression vector of anti-tetanus toxoid human-mouse chimeric antibody.

[0175] (1) Design the upstream and downstream products according to the conventional method, and introduce BamHI single enzyme cutting site at the 5' end of the heavy chain / light chain variable region gene by PCR technology, and introduce EcoR at the 3' end of the ...

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Abstract

The invention relates to a tetanus toxoid monoclonal antibody and a preparation method and application thereof, in particular to a tetanus toxoid monoclonal antibody. A complementary determining region (CDR) of a heavy chain variable region of the monoclonal antibody comprises CDR1 shown as SEQ ID NO:6, CDR2 shown as SEQ ID NO:8 and / or CDR3 shown as SEQ ID NO:10; and a CDR of a light chain variable region of the monoclonal antibody comprises CDR1 shown as SEQ ID NO:12, CDR2 shown as SEQ ID NO:14 and CDR3 shown as SEQ ID NO:16. The invention also provides a deoxyribonucleic acid (DNA) moleculefor coding the sequence, an expression vector and a host cell.

Description

technical field [0001] The invention relates to the field of biological immunology. More specifically, the present invention relates to a monoclonal antibody with neutralizing tetanus toxoid. In addition, the present invention also relates to the preparation method and application of the monoclonal antibody. Background technique [0002] Tetanus (Tetanus) is a serious infectious disease caused by Clostridium tetani (Clostridium tetani). Bacteria grow and reproduce in the wound under anaerobic conditions, producing a large amount of toxins. The toxins invade the central nervous system, causing the patient's generalized muscle tonic spasm, forming tetanus-specific symptoms such as confinement of the teeth, opisthotonus, and severe cases. Died of asphyxiation and systemic failure. At present, tetanus is still a major public health problem in most developing countries. About 1 million people die from tetanus every year, and newborns account for 80% of the total deaths. [00...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/11C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10G01N33/577G01N33/569A61K39/40A61P31/04
Inventor 瞿爱东祝婧烨张燕燕于汉卿梁红远
Owner SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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