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EHEC 0157 shiga-like toxin IIA resisting subunit monoclonal antibody 5F3 light and heavy chain variable region gene and its application

A monoclonal antibody, Shiga-like toxin technology, applied in the direction of antibody, application, gene therapy, etc.

Inactive Publication Date: 2006-10-18
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no antibody drug available for the treatment of O157 infection at home and abroad. After long-term and large-scale clonal screening, we have obtained a hybridoma cell line with high affinity and high specificity for St×2A. It has high application value and development prospect

Method used

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  • EHEC 0157 shiga-like toxin IIA resisting subunit monoclonal antibody 5F3 light and heavy chain variable region gene and its application
  • EHEC 0157 shiga-like toxin IIA resisting subunit monoclonal antibody 5F3 light and heavy chain variable region gene and its application
  • EHEC 0157 shiga-like toxin IIA resisting subunit monoclonal antibody 5F3 light and heavy chain variable region gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation of anti-enterohemorrhagic Escherichia coli (EHEC) O157 Shiga-like toxin II toxin subunit Stx2A monoclonal antibody

[0025] 1. Immunization of Balb / c mice

[0026] Purified target protein Stx2A was used to immunize Balb / c mice (rats aged 6-8 weeks), 100ug / mouse, for the first immunization, 100μL of antigen was mixed with the same amount of complete Freund's adjuvant, and injected into the abdomen and groin of mice for subcutaneous immunization. After 14 days, the rats were immunized again, the dose and route of immunization were the same as the first immunization, and incomplete Freund's adjuvant was used. On the 28th day, additional immunization was given, 150ug / monkey, intraperitoneal injection.

[0027] 2. Preparation of Hybridoma Cells

[0028] 1) Collection of B lymphocytes: 3 days after the booster immunization, the eyes of the mice were extirpated and bled, and the serum was kept as a positive control. Take out the spleen by aseptic opera...

Embodiment 2

[0035] Embodiment 2 Cloning of anti-Stx2A monoclonal antibody 5F3 light and heavy chain variable region genes

[0036] 1. The cell line used is a high-affinity, high-specificity anti-Stx2A monoclonal antibody 5F3 hybridoma cell line obtained by the inventor using the above-mentioned method, and the antibody secreted by it does not interact with various Escherichia coli enterotoxins such as CT and LT. Antigen binding, the subtype of antibody molecules secreted is IgG1k.

[0037] 2. Take the 5F3 hybridoma cells in logarithmic growth phase (2×10 6 ), using guanidine isothiocyanate one-step method (TaKaRa Company) to extract total RNA, and take a small amount for quantification by UV spectrophotometer and detection by 1% agarose gel electrophoresis. Subsequently, oligo(dT)15 (TaKaRa Company) was used as a random primer to generate cDNA by reverse transcription. Then, the light and heavy chain variable region genes of monoclonal antibody 5F3 were amplified specifically using gene...

Embodiment 3

[0099] Example 3 Construction and expression of the ScFv form antibody of the anti-Stx2A monoclonal antibody (using the ScFv expression kit of Pharmacia Company)

[0100] Put VH, VL and Linker(G 4 S) 3 After the primers are equimolarly mixed, the polymerization and filling reactions are carried out, so that VH and VL are linked together by LinkerDNA to form ScFvDNA. Then, restriction enzyme cutting site primers were added to amplify the polymerized ScFv and introduce a Sfi I restriction enzyme cutting site at the 5' end and a Not I restriction enzyme cutting site at the 3' end. Cloned into the modified secretory expression vector pCANTAB6His, transformed into Escherichia coli HB2151 for inducible expression. Identification by restriction enzyme digestion and DNA sequencing analysis confirmed that the gene was constructed correctly. After the expressed protein was purified by affinity chromatography, the expressed product was analyzed and detected by SDS-PAGE electrophoresis...

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Abstract

The present invention belongs to the field of biomedicine preparing technology, and is especially enterohemorrhagic E. coli (EHEC) O157 shiga-like toxin II resisting subunit Stx2A monoclonal antibody 5F3 light and heavy chain variable region gene and the gene coded polypeptide and their application in preparing medicine for diagnosing and treating EHEC O157 infection and its complication. The present inventor adopts common facultative primer to culture one hybridoma cell strain of Stx2A specific monoclonal antibody 5F3 and clones its corresponding light and heavy chain variable region gene. Based on the light and heavy chain variable region gene, several small molecular gene engineering antibodies, such as ScFv antibody, chimeric antibody, antibody fusing protein, etc are constituted and expressed. The present invention provides new way for diagnosing and treating EHEC O157 infection and its complication.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to anti-enterohemorrhagic Escherichia coli (EHEC) O157 Shiga-like toxin II toxin subunit Stx2A monoclonal antibody 5F3 heavy chain and light chain variable region genes and polypeptides, and the genes and polypeptides in Application of the invention in preparing medicines for diagnosing and treating EHEC O157 infection and its complications. Background technique [0002] Since EHEC O157 was recognized as a pathogen in 1982, outbreaks of food poisoning caused by it have occurred all over the world, including China. The infection of this bacterium has become a global public health problem, and it has been widely concerned in both developed and developing countries. my country has listed enterohemorrhagic Escherichia coli as one of the 12 pathogenic microorganisms that may have a significant impact on the health of Chinese people in the 21st century. O157:H7 infection can cause seriou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/13A61K39/395A61K39/40A61K48/00C07K16/18C12N5/20
CPCY02A50/30
Inventor 邹全明马颖毛旭虎杨珺
Owner ARMY MEDICAL UNIV
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