Mouse-derived anti-human CD36 monoclonal antibodies 131-G1 (G2a) and 176-B8 (G1)

A 176-B8, 131-G1 technology, applied in the field of preparation of two monoclonal antibodies, can solve the problem of lack of detection of CD36 antigen

Inactive Publication Date: 2019-04-16
广州血液中心
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is currently no relevant kit and detection method for the detection of CD36 antigen deletion

Method used

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  • Mouse-derived anti-human CD36 monoclonal antibodies 131-G1 (G2a) and 176-B8 (G1)
  • Mouse-derived anti-human CD36 monoclonal antibodies 131-G1 (G2a) and 176-B8 (G1)
  • Mouse-derived anti-human CD36 monoclonal antibodies 131-G1 (G2a) and 176-B8 (G1)

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Embodiment

[0026] (1) Genotype identification of CD36 knockout mice: According to the information provided by The Jackson Laboratory, synthesize primers for identification of CD36 knockout mice. The upstream primer is: 5'- TTGAAGTGCTGATCCTTTCAGA-3', and the downstream primer is 5'- TGTTTGTTTCACCACACTGGA-3', the downstream mutation primer is 5'-CGCCTTCTTGACGAGTTC-3', denatured at 94°C for 1min, annealed at 60°C for 1min, extended at 72°C for 1min, a total of 30 cycles, and finally extended at 72°C for 5min to obtain a 435bp wild-type And / or mutant mouse CD36 gene sequence of 750bp length;

[0027] (2) PCR amplification of human platelet cDNA: Design specific primers according to the cDNA sequence (NM_000072.3) encoding human CD36 in GenBank, the upstream primer is: 5'-GGTGCTTAACACTAATTCACCTCC-3', the downstream primer is: 5'-TTTTATTGTTTTCGATCTGCATGC -3', denatured at 95°C for 30 s, annealed at 54°C for 50 s, and extended at 72°C for 2 min, a total of 35 cycles, and finally extended at 72°...

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Abstract

The invention relates to a preparation method of mouse-derived anti-human CD36 monoclonal antibodies 131-G1 (G2a) and 176-B8 (G1), and belongs to the technical field of genetic engineering. The specific preparation process of the monoclonal antibodies comprises ten steps: identification of a CD36 gene knockout mice genotype, PCR amplification of human platelet cDNA, TA cloning and blue-white spotscreening, cell transfection and G418 screening, flow identification, Western Blot identification, CD36 gene knockout mice immunization, cell fusion and antibody preparation, identification of anti-human CD36 monoclonal antibodies and purification of the monoclonal antibodies. The method can obtain hybridoma cell lines stably secreting mouse-derived anti-human CD36 mAb, wherein the mAb can specifically bind to a human platelet CD36 antigen and can be used for detecting the expression of CD36 antigens in humans.

Description

technical field [0001] The invention relates to a preparation method of two monoclonal antibodies, in particular to a preparation method of a mouse-derived anti-human CD36 monoclonal antibody, and belongs to the field of gene technology. Background technique [0002] Since the first case of platelet transfusion refractory (PTR) patient caused by CD36 antibody was reported in 1989, it has gradually been recognized that CD36-deficient persons (type I) have the risk of inducing CD36 antibody production during blood transfusion or pregnancy, which can cause a series of immune responses. Clinical conditions of acute thrombocytopenia, including fetal / neonatal alloimmune thrombocytopenia (FNAIT), platelet refractory transfusion (PTR) and post-transfusion purpura (PTP), and TRALI. [0003] CD36 is a transmembrane glycoprotein with a molecular weight of 88kD. At present, the expression types of CD36 loss are mainly divided into Type Ⅰ (defective expression of both platelets and monoc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/85C12N15/06G01N33/577
CPCC07K16/2896C12N15/02C12N15/85G01N33/577
Inventor 陈大伟徐秀章夏文杰叶欣付涌水
Owner 广州血液中心
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