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SARS neutralization antibody and application

A SARS virus and specific technology, applied in the field of SARS neutralizing monoclonal antibodies and their preparation, can solve the problems of difficulty in obtaining active neutralizing antibodies, no antibodies, antibody defects, etc.

Inactive Publication Date: 2009-07-29
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although RBD peptides can be directly used for immunization, the antibodies produced may be defective in recognizing the complete natural SARS virus S protein due to structural differences in the full length of the protein and partial peptides, or may obtain some A monoclonal antibody that recognizes the RBD of the receptor binding region but has no binding effect on the full-length intact protein of SARS S protein, so not only the efficiency of obtaining effective antibodies is low, but also it is difficult to obtain neutralizing antibodies with high activity
[0007] In summary, there is no highly active antibody that specifically binds to the SARS virus S protein in the art.

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0065] Example 1: Animal immunization

[0066] The antigen is a SARS pseudovirus vaccine, and the attenuated Ankara virus is obtained by recombining the full-length protein gene of SARS S in its genome. The SARS virus S protein is expressed on the surface of the virus membrane, concentration: 2×10 9 pfu / ml, Balb / c mouse immunization dose: 5×10 7 pfu each time. Multiple intramuscular injections. Dilute with PBS or normal saline before use. Immunization program: 0, 3, 6 three times immunization. Take 5×10 three days before fusion 7 The pfu vaccine stock solution was diluted in PBS to 0.5ml for intraperitoneal injection for memory stimulation.

[0067] Brief introduction of SARS recombinant pseudovirus vaccine: the carrier virus is the live attenuated Ankara vaccine virus (MVA) after transformation, and the preparation method is as follows: with conventional methods, the SARS virus S protein full-length gene is inserted into the No. III deletion region of the genome of the ...

Embodiment 2

[0068] Example 2: Construction of hybridoma cell lines and preparation of monoclonal antibodies

[0069] Fusion was done three days after recall stimulation. The spleen cells of the immunized mice were taken and the mouse myeloma cells NS-1 and SP2 / 0 were fused by conventional fusion method of polyethylene glycol (PEG). After fusion, add HAT selective medium to kill all non-hybridoma cells. The specific steps will be described later.

[0070]Then, screening of hybridomas was carried out. The screening adopts the ELISA method (the specific steps will be described later), and the antigen used for screening is the SARS virus receptor binding domain (Receptor Binding Domain) located at the 318-510 amino acid segment of the S protein expressed by eukaryotes (connected with the Fc segment of the IgG constant region as protein label). The cell supernatant in the grown hybridoma cell sample well is taken out, and used as a sample for ELISA experiment to see if it contains an antib...

Embodiment 3

[0103] Example 3: Cell Fusion Blocking Experiment

[0104] In this example, the cell fusion blocking experiment is used to simulate the membrane fusion process of viruses and cells, so as to detect the neutralizing effect of antibodies. This system realizes the receptor ligand-mediated cell fusion process by transfecting and expressing the membrane protein Spike of SARS virus and its corresponding host cell membrane receptor ACE2 respectively, and the expression occurs after cell fusion through co-transfection The reporter gene system quantitatively expresses its fusion effect, that is, the number of cells that undergo fusion. Adding antibodies or polypeptides to this system, if it has a blocking effect on cell fusion, the size of this blocking effect can be expressed by the decrease in the expression of the reporter gene (such as figure 1 ). pSpike and pACE2 are expression plasmids containing the full-length genes of S protein and ACE2 protein respectively, p13-luc and p15-...

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Abstract

The invention discloses a high-affinity SARS neutralizing monoclonal antibody and a preparation method. The present invention also provides mouse hybridoma cell lines that produce the SARS neutralizing antibody: N-176-15CCTCC-C200507 and S-9-11CCTCC-C200515. The SARS neutralizing antibody of the present invention has a very good SARS virus neutralizing effect.

Description

technical field [0001] The invention relates to a SARS-CoV neutralizing antibody, especially a SARS neutralizing monoclonal antibody and a preparation method thereof. Background technique [0002] The outbreak of infectious atypical pneumonia (SARS) in Guangdong Province, China in autumn 2002 was a serious public health crisis. In just a few months, SARS spread rapidly to more than 25 countries and regions, seriously affecting people's health and normal life, and causing huge losses to China's economic construction and development. In the spring of 2004, atypical pneumonia caused human infection again in Beijing and Anhui due to the operational errors of laboratory researchers, which aroused great concern. So far, SARS has infected about 8,000 people in the world and killed more than 800 people, with a mortality rate of about 10%. [0003] It is currently known that atypical pneumonia is caused by a newly discovered coronavirus (SARS-CoV). Coronaviruses are named for thei...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/00C07K16/28C07K16/10C12N15/12C12N5/12A61K39/395A61P31/14A61P11/00
Inventor 孙兵边超张林琦
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI