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Chemokine-4 resisting monoclonal antibody, chemokine-4 resisting hybridoma cell line and applications of monoclonal antibody

A hybridoma cell line, monoclonal antibody technology, applied in the field of hybridoma cell line and application, anti-chemokine 4 monoclonal antibody, can solve the problem that the research is not very clear and so on

Active Publication Date: 2014-12-31
上海欣生源药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the role of CXCL4, the most abundant chemokine in platelets, in gastrointestinal injury is not very clear.

Method used

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  • Chemokine-4 resisting monoclonal antibody, chemokine-4 resisting hybridoma cell line and applications of monoclonal antibody
  • Chemokine-4 resisting monoclonal antibody, chemokine-4 resisting hybridoma cell line and applications of monoclonal antibody
  • Chemokine-4 resisting monoclonal antibody, chemokine-4 resisting hybridoma cell line and applications of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Preparation of recombinant human anti-chemokine 4 (rhcxcl4-his) and recombinant mouse anti-chemokine 4 (rmcxcl4-his)

[0078] one. gene sequence:

[0079] rhcxcl4-his: NM_002619.3

[0080] rmcxcl4-his: NM_019932.4

[0081] two. Brief introduction of the purification process: resuspension of bacterial cells in bacteriostasis buffer → bacteriostasis → affinity chromatography → denaturation → renaturation → cation exchange chromatography

[0082] three. Purification process:

[0083] a) Purification of rhcxcl4-his:

[0084] Take 20g of fermented cells, add 1× Chinese (his-tag binding buffer) according to the ratio of 10mL bacteriostasis buffer: 1g cells, and suspend the cells on ice;

[0085] Use 500, 700, 800bar high-pressure homogenate to destroy bacteria respectively;

[0086] Centrifuge the bacteriostasis solution at 4°C, 10,000rpm for 30min, take the supernatant and discard the precipitate, keep 1mL of the supernatant sample, and store at 4°C;

[0087] Wash t...

Embodiment 2

[0114] Preparation of anti-human and mouse chemokine 4 (CXCL4) monoclonal antibodies

[0115] 1. Experimental materials

[0116] 1.1 Reagents: RPMI1640 culture medium was purchased from invitrogen, serum was purchased from hyclone, Freund's complete adjuvant and Freund's incomplete adjuvant, HT and HAR were purchased from Sigma, culture plates were Corning products, HRP-labeled goat anti-rat The secondary antibody was purchased from Jackson Company, and both rhcxcl4-his and rmcxcl4-his for immunization were produced in our laboratory.

[0117] 1.2 Cell strains and experimental mice: Human myeloma cell line SP2 / 0 was cultured in suspension in 1640 medium containing 10% FBS, SD rats, 10-12 weeks old, male, purchased from Shanghai Slack Experimental Animal Co., Ltd.

[0118] 2. Experimental method

[0119] 2.1 SD rat immunization: select 3 male SD rats, mix the rhcxcl4-his antigen with an equal volume of Freund's complete adjuvant, and inject it subcutaneously at multiple point...

Embodiment 3

[0122] Identification of Physicochemical Properties of Monoclonal Antibodies

[0123] The invention prepares the Anti-CXCL4 monoclonal antibody capable of neutralizing human and mouse CXCL4 proteins, and performs separation and purification, identification of antibody characteristics, and detection of biological activity. SD rats were immunized with purified recombinant human CXCL4, and a hybridoma cell line secreting anti-recombinant human and mouse CXCL4 monoclonal antibodies was obtained through cell fusion and cloning three times, named 16D6-3. The cell line was continuously cultured in vitro for one month, and the ELISA test proved that the antibody secretion ability was stable. The cell line was injected into nude mice to collect ascites, and the affinity column HiTrap protein G HP (GE Healthcare) was used to purify the antibody in the ascites, and the purity of the antibody as detected by SDS-PAGE reached 98% ( image 3 D-a), and Western blot method was used to detect t...

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Abstract

The invention relates to a chemokine-4 resisting monoclonal antibody, a chemokine-4 resisting hybridoma cell line and applications of the chemokine-4 resisting monoclonal antibody, belongs to the field of biotechnology, aims to immunize a rat by using the recombinant human CXCL4 protein, fuse the spleen cell with the myeloma cell, and screen the formed hybridoma cell and further aims to provide the application of the chemokine-4 resisting monoclonal antibody to the resistance of intestinal mucosa injury caused by chemotherapy. The monoclonal antibody provided by the invention can resist the human chemokine 4 and the rat chemokine 4 and has the effect of preventing and treating the toxic and side effects of tumor chemotherapy.

Description

technical field [0001] The invention relates to an antibody in the field of biotechnology, in particular to an anti-chemokine 4 monoclonal antibody, a hybridoma cell line and applications. Background technique [0002] At present, chemotherapy drugs are one of the main methods to treat malignant tumors. However, while chemotherapy drugs inhibit or kill tumor cells, they also have serious toxic and side effects on normal tissues or organs of the body. Intestinal mucosal injury is a common dose toxicity and complication of anticancer drugs. The rate reaches 50%-80%, and symptoms include nausea, vomiting, anorexia, and diarrhea (Wadler S, Benson AB, 3rd, Engelking C, Catalano R, Field M, Kornblau SM, et al. Recommended guidelines for the treatment of chemotherapy- induced diabetes.J Clin Oncol.1998,16:3169-3178; Benson AB,3rd,Ajani JA,Catalano RB,Engelking C,Kornblau SM,Martenson JA,Jr.,et al.Recommended guidelines for the treatment of cancer treatment- induced diabetes. 2004...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/24C12N15/13C12N15/63A61K39/395A61P35/00A61P1/04
Inventor 韩伟俞雁高婧
Owner 上海欣生源药业有限公司
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