CN106146642A - A kind of for protein microsphere conjugate detecting sperm acrosin activities in assessing and its preparation method and application - Google Patents
A kind of for protein microsphere conjugate detecting sperm acrosin activities in assessing and its preparation method and application Download PDFInfo
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Abstract
nullThe present invention is a kind of for protein microsphere conjugate detecting sperm acrosin activities in assessing and its preparation method and application，Feature is the mono dispersed micrograde microsphere combined by preparation sperm acrosome reaction substrate oocyte zona pellucida protein and fluorescein，Sperm acrosin activities in assessing is detected by the change by fluorescence intensity，The mono-dispersion microballoon being specifically divided into surface carboxyl groups or amination modification prepares、Surface modification、The fluorescent labeling of protein polypeptide、The coupling of fluorescin polypeptide and functional microsphere and the detection of sperm acrosin activities in assessing，Advantage is to be achieved by polymer microsphere technology and protein labeling coupling technology carry out proteolytic cleavage on microsphere and carry out the assessment of enzymatic activity，It is mainly used in the flow cytometer detection of sperm acrosin activities in assessing，The synchronous detecting of sperm acrosome reaction and acrosin activity can be realized，More really and accurately perforatorium function can be evaluated.
Description
Technical field
The present invention relates to sperm acrosin activities in assessing detection technique, especially relate to a kind of for detecting sperm acrosin activities in assessing
Protein microsphere conjugate and its preparation method and application.
Background technology
After human sperm enters female genital tract, it is necessary to through one section of maturation process, it is thus achieved that fertility, sperm during this
The a series of physiological changies occurred become capacitation.Sperm after capacitation passes ovarian cumulus, corona radiata, when arriving zona pellucida, passes through
Essence ovum identification, plasma membrane change, acrosomal enzyme release, zona pellucida enzymolysis, it is molten with nuclear phase that Sperm nuclei enters ovum, and these are a series of
Complicated process constitutes sensu lato acrosome reaction, and narrow sense acrosome reaction mainly include plasma membrane fusion that zona pellucida induce and
Two critical process of the release of acrosomal enzyme.What current laboratory research and clinical diagnosis were paid close attention to most is also the two process
Molecular mechanism and Clinical detection.Wherein plasma membrane fusion predominantly detects method is to be become by in-vitro simulated induction perforatorium film
Changing, and utilize the method for specific stain to observe, acrosomal enzyme release process is primarily upon the Activity determination of the enzyme discharged.
Laboratory has been set up the acrosome reaction detection method of comparative maturity the most both at home and abroad, mainly has Coomassie brilliant blue
Staining, acid phosphatase detection method, and widely used be fluorescent staining method, predominantly chloroteracycline staining method, fluorescence
The pisum sativum agglutinin of labelling or peanut agglatinin coupling method.Wherein pisum sativum agglutinin method proposed with 1993 the earliest, peanut lectin
Element method is also reported the same year, the most gradually improves perfect, coordinates fluorescence microscope and auto Analysis, becomes the most clinical inspection
Survey most common method.
Independent acrosome reaction detection can not reflect acrosin activity, and the activity of acrosomal enzyme is directly connected to zona pellucida
Dissolving, be the important step of sperm-egg fusion.The detection of acrosomal enzyme utilize in early days more acrosomal enzyme antibody carry out radioimmunology or
Fluorescent immune method detects, and judges sperm fertilizing ability by the quantity of acrosomal enzyme in observation perforatorium.Kennedy later
Et al. first reported calculating acrosomal enzyme hydrolyzing N a-benzoyl-DL-arginine-to nitro-amide hydrochlorate (BAPNA) substrate imitate
The method of rate judges the activity of acrosin, and first sperm is cracked by the method, then lysate is placed in addition containing hydrolysis
In the detection pipe of substrate B APNA, the colour developing depth after substrate hydrolysis, can be detected by spectrophotometer, calculate light absorption value
The hydrolysis vigor of detection acrosomal enzyme.The method show that index more can react the physiological function of acrosin, has become standard
Detection method.Because the activity of acrosomal enzyme itself the most a height of μ IU rank in sperm sample, therefore use spectrophotometer or enzyme
There is the high-leveled and difficult problem with accurate quantitative analysis of insufficient sensitivity in the method for mark instrument detection absorbance value.
Summary of the invention
The technical problem to be solved is to provide a kind of sperm acrosome reaction and sperm acrosin activities in assessing of realizing
Synchronous detecting, highly sensitive, detect the fireballing protein microsphere conjugate for detecting sperm acrosin activities in assessing and preparation thereof
Method.
The present invention solves the technical scheme that above-mentioned technical problem used: a kind of for detecting sperm acrosin activities in assessing
Protein microsphere conjugate, its general structure is as follows:
Wherein M is the mono-dispersion microballoon that macromolecular material is made, and R1 is function associative key, and R2 is albumen or polypeptide, and P is glimmering
Optical molecule.
A diameter of 1-50 micron of described mono-dispersion microballoon, material is polystyrene (PS), polymethyl methacrylate
(PMMA), silicon dioxide (SiO2), polylactic acid (PLA) or polylactic-co-glycolic acid polymer (PLGA), described single dispersing is micro-
After outer surface functional modification carboxyl, amino, hydroxyl or the sulfydryl of ball, then its surface is carried out Polyethylene Glycol (PEG), poly-methyl
Acrylic acid methyl ester. (PMMA) or Fe3O4Coating modifying modified.
Described function associative key is the amido link that is combined with amino of carboxyl or hydroxyl and carboxyl condensation key or sulfydryl
With hydroxyl condensation key.
Described R2 be the complete sequence albumen containing arginine, the polypeptide of lysine or oocyte zona pellucida protein, fragment or
The complete sequence albumen of bovine serum albumin, fragment.
Described fluorescence molecule is the fluorescent dye of coupling, described fluorescent dye be Fluorescein isothiocyanate (FITC),
Isothiocyanate rhodamine B (RBITC), RB 200 (RIB200), Tetramethylrhodamine isothiocyanate (TRITC), sieve
Red bright red-X (Red-X), texas Red (Texas Red), phycoerythrin (AlexaFluor 488, PE), phycoerythrin/
Texas Red tandem dye (PE-TR), phycoerythrin/Alexa Fluor 610 tandem dye (PE-Alexa
Fluor610), phycoerythrin/Alexa Fluor647 tandem dye (PE-Alexa Fluor647), phycoerythrin/cyanine dye
Expect 5 tandem dyes (PE-cy5), phycoerythrin/cyanine dye 5.5 tandem dye (PE-cy5.5), phycoerythrin/Alexa
Fluor 700 tandem dye (PE-Alexa Fluor 700), phycoerythrin/cyanine dye 7 tandem dye (PE-Cy7), algae is blue
Albumen (Allophycocyanin, APC), phycocyanin/cyanine dye 5.5 tandem dye (APC-Cy5.5), phycocyanin/flower
Blue or green dyestuff 7 tandem dye (APC-Cy7), cyanine dye (Cy2/3/5).
The preparation method of the above-mentioned protein microsphere conjugate for detecting sperm acrosin activities in assessing, concrete preparation method is such as
Under:
(1) protein fluorescence labelling
Fluorescence molecule Fluorescein isothiocyanate or isothiocyanate rhodamine B or Tetramethylrhodamine isothiocyanate are dissolved in
Dimethyl sulfoxide is configured to the fluorescence molecule solution that concentration is 10mg/mL, by the complete sequence egg of oocyte zona pellucida protein (ZP3)
In vain, the complete sequence albumen of fragment or bovine serum albumin, fragment or be dissolved in carbonate buffer solution containing arginine, the polypeptide of lysine
In be configured to the R2 solution of 10mg/ml, after being mixed with R2 solution by fluorescence molecule solution, in 4 DEG C of lucifuge coupling reactions 16 hours
Obtain fluorescin solution, the fluorescin solution after coupling is added and separates with the glucosan of phosphate buffer washing in advance
On post, then use phosphate buffer to carry out post separation with 20ul/s low speed, collect the liquid of the brown flowed out at first
(495nm has absorption value) obtains fluorescent labeling R2 solution, and quantitative Analysis albumen or peptide concentration；Wherein contained egg in R2 solution
White or peptide molecule is (1: 9)-(1: 11) with the mol ratio of fluorescence molecule in fluorescence molecule solution；
(2) fluorescent marker protein and mono-dispersion microballoon coupling
It is the N-hydroxy-succinamide of 50mg/mL by carbodiimide (EDC) solution and concentration that concentration is 50mg/mL
(NHS), during solution is added separately to the carboxylated mono-dispersion microballoon solution that concentration is 0.01wt%, room temperature lucifuge reaction 10-15 divides
Clock, after then vortex concussion mixes 1 time, continues room temperature lucifuge and reacts 10 minutes, be then centrifuged by reaction solution after removing supernatant and add
Entering the fluorescent labeling R2 solution that concentration is 1mg/mL, 37 DEG C of lucifuge concussions are reacted 3 hours, with the PBS containing 0.02wt% tween 20
Solution, by microsphere centrifuge washing 3 times, obtains the protein microsphere conjugate for detecting sperm acrosin activities in assessing；
Or by sub-to carbodiimide (EDC) solution that concentration is 50mg/mL and the N-hydroxysuccinimidyl acyl that concentration is 50mg/mL
Amine (NHS) solution is added separately in the fluorescent labeling R2 solution that concentration is 1mg/mL, room temperature lucifuge reaction 10-15 minute, so
After rear vortex concussion mixing 1 time, continue room temperature lucifuge and react 10 minutes, then reaction solution is centrifuged after removing supernatant and adds concentration
For in the amination mono-dispersion microballoon solution of 0.01wt%, 37 DEG C of lucifuge concussions are reacted 3 hours, with containing 0.02wt% tween 20
PBS solution by microsphere centrifuge washing 3 times, obtain the protein microsphere conjugate for detecting sperm acrosin activities in assessing；
Its general structure is as follows:
Wherein M is the mono-dispersion microballoon of a diameter of 1-50 micron, material be polystyrene, polymethyl methacrylate, two
Silicon oxide, polylactic acid or polylactic-co-glycolic acid polymer, the outer surface functional modification carboxyl of described mono-dispersion microballoon, ammonia
After base, hydroxyl or sulfydryl, then surface is carried out Polyethylene Glycol, polymethyl methacrylate or Fe3O4Coating modifying modified；R1
For amido link, R2 is albumen or polypeptide, and P is fluorescence molecule.
Described carbodiimide (EDC) solution, described N-hydroxy-succinamide (NHS) solution, described single dispersing
The volume ratio of microsphere and fluorescent labeling R2 solution is 1: 1: 8: 8.
The preparation method of the above-mentioned protein microsphere conjugate for detecting sperm acrosin activities in assessing, concrete preparation method is such as
Under:
(1) protein fluorescence labelling
Weigh 1g RB200 fluorescein and 2g phosphorus pentachloride is placed on after grinding 5min in fume hood in mortar, add 10ml
Anhydrous propanone, is stirred continuously, and filters, be marked labelled protein or polypeptide with filtrate, obtain fluorescence molecule solution after 5min；Will
Concentration be the oocyte zona pellucida protein solution of 20mg/ml, normal saline and 0.1mol/L pH 9.0 carbonate buffer solution by volume
R2 solution is obtained after ratio mixing than 1: 1: 1；Being mixed with R2 solution by fluorescence molecule solution, the RB200 adding 0.1ml is glimmering
Light cellulose solution, the stirring of dropping limit, limit, in 4 DEG C, coupling reaction obtains fluorescin solution in 16 hours；By the fluorescence egg after coupling
White solution adds on the glucosan detached dowel washed with phosphate buffer in advance, then uses phosphate buffer with 20ul/s
Low speed carries out post separation, and the liquid collecting the brown flowed out at first obtains fluorescent labeling R2 solution, and quantitative Analysis albumen or many
Peptide concentration；Wherein in R2 solution, contained albumen or peptide molecule are 1: 9-1 with the mol ratio of fluorescence molecule in fluorescence molecule solution:
11。
(2) fluorescent marker protein and mono-dispersion microballoon coupling
Carbodiimide solution and the N-hydroxy-succinamide solution that concentration is 50mg/mL that concentration is 50mg/mL are divided
Not joining in the carboxylated mono-dispersion microballoon solution that concentration is 0.01wt%, room temperature lucifuge is reacted 10-15 minute, then vortex
Shaking after mixing 1 time, continue room temperature lucifuge and react 10 minutes, then reaction solution is centrifuged addition concentration after removing supernatant is 1mg/
The fluorescent labeling R2 solution of mL, 37 DEG C of lucifuges concussion reaction 3 hours, with the PBS solution containing 0.02wt% tween 20 by microsphere from
The heart washs 3 times, obtains the protein microsphere conjugate for detecting sperm acrosin activities in assessing；
Or by molten to Carbodiimide solution and the N-hydroxy-succinamide that concentration is 50mg/mL that concentration is 50mg/mL
Liquid is added separately in the fluorescent labeling R2 solution that concentration is 1mg/mL, and room temperature lucifuge is reacted 10-15 minute, then vortex concussion
After mixing 1 time, continuing room temperature lucifuge and react 10 minutes, then reaction solution is centrifuged addition concentration after removing supernatant is 0.01wt%
Amination mono-dispersion microballoon solution in, 37 DEG C of lucifuges concussion reaction 3 hours, will by the PBS solution containing 0.02wt% tween 20
Microsphere centrifuge washing 3 times, obtains the protein microsphere conjugate for detecting sperm acrosin activities in assessing；
Its general structure is as follows:
Wherein M is the mono-dispersion microballoon of a diameter of 1-50 micron, material be polystyrene, polymethyl methacrylate, two
Silicon oxide, polylactic acid or polylactic-co-glycolic acid polymer, the outer surface functional modification carboxyl of described mono-dispersion microballoon, ammonia
After base, hydroxyl or sulfydryl, then surface is carried out Polyethylene Glycol, polymethyl methacrylate or Fe3O4Coating modifying modified；R1
For amido link, R2 is albumen or polypeptide, and P is fluorescence molecule.
Described Carbodiimide solution, described N-hydroxy-succinamide solution, described mono-dispersion microballoon and fluorescence
The volume ratio of labelling R2 solution is 1: 1: 8: 8.
The application of the above-mentioned protein microsphere conjugate for detecting sperm acrosin activities in assessing, for detecting acrosin activity
Specifically comprise the following steps that after fresh semen sample is liquefied completely, take 1mL in test tube, and be slowly added to people's fallopian tube on upper strata
After substantially layering occurs in liquid 1.2mL, test tube inclination constant temperature 37 DEG C is hatched 30 minutes and is carried out upstream preferably, then takes out higher slice liquid
Body, adds derivant Calcium ionophore and carries out acrosome reaction, and constant temperature lucifuge 37 DEG C adds acrosome film dyeing liquor after reacting 15 minutes
PSA-FITC and for detecting the protein microsphere conjugate of sperm acrosin activities in assessing, abundant endonuclease reaction carried out streaming after 15 minutes
Detection, the weakening degree and can represent the activity of acrosin of the protein microsphere conjugate fluorescence intensity that streaming is identified.
Cleaning Principle is as follows: sperm, in upstream and after inducing acrosome reaction, will discharge acrosin, and acrosomal enzyme has pancreas
Protease sample hydrolytic enzyme activities, will identify arginine and the lysine sites on protein microsphere surface, thus endonuclease reaction will occur, water
Solving coated fluorescent marker protein, cause coming off of fluorescin, the reaction that comes off of fluorescin in various degree is different strong and weak
Enzyme hydrolysis activity.Therefore, the protein microsphere conjugate fluorescence intensity that flow cytometer is identified weaken degree can represent essence
The activity of sub-acrosomal enzyme.
Compared with prior art, it is an advantage of the current invention that: a kind of egg for detecting sperm acrosin activities in assessing of the present invention
Bai Weiqiu conjugate and application thereof, the single dispersing combined by preparation sperm acrosome reaction substrate oocyte zona pellucida protein and fluorescein
Micron order microsphere, sperm acrosin activities in assessing is detected by the change by fluorescence intensity.Be specifically divided into surface carboxyl groups or
Mono-dispersion microballoon preparation, surface modification, the fluorescent labeling of protein polypeptide, fluorescin polypeptide and the functional microsphere that amination is modified
Coupling and the detection of sperm acrosin activities in assessing.The present invention is achieved by polymer microsphere technology and protein labeling coupling technology
Microsphere carry out proteolytic cleavage and carries out the assessment of enzymatic activity, being mainly used in the flow cytometer detection of sperm acrosin activities in assessing, can
To realize the synchronous detecting of sperm acrosome reaction and acrosin activity, thus can more really and accurately perforatorium function be carried out
Evaluate, to diagnosis male infertility sterility disease because of, and science, comprehensive assessment motility of sperm are had great importance.
In sum, the present invention, for detecting protein microsphere conjugate and the application thereof of sperm acrosin activities in assessing, uses micro-
The method of ball simulation ovum, it is achieved external discharges acrosome reaction with sperm, and uses sensitiveer fluorescent quantitation side
Method detects, and sensitivity is higher, and detection speed is faster.
Detailed description of the invention
Below in conjunction with preferred embodiment, the present invention is described in further detail.
Specific embodiment one
A kind of protein microsphere conjugate for detecting sperm acrosin activities in assessing, its general structure is as follows:
Wherein M is the mono-dispersion microballoon that macromolecular material is made, and R1 is function associative key, and R2 is albumen or polypeptide, and P is glimmering
Optical molecule.
A diameter of 1-50 micron of above-mentioned mono-dispersion microballoon, material is polystyrene (PS), polymethyl methacrylate
(PMMA), silicon dioxide (SiO2), polylactic acid (PLA) or polylactic-co-glycolic acid polymer (PLGA), this mono-dispersion microballoon
After outer surface functional modification carboxyl, amino, hydroxyl or sulfydryl, then its surface is carried out Polyethylene Glycol (PEG), polymethyl
The coating modifying of acid methyl ester (PMMA) or Fe3O4 is modified.Above-mentioned all surface is modified and further through the single dispersing of coating modifying
Microsphere all can think happy chromatographic technique development centre, Suzhou Zhi Yi microsphere Science and Technology Ltd. and Aladdin etc. by Tianjin again
Directly buy acquisition.
Above-mentioned functions associative key be the amido link that is combined with amino of carboxyl or hydroxyl with carboxyl condensation key or sulfydryl with
Hydroxyl condensation key.
Above-mentioned R2 be containing arginine, the polypeptide of lysine or the complete sequence albumen of oocyte zona pellucida protein (ZP3), fragment or
The complete sequence albumen of person's bovine serum albumin, fragment.
Above-mentioned fluorescence molecule is the fluorescent dye of coupling, and fluorescent dye is that FITC (Fluorescein isothiocyanate), RBITC are (different
Rhodanate rhodamine B) or other structures such as RIB200 (RB 200) of this type of fluorescein, (tetramethyl is different for TRITC
Hydrogen thiocyanate rhodamine, TetramethylRhodaminIsothiocyanate), RRX (Rhodamine Red-X, rhodamine is red-
X), TR (Texas Red, texas Red), AlexaFluor 488, PE (phycoerythrin), PE-TR (phycoerythrin/De Kesa
This red tandem dye), PE-Alexa Fluor 610 (phycoerythrin/Alexa Fluor 610 tandem dye), PE-Alexa
Fluor 647 (phycoerythrin/Alexa Fluor 647 tandem dye), PE-cy5 (phycoerythrin/cyanine dye 5 series connection dye
Material), PE-cy5.5 (phycoerythrin/cyanine dye 5.5 tandem dye), PE-Alexa Fluor 700 (phycoerythrin/Alexa
Fluor 700 tandem dye), PE-Cy7 (phycoerythrin/cyanine dye 7 tandem dye), APC (Allophycocyanin, algae
Azurin), Alexa Fluor 647, APC-Cy5.5 (phycocyanin/cyanine dye 5.5 tandem dye), (algae is blue for APC-Cy7
Albumen/cyanine dye 7 tandem dye), Cy2/3/5 (Cyanine Dyes, cyanine dye) etc. may be used for flow cytometer inspection
The fluorescent dye surveyed.
Specific embodiment two
The preparation method of a kind of protein microsphere conjugate for detecting sperm acrosin activities in assessing, specifically comprises the following steps that
(1) acquisition of carboxylic polystyrene microsphere
Polystyrene microsphere is purchased from commercialization microsphere, and outer surface functional modification is carboxyl, and surface has carried out the painting of Fe3O4
Layer modification, Tianjin thinks happy chromatographic technique development centre, Suzhou Zhi Yi microsphere Science and Technology Ltd. and Aladdin again
Offer meets microsphere used in this example；
(2) protein fluorescence labelling
By fluorescence molecule Fluorescein isothiocyanate (FITC) or isothiocyanate rhodamine B (RBITC) or the different sulfur of tetramethyl
Cyanic acid rhodamine (TRITC) is dissolved in dimethyl sulfoxide (DMSO) and is configured to the fluorescence molecule solution that concentration is 10mg/mL, by ovum
The complete sequence albumen of zona pellucida protein (ZP3), fragment or the complete sequence albumen of bovine serum albumin (BSA), fragment or containing essence ammonia
Acid, the polypeptide of lysine are dissolved in carbonate buffer solution the R2 solution being configured to 10mg/ml, by fluorescence molecule solution and R2 solution
After mixing, within 16 hours, obtain fluorescin solution in 4 DEG C of lucifuge coupling reactions, the fluorescin solution after coupling is added in advance
On the glucosan detached dowel washed with phosphate buffer (PBS), phosphate buffer is then used to carry out with 20ul/s low speed
Post separates, and the liquid (495nm has absorption value) collecting the brown flowed out at first obtains fluorescent labeling R2 solution, and quantitative Analysis egg
White or peptide concentration；Wherein in R2 solution, contained albumen or peptide molecule with the mol ratio of fluorescence molecule in fluorescence molecule solution are
1∶10；(may be otherwise is 1: 9 or 1: 11)；
(3) fluorescent marker protein and carboxylated mono-dispersion microballoon coupling
It is the N-hydroxy-succinamide of 50mg/mL by carbodiimide (EDC) solution and concentration that concentration is 50mg/mL
(NHS) during solution is added separately to the carboxylated mono-dispersion microballoon solution (solvent is PBS buffer solution) that concentration is 0.01wt%,
Room temperature lucifuge is reacted 10-15 minute, after then vortex concussion mixes 1 time, continues room temperature lucifuge and reacts 10 minutes, then will reaction
Solution centrifugal adds the fluorescent labeling R2 solution that concentration is 1mg/mL after removing supernatant, 37 DEG C of lucifuge concussions are reacted 3 hours, with containing
The PBS solution of 0.02wt% tween 20, by microsphere centrifuge washing 3 times, obtains the albumen for detecting sperm acrosin activities in assessing micro-
Ball conjugate.
Its general structure is as follows:
Wherein M is the carboxyl polystyrene microsphere of surface modification；R1 is amido link, and R2 is albumen or polypeptide, and P is that fluorescence divides
Son, carbodiimide (EDC) solution, N-hydroxy-succinamide (NHS) solution, the carboxyl polystyrene microsphere solution of surface modification
It is 1: 1: 8: 8 with the volume ratio of fluorescent labeling R2 solution.
Specific embodiment three
With above-mentioned specific embodiment two, its difference is in step (1) protein fluorescence labelling to use RB 200
(RB200) being marked with albumen or polypeptide, concrete grammar is as follows:
(1) weigh 1g RB200 fluorescein and 2g phosphorus pentachloride is placed on after grinding 5min in fume hood in mortar, add
10ml anhydrous propanone, is stirred continuously, and filters, be marked labelled protein or polypeptide with filtrate, obtain fluorescence molecule molten after 5min
Liquid；
(2) by oocyte zona pellucida protein solution that concentration is 20mg/ml, the carbonate of normal saline and 0.1mol/LpH9.0
Buffer by volume 1: 1: 1 ratio mixing after obtain R2 solution；
(3) being mixed with R2 solution by fluorescence molecule solution, add the RB200 luciferin solution of 0.1ml, dropping limit, limit is stirred
Mixing, in 4 DEG C, coupling reaction obtains fluorescin solution in 16 hours, is added by the fluorescin solution after coupling and uses phosphoric acid in advance
On the glucosan detached dowel that salt buffer (PBS) washs, phosphate buffer is then used to carry out post separation with 20ul/s low speed,
The liquid (495nm has absorption value) collecting the brown flowed out at first obtains fluorescent labeling R2 solution, and quantitative Analysis albumen or many
Peptide concentration；Wherein in R2 solution, contained albumen or peptide molecule are 1: 9-1 with the mol ratio of fluorescence molecule in fluorescence molecule solution:
11。
Specific embodiment four
With above-mentioned specific embodiment two, its difference is in step (2) protein fluorescence labelling to use Rhodamine Red-X
(RRX), Texas Red (TR), Alexa Fluor 647, Cyanine Dyes (Cy2/3/5) mark with albumen or polypeptide
Note.
Specific embodiment five
With above-mentioned specific embodiment two, its difference is in step (2) protein fluorescence labelling to use PE, PE-TR, PE-
Alexa Fluor 610, PE-Alexa Fluor 647, PE-cy5 (TRI-COLOR, TC), PE-cy5.5, PE-Alexa
Fluor 700, PE-Cy7, APC (Allophycocyanin), APC-Cy5.5, APC-Cy7 are marked with albumen or polypeptide.
Specific embodiment six
With above-mentioned specific embodiment two, its difference is fluorescent marker protein and amination mono-dispersion microballoon in step (3)
Coupling, specifically comprises the following steps that
It is the N-hydroxy-succinamide of 50mg/mL by carbodiimide (EDC) solution and concentration that concentration is 50mg/mL
(NHS) during solution is added separately to the fluorescent labeling R2 solution that concentration is 1mg/mL, room temperature lucifuge reaction 10-15 minute, then
Vortex shakes after mixing 1 time, continues room temperature lucifuge and reacts 10 minutes, then reaction solution is centrifuged addition concentration after removing supernatant and is
In the amination polystyrene microsphere solution (solvent is PBS buffer solution) of 0.01wt%, 37 DEG C of lucifuge concussions are reacted 3 hours,
By the PBS solution containing 0.02wt% tween 20 by microsphere centrifuge washing 3 times, obtain the egg for detecting sperm acrosin activities in assessing
Bai Weiqiu conjugate.
Specific embodiment seven
Acrosin activity detecting step is as follows:
After fresh semen sample is liquefied completely, take 1mL in test tube, and be slowly added to HOF on upper strata
After substantially layering occurs in 1.2mL, test tube inclination constant temperature 37 DEG C is hatched 30 minutes and is carried out upstream preferably, then takes out higher slice liquid
Body, adds derivant Calcium ionophore and carries out acrosome reaction, and constant temperature lucifuge 37 DEG C adds acrosome film dyeing liquor after reacting 15 minutes
For detecting the protein microsphere conjugate of sperm acrosin activities in assessing, abundant endonuclease reaction 15 in PSA-FITC and specific embodiment one
Flow cytometer detection is carried out after minute.
Flow cytometer use forward scattering light and two parameter circle doors of side scattered light distinguish spermatid group
With protein microsphere conjugate group, detection protein microsphere conjugate group's green fluorescence and green fluorescence value, protein microsphere conjugate is anti-
Fluorescent value before and after should weaken degree, the activity of acrosin can be represented.
Claims (10)
1. the protein microsphere conjugate being used for detecting sperm acrosin activities in assessing, it is characterised in that its general structure is as follows:
Wherein M is the mono-dispersion microballoon that macromolecular material is made, and R1 is function associative key, and R2 is albumen or polypeptide, and P is that fluorescence divides
Son.
A kind of protein microsphere conjugate for detecting sperm acrosin activities in assessing the most according to claim 1, its feature exists
In a diameter of 1-50 micron of described mono-dispersion microballoon, material be polystyrene, polymethyl methacrylate, silicon dioxide,
Polylactic acid or polylactic-co-glycolic acid polymer, the outer surface functional modification carboxyl of described mono-dispersion microballoon, amino, hydroxyl
Or after sulfydryl, then its surface is carried out Polyethylene Glycol, polymethyl methacrylate or Fe3O4Coating modifying modified.
A kind of protein microsphere conjugate for detecting sperm acrosin activities in assessing the most according to claim 1, its feature exists
It is the amido link that is combined with amino of carboxyl or hydroxyl and carboxyl condensation key or sulfydryl and hydroxyl in: described function associative key
Condensation key.
A kind of protein microsphere conjugate for detecting sperm acrosin activities in assessing the most according to claim 1, its feature exists
In: described R2 is complete sequence albumen, fragment or the Ox blood serum containing arginine, the polypeptide of lysine or oocyte zona pellucida protein
Albuminous complete sequence albumen, fragment.
A kind of protein microsphere conjugate for detecting sperm acrosin activities in assessing the most according to claim 1, its feature exists
In: described fluorescence molecule is the fluorescent dye of coupling, and described fluorescent dye is Fluorescein isothiocyanate, isothiocyanate sieve
Red bright B, RB 200, Tetramethylrhodamine isothiocyanate, the red-X of rhodamine, texas Red, phycoerythrin, algae red eggs
In vain/texas Red tandem dye, phycoerythrin/Alexa Fluor 610 tandem dye, phycoerythrin/Alexa Fluor
647 tandem dyes, phycoerythrin/cyanine dye 5 tandem dye, phycoerythrin/cyanine dye 5.5 tandem dye, phycoerythrin/
Alexa Fluor 700 tandem dye, phycoerythrin/cyanine dye 7 tandem dye, phycocyanin, phycocyanin/cyanine dye
5.5 tandem dyes, phycocyanin/cyanine dye 7 tandem dye, cyanine dye.
6. a preparation method for the protein microsphere conjugate for detecting sperm acrosin activities in assessing described in claim 1, its
It is characterised by that concrete preparation method is as follows:
(1) protein fluorescence labelling
Fluorescence molecule Fluorescein isothiocyanate or isothiocyanate rhodamine B or Tetramethylrhodamine isothiocyanate are dissolved in diformazan
Base sulfoxide is configured to the fluorescence molecule solution that concentration is 10mg/mL, by complete sequence albumen, fragment or the cattle of oocyte zona pellucida protein
Sero-abluminous complete sequence albumen, fragment or be dissolved in carbonate buffer solution being configured to containing arginine, the polypeptide of lysine
The R2 solution of 10mg/ml, after being mixed with R2 solution by fluorescence molecule solution, obtains fluorescence in 16 hours in 4 DEG C of lucifuge coupling reactions
Protein solution, adds the fluorescin solution after coupling on the glucosan detached dowel washed with phosphate buffer in advance, so
Rear employing phosphate buffer carries out post separation with 20ul/s low speed, and the liquid collecting the brown flowed out at first obtains fluorescent labeling
R2 solution, and quantitative Analysis albumen or peptide concentration；Wherein contained albumen or peptide molecule and fluorescence molecule solution in R2 solution
The mol ratio of middle fluorescence molecule is (1: 9)-(1: 11)；
(2) fluorescent marker protein and mono-dispersion microballoon coupling
Carbodiimide solution and the N-hydroxy-succinamide solution that concentration is 50mg/mL that concentration is 50mg/mL are added respectively
Enter to concentration be 0.01wt% carboxylated mono-dispersion microballoon solution in, room temperature lucifuge react 10-15 minute, then vortex concussion
After mixing 1 time, continuing room temperature lucifuge and react 10 minutes, adding concentration after then reaction solution being centrifuged supernatant is 1mg/mL's
Fluorescent labeling R2 solution, 37 DEG C of lucifuge concussions are reacted 3 hours, wash centrifugal for microsphere by the PBS solution containing 0.02wt% tween 20
Wash 3 times, obtain the protein microsphere conjugate for detecting sperm acrosin activities in assessing；
Or Carbodiimide solution and the N-hydroxy-succinamide solution that concentration is 50mg/mL that concentration is 50mg/mL are divided
Not joining in the fluorescent labeling R2 solution that concentration is 1mg/mL, room temperature lucifuge is reacted 10-15 minute, then vortex concussion mixing
After 1 time, continuing room temperature lucifuge and react 10 minutes, adding concentration after then reaction solution being centrifuged supernatant is the ammonia of 0.01wt%
In base mono-dispersion microballoon solution, 37 DEG C of lucifuge concussions are reacted 3 hours, by the PBS solution containing 0.02wt% tween 20 by microsphere
Centrifuge washing 3 times, obtains the protein microsphere conjugate for detecting sperm acrosin activities in assessing；
Its general structure is as follows:
Wherein M is the mono-dispersion microballoon of a diameter of 1-50 micron, and material is polystyrene, polymethyl methacrylate, titanium dioxide
Silicon, polylactic acid or polylactic-co-glycolic acid polymer, the outer surface functional modification carboxyl of described mono-dispersion microballoon, amino,
After hydroxyl or sulfydryl, then surface is carried out Polyethylene Glycol, polymethyl methacrylate or Fe3O4Coating modifying modified；R1 is acyl
Amine key, R2 is albumen or polypeptide, and P is fluorescence molecule.
The preparation side of a kind of protein microsphere conjugate for detecting sperm acrosin activities in assessing the most according to claim 6
Method, it is characterised in that: described Carbodiimide solution, described N-hydroxy-succinamide solution, described mono-dispersion microballoon
It is 1: 1: 8: 8 with the volume ratio of fluorescent labeling R2 solution.
8. a preparation method for the protein microsphere conjugate for detecting sperm acrosin activities in assessing described in claim 1, its
It is characterised by that concrete preparation method is as follows:
(1) protein fluorescence labelling
Weigh 1g RB200 fluorescein and 2g phosphorus pentachloride is placed on after grinding 5min in fume hood in mortar, add 10ml anhydrous
Acetone, is stirred continuously, and filters, be marked labelled protein or polypeptide with filtrate, obtain fluorescence molecule solution after 5min；By concentration
For the oocyte zona pellucida protein solution of 20mg/ml, the carbonate buffer solution by volume 1: 1 of normal saline and 0.1mol/LpH 9.0
: obtain R2 solution after the ratio mixing of 1；Fluorescence molecule solution is mixed with R2 solution, adds the RB200 fluorescein of 0.1ml
Solution, the stirring of dropping limit, limit, in 4 DEG C, coupling reaction obtains fluorescin solution in 16 hours；By molten for the fluorescin after coupling
Liquid adds on the glucosan detached dowel washed with phosphate buffer in advance, then uses phosphate buffer with 20ul/s low speed
Carry out post separation, collect the liquid of the brown flowed out at first and obtain fluorescent labeling R2 solution, and quantitative Analysis albumen or polypeptide dense
Degree；Wherein in R2 solution, contained albumen or peptide molecule are 1: 9-1: 11 with the mol ratio of fluorescence molecule in fluorescence molecule solution.
(2) fluorescent marker protein and mono-dispersion microballoon coupling
Carbodiimide solution and the N-hydroxy-succinamide solution that concentration is 50mg/mL that concentration is 50mg/mL are added respectively
Enter to concentration be 0.01wt% carboxylated mono-dispersion microballoon solution in, room temperature lucifuge react 10-15 minute, then vortex concussion
After mixing 1 time, continuing room temperature lucifuge and react 10 minutes, adding concentration after then reaction solution being centrifuged supernatant is 1mg/mL's
Fluorescent labeling R2 solution, 37 DEG C of lucifuge concussions are reacted 3 hours, wash centrifugal for microsphere by the PBS solution containing 0.02wt% tween 20
Wash 3 times, obtain the protein microsphere conjugate for detecting sperm acrosin activities in assessing；
Or Carbodiimide solution and the N-hydroxy-succinamide solution that concentration is 50mg/mL that concentration is 50mg/mL are divided
Not joining in the fluorescent labeling R2 solution that concentration is 1mg/mL, room temperature lucifuge is reacted 10-15 minute, then vortex concussion mixing
After 1 time, continuing room temperature lucifuge and react 10 minutes, adding concentration after then reaction solution being centrifuged supernatant is the ammonia of 0.01wt%
In base mono-dispersion microballoon solution, 37 DEG C of lucifuge concussions are reacted 3 hours, by the PBS solution containing 0.02wt% tween 20 by microsphere
Centrifuge washing 3 times, obtains the protein microsphere conjugate for detecting sperm acrosin activities in assessing；
Its general structure is as follows:
Wherein M is the mono-dispersion microballoon of a diameter of 1-50 micron, and material is polystyrene, polymethyl methacrylate, titanium dioxide
Silicon, polylactic acid or polylactic-co-glycolic acid polymer, the outer surface functional modification carboxyl of described mono-dispersion microballoon, amino,
After hydroxyl or sulfydryl, then surface is carried out Polyethylene Glycol, polymethyl methacrylate or Fe3O4Coating modifying modified；R1 is acyl
Amine key, R2 is albumen or polypeptide, and P is fluorescence molecule.
The preparation side of a kind of protein microsphere conjugate for detecting sperm acrosin activities in assessing the most according to claim 8
Method, it is characterised in that: described Carbodiimide solution, described N-hydroxy-succinamide solution, described mono-dispersion microballoon
It is 1: 1: 8: 8 with the volume ratio of fluorescent labeling R2 solution.
10. an application for the protein microsphere conjugate for detecting sperm acrosin activities in assessing according to any one of right 1-5,
It is characterized in that, after detecting the specifically comprising the following steps that and liquefied completely by fresh semen sample of acrosin activity, taking 1mL in examination
Guan Zhong, and after upper strata is slowly added to HOF 1.2mL appearance substantially layering, test tube tilts constant temperature 37 DEG C and hatches 30 minutes
Carry out upstream preferred, then take out higher slice liquid, add derivant Calcium ionophore and carry out acrosome reaction, constant temperature lucifuge 37 DEG C
Acrosome film dyeing liquor PSA-FITC is added and for detecting the protein microsphere conjugate of sperm acrosin activities in assessing after reacting 15 minutes,
Fully endonuclease reaction carried out flow cytometer detection after 15 minutes, the protein microsphere conjugate fluorescence intensity that streaming is identified weaken degree
The activity of acrosin can be represented.
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