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Humanized neutralizing antibody against rabies virus, method for preparing same and use

A rabies virus and antibody technology, applied in the field of human monoclonal antibodies, can solve problems such as poor stability, achieve strong binding, clear mechanism of action, and inhibit cytopathic effects

Active Publication Date: 2011-04-06
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the problem of poor stability of small molecule antibodies such as single chain antibodies, the present invention also provides a genetically engineered small molecule stability variant (3d-dsFv) of an anti-rabies virus neutralizing human monoclonal antibody , the three-domain disulfide bond-stabilizing Fvs

Method used

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  • Humanized neutralizing antibody against rabies virus, method for preparing same and use
  • Humanized neutralizing antibody against rabies virus, method for preparing same and use
  • Humanized neutralizing antibody against rabies virus, method for preparing same and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction and Screening of Human Anti-rabies Virus Phage Immune Library

[0040] 1. Materials:

[0041] 1. Bacterial strains: the helper phage M13K07 was purchased from Biolab Company; the host strain E.coli TG1 was a product of Pharmacia Company.

[0042] 2. Reagents: NotI, SfiI and other restriction enzymes are all products of Promega; FicollPlus Pague and anti M13-HRP are products of Pharmacia; total RNA extraction kit, RT reverse transcription kit, PCR amplification reagent are products of TAKARA company ; BSA (bovine serum albumin fraction V) is a product of GIBCO; DEPC, carbenicillin, tetracycline and kanamycin sulfate were purchased from SIGMA.

[0043] 3. Vector: The phage vector pCANTAB-5E is a product of Pharmacia.

[0044] 2. Method results:

[0045] 1. Volunteer immunization with rabies aG vaccine

[0046] Three volunteers were first immunized with aG vaccine (Vilbo, France), followed by two booster immunizations with aG vaccine on days 7 an...

Embodiment 2

[0068] Example 2 Recombinant Preparation of Human Anti-rabies Virus Neutralizing Antibody

[0069] 1. Materials:

[0070]1. Strains: the host strain E.coli HB2151 is a product of Pharmacia; E.coli BL21(DE3) is a product of Novagen.

[0071] 2. Reagents: E-tag affinity chromatography purification medium and Histrap HP prepacked column for Ni-NTA affinity purification were purchased from Amersham Pharmacia; PCR amplification reagents were products from TAKARA; carbenicillin was purchased from SIGMA; imidazole Purchased from Amresco Corporation.

[0072] 3. Vector: The phage recombinant expression vector pCANTAB-5E is a product of Pharmacia; the expression vector pET22b is a product of Novagen.

[0073] 2. Method results:

[0074] 1. Recombinant preparation of human ScFv

[0075] 1.1 Soluble expression of human ScFv in Escherichia coli

[0076] The recombinant expression plasmid pCan-ScFv and the empty vector of the positive clone obtained by screening were transformed into ...

Embodiment 3

[0091] Example 3 Biological Activity Analysis of Human Anti-rabies Virus Neutralizing Genetic Engineering Antibody

[0092] 1. Materials:

[0093] 1. Reagents: anti His-HRP is the product of Pierce Company; FITC-labeled series antibodies are products of Promega Company; BSA (bovine serum albumin component V), DMEM medium and fetal bovine serum are products of GIBCO Company; ammonium thiocyanate was purchased from From Beijing Biochemical Reagent Company; anti-botulinum horse serum was purchased from China Pharmaceutical and Biological Products Identification Institute; reagents for affinity determination such as HBS solution, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide ( EDC), N hydroxysuccinimine (NHS), and ethanolamine are all products of Pharmacia.

[0094] 2. Materials: CM5 chips are products of Pharmacia; Balb / C mice were purchased from the Experimental Animal Center of Academy of Military Medical Sciences.

[0095] 3. Instrument: Biacore3000 is a product of GE.

[...

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Abstract

The invention discloses a human anti-rabies virus neutralizing antibody as well as the preparation method and the application thereof. The heavy chain variable region and the light chain variable region of the antibody respectively have an amino acid sequence represented by sequence 2 and sequence 4 in a sequence list, and the heavy chain variable region and the light chain variable region of a modified body of the antibody irrespectively have an amino acid sequence represented by sequence 8 and sequence 10 in the sequence list. The antibody is prepared according to the following steps: firstly, preparing a CDR region, a variable region, IgG section or the whole genes of the antibody by molecular biology or other methods; expressing in prokaryotic cells such as Escherichia coli, eukaryotic cells such as yeast, insect cells, plant cells or mammal cells such as CHO; and purifying to obtain the antibody. The antibody has the advantages of high specific neutralization activity, high affinity and no toxic and side effects, suits prevention and emergency treatment of rabies as well as detection of rabies virus, and has excellent application prospect.

Description

technical field [0001] The invention relates to a human monoclonal antibody, in particular to a human neutralizing anti-rabies virus antibody, and also relates to a preparation method and application of the antibody. Background technique [0002] Rabies virus (Rabies Virus) is Rhabdoviridaes (Rhabdoviridaes) serotype I virus of the genus Lyssavirus, which has a typical bullet-shaped structure. The size is about 75×180nm. Its genome size is about 12Kb, and it is a single-stranded non-segmented negative-strand RNA. Its genome encodes five structural proteins including NP (nucleoprotein), M1P (capsid matrix protein), M2P (envelope matrix protein), GP (glycoprotein), and LP (transcriptase large protein). These five proteins are all antigenic. Among them, NP, M1P and LP form a nucleocapsid (RNP) complex together with viral RNA. Carbohydrates in virus particles mainly exist in the form of glycoproteins and liposaccharides. G protein is located on the surface of the virion and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/63A61K39/42A61P31/14G01N33/577
Inventor 王慧蔡昆史晶荫俊侯晓军包士中
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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