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Hybrid tumor generating anti-AMP-18 (Antrum Mucosalprotein-18) monoclonal antibody as well as anti-AMP-18 monoclonal antibody and application thereof in gastric carcinoma detection

A technology of AMP-18 and monoclonal antibody, applied in the direction of anti-animal/human immunoglobulin, microorganism, measuring device, etc.

Active Publication Date: 2012-11-21
BEIJING PROTEIN INNOVATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, we could only detect the expression of AMP-18 in the normal gastric mucosal cell line GES, while the expression of MP-18 in the six gastric cancer cell lines were all negative

Method used

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  • Hybrid tumor generating anti-AMP-18 (Antrum Mucosalprotein-18) monoclonal antibody as well as anti-AMP-18 monoclonal antibody and application thereof in gastric carcinoma detection
  • Hybrid tumor generating anti-AMP-18 (Antrum Mucosalprotein-18) monoclonal antibody as well as anti-AMP-18 monoclonal antibody and application thereof in gastric carcinoma detection
  • Hybrid tumor generating anti-AMP-18 (Antrum Mucosalprotein-18) monoclonal antibody as well as anti-AMP-18 monoclonal antibody and application thereof in gastric carcinoma detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Preparation of AMP-18 antigen

[0016] AMP-18 antigen is based on normal gastric tissue cDNA as a template, and specific primers are designed according to the AMP-18 sequence (SEQ ID No.1), and both ends of the primers are connected to BamHI and SalI restriction sites

[0017] Upstream primer: 5′-TGAAGGATCCAACTATAATATCAACGTC-3′ (SEQ ID NO.2)

[0018] Downstream primer: 5'-AAATGTCGACGTTTCTCCACCGTGTCTCC-3' (SEQ ID NO.3)

[0019] The gene that removed the AMP-18 signal peptide sequence was amplified by PCR (PCR parameters: 95°C for 5 minutes; 95°C for 30s, 55°C for 30s, 72°C for 60s, a total of 40 cycles; 72°C and then extended for 10 minutes) by BamHI and SalI double enzymes After cutting, it was ligated with pQE30a, and the competent cell JM109 was chemically transformed, and the single clone was picked to extract the plasmid for sequencing, and the sequence was verified to be correct. Select positive bacteria with correct sequencing and induce expression wi...

Embodiment 2

[0021] Embodiment 2: Preparation of anti-AMP-18 hybridoma

[0022] 1) immunity

[0023] The AMP-18 protein purified by Ni-NAT affinity chromatography was subcutaneously immunized in the abdomen (20 μL serum was taken from the orbit before immunization as a negative control) 4-6 week old female Balb / c mice; the dose was 60 μg per mouse Protein+normal saline to 200μL+CFA200μL. Subcutaneous booster immunization once every 14 days, the dose was 30 μg protein + normal saline to 200 μL + IFA 200 μL per mouse. 7 days after the third booster immunization, blood was taken from the orbit to measure the titer. For those who met the requirements, 50 μg protein + normal saline was added to 100 μL; the tail vein was injected, and it was fused after 3 days.

[0024] 2) Fusion

[0025] The freshly excised mouse spleen was crushed and filtered on a cell sieve, mixed with sp2 / 0 cells at a ratio of 1:5, and centrifuged at 1500 rpm for 5 minutes. Put the centrifuge tube containing the centrif...

Embodiment 3

[0034] Example 3: Preparation of anti-AMP-18 by hybridoma cell line with deposit number CGMCC 2839

[0035] Monoclonal antibodies

[0036] The inventor used the hybridoma (70110-4, date of deposit on December 30, 2008, deposit number CGMCC 2839, place of deposit: CGMCC, General Microbiology Center, China Committee for Culture Collection of Microorganisms) to prepare mouse anti-AMP-18 monoclonal Antibody, the specific operation steps are as follows:

[0037] 1) Cell recovery

[0038] Quickly take out the cryopreservation tube from the -80°C refrigerator or liquid nitrogen tank; quickly put it into a 37°C water bath and stir quickly to make the cryopreservation solution melt into a liquid within 2 minutes. Add 3mL of serum medium to a 15mL centrifuge tube, draw the cryopreserved solution into the centrifuge tube, and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, suspend the cells with complete medium, and culture in a 6-well plate (3 mL) or bottle (5 mL).

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Abstract

The invention aims at preparing a hybrid tumor of an anti-antrum mucosalprotein-18 (AMP-18) monoclonal antibody and relates to application thereof in preparing a tumor clinical detection reagent. Particularly, in the invention, a hybrid tumor cell (70110-4) capable of secreting the monoclonal antibody specifically recognizing the AMP-18 is screened through a molecular cloning technology and a monoclonal antibody technology. The monoclonal antibody secreted by the 70110-4 can be used for the clinical detection of carcinoma lesion in gastric tissues of human bodies.

Description

technical field [0001] The invention relates to the fields of immunochemistry, tumor biology and cell biology. Specifically, the purpose of the present invention is to prepare a hybridoma of a monoclonal antibody combined with the natural gastric antrum mucosa secreted protein-AMP-18 (Antrum mucosal protein-18) secreted by the normal gastric mucosa of the human body; The anti-AMP-18 monoclonal antibody produced by the hybridoma and its corresponding products can be used as diagnostic reagents for human gastric cancer or related fields. technical background [0002] Gastric cancer is one of the most common tumors that seriously endanger the health of the population. Its morbidity and mortality rate are only listed after lung cancer, ranking second in the cause of cancer death. At present, the main methods of treating tumors in the world are surgery, radiotherapy, chemotherapy and biological therapy. From a medical point of view, there is no completely effective method for ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18G01N33/577C12N5/20
Inventor 邢蕊张军康滨韦汉福徐宁志刘国振吴琳吕有勇刘斯奇
Owner BEIJING PROTEIN INNOVATION
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