Sequences of variable regions of anti-GFAP (glial fibrillary acidic protein) monoclonal antibody and method for preparing same

A monoclonal antibody and variable region technology, applied in the field of biomedicine, can solve problems such as the reduction of GFAP expression level

Inactive Publication Date: 2013-06-26
百奇生物科技(苏州)有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

In addition, GFAP expression levels are reduced in acute infection and neurodegeneration

Method used

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  • Sequences of variable regions of anti-GFAP (glial fibrillary acidic protein) monoclonal antibody and method for preparing same
  • Sequences of variable regions of anti-GFAP (glial fibrillary acidic protein) monoclonal antibody and method for preparing same
  • Sequences of variable regions of anti-GFAP (glial fibrillary acidic protein) monoclonal antibody and method for preparing same

Examples

Experimental program
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Embodiment 1

[0028] Example 1. Preparation and Identification of Anti-GFAP Monoclonal Antibody

[0029] 1. Preparation of hybridoma cells

[0030] (1) Animal immunization: Simultaneously immunize 3 healthy female BALB / c mice aged 6-8 weeks with GFAP protein antigen. After the 3 times of immunization, blood was collected once a week, and the OD value detected by ELISA at 1:4000 dilution was selected to be greater than The serum of 1.0 and BALB / c mice with positive serum WB test were used for fusion, and 3 days before the fusion, the antigen without adjuvant was injected intraperitoneally, and the injection dose was 50ug / mouse.

[0031] (2) Collect B lymphocytes: 3 days after the booster immunization, take blood from the mice, centrifuge the blood, and keep the serum as a positive control; then take the spleen of the mouse under sterile conditions, and put the spleen in 10ml pre-warmed incomplete In the culture medium, peel off the surrounding connective tissue, place it in a 100-mesh stain...

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Abstract

The invention relates to sequences of variable regions of an anti-GFAP (glial fibrillary acidic protein) monoclonal antibody. The sequences comprise a nucleotide sequence CM10330-VH and a nucleotide sequence CM10330-VL. A method for preparing the sequences comprises the following steps of: immunizing a mouse by taking a GFAP protein as an antigen, detecting to obtain the mouse of which the corresponding antibody expression is positive, separating the spleen cell of the mouse, fusing the spleen cell with the myeloma cell, culturing the spleen cell and the myeloma cell in an HAT culture medium, detecting to obtain the positive antibody-expression cloning, extracting the total RNA (ribonucleic acid) of the positive hybridoma cell, carrying out inverse transcription by taking the mRNA (messenger ribonucleic acid) in the total RNA as a template to obtain the cDNA (complementary deoxyribonucleic acid) of the gene of the corresponding antibody, obtaining the heavy-chain variable region and the light-chain variable region of the corresponding antibody by using a specific primer through PCR (polymerase chain reaction), and cloning and testing the sequences.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, and more specifically, to the variable region sequence of an anti-GFAP monoclonal antibody and a preparation method thereof. Background technique [0002] Glial fibrillary acidic protein (GFAP) is an intermediate fibrous protein specifically expressed by astrocytes in the central nervous system, and plays an important role in intercellular signal transduction and brain-blood barrier function. The expression level of GFAP increases during cell mitosis, which is compatible with the increase of intracellular fiber network during mitosis. Studies have shown that GFAP gene knockout mice have mutations in myelin and severe damage to the structure and function of the brain-blood barrier. [0003] Abnormal expression levels of GFAP are associated with various genetic diseases and psychiatric disorders, such as Alexander disease, Down syndrome, schizophrenia, affective bipolar disorder and de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/13C07K16/18C12N15/10
Inventor 吴纯李静文邹建炫汪伟杨春花周延庆李顺玲孙其玲洪扬陈媛
Owner 百奇生物科技(苏州)有限公司
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