Monoclonal antibody for resisting CA125 protein and cell strain, preparation method and application thereof

A monoclonal antibody, CA125 technology, applied in the field of biomedical engineering to achieve high specificity

Active Publication Date: 2021-07-02
FUZHOU MAIXIN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some researchers have tested patients with early ovarian cancer, and the fixed specificity is 98%, and the sensitivity of single detection is 48%.

Method used

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  • Monoclonal antibody for resisting CA125 protein and cell strain, preparation method and application thereof
  • Monoclonal antibody for resisting CA125 protein and cell strain, preparation method and application thereof
  • Monoclonal antibody for resisting CA125 protein and cell strain, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The preparation of embodiment 1 recombinant CA125 protein fragment

[0021] 1. Gene optimization and synthesis

[0022] According to the protein sequence with the accession number Q8WXI7 in the Uniprot database, CA125 selects the protein fragment at positions 13837-14184, 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.

[0023] 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 positi...

Embodiment 2

[0028] The establishment of embodiment 2 hybridoma cell lines

[0029] 1. Immunity

[0030] 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 20 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.

[0031] 2. Cell Fusion

[0032] 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 super...

Embodiment 3

[0035] Example 3 Preparation of Monoclonal Antibody by Ascites Induction Method

[0036] 1. Ascites preparation

[0037] 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.

[0038] 2. Purification of monoclonal antibodies

[0039] 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 CA125 antigen, a secretory cell strain, a preparation method of the monoclonal antibody and application of the secretory cell strain in immunodetection. According to the technical scheme, 13837-14184 amino acids at the C tail end of the CA125 protein are selected as the antigen peptide, codon optimization is carried out, a gene segment suitable for being expressed in escherichia coli BL21 (DE3) is formed, and the finally obtained recombinant protein contains a CA125 protein segment and a histidine protein tag. A mouse is immunized by the recombinant protein, and the mouse hybridoma cell strain 20E3 capable of efficiently secreting the anti-CA125 protein monoclonal antibody and the anti-CA125 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 CA125 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-CA125 protein monoclonal antibody and its cell line, preparation method and application. Background technique [0002] CA125 is a transmembrane macromolecular glycoprotein expressed by a gene located in the 19pl3.2 region of chromosome 19pl3. Ovarian cancer-associated antigen, which has a high detection rate in epithelial ovarian cancer, is one of the important markers of epithelial ovarian cancer at present, and has obtained many promising results in the prevention, screening, disease monitoring, and prognosis of ovarian cancer. meaningful results. With the extensive application and research of CA125, it is found that not only ovarian cells and lung cancer cells are produced, but also in normal human serosa epithelial cells (such as pleura, pericardium, peritoneum, endometrium, endocervix and normal lung tissue including respiratory tract). Mucous membrane columna...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30G01N33/574C12N5/20C12N15/70C07K19/00C12R1/19
CPCC07K16/3069G01N33/57449G01N33/57484C07K14/4748C12N15/70C07K2317/56C07K2319/21C12N2800/22
Inventor 高惠然杨清海王小亚陈惠玲林洋李萍
Owner FUZHOU MAIXIN BIOTECH CO LTD
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