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AP-2alpha monoclonal antibody and application to preparation of drugs for treating cervical cancers

An ap-2alpha, monoclonal antibody technology, applied in the direction of antibodies, anti-tumor drugs, drug combinations, etc., can solve the problem of less antibody research and achieve the effect of inhibiting cell proliferation

Active Publication Date: 2020-07-10
湖南金朋医疗器械股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still very few studies on high-affinity specific monoclonal antibodies against AP-2alpha

Method used

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  • AP-2alpha monoclonal antibody and application to preparation of drugs for treating cervical cancers
  • AP-2alpha monoclonal antibody and application to preparation of drugs for treating cervical cancers
  • AP-2alpha monoclonal antibody and application to preparation of drugs for treating cervical cancers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Screening and expression of AP-2alpha antigenic peptide fragments

[0033] According to the gene and amino acid sequence of AP-2alpha on NCBI, the inventor screened and obtained the AP-2alpha antigen fragment with high immunogenicity and isoelectric point through optimized screening of antigenic epitopes. Its amino acid sequence is shown as SEQ ID NO: As shown in 1, the isoelectric point pH 6.60 is suitable for prokaryotic expression, and the molecular weight of the protein is about 12.47KD.

[0034] SEQ ID NO: 1:

[0035] 1 QSQESGLLHT HRGLPHQLSG LDPRRDYRRH EDLLHGPHAL SSGLGDLSIH SLPHAIEEVPHVEDPGINIP DQTVIKKGPV

[0036] 81 SLSKSNSNAV SAIPINKDNL FGGVVNPNEV FCSVPG

[0037] After codon optimization, the corresponding nucleotide sequence was obtained, as shown in SEQ ID NO:2.

[0038] SEQ ID NO: 2:

[0039] 1 CAATCCCAGG AATCTGGTCT GCTGCATACT CATCGTGGTC TGCCTCATCA GCTGAGCGGTCTGGACCCTC

[0040] 71 GCCGTGATTA TCGTCGTCAC GAAGACCTGC TGCACGGTCC ACACGCACTG TCCAGCGGTC...

Embodiment 2

[0045] The preparation of embodiment 2 monoclonal hybridomas

[0046] 1. Immunization of mice:

[0047] The immunogen human AP-2alpha (prepared in Example 1) was emulsified with the volume of antigen and adjuvant at a ratio of 1:1, and Freund's complete adjuvant was used to emulsify the antigen for the first immunization, and the second immunization was started after 2 weeks , the antigen was emulsified with incomplete Freund's adjuvant, and injected subcutaneously at 2 points, the amount of antigen injected per mouse was 10 μg, and the volume injected at each injection point was 20 μL, a total of 10 mice.

[0048] Three days after the second immunization, blood was collected from the eyes of the mice, and a small amount of blood samples were taken for serum titer detection. The mice whose serum titer titer reached 1:150,000 were detected by indirect ELISA were strengthened. immunity.

[0049] 2. Preparation of feeder cells and myeloma cells

[0050] For the preparation of ...

Embodiment 3

[0065] Example 3 Detection of monoclonal antibody titer

[0066] Use the indirect ELIS A method to detect the titer of the ascites obtained. The specific operation method is: use 28a-AP-2alpha purified protein and pET-28a empty carrier protein to coat the microtiter plate, and start to multiply the ascites at 1: 200 times Diluted as the primary antibody, and HRP-labeled goat anti-mouse IgG as the secondary antibody. When the OD630 value is greater than 1, the maximum dilution factor of the hybridoma cell ascites is the ELISA titer.

[0067] The results of indirect ELISA detection are shown in the figure ( figure 2 ), the ascites titer of AP-2alpha-3B6 antibody was 1:409600.

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Abstract

The present invention relates to an AP-2alpha monoclonal antibody and an application to preparation of drugs for treating cervical cancers. The monoclonal antibody has the relatively good cervical cancer cell proliferation inhibiting effect, thereby having the wide application prospect.

Description

technical field [0001] The invention relates to the field of antibodies, in particular to a monoclonal antibody of AP-2alpha and its use in the preparation of medicines for treating cervical cancer. Background technique [0002] Cervical cancer is an important disease that threatens the majority of women. According to statistics, there are approximately 500,000 new cases and 240,000 deaths worldwide each year. my country is one of the high-incidence areas of cervical cancer in the world, with about 131,500 new cervical cancer patients every year, accounting for 28% of the total number of new cervical cancer cases in the world. With the widespread implementation of cervical cytology screening, the mortality rate of cervical cancer in the United States has decreased significantly in the past 50 years. However, due to economic reasons, cervical cancer screening in developing countries lags far behind developed countries, so cervical cancer has become a common type of cancer i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/70C12N1/21A61K39/395A61P35/00C12R1/19
CPCA61K2039/505A61P35/00C07K16/18C07K2317/56C12N15/70
Inventor 刘欢朱小明
Owner 湖南金朋医疗器械股份有限公司
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