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Epididymal protease inhibitor protein monoclonal antibody and application thereof

A technology of monoclonal antibody and protease inhibition, applied in the field of preparation of anti-fertility research biological products, male infertility diagnostic reagents, monoclonal antibody, epididymis protease inhibitory protein monoclonal antibody

Inactive Publication Date: 2009-09-02
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there is no male contraceptive that directly acts on the epididymis and is available for clinical trials, studies on the function of the epididymis and the maturation of sperm in the epididymis have shown that the epididymis is likely to become the most ideal target organ for male anti-fertility

Method used

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  • Epididymal protease inhibitor protein monoclonal antibody and application thereof
  • Epididymal protease inhibitor protein monoclonal antibody and application thereof
  • Epididymal protease inhibitor protein monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Preparation of anti-Eppin monoclonal antibody

[0049] 1. The software analyzes and selects the epitope of a human Eppin protein ( figure 1 )

[0050] Chemical synthesis of 14aa polypeptide (SEQ ID No. 1)

[0051] 2. Immunize BALB / c mice with Eppin protein

[0052] For the first immunization, take 0.5ml of purified chemically synthesized Eppin polypeptide (SEQ ID No.1) (concentration 100μg / ml) and mix it with an equal volume of Freund’s complete adjuvant, and grind it in a mortar until it is fully emulsified (ie, water-in-oil). After state), it was injected into the abdominal cavity of the mouse (0.1ml / mouse). The second booster immunization was performed after an interval of 2 weeks, and Freund’s incomplete adjuvant was used to fully emulsify the antigen. The injection dose and location were the same as before. The third booster immunization was carried out after an interval of 2 weeks. The antigen solution was used directly, and the injection dose and location...

Embodiment 2

[0065] Example 2: Determination of the titer of anti-Eppin monoclonal antibody

[0066] Using ELISA method. Eppin polypeptide (1mg / L) was used to coat the microwells (100μl / well) of polystyrene plates at 4°C overnight. The next day, the plate was washed with PBS-T 3 times, 200μl / well was added with 50g / L skimmed milk powder and blocked at 37°C for 2 hours, and the plate was washed 3 times with PBS-T. Add the test sample (culture supernatant of hybridoma cell line, or serially diluted mouse ascites monoclonal antibody), and incubate at 37°C for 1 hour. Wash the plate 3 times with PBS-T, add HRP-goat anti-mouse IgG (1:3000 dilution), and incubate at 37°C for 1 hour. Wash the plate 3 times with PBS-T, add TMB-H to each well 2 O 2 Incubate the solution at 37°C for 10 minutes. Add 2 drops of 1mol / L H 2 SO 4 The reaction was terminated, and the color was compared with an enzyme-linked immunoassay at a wavelength of 450nm. Results: The antibody titers of the culture supernatant of hybrid...

Embodiment 3

[0070] Example 3: Eppin cloning and prokaryotic expression

[0071] In order to verify the specificity of monoclonal antibodies, we must first clone and express the Eppin gene. We used RT-PCR to clone the Eppin gene from the testis tissues of normal fertile males, and then DNA sequencing (SEQ ID No. 2) was performed after PCR and double enzyme digestion identification. Then, the recombinant expression vector pET-28a(+) / Eppin was constructed. After PCR and double enzyme digestion, it was transformed into E. coli BL21(DE3), and the recombinant Sp17 was detected by IPTG induction and SDS-PAGE, and purified by nickel ion affinity chromatography. Recombinant protein.

[0072] 1.1 Experimental specimen: human testicular tissue

[0073] 1.2 Preparation of Eppin double-stranded cDNA fragment

[0074] 1.2.1 Primer design

[0075] Designed with DNAMAN analysis software, PCR primer sequences are as follows:

[0076] Upstream primer (primer 1):

[0077] 5′-GTTC CCATGG TCCAGGGACCTGGTCTGA-3'(S...

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Abstract

The invention discloses an epididymal protease inhibitor protein monoclonal antibody and application thereof, which belong to the field of biological engineering. The monoclonal antibody is secreted by hybridoma preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C200849. The monoclonal antibody can be combined to the epididymal protease inhibitor protein Eppin protein of specific expression in a testis and epididymis tissue by specificity. The monoclonal antibody can be used for preparing male infertility diagnostic reagents, male contraceptives and anti-fertility study biological products.

Description

Technical field [0001] The invention belongs to the fields of bioengineering, cell engineering and immunodiagnosis, and relates to an epididymal protease inhibitor monoclonal antibody and its use. More specifically, the present invention relates to a monoclonal antibody that specifically recognizes an epididymal protease inhibitor protein, which is an epididymal protease inhibitor protein specifically expressed in testis and epididymal tissue, and is mainly expressed in sperm Surface; The present invention also relates to the preparation method of the protein-specific monoclonal antibody and the application of the monoclonal antibody in the preparation of anti-fertility research biological products and diagnostic reagents for male infertility. Background technique [0002] The epididymis is the place for sperm capacitation and maturation. Sperm obtains fertilization and fertility through structural and functional changes in the epididymal microenvironment. Although there is no ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/38C12N5/20A61K39/395A61P15/16G01N33/577C12R1/91
Inventor 王心如夏彦恺张洁王守林顾爱华
Owner NANJING MEDICAL UNIV
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