Monoclonal antibody against DOG1 protein and cell strain, preparation method and application thereof

A monoclonal antibody, hybridoma cell line technology, applied in the field of biomedical engineering, can solve the problem of less research on DOG1 protein and achieve high specificity

Active Publication Date: 2021-06-29
FUZHOU MAIXIN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, most of the research on DOG1 protein focuses on its relationship with gastrointestinal stromal tumors, while the relationship between DOG1 protein and other tumors is currently less studied

Method used

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  • Monoclonal antibody against DOG1 protein and cell strain, preparation method and application thereof
  • Monoclonal antibody against DOG1 protein and cell strain, preparation method and application thereof
  • Monoclonal antibody against DOG1 protein and cell strain, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 Preparation of recombinant DOG1 protein fragment

[0023] 1. Gene optimization and synthesis

[0024] According to the protein sequence with the accession number Q5XXA6 in the Uniprot database, DOG1 selects a protein fragment with 53-291 amino acid residues, and directly optimizes it into a gene fragment suitable for expression in Escherichia coli BL21 (DE3). During the PCR process, EcoR I and XhoI restriction sites were added to the 5' and 3' ends of the gene, respectively.

[0025] The PCR product was separated by agarose gel electrophoresis and recovered. The recovered fusion protein gene and the plasmid vector Pet30a used for expression were digested with EcoRI and XhoI respectively, recovered by electrophoresis again, and ligated with T4 DNA ligase. The ligation product was transformed into Escherichia coli competent cell BL21(DE3), and the clones on the plate were picked and inoculated, and the bacteria liquid PCR was identified. Clones with positiv...

Embodiment 2

[0030] The establishment of embodiment 2 hybridoma cell lines

[0031] 1. Immunity

[0032] The cross-linked polypeptide in Example 1 was emulsified with complete Freund's adjuvant (Sigma, F5881), and immunized with 4-6 week-old female ICR mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), subcutaneously in the abdomen. Inject each mouse at 6 points with a dose of 60 μg / mouse. Immunization was boosted once every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Company, F5506) at a dose of 30 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. 50μg / only.

[0033] 2. Cell Fusion

[0034]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. Aft...

Embodiment 3

[0037] Example 3 Preparation of Monoclonal Antibody by Ascites Induction Method

[0038] 1. Ascites preparation

[0039] 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.

[0040] 2. Purification of monoclonal antibodies

[0041] Antibody was purified from ascitic fluid by HiTrap rProtein A FF (GE Company) affinity chromatography according to the manual. 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 a human DOG1 antigen, a secretory cell strain, a preparation method and application thereof in immunological detection. According to the technical scheme, amino acids at the positions 53-291 at C-terminal of the DOG1 protein are selected as antigen peptides, codon optimization is carried out, a gene fragment suitable for being expressed in escherichia coli BL21 (DE3) is formed, and finally an obtained recombinant protein contains a DOG1 protein fragment and a histidine protein tag. Mice are immunized by the recombinant protein, and a mouse hybridoma cell strain 19F2 capable of efficiently secreting the anti-DOG1 protein monoclonal antibody and the anti-DOG1 protein monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing the DOG1 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-DOG1 protein monoclonal antibody and its cell line, preparation method and application. Background technique [0002] DOG1 (discovered on gastrointestinal stromal tumors protein 1) protein is a newly discovered membrane protein, DOG1, the gene encoding the protein is a member of the TMEM family, contains 26 exons, located in 11q13. This region is one of the most frequently amplified regions in human cancers, mainly regulating cell cycle, proliferation and apoptosis, surrounded by famous oncogenes, such as FGF4, FGF9, CCND1, FADD, CTIN, etc. The protein is mainly located on the cell membrane, has eight transmembrane structures, and the N-terminal and C-terminal are both in the cytoplasm. It is a calcium ion-activated chloride channel protein, and its function may be a transporter. In recent years, many studies have found that this protein is mainly selectively highly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N5/20C12N15/70C07K19/00C07K14/47G01N33/577
CPCC07K16/18C07K14/47C12N15/70G01N33/577C07K2317/56C07K2317/92C07K2319/21
Inventor 程本亮蒋宁城杨清海陈惠玲王小亚
Owner FUZHOU MAIXIN BIOTECH CO LTD
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