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Anti-CK20 protein monoclonal antibody and cell line and preparing method and application thereof

A monoclonal antibody, CK20 technology, applied in biochemical equipment and methods, anti-animal/human immunoglobulin, and microbial-based methods, etc., can solve the problems of high detection cost, unclear background information of monoclonal antibodies, and dependence

Active Publication Date: 2019-05-10
FUZHOU MAIXIN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the current high-quality CK20 monoclonal antibodies rely on foreign imports, the cost of testing is high, and the background information for monoclonal antibodies is not clear

Method used

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  • Anti-CK20 protein monoclonal antibody and cell line and preparing method and application thereof
  • Anti-CK20 protein monoclonal antibody and cell line and preparing method and application thereof
  • Anti-CK20 protein monoclonal antibody and cell line and preparing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The preparation of embodiment 1 recombinant CK20 protein fragment

[0021] 1. Gene cloning

[0022] The CK20 protein sequence numbered P35900 was selected from the Uniprot database (http: / / www.uniprot.org) as a standard sequence. According to its DNA-binding structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids, and secondary structure, a region with a suitable length and special antigenicity that is different from other similar proteins is selected as the antigenic peptide. The 411th to 424th amino acid sequence of the selected CK20 amino acid sequence is selected, and its corresponding nucleotide sequence is SEQ ID No.1. Synthesize the above CK20 protein, add sulfhydryl modification to its N-terminus and couple with the carrier protein KLH to improve its immunogenicity, and use it as an immunogen for future use.

Embodiment 2

[0023] Example 2 Establishment of 10F6B12A9 hybridoma cell line

[0024] 1. Immunity

[0025] The CK20 protein-KLH conjugate obtained in Example 1 was diluted to 1 mg / mL, and then mixed and emulsified with an equal volume of complete Freund's adjuvant (CFA, Sigma Company), for Balb / c mice of 18-20 g ( (purchased from Fuzhou Wu's Experimental Animals) for abdominal injection immunization, the injection dose was 50 μg / animal. Immunization was boosted every 14 days thereafter, and the antigen was emulsified with incomplete Freund's adjuvant (IFA, Sigma Company) at a dose of 25 μg per mouse. 14 days after the second booster immunization, indirect ELISA (wavelength 450nm) was used to detect the multi-antibody titer against the immunogen in the mouse serum. 50μg / only.

[0026] 2. Cell Fusion

[0027] Aseptically prepare the mouse splenocyte suspension that meets the immune standard, mix it with mouse myeloma cell sp2 / 0 (ATCC) at a ratio of 5:1, centrifuge at 1000rpm for 10min, d...

Embodiment 3

[0031] Example 3 Preparation of Monoclonal Antibody by Ascites Induction Method

[0032] 1. Ascites preparation

[0033] After culturing the cell line to the logarithmic growth phase, wash with serum-free medium and resuspend, count 2×10 6individual / mL. The suspended cell solution was intraperitoneally injected into BALB / C mice sensitized with paraffin oil for 10 days, and each mouse was injected with 0.5 mL. Ascites was collected 7 days after feeding the mice. The collected ascites was centrifuged at 4°C and 8000rpm for 10 minutes, the supernatant solution was collected in a centrifuge tube, which was ascites, and stored at 4°C or -20°C.

[0034] 2. Purification of monoclonal antibodies

[0035] Using rProtein A sepharose Fast Flow (GE Company) affinity chromatography column to purify antibody from ascitic fluid is mainly divided into: ① Packing column, packing the protein A stuffing that buys in right amount in the gravity chromatography column and equilibrating buffer s...

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Abstract

The invention relates to the field of biological detection, provides an anti-CK20 protein monoclonal antibody, further provides a preparing method of the anti-CK20 protein monoclonal antibody, and further provides a hybridoma cell line for secreting anti-CK20 proteins. A heavy-chain variable region and a light-chain variable region of the antibody have the amino acid sequence shown in SEQ ID NO.4and the amino acid sequence shown in SEQ ID NO.5. The 411th-site to 424th-site proteins of the human CK20 amino acid are selected to be coupled with KLH to form an immunogen. The cell line is a rat hybridoma cell line 10F6B12A9 and has the preservation number of CGMCC NO.16204. The anti-CK20 protein monoclonal antibody has high specificity and sensitivity, can specifically recognize cells of expressing CK20 proteins, and is suitable for immunological detection, especially immuno-histochemistry.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to an anti-CK20 protein monoclonal antibody, a cell line and a preparation method and application thereof. Background technique [0002] Cytokeratin (CK) is an intermediate filament distributed in cells of ectoderm origin, and is one of the components of the cytoskeleton. Divided into 20 kinds such as CK1~CK20. Cytokeratin is mainly distributed in upper tissue cells, but its expression ability is weak in bone marrow, blood, lymph nodes, etc. Tumor cells can retain the germ layer characteristics of their origin. Cytokeratin has become a more sensitive and specific marker of tumor cells, and has been Used to detect malignant tumor cells. Protein cytokeratin 20 (Cytokeratin 20, CK20) is a kind of type I cytokeratin, with a relative molecular mass of 48,000. The total length of the coding DNA is 18kb, including 8 exons and 7 introns. [0003] The synthesis of CK20 first appeared i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N5/20G01N33/68G01N33/577C12R1/91
Inventor 李恢波吴茂李玲玲黄信超
Owner FUZHOU MAIXIN BIOTECH CO LTD
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