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Anti-double-chain DNA monoclone antibody with antineoplastic activity and preparation method thereof

A monoclonal antibody, anti-tumor activity technology, applied in the field of bioengineering, can solve problems such as large side effects and poor tumor treatment effect, and achieve obvious effects, significant cytotoxicity, and inhibit tumor growth.

Inactive Publication Date: 2009-05-13
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a kind of anti-double-stranded DNA monoclonal antibody with anti-tumor activity and its preparation method, and described this anti-double-stranded DNA monoclonal antibody with anti-tumor activity and its preparation method should solve existing The anti-tumor drugs in the technology are not effective in treating tumors, and the technical problems of large side effects

Method used

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  • Anti-double-chain DNA monoclone antibody with antineoplastic activity and preparation method thereof
  • Anti-double-chain DNA monoclone antibody with antineoplastic activity and preparation method thereof
  • Anti-double-chain DNA monoclone antibody with antineoplastic activity and preparation method thereof

Examples

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Embodiment 1

[0047] Example 1 Extraction and purification of dsDNA from mouse tumor cells:

[0048] Collect mouse myeloma cells SP2 / 0, centrifuge, wash 3 times with PBS, and extract genomic DNA, the process is as follows: add cell lysate (100μg / ml proteinase K, 20μg / ml RNase A, 10mM Tris-CL pH8.0 , 15mM NaCl, 10mM EDTA pH8.0, 0.4% SDS), at 37°C for 12-24 hours. Add balanced phenol, mix well and centrifuge at 5000g for 10 minutes; transfer the upper aqueous phase, repeat the phenol extraction process once; transfer the upper aqueous phase, add chloroform / isopropanol (V / V=24:1) for extraction, mix well and extract Twice, centrifuge at 5000g for 10min; transfer the upper aqueous phase, add 1 / 10 volume of sodium acetate (pH5.2) and 2.5 times the volume of pre-cooled absolute ethanol, mix well, act at -70°C for 1 hour, centrifuge at 10000g for 15min; Wash once with ethanol, dry in the air, and dissolve the precipitate in triple distilled water. Then add S1 nuclease, act at 37°C for 3 hours, d...

Embodiment 2

[0049] Example 2 Immunization of BALB / c mice with dsDNA

[0050] BALB / c mice were immunized with 50 μg DNA / mouse / time, and immunized 4 times in total: 50 μg / 0.1ml DNA was taken for the first immunization, mixed with an equal volume of Freund’s complete adjuvant to form a homogeneous emulsion, and injected subcutaneously at multiple points; Weeks later, the immune system was re-immunized with incomplete adjuvant mixed with dsDNA; on the 28th and 38th day, no adjuvant was added, and only dsDNA was used to boost immunization twice. Blood is drawn and serum is separated. Store at -20°C for later use.

Embodiment 3

[0051] Example 3 Detection of Anti-dsDNA Antibodies in Serum of Immunized Mice

[0052] Mice were immunized with dsDNA derived from tumor cells, and mouse serum was collected every two weeks, and the production and titer of anti-dsDNA antibodies in mouse serum were detected by ELISA method. Detection by conventional ELISA method: treat 96-well ELISA plate with 0.5% protamine sulfate, 37°C for 1 hour; wash once with washing solution (PBS-0.05% Tween-20pH7.4); dsDNA (50 μg / ml, coating solution 0.1 M Na 2 CO 3 , pH9.6, 0.1% BSA) to coat the antigen, 37°C for 2h and then set at 4°C overnight; wash 3 times; add blocking solution (0.05M pH9.6 coating solution containing 10% goat serum) and incubate at 37°C for 2h, Wash once; dilute the serum to be tested with diluent (PBS-T pH7.4 containing 10% calf serum and 1-5% sheep serum) 1:50, add sample, wash 3 times at 37°C for 2 hours, add Goat anti-mouse IgG-HRP diluted 1:8000, washed 4 times at 37°C for 2h; add substrate solution (o-ph...

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Abstract

The invention discloses an anti-double-stranded DNA monoclonal antibody with antitumor activity, as well as a preparation method thereof. The monoclonal antibody is specifically bound to double-stranded DNA or can express tumor cells of the double-stranded DNA, and the double-stranded DNA comes from a BALB / c mouse complete genome DNA sequence. The method comprises the following steps: double-stranded DNA derived from BALB / c mouse tumor cells is purified; a BALB / c mouse is immunized by the purified double-stranded DNA derived from the BALB / c mouse tumor cells; and mouse spleen cells secreting high-titer anti-dsDNA antibodies and mouse B cell lymphoma are fused, screened and repeatedly cloned, so as to obtain hybridoma cell lines capable of stably secreting anti-dsDNA monoclonal antibodies. The anti-dsDNA monoclonal antibody can be specifically bound to dsDNA, can be bound to the surfaces of tumor cells, can effectively inhibit tumor growth in vitro and in vivo, and has remarkable effect of treating tumor-bearing animals.

Description

Technical field: [0001] The invention relates to the fields of bioengineering and tumor immunotherapy, and relates to a monoclonal antibody, in particular to an anti-double-stranded DNA monoclonal antibody with anti-tumor activity and a preparation method thereof. Background technique: [0002] Tumor biological immunotherapy is a new type of tumor treatment after surgical treatment, radiotherapy and chemical drug treatment. Among them, monoclonal antibody (monoclonal antibody, mAb) developed against tumor antigens is one of the hot spots in tumor biological treatment. The specificity of the antigen-antibody reaction determines that the monoclonal antibody against the tumor-specific target antigen will preferentially bind to the corresponding tumor tissue instead of recognizing normal tissue. Monoclonal antibodies not only directly induce tumor cell apoptosis, but also can be coupled with radiotherapy and chemotherapy drugs to reduce the non-specific damage of drugs to normal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N5/18G01N33/577
Inventor 熊思东曹清华徐薇
Owner FUDAN UNIV
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