Albumen microsphere conjugate for detecting acrosin activity and preparation method and application of albumen microsphere conjugate

A technology of acrosome enzyme and conjugate, which is applied in the detection of sperm acrosome enzyme activity protein microsphere conjugate and its preparation field, can solve the problems of difficult accurate quantification and insufficient sensitivity, and achieve high sensitivity and fast detection speed Effect

Inactive Publication Date: 2016-11-23
ZHEJIANG CELLPRO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the activity of acrosome enzyme in sperm samples is not as high as μIU level, the method of detecting light absorption value with spectrophotometer or microplate reader has the problem that the sensitivity is not high enough and it is difficult to accurately quantify

Method used

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  • Albumen microsphere conjugate for detecting acrosin activity and preparation method and application of albumen microsphere conjugate
  • Albumen microsphere conjugate for detecting acrosin activity and preparation method and application of albumen microsphere conjugate
  • Albumen microsphere conjugate for detecting acrosin activity and preparation method and application of albumen microsphere conjugate

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specific Embodiment 1

[0038] A protein microsphere conjugate for detecting the activity of sperm acrosome, the general structural formula of which is as follows:

[0039]

[0040] Among them, M is a monodisperse microsphere made of polymer material, R1 is a functional bond, R2 is a protein or polypeptide, and P is a fluorescent molecule.

[0041] The diameter of the above-mentioned monodisperse microspheres is 1-50 microns, and the material is polystyrene (PS), polymethyl methacrylate (PMMA), silicon dioxide (SiO2), polylactic acid (PLA) or polylactic acid glycolic acid polymerized (PLGA), the outer surface of the monodisperse microspheres is functionally modified with carboxyl, amino, hydroxyl or mercapto groups, and then the surface is treated with polyethylene glycol (PEG), polymethyl methacrylate (PMMA) or Fe3O4 Coating modification. All the above surface-modified and further coated monodisperse microspheres can be directly purchased through Tianjin Basele Chromatography Technology Developm...

specific Embodiment 2

[0045] A method for preparing a protein microsphere conjugate for detecting sperm acrosome enzyme activity, the specific steps are as follows:

[0046] (1) Obtaining of carboxylated polystyrene microspheres

[0047] Polystyrene microspheres were purchased from commercial microspheres, the outer surface of which was functionally modified with carboxyl groups, and the surface had been modified by Fe3O4 coating. Tianjin Beisile Chromatography Technology Development Center, Suzhou Zhiyi Microsphere Technology Co., Ltd. and Aladdin can provide the microspheres used in this example;

[0048] (2) Protein fluorescent labeling

[0049] The fluorescent molecule fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate (RBITC) or tetramethylrhodamine isothiocyanate (TRITC) was dissolved in dimethyl sulfoxide (DMSO) to prepare Fluorescent molecule solution with a concentration of 10mg / mL, dissolve the full sequence protein, fragment of zona pellucida protein (ZP3) or bovine serum ...

specific Embodiment 3

[0055] Same as the above-mentioned specific embodiment 2, the difference is that in step (1) protein fluorescent labeling, tetraethylrhodamine (RB200) and protein or polypeptide are used for labeling, and the specific method is as follows:

[0056] (1) Weigh 1g RB200 fluorescein and 2g phosphorus pentachloride, put them in a mortar, grind them in a fume hood for 5min, add 10ml of anhydrous acetone, stir continuously, filter after 5min, and use the filtrate to mark proteins or polypeptides, Obtain a fluorescent molecule solution;

[0057] (2) The Zona pellucida protein solution with a concentration of 20 mg / ml, physiological saline and carbonate buffer solution of 0.1mol / L pH9.0 are mixed in a volume ratio of 1:1:1 to obtain R2 solution;

[0058] (3) Mix the fluorescent molecule solution with the R2 solution, then add 0.1ml of RB200 fluorescein solution, stir while adding dropwise, and perform a coupling reaction at 4°C for 16 hours to obtain a fluorescent protein solution. The...

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Abstract

The invention discloses an albumen microsphere conjugate for detecting acrosin activity and a preparation method and application of the albumen microsphere conjugate. The albumen microsphere conjugate is characterized in that monodisperse micron microspheres obtained by combining fluorescein and the acrosome reaction substrate, namely zona pellucida protein are prepared, the acrosin activity is detected by aid of change in fluorescence intensity, and the specific steps include preparation of monodisperse microspheres modified through surface carboxylation or amination, surface modificaction, fluorescence labeling of protein polypeptide, conjugation of fluorescent protein polypeptide and functional microspheres and detection of the acrosin activity. The albumen microsphere conjugate has the advantages that protease digestion on the microspheres is realized through the polymer microsphere technology and the protein labeling and conjugating technology, and enzymatic activity is evaluated; the albumen microsphere conjugate is mainly applied to flow detection of the acrosin activity, synchronous detection of an acrosome reaction and the acrosin activity can be realized, and acrosome functions can be evaluated more truthfully and accurately.

Description

technical field [0001] The invention relates to a technique for detecting sperm acrosome activity, in particular to a protein microsphere conjugate for detecting sperm acrosome activity, a preparation method and application thereof. Background technique [0002] After human sperm enter the female reproductive tract, they must go through a period of maturation to obtain the ability to fertilize. During this period, a series of physiological changes in sperm become capacitation. After capacitation, the sperm passes through the cumulus and corona radiata, and when it reaches the zona pellucida, it undergoes sperm-egg recognition, changes in the plasma membrane, release of acrosome enzyme, enzymatic hydrolysis of the zona pellucida, and the nucleus of the sperm cell enters the egg cell and dissolves with the nucleus. The process constitutes the acrosome reaction in the broad sense, while the acrosome reaction in the narrow sense mainly includes two key processes: plasma membrane...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C07K14/765G01N21/64C09K11/06
CPCC07K14/47C07K14/765C09K11/06G01N21/6428
Inventor 曾桥许毅李翼飞李德才房海燕薛金锋
Owner ZHEJIANG CELLPRO BIOTECH
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