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Fully-humanized rabies virus resisting neutralizing antibody

A rabies virus, fully human technology, applied in the fields of genetic engineering and cellular immunology, can solve problems such as failure to complete preclinical research, complex antibody process, chimeric antibodies and humanized antibodies can not completely solve the HAMA reaction and other problems

Active Publication Date: 2017-02-22
CHANGCHUN BCHT BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the early genetically engineered antibodies also had disadvantages that cannot be ignored, such as the gene is taken from hybridoma cells, the process of producing antibodies is complicated, chimeric antibodies and humanized antibodies cannot completely solve the HAMA reaction, and it is difficult to modify antibodies to obtain high-affinity antibodies Wait
Mab57 thus has not been able to complete preclinical studies

Method used

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  • Fully-humanized rabies virus resisting neutralizing antibody
  • Fully-humanized rabies virus resisting neutralizing antibody
  • Fully-humanized rabies virus resisting neutralizing antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Lymphocyte Separation and Single Memory B Cell Sorting

[0060] 1. Materials:

[0061] 1. Reagent: rabies vaccine (Novartis, batch number: 1928). Ficoll lymphocyte separation medium is a product of Cedarlane Company. CD3-APC, CD14-APC, CD16-PE, CD20-APC, CD27-PE, IgD-FITC, etc. are all products of Invirogen.

[0062] 2. Method results

[0063] 1. Volunteer immunization with rabies vaccine

[0064] Two 25-year-old healthy volunteers who were inoculated with rabies vaccine were selected, and they received booster vaccinations on the 7th day and 21st day respectively, and the quantitative detection of 28d IgG was positive; 100 mL of their peripheral blood was drawn for anticoagulation.

[0065] 2. Lymphocyte isolation and single memory B cell sorting

[0066] Separate 100mL blood sample with Ficoll, absorb the mononuclear cell (PBMC) layer suspension, wash with PBS 3 times, and discard the supernatant. Set single dye tube: add AqVd-AmCyan, CD3-PE-Cy5, IgD-P...

Embodiment 2

[0069] Example 2 Single B cell RT-PCR isolation antibody variable region gene

[0070] 1. Materials

[0071] 1. Reagents: primers (synthesized by Invitrogen), Superscript III reverse transcriptase, HotStarTaq Plus enzyme (Invitrogen, Carlsbad, CA), etc.

[0072] 2. Method results

[0073] 1. Single B cell RT-PCR (synthesis of the first strand of cDNA)

[0074] Add 0.5uM constant region primers of heavy and light chains of each subtype to a 96-well plate containing a single B cell and Superscript III reverse transcriptase, and incubate at 37°C for 1 hour; carry out PCR amplification under the following conditions: 95°C for 15min ; 95°C for 1min, 55°C for 1min, 72°C for 1min, 30 cycles; 72°C for 10min; 4°C for 5min. The product cDNA was stored at -20°C.

[0075] 2. Nest-PCR (isolation of antibody gene)

[0076] Each single cell amplifies the heavy chain and light chain sequences separately. The 50uL system contains 5uL of RT reaction products, HotStarTaq Plus enzyme, dNTPs...

Embodiment 3

[0077] Example 3PCR product clone identification and antibody expression

[0078] 1. Materials

[0079] 1. Reagents: Agarose, Tris, LB, Ampicillin, PolyFect (Qiagen, Valencia, CA), FCS, DMEM, PBS, etc.

[0080] 2. Vector: pcDNA3.3 vector

[0081] 3. Strain: Escherichia coli DH5α

[0082] 2. Method results

[0083] 1.PCR product purification clone identification

[0084] Take 2 μL of the amplification product and detect it by 1.2% agarose gel electrophoresis, and the target fragment is about 400 bp. The gel electrophoresis was identified as positive, and the PCR product of the antibody variable region gene whose heavy chain and light chain could be paired was connected to the pcDNA3.3 vector (which has been transformed and contains the antibody leader and constant region) by the method of TA cloning, Transform the ligation product into DH5α-competent bacteria, culture on a plate containing ampicillin at 37°C overnight, then pick 10 single colonies and perform PCR with spec...

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Abstract

The invention discloses a fully-humanized rabies virus resisting neutralizing antibody. DNA sequences for encoding the antibody and binding fragments thereof, host cells containing DNA, and an expression vector are included. The invention further discloses a preparation method of the fully-humanized rabies virus resisting neutralizing antibody with high affinity and application of the antibody in medicine for treating and / or preventing rabies.

Description

technical field [0001] The present invention belongs to the fields of cellular immunology and genetic engineering, and relates to a fully humanized neutralizing antibody against rabies virus. Background technique [0002] Rabies passive immunization preparations can neutralize the local rabies virus in the wound, prevent the virus from spreading and invading the nervous system, and its half-life is as long as 14 to 21 days, which can effectively play a role in the treatment or emergency prevention of rabies. [0003] The earliest example of using rabies passive immunization to cure patients can be traced back to 1891, but it took decades before humans produced the first commercial anti-rabies immune serum for humans. This anti-rabies immune serum for humans is derived from horses, and since it is a heterologous serum, adverse reactions after use can be as high as more than 40%. At present, the main product on the market is horse-derived anti-rabies serum globulin (ERIG). Al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14G01N33/577G01N33/569
CPCA61K2039/505C07K16/10C07K2317/21C07K2317/565C07K2317/76C07K2317/92G01N33/56983G01N33/577
Inventor 廖化新袁晓辉王月明
Owner CHANGCHUN BCHT BIOTECH
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