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Monoclonal antibody for resisting Cyr61 protein and application thereof

A monoclonal antibody and protein technology, applied in the direction of antibodies, anti-animal/human immunoglobulins, anti-inflammatory agents, etc., can solve the problem of high technical difficulty of monoclonal antibodies and achieve the effect of inhibiting proliferation

Inactive Publication Date: 2010-05-19
SHANGHAI INST OF IMMUNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since the Cyr61 protein is rich in cysteine, it is easy to form disulfide bonds during expression, resulting in various conformations. Moreover, as an important matrix protein, Cyr61 is highly conserved and has a high homology among mammals , especially the Cyr61 homology with mouse is as high as 93%, therefore, the technical difficulty of preparing monoclonal antibody is relatively high

Method used

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  • Monoclonal antibody for resisting Cyr61 protein and application thereof
  • Monoclonal antibody for resisting Cyr61 protein and application thereof
  • Monoclonal antibody for resisting Cyr61 protein and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1 Constructing and expressing human Cyr61 full-length (26-381) protein plasmid and protein expression

[0047] According to the principles of primer design and the 2295 bp full-length cDNA sequence of human Cyr61 published in GeneBank (NM 001554), the primer design software Primer 5.0 was used to design, remove the signal peptide part, and add corresponding restriction enzyme sites at both ends.

[0048] Primers for Cyr61 full-length (26-381) protein:

[0049] Upstream primer: 5'-TT GGATCC TGCCCCGCTGCCTGCCACT-3'

[0050] (the underline is the BamH I restriction site);

[0051] Downstream primer: 5'-TT AAGCTT CTAGTCCCCTAAATTTGTGAATGTCATT-3'

[0052] (The underline is the Hind III restriction site).

[0053] Engineering bacteria: BL21(DE3), expression vector: pET28(a).

[0054] The plasmid pET28(a) was co-digested with restriction endonucleases BamH I and Hind III respectively, and the amplified human full-length Cyr61 (hCyr61-FL) was ligated into the pET2...

Embodiment 2

[0058] Example 2 Constructing and expressing human Cyr61 full-length (103-381) protein plasmid and protein expression

[0059] Primer for the C-terminal fragment of Cyr61 protein (Cyr61-XC-103-381aa)

[0060] Upstream primer: 5'-TT GGATCC GGCTGTCCCAACCCTCGGCTG-3'

[0061] (the underline is the BamH I restriction site);

[0062] Downstream primer: 5'-TT AAGCTT CTAGTCCCCTAAATTTGTGAATGTCATT-3'

[0063] (The underline is the Hind III restriction site);

[0064] Engineering bacteria: BL21(DE3), expression vector: pET28(a).

[0065] Plasmid pET28(a) was co-digested with restriction endonucleases BamH I and Hind III respectively, and the amplified human Cyr61 near C-terminal 2 / 3 fragment (hCyr61-XC-103-381aa) was respectively Insert into the pET28(a) vector to obtain the corresponding recombinant expression plasmid: pET28a-hCyr61-XC (see Figure 5 ).

[0066] Identification of the recombinant plasmid of the His-Cyr61 C-terminal fragment: Extract the total RNA of synoviocyte...

Embodiment 3

[0069] Example 3 Preparation of mouse anti-human Cyr61 monoclonal antibody and analysis of binding sites

[0070] BABC / c mice: female, 4 mice aged 6-8 weeks and 50 mice aged 8-10 weeks, provided by the Animal Center of Shanghai Academy of Sciences, Chinese Academy of Sciences.

[0071] Mouse myeloma cells SP20 / Ag-14, preserved for passage.

[0072] It was prepared according to the conventional monoclonal antibody preparation method, and the Cyr61 protein used for identification was a commercially available recombinant human Cyr61 protein (PeproTech Inc. New Jersey, USA, Cat: 120-25).

[0073] According to conventional immunization methods, BABL / c mice were immunized with self-made human His-hCyr61 fusion protein, and mouse splenocytes were collected, and cell fusion was carried out according to classical methods. After 5 consecutive subcloning and ELISA screening, 9 hybridoma cells can stably secrete specific anti-human Cyr61 antibody (only react with purified or His-hCyr61, ...

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Abstract

The invention discloses a monoclonal antibody for resisting Cyr61 protein, which is prepared from a hybridoma cell line CGMCC3299 or CGMCC3300 and can be specifically combined with an N end SEQ ID No 1of the Cyr61 protein. The monoclonal antibody for resisting Cyr61 protein can be used for inhibiting proliferation, adhesion and protraction of rheumatoid arthritis synovial cells and malignant tumor cells as well as in vitro detection of the Cyr61 protein.

Description

technical field [0001] The present invention relates to a monoclonal antibody, in particular to an anti-Cyr61 protein monoclonal antibody, which can combine with Cyr61 protein and have the effect of inhibiting the proliferation, adhesion and persistence of rheumatoid synoviocytes and malignant tumor cells. It can also be used for in vitro detection of Cyr61 protein. Background technique [0002] Human Cyr61 protein (hCyr61) consists of 381 amino acid residues, rich in cysteine ​​and proline residues, with a relative molecular mass of 42kDa. It is currently known that Cyr61 is widely expressed in a variety of normal tissue cells, including fibroblasts, epithelial cells, endothelial cells, bladder and vascular smooth muscle cells, mesenchymal cells, chondrocytes, osteoblasts, and nerve cells. In addition, Cyr61 is also highly expressed in some tumor cells, currently known as breast cancer cells, liver cancer cells, prostate cancer cells, gastric cancer cells, melanoma cells, ...

Claims

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Application Information

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IPC IPC(8): C07K16/18G01N33/577A61K39/395A61P19/04A61P29/00A61P35/00
Inventor 李宁丽沈佰华王利林锦骠
Owner SHANGHAI INST OF IMMUNOLOGY
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