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Hybridoma cell strain secreting tetanus exotoxin monoclonal antibody, monoclonal antibody prepared by same, Fab antibody and application

A hybridoma cell line and monoclonal antibody technology, applied in the field of medicine and biology, can solve the problems of difficulty in immunizing human plasma sources, death, and limited production.

Inactive Publication Date: 2012-09-26
ARMY MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

TAT is a liquid or freeze-dried antitoxin globulin preparation obtained by purifying the serum of horses immunized with tetanus toxoid after digestion with gastric enzymes. Death; HIGT is derived from the plasma of healthy people immunized with tetanus vaccine or tetanus toxoid. It is the same protein of human origin and is not easy to cause allergic reactions. The potential safety hazard of exogenous virus contamination such as HCV has greatly restricted its industrial production and clinical use
At present, the production of HIGT at home and abroad cannot meet the market demand

Method used

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  • Hybridoma cell strain secreting tetanus exotoxin monoclonal antibody, monoclonal antibody prepared by same, Fab antibody and application
  • Hybridoma cell strain secreting tetanus exotoxin monoclonal antibody, monoclonal antibody prepared by same, Fab antibody and application
  • Hybridoma cell strain secreting tetanus exotoxin monoclonal antibody, monoclonal antibody prepared by same, Fab antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Preparation and Identification of Monoclonal Antibody Neutralizing Tetanus Exotoxin

[0057] 1. Animal immunization

[0058] Simultaneously immunize five female Balb / c mice aged 6-8 weeks (purchased from the Experimental Animal Center of Third Military Medical University) with formaldehyde-inactivated tetanus toxoid as the antigen, inject 0.5ml each, containing 100μg of antigen, and immunize The time was 0, 3, 5, and 6 weeks, 4 times in total. The first time was added with Freund's complete adjuvant for subcutaneous multi-point injection, the second time was added with Freund's incomplete adjuvant, and the third time was intraperitoneally injected with the same dose without adjuvant After immunization, the tail blood was taken one week later and the serum titer of the immunized Balb / c mice was detected by indirect ELISA method. The splenocytes with the highest titer were used for fusion. Three days before the fusion, intraperitoneal booster immunization was ...

Embodiment 2

[0084] Example 2 Cloning of κ Light Chain and Fd Chain Genes of Tetanus Exotoxin Neutralizing Monoclonal Antibody TT5C4

[0085] 1. The cell line used is the hybridoma cell line obtained by the inventors using the above method, which can secrete high-affinity, high-specificity monoclonal antibody TT5C4 neutralizing the toxic effect of tetanus exotoxin, and the preservation number is CCTCC NO.C201257 , the subclass and subtype of antibody molecules secreted are IgG1 type.

[0086] 2. Take the TT5C4 hybridoma cells in logarithmic growth phase (2×10 6 ), using TRIZOL one-step method (Invitrogen Company) to extract total RNA, and take a small amount for quantification by UV spectrophotometer and detection by 1% agarose gel electrophoresis. Then, the One step PT-PCR kit (TaKaRa Company) was used to amplify the κ light chain (VL+CL) and Fd chain (VH+CH1) genes of monoclonal antibody TT5C4. The amplified products containing the κ chain and Fd chain of TT5C4 were subjected to 1% a...

Embodiment 3

[0161] Example 3 Construction and Soluble Expression of Fab Antibody of Tetanus Exotoxin Neutralizing Monoclonal Antibody TT5C4

[0162] First, clone the correctly analyzed TT5C4κ chain and TT5C4Fd chain genes into the vector pcomb3X (purchased from Chongqing Jinmai Biotechnology Co., Ltd.) that secretes and expresses Fab antibodies, and construct the pcomb3X-TT5C4Fdκ plasmid. The coat protein III gene was self-ligated to construct the pcomb3X-TT5C4Fab expression plasmid, which was confirmed by restriction enzyme digestion and DNA sequencing analysis, and then transformed into Escherichia coli XL-blue (purchased from Shanghai Jereh Bioengineering Company) , Constructed into pcomb3X-TT5C4Fab / XL-blue recombinant, IPTG induces the expression of TT5C4Fab antibody protein. SDS-PAGE electrophoresis ELISA and Western-blot analysis showed the expression of Fab gene in E. coli. The specific steps of the operation are as follows:

[0163]1. Plasmid DNA extraction (using Omega plasmi...

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Abstract

The invention relates to a mouse hybridoma cell strain secreting a clostridium tetani bacillus exotoxin monoclonal antibody, wherein the strain has a preservation number of CCTCC (China Center for Type Culture Collection) NO. C201257. The invention further relates to a clostridium tetani bacillus exotoxin monoclonal antibody prepared by the mouse hybridoma cell strain; the invention further provides an Fab antibody, comprising a kappa chain and an Fd chain; the kappa chain and the Fd chain are obtained by amplifying from total RNA (ribonucleic acid) of the hybridoma cell strain. The invention further relates to a medicine for preventing or treating clostridium tetani bacillus infection, comprising the clostridium tetani bacillus exotoxin monoclonal antibody and / or the Fab antibody, and a pharmaceutically acceptable carrier. According to the invention, a monoclonal antibody for effectively neutralizing tetanus exotoxin is screened with natural tetanus exotoxin, and an Fab gene engineering antibody which is produced by large scale in vitro and which has toxin neutralizing effect is prepared with a gene engineering antibody technology on the basis of the monoclonal antibody.

Description

technical field [0001] The invention relates to the field of medical biotechnology, in particular to a hybridoma cell strain secreting a monoclonal antibody of Clostridium tetani exotoxin, a monoclonal antibody, a Fab antibody prepared therefrom and applications thereof. Background technique [0002] Tetanus toxin (TT) is a neurotoxin secreted by Clostridium tetani under hypoxic conditions after wound infection, which can lead to death of patients due to laryngospasm or secondary severe lung infection. The mortality rate can be as high as 20% to 50%. About 50,000 people worldwide die from tetanus every year. Antibiotics cannot eliminate the toxic effect of tetanus exotoxin, and anti-tetanus exotoxin antibodies with neutralizing toxin effects can bind to free toxins to block the binding of toxins to target cells. Therefore, anti-tetanus exotoxin antibodies are important for prevention and Drug of choice for the treatment of tetanus. [0003] The current clinical antibody d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C12N15/11C12N15/63C12N1/15C12N1/19C12N1/21C07K16/12A61K39/40A61P31/04C12R1/91
Inventor 罗萍邹全明毛旭虎顾江秦利燕陈立余抒刘唯
Owner ARMY MEDICAL UNIV
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