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Anti-human YKL-40 neutralizing monoclonal antibody as well as preparation and application thereof

A technology of YKL-40, monoclonal antibody, applied in the field of medical bioengineering

Active Publication Date: 2021-02-19
中国人民解放军海军军医大学第三附属医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there is no report about an anti-human YKL-40 neutralizing monoclonal antibody, its antibody sequence information and the application of this monoclonal antibody in neutralizing YKL-40 secreted by lung epithelial cells and inhibiting lung metastasis of liver cancer.

Method used

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  • Anti-human YKL-40 neutralizing monoclonal antibody as well as preparation and application thereof
  • Anti-human YKL-40 neutralizing monoclonal antibody as well as preparation and application thereof
  • Anti-human YKL-40 neutralizing monoclonal antibody as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: YKL-40 protein antigen screening

[0038] Antigen screening: Human YKL-40 recombinant proteins from different merchants were selected, and the transwell cell migration system was used to detect the effect of the protein on the migration ability of liver cancer cell MHCC-LM3, and different YKL-40 recombinant proteins were added to the lower chamber of the transwell, and the screening criteria In order to significantly promote the migration of liver cancer cells MHCC-LM3. according to figure 2 , it can be seen that different YKL-40 recombinant proteins have different effects on the migration of liver cancer cells. The YKL-40 recombinant protein we selected can significantly promote the migration ability of liver cancer cells. The antigenic sequence of the YKL-40 recombinant protein is Tyr 22-Thr 383 Structural domain, which has been verified by experiments to be a functional domain that can play a chemotaxis role in liver cancer cells.

Embodiment 2

[0039] Example 2: Application of anti-human YKL-40 neutralizing monoclonal antibody in inhibiting lung metastasis of liver cancer cells

[0040] 1) Neutralizing monoclonal antibody can significantly reduce the concentration of YKL-40 in the serum of patients with lung metastasis of liver cancer

[0041] The present invention's study found that the concentration of YKL-40 in the serum of patients with liver cancer combined with lung metastasis increased significantly (attached figure 1 ). Take the serum of 3 patients with lung metastases from liver cancer, dilute the serum 1:100 with PBS in advance, add YKL-40 neutralizing monoclonal antibody at a concentration of 10ug / mL, use IgG (10ug / mL) as the control group, and incubate overnight at 4°C , ELISA detects YKL-40 concentration in serum, the results are as follows image 3 As shown, the anti-human YKL-40 neutralizing monoclonal antibody can bind YKL-40 and reduce the detectable concentration of YKL-40 in the patient's serum t...

Embodiment 3

[0052] Embodiment 3: monoclonal antibody sequencing

[0053] The YKL-40 neutralizing monoclonal antibody obtained by screening was sequenced to obtain its full-length sequence. The specific process was as follows: 1) using the TRIZol method to extract RNA from the hybridoma cell line of the monoclonal antibody;

[0054] 2) Using SMART Race technology to reverse transcribe RNA into cDNA;

[0055] 3) PCR amplification to obtain the full length of heavy chain and light chain;

[0056] 4) Use ligase to connect the target fragment to the vector, and transform the ligated product into E. coli competent cells, and then pick a single clone for sequencing;

[0057] 5) Analyze and annotate the sequencing results.

[0058] 6) The sequence information (base and amino acid) of the light and heavy chain variable regions of the monoclonal antibody is as follows:

[0059] The heavy chain variable region base sequence, as shown in SEQ ID NO: 3;

[0060] The base sequence of the light chain...

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Abstract

The invention relates to the technical field of medical bioengineering, in particular to an anti-human YKL-40 neutralizing monoclonal antibody. The amino acid sequence of a heavy chain variable regionof the monoclonal antibody is shown as SEQ ID NO:1, and the amino acid sequence of a light chain variable region of the monoclonal antibody is shown as SEQ ID NO:2. According to the invention, uniquesequence information of the monoclonal antibody is provided, and a hybridoma cell strain secreting the monoclonal antibody and application of the monoclonal antibody and the hybridoma cell strain inpreparation of drugs for inhibiting lung metastasis of liver cancer are also provided.

Description

technical field [0001] The invention relates to the technical field of medical bioengineering, in particular to an anti-human YKL-40 neutralizing monoclonal antibody and its preparation and application. Background technique [0002] YKL-40 is a secreted glycoprotein, also called Chi3l1, BRP39, encoded by the CHI3L1 gene located on chromosome 1, and belongs to the 18-glycosyl hydrolase gene family. In recent years, it has attracted more and more attention from the scientific community because of its wide range of biological effects. The current study found that YKL-40 is mainly involved in two types of reactions, one is inflammatory response, especially lung inflammation; the other is involved in tumor progression, which is related to the poor prognosis of patients. [0003] YKL-40 is an important cytokine in the lung inflammatory response, which can be secreted by different types of cells, such as smooth muscle cells, lung epithelial cells, macrophages, etc. The expression...

Claims

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Application Information

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IPC IPC(8): C07K16/40C12N15/13C12N5/20A61K39/395A61P35/04
CPCA61K2039/505A61P35/04C07K16/40C07K2317/56C07K2317/76
Inventor 王红阳文文于晗陈淑桢唐昊缪瑜姗
Owner 中国人民解放军海军军医大学第三附属医院
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