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Heavy and light chain variable region gene of monoclonal antibody, encoding polypeptide thereof and use

A monoclonal antibody and gene-encoded technology, which can be used in antibody, gene therapy, genetic engineering, etc., can solve the problems of inability to resist EHEC0157 infection and low secretion

Inactive Publication Date: 2009-05-20
JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

StxII is more likely to cause HUS than StxI, and in StxII, StxII protoxin and StxIIc, StxIIvha and other variants are the most virulent, and the monoclonal antibody provided in this application is only against StxII protoxin, and has no effect on StxII variants, so it cannot resist EHEC 0157 infection caused by StxII variants, and Stx secretion in the human body is very low, which is not enough to stimulate the body to produce sufficient antibodies to resist reinfection. Therefore, blocking the binding of StxII toxin and its variants to its receptor Gb3 prevents the occurrence of HUS It has become a bottleneck urgently needed to overcome EHEC infection

Method used

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  • Heavy and light chain variable region gene of monoclonal antibody, encoding polypeptide thereof and use
  • Heavy and light chain variable region gene of monoclonal antibody, encoding polypeptide thereof and use
  • Heavy and light chain variable region gene of monoclonal antibody, encoding polypeptide thereof and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] 1. Preparation of monoclonal antibody S2C4 against EHEC Shiga toxin type II A subunit

[0015] (1) Immunization of Balb / c mice

[0016] Immunize 5-week-old female Balb / c mice with the fusion protein Stx2A-GST, 50ug / mouse, for the first immunization, mix 100ul antigen with an equal volume of Freund's complete adjuvant, and inject intraperitoneally; after the third week, the antigen and incomplete adjuvant Intraperitoneal immunization after mixing equal doses; the third immunization in the 5th week without adjuvant.

[0017] (2) Fusion of splenocytes and myeloma cells

[0018] One week before fusion, resuscitate mouse myeloma cells sp2 / 0 into OPTI-MEM medium (containing 10% fetal bovine serum) and place at 37°C, 5% CO 2 The cells were cultured in an incubator, and the cells were passaged once 3 days before fusion. On the day of fusion, harvest myeloma cells, count, and put 5×10 7 Myeloma cells were washed twice with serum-free medium for later use. 3-5 days after the...

Embodiment 2

[0165] LD 100 Determination: EHEC StxII toxin was diluted with sterilized PBS, intraperitoneally inoculated with 6-week-old BALB / c mice, and within 12 days of the experiment, the lowest dose of toxin that could kill all mice in the test group was 5 ng / mouse.

[0166] The mice were first intraperitoneally inoculated with 5ng / mouse of toxin, and 16 hours later, the intraperitoneal inoculation of S2C4 single-chain antibody ScFv, the dosage was 60, 30, 15, 8, 4ug / mouse, respectively, with S2C4 full molecule antibody and PBS as Positive, negative reference. The number of dead mice was recorded every day, and the whole experiment ended on the 12th day. It was found that when the dose of S2C4 single-chain antibody ScFv was 60-15ug / mouse, the mice could be completely protected from toxin attack; while the dose of ScFv was 8 and 4ug / mouse, the protective efficiency was only 60%. % and 20%, the results are shown in Table 1 and Table 2.

[0167] animal grouping number of a...

Embodiment 3

[0173] Get the crude extracts of three subtypes of EHEC StxII toxin: StxIIc, StxIId and StxIIvha were respectively intraperitoneally inoculated with 6-week-old BALB / c mice (100ul / only), 16 hours later, intraperitoneally inoculated with S2C4 10ug / mouse, recorded every day The number of dead mice, the whole experiment ended on the 12th day. It was found that, like StxII, S2C4 could completely neutralize the toxic effects of the three subtypes of StxII and protect mice from toxin challenge. The results are shown in Table 3.

[0174]

[0175] table 3

[0176] Conclusion: S2C4 has a strong neutralizing spectrum, and the epitope it targets may be quite conserved between StxII and its subtypes, which indicates that S2C4 is a very promising antibody drug candidate for the treatment of EHEC infection.

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Abstract

The invention relates to a monoclonal antibody and usage thereof, in particular to the monoclonal antibody of Shiga toxin II (StxII) and usage thereof, and belongs to the technical field of biopharming. The invention can obtain genes of heavy chain and light chain variable regions of the antibody from a hybridoma cell line S2C4 that can secrete anti-StxII monoclonal antibody with the PCR method clone, can express an ScFv antibody active segment with StxII protein specificity recognition by recombining two genes with genetic engineering methods, and can be used for preparing medicaments with EHEC infection and complicating diseases resistance. The monoclonal antibody has the advantages that the monoclonal antibody S2C4 has strong neutralizing activity not only to StxII protoxin but also to multiple StxII variants. Experiments prove that the monoclonal antibody S2C4 has wide neutralizing spectrum, can protect mice from the attack of StxII toxin and subtypes with lethal dosage, and is a medicament candidate with wide application prospect.

Description

technical field [0001] The invention relates to a monoclonal antibody and its use, in particular to the monoclonal antibody of Shiga toxin type II A subunit and its use, and belongs to the technical field of biopharmaceuticals. Background technique [0002] Enterohemorrhagic Escherichia coli (Enterohemorrhagic Escherichia coli., EHEC) infection is an important infectious disease, which has outbreaks of different scales all over the world and has become a global public health problem. There are many EHEC serotypes, including 0157, 026, 091, 0103 and 0111, among which 0157 caused the most outbreaks. EHEC0157:H7 infectious diarrhea combined with acute renal failure broke out in parts of Jiangsu, Anhui, and Henan provinces from 1999 to 2000, resulting in significant human and property losses. EHEC0157: H7 infection also occurred in the United States from September to October 2006 due to eating contaminated spinach, which spread to 26 states, and many people caused hemolytic ure...

Claims

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Application Information

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IPC IPC(8): C12N15/13C07K16/12A61K48/00A61K39/40A61P31/04
CPCY02A50/30
Inventor 焦永军郭喜玲崔仑标曾晓燕吴涛史智扬
Owner JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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