Human prolactin receptor antibody and application thereof

A prolactin receptor and antibody technology, applied in the field of genetic engineering technology and biological products, can solve the problem of breast cancer without effective treatment methods, and achieve the effect of highly targeted application

Inactive Publication Date: 2011-11-23
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Breast cancer is the most common malignant tumor in women. Although breast cancer surgery, radiotherapy, chemotherapy a

Method used

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  • Human prolactin receptor antibody and application thereof
  • Human prolactin receptor antibody and application thereof
  • Human prolactin receptor antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Separation of human peripheral blood lymphocytes and extraction of total RNA

[0039] Collect 2 to 40 samples of anticoagulated peripheral blood in batches from patients aged 20 to 60 years old and clinically diagnosed with breast cancer. Collect peripheral blood lymphocytes with lymphocyte separation medium to obtain peripheral blood lymphocytes, mix them, and wash with sterilized PBS. The cells were resuspended, and the total RNA of peripheral blood lymphocytes was extracted with the total RNA purification kit.

Embodiment 2

[0040] Example 2: Amplification of human immunoglobulin G (IgG) light chain and heavy chain Fd segment genes

[0041] First, the total RNA of lymphocytes is reverse-transcribed into cDNA, and then a group that almost covers the human immunoglobulin G (IgG) light chain or heavy chain Fd segment V H and C H 1 Specific primers for the coding sequences of the variable region and the constant region of the gene fragment, and PCR was performed on the above reaction products to amplify human immunoglobulin G (IgG) respectively, using 4 pairs of κ chain gene primers and 9 pairs of λ chain gene primers respectively And 6 pairs of heavy chain gene primers to amplify the variable region. The amplification conditions were 94°C for 5 minutes, 94°C for 1-2 minutes, 50-60°C for 1-2 minutes, and 72°C for 1-2 minutes, a total of 30 cycles, and the final extension was 72°C for 10 minutes. PCR products were identified by 2.0% agarose gel electrophoresis. The amplification results of 6 pairs o...

Embodiment 3

[0061] Example 3: Construction of natural human anti-breast cancer IgG Fab phage antibody library

[0062] Using the strategy of first cloning the light chain gene and then the heavy chain gene, Fab fragments targeting breast cancer patients with good diversity were obtained by overlapping PCR ( Figure 4 ), the Fab length is about 1600bp, the Fd segment of the heavy chain and the light chain gene are about 750bp, and the variable and constant regions of the light chain and the heavy chain are about 350bp.

[0063] Then the Fab fragment and plasmid pcomb3XSS were digested with Sfi I, and the fragment of the PCR product was digested with the phage vector pCom3XSS (gifted by Scripps Institute, Figure 5 ) connection, after electroporation into competent cells, spread the transformed bacteria evenly on LB plates containing 100 μg / ml ampicillin, collect the plasmid DNA of all clones, and construct a library with a capacity of 1×10 9 Human breast cancer IgG Fab phage antibody libr...

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Abstract

The invention discloses a human prolactin receptor antibody, which comprises a heavy chain Fd fragment variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain Fd fragment variable region is shown as SEQ ID No.2, and the amino acid sequence of the light chain variable region is shown as SEQ ID No.4. The invention also discloses a gene for encoding the antibody and application of the antibody in preparation of medicines for treating human breast cancer. A human prolactin receptor hPRLR antibody with high specificity and high affinity is obtained and has potential application prospect in biological immunotherapy of breast cancer.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and biological products, in particular to a human anti-prolactin receptor antibody (hPRLR-Fab) and its application. Background technique [0002] Antibodies play an important role in the diagnosis, treatment and prevention of diseases. At present, antibody drugs account for 30% of the biopharmaceuticals used in clinical practice worldwide. Among them, monoclonal antibodies have the advantages of targeting specificity. Therapeutic antibodies show broad application prospects and have gradually developed into a large class of independent pharmaceutical products [1] . Most of the monoclonal antibodies developed at present are of murine origin, and murine monoclonal antibodies, as heterologous proteins, can induce the production of anti-mouse antibody (Human Anti-Mouse Antibody, HAMA) in the human body, making it useful in clinical treatment. The application has been greatly limited, so ...

Claims

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Application Information

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IPC IPC(8): C07K16/30C12N15/13A61K39/395A61P35/00
Inventor 魏钦俊曹新姚俊鲁雅洁
Owner NANJING MEDICAL UNIV
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