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Anti-cd5 protein monoclonal antibody and its cell line, preparation method and application

A monoclonal antibody and protein technology, applied in the field of biomedical engineering, can solve problems such as unclear mechanism and achieve high specificity

Active Publication Date: 2022-04-08
FUZHOU MAIXIN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The exact mechanism leading to overexpression of CD5 in DLBCL remains unclear

Method used

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  • Anti-cd5 protein monoclonal antibody and its cell line, preparation method and application
  • Anti-cd5 protein monoclonal antibody and its cell line, preparation method and application
  • Anti-cd5 protein monoclonal antibody and its cell line, preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0018] The preparation of embodiment 1 recombinant CD5 protein fragment

[0019] 1. Gene optimization and synthesis

[0020] According to the protein sequence with accession number O43866 in the Uniprot database, CD5 selects the protein fragment at positions 398-495, which is the amino acid sequence shown in SEQ ID NO.1; it is directly optimized into a gene fragment suitable for expression in Escherichia coli BL21. During the PCR process, BamH I and XhoI restriction sites were added to the 5' and 3' ends of the gene, respectively.

[0021] The PCR products were separated by agarose gel electrophoresis and recovered. The recovered fusion protein gene and the plasmid vector Pet30a used for expression were digested with BamHI and XhoI respectively, recovered by electrophoresis again, and ligated with T4 DNA ligase. The ligation product was transformed into Escherichia coli competent cell BL21, and the clones on the plate were picked and inoculated, and the PCR identification of ...

Embodiment 2

[0025] The establishment of embodiment 2 hybridoma cell lines

[0026] 1. Immunity

[0027] The recombinant protein in Example 1 was emulsified with complete Freund's adjuvant (Sigma, F5881), immunized 4-6 weeks old SPF female mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and injected subcutaneously into the abdomen 6 points per mouse, the dose is 60 μg / mouse. Immunization was boosted every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma, F5506) at a dose of 20 μg per mouse. 7 days after the third booster immunization, indirect ELISA (wavelength 450nm) was used to detect the polyantibody titer of the anti-immunogen in the mouse serum. 20μg / only.

[0028] 2. Cell Fusion

[0029] Aseptically prepare the mouse splenocyte suspension that reaches the immune standard, mix it with mouse myeloma cell sp2 / 0 (ATCCNumberCRL-8287) at a ratio of 5:1, and centrifuge at 1500rpm for 5min. After the supernatant was d...

Embodiment 3

[0032] Example 3 Preparation of Monoclonal Antibody by Ascites Induction Method

[0033] 1. Ascites preparation

[0034] The cells in the logarithmic growth phase were washed with serum-free medium and suspended, counted about 5×10 5 , 1ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The removed ascites was centrifuged at 4000rpm for 10min at 4°C. Carefully suck out the ascites in the middle and collect in a centrifuge tube, and store at 4°C or -20°C.

[0035]2. Purification of monoclonal antibodies

[0036] Antibody was purified from ascitic fluid by HiTrap rProtein A FF (GE Company) affinity chromatography according to the instructions. The purity was identified by SDS-PAGE gel, and the concentration was determined by Bradford method. Purified antibodies were stored at -20°C.

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Abstract

The invention relates to a monoclonal antibody capable of recognizing human CD5 antigen, a secreting cell line, its preparation method and its application in immunoassay. The above technical scheme selects the 398th to 495th amino acid at the C-terminus of the CD5 protein as the antigenic peptide, and performs codon optimization to become a gene fragment suitable for expression in Escherichia coli BL21. The resulting recombinant protein contains the CD5 protein fragment and histidine protein tag. The recombinant protein is used to immunize mice, and through cell fusion, screening and subcloning, the mouse hybridoma cell line 11H3 that efficiently secretes anti-CD5 protein monoclonal antibody, and the anti-CD5 protein monoclonal antibody secreted by the cell line are obtained Antibody. The antibody obtained by this protocol has high specificity and sensitivity, can specifically recognize cells expressing CD5 protein, and is suitable for immunological detection, especially immunohistochemical detection.

Description

technical field [0001] The invention relates to the field of biomedical engineering, in particular to an anti-CD5 protein monoclonal antibody and its cell line, preparation method and application. Background technique [0002] The human CD5 gene is located on chromosome 11q12.2. The CD5 molecule is a 67KD transmembrane glycoprotein expressed in a subclass of T cells and virgin B cells. CD5 is a macrophage scavenger receptor rich in cysteine (SRCR) Domain homology domain receptor family members, these CD5-positive B cells can produce autoantibodies, and can also spread in autoimmune diseases and lymphoid malignancies, such as leukemia, MCL and CD5-positive The DLBCL. Unlike mouse B1 cells that express CD5, human B1 cells do not have CD5 expression. Experiments in chicken egg lysozyme transgenic models in mice and chicken CD5-knockout cells showed that CD5 expression promotes tolerance to self-reactive antigens in vitro, while the absence of CD5 molecules leads to enhanced c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N5/20C12N15/13G01N33/68G01N33/577C12R1/91
CPCC07K16/2896G01N33/6872G01N33/577C07K2317/56C07K2317/20C07K2317/92G01N2333/70596
Inventor 杨清海陈惠玲王小亚李婷赵普
Owner FUZHOU MAIXIN BIOTECH CO LTD
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