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Monoclonal antibody, Fab antibody and application for neutralizing enterohemorrhagic escherichia coli o157:H7 shiga toxin II

A monoclonal antibody, enterohemorrhagic technology, applied in the field of medicine and biology, can solve the problems of long half-life, low uptake percentage, and large molecular weight of monoclonal antibody

Inactive Publication Date: 2009-02-04
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005]Because the mouse monoclonal antibody can cause human anti-mouse antibody reaction in the human body; the complete monoclonal antibody has a large molecular weight, a low percentage of tissue uptake, and a long half-life in vivo, so make its application in the human body limited

Method used

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  • Monoclonal antibody, Fab antibody and application for neutralizing enterohemorrhagic escherichia coli o157:H7 shiga toxin II
  • Monoclonal antibody, Fab antibody and application for neutralizing enterohemorrhagic escherichia coli o157:H7 shiga toxin II
  • Monoclonal antibody, Fab antibody and application for neutralizing enterohemorrhagic escherichia coli o157:H7 shiga toxin II

Examples

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Embodiment 1

[0064] Implementation 1 Preparation and identification of monoclonal antibody neutralizing EHEC O157:H7 Stx2

[0065] 1. Animal immunization

[0066] Conjugate KLH with the B cell epitope peptide P1 (SEQ ID NO: 5_DIRGLDVYQARFDHLRLIIEQNNLYVAG) from the EHEC O157: H7 Stx2 A1 subunit and immunize five female healthy BALB / C mice aged 6-8 weeks at the age of 17g-22g at the same time. The method, route, and dosage are the same as those in the first part. The antibody titer was measured by tail blood collection at the last immunization, and the BALB / C mice with a serum antibody titer of 1:8000 or more detected by ELISA were selected for fusion. 3 days before fusion, no adjuvant antigen was added. Intraperitoneal booster injection 1 time, 100μg / rat.

[0067] 2 Preparation of hybridoma cells

[0068] 2.1 Collection of B lymphocytes

[0069] Three days after the booster immunization, the eyes of the mice were extirpated and bled, and the serum was kept as a positive control. Take...

Embodiment 2

[0104] Example 2 Cloning of κ Light Chain and Fd Chain Genes of Stx2 Neutralizing Monoclonal Antibody 1F2

[0105] 1. The cell strain used is a hybridoma cell strain that can secrete high-affinity and high-specificity monoclonal antibody 1F2 that neutralizes the toxic effect of Stx2 obtained by the inventor using the above-mentioned method, and the antibody molecule subclasses and subclasses it secretes The type is IgG1κ.

[0106] 2. Take the 1F2 hybridoma cells in logarithmic growth phase (2×10 6 ), using the iso-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. The κ light chain (VL+CL) and Fd chain (VH+CH1) genes of mAb 1F2 were then amplified using the One step PT-PCR kit (TaKaRa Company). After the amplified products containing κ chain and Fd chain of 1F2 were subjected to 1% agarose gel electrophoresis, the target fragments were separat...

Embodiment 3

[0189] Example 3 Construction and Soluble Expression of Stx2 Neutralizing Monoclonal Antibody 1F2 in Fab Form

[0190] Firstly, the correctly analyzed 1F2κ chain and 1F2Fd chain genes are cloned into the vector pcomb3X that secretes and expresses Fab antibody, and the pcomb3X-1F2Fdκ plasmid is constructed. The pcomb3X-1F2Fab expression plasmid was identified by restriction enzyme digestion and DNA sequencing analysis, and it was confirmed that the gene was constructed correctly and transformed into Escherichia coli XL-blue to construct a pcomb3X-1F2Fab / XL-blue recombinant, and IPTG induced the expression of 1F2Fab 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:

[0191] 1. Plasmid DNA extraction (using Omega plasmid extraction kit)

[0192] Pick the well-separated colonies on the plate and inoculate them in LB culture medium with corresponding ant...

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Abstract

The invention belongs to the technological field of medicine bioengineering, which more particularly relates to a monoclonal antibody neutralizing enterohemorrhagic escherichia coli O157:H7 shiga toxin II, Fab antibody sequences and the application. The monoclonal antibody is prepared by hybridoma cell lines with the preservation number of CCTCC: C200822. The antibody of the invention can be used for preparing medicines for diagnosing and treating EHEC O157 infections and the complicating diseases thereof.

Description

technical field [0001] The invention relates to the field of medical biotechnology, in particular to a monoclonal antibody, a Fab antibody and an application thereof for neutralizing enterohaemorrhagic Escherichia coli O157:H7 Shiga toxin II. Background technique [0002] Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection is a global public health problem. The food poisoning caused by it has large-scale outbreaks all over the world. The 2006 outbreak of poisonous spinach in the United States involving 26 states was caused by this bacteria. From the end of 1999 to the beginning of 2000, the world's largest outbreak of O157: H7 infection occurred in some parts of eastern my country. Due to the ease of cultivation, strong infectivity, and diverse transmission routes, the bacterium is very likely to be used as a bacterial weapon and bioterrorist agent in future military struggles. The U.S. Centers for Disease Control (CDC) has listed EHECO157 bacteria as a Class B bio...

Claims

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Application Information

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IPC IPC(8): C07K16/12C12N15/13C12N5/18C12N15/63C12N1/15C12N1/19C12N1/21A61K39/395A61P31/04
Inventor 邹全明罗萍曾浩毛旭虎石云张卫军刘璐
Owner ARMY MEDICAL UNIV
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