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Method for preparing chimeric monoclonal antibody capable of neutralizing EV71 (enterovirus 71)

A monoclonal antibody and enterovirus technology, applied in the direction of antibodies, antiviral agents, antiviral immunoglobulin, etc., can solve the problem of no corresponding vaccines and drugs approved for marketing

Inactive Publication Date: 2015-05-13
BEIJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the domestic EV71 virus inactivated vaccine has completed phase III clinical research, but there are still no corresponding vaccines and drugs approved for marketing. In addition, effective vaccines and drugs have not yet been developed for CVA16 virus, another major antigen that causes hand, foot and mouth disease. Finding a drug that can neutralize both EV71 virus and CVA16 virus is of great significance for the current fight against hand, foot and mouth disease

Method used

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  • Method for preparing chimeric monoclonal antibody capable of neutralizing EV71 (enterovirus 71)
  • Method for preparing chimeric monoclonal antibody capable of neutralizing EV71 (enterovirus 71)
  • Method for preparing chimeric monoclonal antibody capable of neutralizing EV71 (enterovirus 71)

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1, the polypeptide particle HBcAg-VP4 protein that carries EV71VP4 protein immunizes animals

[0045] Experimental animals: 5 female healthy BALB / c mice (Beijing Huafukang Biotechnology Co., Ltd., experimental animal quality certificate license number: SCXK (Beijing) 2009-0007), the age of mice is 6-8 weeks, They were kept in the animal room of the New Drug Research and Development Center of the Institute of Chinese Materia Medica, Academy of Chinese Medical Sciences.

[0046] For the first immunization (week 0), each mouse was injected with 50 micrograms (50 microliters) of purified HBcAg-VP4 protein in an equal volume of Freund's complete adjuvant (Sigma-Aldrich, catalog number: F5506) mix. Afterwards, booster immunization (2 weeks, 5 weeks) with the same dose as above, and the injection methods were all subcutaneous injections. At 0, 2, 4, 6, 8, 10, and 12 weeks after the third immunization, blood was collected from the inner canthus vein, and the polyan...

Embodiment 2

[0047] Example 2. Extraction and isolation of antibody-secreting lymphocytes and synthesis of cDNA

[0048] Spleen was taken aseptically, ground to prepare cell suspension, lymphocytes were separated, mRNA was extracted by Trizol method, and cDNA was synthesized by reverse transcription.

Embodiment 3

[0049] Example 3, Construction of ScFv single-chain antibody phage display library

[0050] 1. Preparation of Escherichia coli TG1 and HB2151 host bacteria

[0051] From the TG1 / HB2151 strain preservation medium (purchased from Pharmacia, the product catalog number is 279401-01), the bacterial liquid was picked and streaked on a 2×YT (yeast extract-peptone medium) plate, and cultured overnight at 37°C, again Pick a single colony and streak it on a Minimal Medium plate (refer to the instruction manual for the preparation method), and culture it overnight at 37°C before use;

[0052] 2. Transformation of pCANTAB5E plasmid

[0053] Take 1 μg of pCANTAB5E plasmid (a phagemid display system vector plasmid capable of inserting ScFv, purchased from Pharmacia, catalog number 279401-01) with a sterile tip and add it to 200 μl of TG1 competent cells prepared in step 1 above, Gently swirl to mix, ice bath for 30min. Gently add the mixture along the tube wall into the electric cup with...

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Abstract

The invention relates to a method for preparing a chimeric monoclonal antibody capable of neutralizing EV71 (enterovirus 71). The chimeric monoclonal antibody for neutralizing EV71 or a fragment of the chimeric monoclonal antibody is characterized in that a heavy chain variable region of the chimeric monoclonal antibody or the fragment of the chimeric monoclonal antibody has an amino acid sequence shown in SEQ ID NO:1; a light chain variable region has an amino acid sequence shown in SEQ ID NO:2. EV71 VP4 (viral protein 4) protein is fused into an HBcAg (hepatitis B core antigen) virus-like particle to be prepared into fusion protein, and the fusion protein is taken as an antigen to endow a Balb / c mouse with immunity. A phage display antibody library technique is used for screening to obtain the monoclonal antibody capable of specifically neutralizing EV71 VP4 protein. The invention provides the method for preparing a potential monoclonal antibody medicine with broad spectrum neutralization activity and for preventing and treating a hand foot mouth disease.

Description

technical field [0001] The present invention relates to a monoclonal antibody for recognizing enterovirus 71 type and Coxsackie virus type A16 VP4 protein and its preparation method and application, in particular to a kind of EV71 VP4 protein chimerized into HBcAg virus-like particles prepared The fusion protein was used as an antigen to immunize Balb / c mice. The monoclonal antibody that can specifically neutralize EV71VP4 protein was screened by phage display antibody library technology. Background technique [0002] Hand foot mouth disease (HFMD) is a common infectious disease in infants and young children caused by different enteroviruses. It is characterized by fever and herpes on the hands, feet, and mouth. Injury, pulmonary edema, pulmonary hemorrhage, circulatory failure and heart damage, some of them died due to serious complications. In recent decades, hand, foot and mouth disease has broken out and become popular in many parts of the world, involving a wide range...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/63C12N5/10A61K39/42A61P31/14
Inventor 盛望张潇白玉赵淼曾毅李泽琳刘伟
Owner BEIJING UNIV OF TECH
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