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Method for preparing monoclonal antibody to respiratory syncytial virus

A technology of monoclonal antibody and syncytial virus, applied in botany equipment and methods, biochemical equipment and methods, antiviral immunoglobulin, etc., can solve the problems of lack of in vitro expression methods of respiratory syncytial virus monoclonal antibody, etc. to save time

Inactive Publication Date: 2017-05-17
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims at the technical defects of the prior art, and provides a method for preparing a monoclonal antibody against respiratory syncytial virus, so as to solve the technical problem in the prior art that there is no method for expressing a monoclonal antibody against respiratory syncytial virus in vitro

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] A. Activation of Typhimurium VNP20009 bacteria: Take out Typhimurium VNP20009 bacteria frozen in 1.5ml EP tube from -80℃ refrigerator, draw 100μl after thawing and inoculate them in a sterilized centrifuge tube filled with 5ml LB culture medium, and put them in the constant temperature incubator sink 220r / min, culture at 37°C for 12-14h;

[0035] B. Preparation of bacterial competence of Typhimurium VNP20009:

[0036] 1. Streak inoculate LB5000 and SL3261 glycerol bacteria stored at -80°C on non-resistant LB plates, and culture at 37°C for 18 hours;

[0037]2. Pick a single colony and place it in 2ml LB, shake and culture at 37°C for 12h;

[0038] 3. Inoculate in 50ml LB at a ratio of 1:100 and culture with shaking until the OD600 of the bacteria is about 0.4;

[0039] 4. Pour into a 50ml sterile centrifuge tube for ice bath for 20min, centrifuge at 4°C, 3000rpm for 10min;

[0040] 5. Discard the supernatant, add pre-cooled sterile deionized water to fully resuspend ...

Embodiment 2

[0080] A method for preparing a monoclonal antibody against respiratory syncytial virus, comprising the following steps:

[0081] 1) Extract memory B cells from the serum of patients who have recovered from respiratory syncytial virus infection, and screen RSV antibody-positive cells by ELISA after activation and culture;

[0082] 2) extract the RNA of RSV antibody-positive cells, and amplify the antibody heavy chain variable region gene VH and light chain variable region gene Vκ or Vλ by one-step RT-PCR method respectively;

[0083]3) Ligate the VH gene with the constant region vector plasmid pIgH by enzyme digestion to obtain the heavy chain variable region gene recombination plasmid, connect the Vκ gene with the constant region vector plasmid pIgκ by enzyme digestion or connect the Vλ gene with the constant region vector by enzyme digestion Region vector plasmid pIgλ was connected to obtain light chain variable region gene recombination plasmid;

[0084] 4) amplifying the ...

Embodiment 3

[0102] A method for preparing a monoclonal antibody against respiratory syncytial virus, comprising the following steps:

[0103] 1) Extract memory B cells from the serum of patients who have recovered from respiratory syncytial virus infection, and screen RSV antibody-positive cells by ELISA after activation and culture;

[0104] 2) extract the RNA of RSV antibody-positive cells, and amplify the antibody heavy chain variable region gene VH and light chain variable region gene Vκ or Vλ by one-step RT-PCR method respectively;

[0105] 3) Ligate the VH gene with the constant region vector plasmid pIgH by enzyme digestion to obtain the heavy chain variable region gene recombination plasmid, connect the Vκ gene with the constant region vector plasmid pIgκ by enzyme digestion or connect the Vλ gene with the constant region vector by enzyme digestion Region vector plasmid pIgλ was connected to obtain light chain variable region gene recombination plasmid;

[0106] 4) amplifying the...

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Abstract

The invention provides a method for preparing a monoclonal antibody to a respiratory syncytial virus. According to the method for preparing the monoclonal antibody to the respiratory syncytial virus provided by the invention, memory B cells are separated from peripheral blood of a patient who is infected with and recovered from the respiratory syncytial virus recently to further determine genes of a heavy chain variable region and a light chain variable region to express the antibody; murine typhia competent cell VNP20009 is utilized to directly transfect a 293T cell and enable expression of the monoclonal antibody to the respiratory syncytial virus in vitro; neutralization capacity and killing competence of the monoclonal antibody to the respiratory syncytial virus is evaluated after appraisal, so as to screen high-efficiency and scale production-available monoclonal antibodies to the respiratory syncytial virus. According to the method for preparing the monoclonal antibody to the respiratory syncytial virus provided by the invention, as the murine typhia competent cell VNP20009 is utilized to directly transfect the 293T cell, compared with the conventional methods, not only time is saved, but also the application a lot of transfection reagent can be avoided. As transfection of exogenous plasmids to an expression system is a key link of in-vitro expression of the monoclonal antibody, the invention provides a strong support to industrialized application of the technology.

Description

technical field [0001] The invention relates to the technical field of biological products, and further relates to the in vitro expression technology of monoclonal antibodies, in particular to a method for preparing monoclonal antibodies to respiratory syncytial virus. Background technique [0002] According to the definition of the World Health Organization (WHO), respiratory syncytial virus infection is a contagious viral disease and is the most important cause of severe respiratory diseases in infants and young children. The condition manifests primarily as bronchitis, inflammation of the small airways of the lungs, or viral pneumonia. [0003] Respiratory syncytial virus has become the leading cause of hospitalization in infants in developed countries, but there is no vaccine to prevent it. There are currently only two drugs (palivizumab and ribavirin) available to patients, and these two drugs also have great drawbacks. [0004] Respiratory syncytial virus drugs are u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/13C07K16/10
Inventor 陈廷涛辛洪波孟凡景王鑫
Owner NANCHANG UNIV
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