CN106896016A - The rapid extracting method of acrosin and its flow cytometer detection method of activity - Google Patents
The rapid extracting method of acrosin and its flow cytometer detection method of activity Download PDFInfo
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- CN106896016A CN106896016A CN201710302258.0A CN201710302258A CN106896016A CN 106896016 A CN106896016 A CN 106896016A CN 201710302258 A CN201710302258 A CN 201710302258A CN 106896016 A CN106896016 A CN 106896016A
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- acrosin
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- 108090000107 Acrosin Proteins 0.000 title claims abstract description 85
- 102100026041 Acrosin Human genes 0.000 title claims abstract description 82
- 230000000694 effects Effects 0.000 title claims abstract description 49
- 238000001514 detection method Methods 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 38
- 239000007788 liquid Substances 0.000 claims abstract description 42
- 239000000284 extract Substances 0.000 claims abstract description 35
- 239000012528 membrane Substances 0.000 claims abstract description 33
- 102000004190 Enzymes Human genes 0.000 claims abstract description 29
- 108090000790 Enzymes Proteins 0.000 claims abstract description 29
- 230000029087 digestion Effects 0.000 claims abstract description 29
- 229920001184 polypeptide Polymers 0.000 claims abstract description 17
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 17
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 17
- 239000006228 supernatant Substances 0.000 claims abstract description 17
- 238000005119 centrifugation Methods 0.000 claims abstract description 15
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 239000004005 microsphere Substances 0.000 claims abstract description 11
- 239000008188 pellet Substances 0.000 claims abstract description 11
- 239000000758 substrate Substances 0.000 claims abstract description 11
- 239000000725 suspension Substances 0.000 claims abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims abstract description 10
- 210000000582 semen Anatomy 0.000 claims abstract description 7
- 230000008878 coupling Effects 0.000 claims abstract description 5
- 238000010168 coupling process Methods 0.000 claims abstract description 5
- 238000005859 coupling reaction Methods 0.000 claims abstract description 5
- 238000007670 refining Methods 0.000 claims abstract description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 40
- 239000000243 solution Substances 0.000 claims description 27
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- 239000011780 sodium chloride Substances 0.000 claims description 12
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 10
- 229910052739 hydrogen Inorganic materials 0.000 claims description 10
- 239000001257 hydrogen Substances 0.000 claims description 10
- 229910052708 sodium Inorganic materials 0.000 claims description 10
- 239000011734 sodium Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000008367 deionised water Substances 0.000 claims description 9
- 229910021641 deionized water Inorganic materials 0.000 claims description 9
- 238000001556 precipitation Methods 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
- 238000005374 membrane filtration Methods 0.000 claims description 8
- OOSZCNKVJAVHJI-UHFFFAOYSA-N 1-[(4-fluorophenyl)methyl]piperazine Chemical compound C1=CC(F)=CC=C1CN1CCNCC1 OOSZCNKVJAVHJI-UHFFFAOYSA-N 0.000 claims description 7
- 229940074545 sodium dihydrogen phosphate dihydrate Drugs 0.000 claims description 7
- 239000004475 Arginine Substances 0.000 claims description 6
- 101001091385 Homo sapiens Kallikrein-6 Proteins 0.000 claims description 6
- 102100034866 Kallikrein-6 Human genes 0.000 claims description 6
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 6
- PXXJHWLDUBFPOL-UHFFFAOYSA-N benzamidine Chemical compound NC(=N)C1=CC=CC=C1 PXXJHWLDUBFPOL-UHFFFAOYSA-N 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 6
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 6
- 239000004472 Lysine Substances 0.000 claims description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 5
- 229920003229 poly(methyl methacrylate) Polymers 0.000 claims description 5
- 239000004926 polymethyl methacrylate Substances 0.000 claims description 5
- 239000003643 water by type Substances 0.000 claims description 5
- 102000004533 Endonucleases Human genes 0.000 claims description 4
- 108010042407 Endonucleases Proteins 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 4
- 150000002431 hydrogen Chemical class 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 239000004793 Polystyrene Substances 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims description 3
- 230000006037 cell lysis Effects 0.000 claims description 3
- 230000002255 enzymatic effect Effects 0.000 claims description 3
- 238000002372 labelling Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 229910052751 metal Inorganic materials 0.000 claims description 3
- 239000002184 metal Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 3
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 3
- 229920000642 polymer Polymers 0.000 claims description 3
- 238000004062 sedimentation Methods 0.000 claims description 3
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 3
- ISIJQEHRDSCQIU-UHFFFAOYSA-N tert-butyl 2,7-diazaspiro[4.5]decane-7-carboxylate Chemical compound C1N(C(=O)OC(C)(C)C)CCCC11CNCC1 ISIJQEHRDSCQIU-UHFFFAOYSA-N 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 2
- XONPDZSGENTBNJ-UHFFFAOYSA-N molecular hydrogen;sodium Chemical compound [Na].[H][H] XONPDZSGENTBNJ-UHFFFAOYSA-N 0.000 claims description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims 3
- 238000001914 filtration Methods 0.000 claims 1
- DJFBJKSMACBYBD-UHFFFAOYSA-N phosphane;hydrate Chemical compound O.P DJFBJKSMACBYBD-UHFFFAOYSA-N 0.000 claims 1
- CKRORYDHXIRZCH-UHFFFAOYSA-N phosphoric acid;dihydrate Chemical compound O.O.OP(O)(O)=O CKRORYDHXIRZCH-UHFFFAOYSA-N 0.000 claims 1
- 239000004626 polylactic acid Substances 0.000 claims 1
- 229920002223 polystyrene Polymers 0.000 claims 1
- 239000000377 silicon dioxide Substances 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000001215 fluorescent labelling Methods 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 239000007850 fluorescent dye Substances 0.000 abstract description 2
- 239000002245 particle Substances 0.000 abstract 1
- 230000008569 process Effects 0.000 description 9
- 235000013601 eggs Nutrition 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 230000008859 change Effects 0.000 description 5
- 210000000170 cell membrane Anatomy 0.000 description 3
- 230000006641 stabilisation Effects 0.000 description 3
- 238000011105 stabilization Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 102000010911 Enzyme Precursors Human genes 0.000 description 2
- 108010062466 Enzyme Precursors Proteins 0.000 description 2
- DEOKFPFLXFNAON-UHFFFAOYSA-N N-α-Benzoyl-DL-arginine 4-nitroanilide hydrochloride Chemical compound Cl.C=1C=C([N+]([O-])=O)C=CC=1NC(=O)C(CCCN=C(N)N)NC(=O)C1=CC=CC=C1 DEOKFPFLXFNAON-UHFFFAOYSA-N 0.000 description 2
- 230000030120 acrosome reaction Effects 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000034217 membrane fusion Effects 0.000 description 2
- -1 nitro acyl Amine Chemical class 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 230000008010 sperm capacitation Effects 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 239000006173 Good's buffer Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000005002 female reproductive tract Anatomy 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 230000007903 penetration ability Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- NFIYTPYOYDDLGO-UHFFFAOYSA-N phosphoric acid;sodium Chemical compound [Na].OP(O)(O)=O NFIYTPYOYDDLGO-UHFFFAOYSA-N 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000002133 sample digestion Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Engineering & Computer Science (AREA)
- Dispersion Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses the rapid extracting method and its flow cytometer detection method of activity of acrosin,Feature is that extracting method includes being added to seminal fluid in centrifuge tube,Centrifugation sucks refining partly,Retain lower floor's Sperm pellets,It is subsequently adding rupture of membranes liquid,After Sperm pellets is rolled into a ball even suspension,Supernatant is removed in centrifugation,It is subsequently adding extract solution,Make sperm suspension,3 collection supernatants are centrifuged again,Obtain acrosome enzyme extract,Its active flow cytometer detection method is by polypeptide or protein microsphere coupling technology and polypeptide or protein fluorescence labelling technique,Using being connected on microballoon after acrosin digestion substrate is carried out into fluorescence labeling,The fluorescent microsphere of certain particle diameter can be recognized by flow cytometer,The height of acrosin activity in fluorescence power direct reaction sample,Advantage is combined by rapid extraction and flow cytometer detection,So that detection efficiency is greatly improved,Increased the sensitivity of detection.
Description
Technical field
The present invention relates to a kind of extractive technique of acrosin, more particularly, to a kind of rapid extraction of acrosin
The flow cytometer detection method of method and its activity.
Background technology
After human sperm enters female genital tract, it is necessary to by one section of maturation, fertility, sperm during this are obtained
A series of physiological changes referred to as capacitation for being occurred.Sperm after capacitation passes through ovarian cumulus, corona radiata, when reaching oolemma, passes through
Smart ovum identification, plasma membrane change, acrosome enzyme r e lease, oolemma enzymolysis, Sperm nuclei are molten with nuclear phase into egg cell, and this is a series of
Complicated process constitutes sensu lato acrosome reaction, and narrow sense acrosome reaction mainly include plasma membrane fusion that oolemma induces with
Two critical process of release of acrosin.What current laboratory research and clinical diagnosis was most paid close attention to is also the two processes
Molecular mechanism and clinical detection.Wherein plasma membrane fusion predominantly detects method is become by in-vitro simulated induction perforatorium film
Change, and method using specific stain is observed, acrosome enzyme r e lease process is primarily upon the Activity determination of the enzyme for discharging.
Acrosin is present on perforatorium inner membrance and ambitus film, and it is important proteolytic enzyme in fertilization process, generally with nothing
The zymogen forms of activity are present in acrosome.When sperm head enters egg vitellary membrane, acrosome proenzyme is activated being acrosin water
Solution egg vitellary membrane, makes sperm pass through egg vitellary membrane finally and egg fusion.Acrosin activity is to judge mankind spermatozoon function and life
Educate the strong and weak important evaluation index of power.Meanwhile, because acrosin activity is higher, Sperm penetration ability is stronger, therefore as auxiliary
Help one of evaluation index that reproductive technology (ART) helps pregnant mode to select.Acrosin activity quantitative determination is that supplementary reproduction center is normal
Detection project.
The detection of acrosin is most initially using acrosome enzyme antibody to carry out radioimmunology or fluorescent immune method is detected,
The ability of sperm fertilization is judged by the quantity for observing acrosin in perforatorium.Because the method has many limitation, after
Come Kennedy et al. be found that it is a kind of can be by the substrate of acrosin institute digestion, Na- benzoyl-DL-arginines-to nitro acyl
Amine hydrochlorate (BAPNA), this substrate can be developed the color after acrosome enzyme hydrolysis, be extracted by by acrosin, then digestion
BAPNA substrates, the height of acrosin activity is judged by the depth of end reaction system color.The index that the method draws is more
The physiological function of acrosin can be reacted, the universal detection method as andrology laboratory.But due to sperm sample
Middle acrosin activity in itself is not high, usually μ IU ranks, therefore the sample relatively low for some enzyme activity has sensitivity not
Enough high-leveled and difficult problems with accurate quantitative analysis.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of quick release for realizing acrosin and separate essence
The rapid extracting method of sub- acrosin, while providing, a kind of sensitivity is high, the streaming of the fast sperm acrosin activities in assessing of detection speed
Detection method.
The present invention solve the technical scheme that is used of above-mentioned technical problem for：
1st, a kind of rapid extracting method of acrosin, comprises the following steps：
(1) 7.5*10 will be contained6The seminal fluid of individual sperm number is added in centrifuge tube, and 2min is centrifuged in 10000rpm, is inhaled
Refining after going to be centrifuged in pipe partly retains lower floor's Sperm pellets；
(2) 200uL rupture of membranes liquid is added in the centrifuge tube containing Sperm pellets, after Sperm pellets is rolled into a ball even suspension, in
After 7000rpm centrifugations 2min, remove supernatant, be subsequently adding 400uL extract solutions, make sperm in 37 DEG C of suspension 10min, then at
After 12000rpm centrifugations 3min, supernatant is collected, that is, obtain acrosome enzyme extract, wherein phosphorous acid dihydride in per 100ml rupture of membranes liquid
Sodium 1.2g, Triton-1000.2-0.4ml and Sodium azide 0.05g；1g containing BSA in per 100ml extract solutions, sodium chloride 0.35g,
Disodium-hydrogen 1.19g, sodium dihydrogen phosphate dihydrate 0.25g and Sodium azide 0.05g.
The compound method of described rupture of membranes liquid is as follows：Precise sodium dihydrogen phosphate 1.2g, Sodium azide 0.05g and 0.2-
0.4ml Triton-100 are dissolved in 80 milliliter deionized waters, after adjusting pH to 4-6 with 1N HCl, are settled to deionized water
100 milliliters, rupture of membranes liquid is obtained with after 0.22 micron of membrane filtration.Rupture of membranes liquid employs Triton-100 and phosphate-buffered
Liquid, the rate of release that can accelerate enzyme and the stabilization for promoting enzyme, triton-100 destruction sperm membranes, are easy to the release of acrosin,
Phosphate buffer maintains low pH, keeps enzyme activity；Sodium azide is preservative.
The compound method of described extract solution is as follows：Precise bovine serum albumin(BSA) (BSA) 1g, sodium chloride 0.35g, phosphorus
One hydrogen sodium 1.19g, sodium dihydrogen phosphate dihydrate 0.25g and Sodium azide 0.05g of acid, is dissolved in 80 milliliter deionized waters, uses 1N hydrogen-oxygens
Change sodium and adjust pH to 7.0-8.0, be then settled to 100 milliliters with deionized water, carried with after 0.22 micron of membrane filtration
Take liquid.Extract solution employs BSA and phosphate buffer, can accelerate the rate of release of enzyme and the stabilization of promotion enzyme, phosphate
Buffer solution maintains constant pH, is easy to the release of acrosin, and BSA keeps the enzyme activity of stabilization；Sodium azide is preservative.
2nd, a kind of flow cytometer detection method of sperm acrosin activities in assessing, comprises the following steps：
(1) sample is collected：By fresh collection, complete sperm sample is liquefied after Spermatozoon counter is counted, take 7.5*
106The seminal fluid of individual sperm number, 2min is centrifuged in 10000rpm, is inhaled with pipette tips and is abandoned supernatant, leaves sperm sample precipitation stand-by；
(2) cell lysis：200uL rupture of membranes liquid is added in being precipitated to the spermatoblast obtained in step (1), it is upper and lower with pipette tips
Piping and druming cell, makes spermatoblast fully resuspended, by (no more than 5min is otherwise by shadow after re-suspension liquid 7000rpm centrifugations 2-4min
Ring acrosin activity), supernatant is sucked with pipette tips, leave precipitation stand-by；
(3) acrosin is dissolved：400uL extract solutions are added in being rolled into a ball to the sedimentation cell in step (2), is inhaled up and down with pipette tips
Beat, fully mix precipitation, after being incubated 10min on 37 degrees Celsius of metal baths, 12000rpm centrifugation 2min, Aspirate supernatant is
Body zyme extract；Temperature is too high or too low, overlong time or it is too short can all influence extract acrosin activity；
(4) digestion fluorescent microsphere：The acrosome zyme extract 100uL obtained in aspiration step (3), adds 100uLPB bufferings
Solution, adds and contains 5*104The solution of individual acrosin Substrate fluorescence microballoon, makes digestion system for 250uL, 24 degrees Celsius of incubations
After 1 hour, terminator terminating reaction 1-2min is added；
(5) flow cytometer detection：Mixed liquor after endonuclease reaction in step (4) is detected on flow cytometer；
(6) enzyme activity is calculated：According to the fluorescent value obtained on flow cytometer, fluorescence decay rate is calculated, decayed according to enzyme activity
Rate standard curve y=0.0001X2+ 0.0355X+67.324, calculates the enzyme activity for obtaining acrosin, and wherein y represents acrosin enzyme activity
Property, unit is μ IU, and X represents fluorescence decay rate.
Rupture of membranes formula of liquid described in step (2) is：Phosphoric acid sodium dihydrogen 1.2g, TritonX- in per 100ml rupture of membranes liquid
1000.2-0.4ml and Sodium azide 0.05g.
Extract recipe described in step (3) is：1g containing BSA, sodium chloride 0.35g, phosphoric acid in per 100ml extract solutions
One hydrogen sodium 1.19g, sodium dihydrogen phosphate dihydrate 0.25g and Sodium azide 0.05g.
The formula of PB cushioning liquid is described in step (4)：0.1M NaH2PO4, 0.5M NaCl, pH value is 7-8, configuration
Good buffer solution is with 0.22 micron of membrane filtration.
Terminator described in step (4) is the deionized water solution containing benzenecarboximidamide, is contained in every 100ml terminate liquids
17.46g benzenecarboximidamides.
Acrosin Substrate fluorescence microballoon described in step (4) includes a diameter of micron-sized microballoon, on described microballoon
Coupling has can be by the polypeptide of acrosin digestion or protein, and described polypeptide or protein labeling has fluorescence molecule group, described
The fluorescence molecule group fluorescence launched that is excited on flow cytometer after light irradiation can be detected by flow cytometer.
The material of described microballoon is polystyrene (PS), polymethyl methacrylate (PMMA), silica (SiO2)、
PLA (PLA) or polylactic-co-glycolic acid polymer (PLGA)；Described polypeptide refer to containing can by acrosin recognize
The polypeptide of arginine or lysine, described protein refers to contain the arginine that can be recognized by acrosin or the egg of lysine
White matter
Specific Cleaning Principle is as follows：After spermatoblast is collected by centrifugation, the rupture of membranes liquid weight containing TritonX-100 is added
Outstanding, the TritonX-100 of low concentration can increase membrane passage so that acrosin can be good at being discharged into cell
Outward, after adding extract solution, the acrosin in spermatoblast is discharged well, and the salt ion in extract solution can keep enzyme
Activity higher.After adding digestion Substrate fluorescence microballoon, acrosin can cut away the polypeptide or egg marked on microballoon
In vain, fluorescence molecule is made to be split away off from microballoon, the system after digestion will reduce through flow cytomery, fluorescent value.Subtract
Go after the fluorescent value of control group just calculate the fluorescence decay rate of this digestion, substitute into standard curve y=0.0001X2+
The enzyme activity of acrosin can be just obtained after 0.0355X+67.324.
Compared with prior art, the advantage of the invention is that：Present invention firstly discloses the rapid extraction of acrosin
The flow cytometer detection method of method and its activity, rupture of membranes liquid promotes sperm release acrosin in rapid extracting method, is subsequently adding and carries
Take liquid extract sperm in acrosin, rupture of membranes liquid using Triton-100 it is gentle make spermatoblast rupture of membranes, be subsequently adding extraction
Liquid enables acrosin to discharge and keeps acrosin enzyme activity, and whole experiment process being capable of the interior completion in 15min.Existing document
The acid treatment extracting mode of report is, it is necessary to more than 16h.With time saving, process gentle, the characteristics of safe.Additionally, the present invention is extracted
Acrosin can be kept under the conditions of -20 DEG C one week, meet some projects to original acrosin (without spermatoblast)
Demand.
Meanwhile, the invention provides a kind of flow cytometer detection method of sperm acrosin activities in assessing, first passage microballoon polypeptide egg
White coupling technology and polypeptide protein fluorescent labelling techniques realize the activity of flow cytomery acrosin, according to fluorescent value
Power directly judges the height of acrosin activity, the laboratory operating procedures in detection process is simplified, while being suitable for high flux
The continuous detection of multisample, greatly improves the efficiency of detection, and the flow cytometer for realizing acrosin in sample is quantitatively examined
Survey..In addition, the present invention extracts formula by optimizing, acrosin more in spermatoblast can be extracted so that some enzyme activity are very
Low sample can also be detected well, improve the sensitivity of detection, while being terminated using terminator of the present invention
After reaction, after room temperature 2 hours, up flow type detection finds that testing result, without substantially change, illustrates that this digestion system is more steady
It is fixed, the limitation detected immediately after flow cytometer detection will react is relieved, significantly widen the application scenario of flow cytometer detection and answered
Use scope.The present invention is combined by rapid extraction and flow cytometer detection so that detection efficiency is greatly improved.
Brief description of the drawings
After Fig. 1 is the digestion sample flow cytometer of specific embodiment two, SSC-A and FSC-A intensity dot plots and R1；
Fig. 2 is the front and rear peak fluorescence intensity on flow cytometer of the fluorescent microsphere digestion of specific embodiment two.
Specific embodiment
The present invention is described in further detail below in conjunction with accompanying drawing embodiment.
Specific embodiment one
A kind of rapid extracting method of acrosin, comprises the following steps：
(1) 7.5*10 will be contained6The seminal fluid of individual sperm number is added in centrifuge tube, and 2min is centrifuged in 10000rpm, is inhaled
Refining after going to be centrifuged in pipe partly retains lower floor's Sperm pellets；
(2) 200uL rupture of membranes liquid is added in the centrifuge tube containing Sperm pellets, after Sperm pellets is rolled into a ball even suspension, in
After 7000rpm centrifugations 2min, remove supernatant, be subsequently adding 400uL extract solutions, make sperm in 37 DEG C of suspension 10min, then at
After 12000rpm centrifugations 3min, supernatant is collected, that is, obtain acrosome enzyme extract, wherein phosphorous acid dihydride in per 100ml rupture of membranes liquid
Sodium 1.2g, Triton-1000.2-0.4ml and Sodium azide 0.05g；1g containing BSA in per 100ml extract solutions, sodium chloride 0.35g,
Disodium-hydrogen 1.19g, sodium dihydrogen phosphate dihydrate 0.25g and Sodium azide 0.05g.
The acrosin of extraction is stored in -20 DEG C and can preserve one week
The compound method of above-mentioned rupture of membranes liquid is as follows：Precise sodium dihydrogen phosphate 1.2g, Sodium azide 0.05g and 0.2-
0.4ml Triton-100 are dissolved in 80 milliliter deionized waters, after adjusting pH to 4-6 with 1N HCl, are settled to deionized water
100 milliliters, rupture of membranes liquid is obtained with after 0.22 micron of membrane filtration.
The compound method of said extracted liquid is as follows：Precise bovine serum albumin(BSA) (BSA) 1g, sodium chloride 0.35g, phosphoric acid
One hydrogen sodium 1.19g, sodium dihydrogen phosphate dihydrate 0.25g and Sodium azide 0.05g, is dissolved in 80 milliliter deionized waters, uses 1N hydroxides
Sodium adjusts pH to 7.0-8.0, is then settled to 100 milliliters with deionized water, is extracted with after 0.22 micron of membrane filtration
Liquid.
Specific embodiment two
A kind of flow cytometer detection method of sperm acrosin activities in assessing, comprises the following steps：
(1) sample is collected：By fresh collection, complete sperm sample is liquefied after Spermatozoon counter is counted, take 7.5*
106The seminal fluid of individual sperm number, 2min is centrifuged in 10000rpm, is inhaled with pipette tips and is abandoned supernatant, leaves sperm sample precipitation stand-by；
(2) cell lysis：200uL rupture of membranes liquid is added in being precipitated to the spermatoblast obtained in step (1), it is upper and lower with pipette tips
Piping and druming cell, makes spermatoblast fully resuspended, by (no more than 5min is otherwise by shadow after re-suspension liquid 7000rpm centrifugations 2-4min
Ring acrosin activity), supernatant is sucked with pipette tips, leave precipitation stand-by；Wherein rupture of membranes formula of liquid is：Per in 100ml rupture of membranes liquid
Phosphoric acid sodium dihydrogen 1.2g, Triton-1000.2-0.4ml and Sodium azide 0.05g；
(3) acrosin is dissolved：400uL extract solutions are added in being rolled into a ball to the sedimentation cell in step (2), is inhaled up and down with pipette tips
Beat, fully mix precipitation, after being incubated 10min on 37 degrees Celsius of metal baths, 12000rpm centrifugation 2min, Aspirate supernatant is
Body zyme extract；Wherein extract recipe is：1g containing BSA, sodium chloride 0.35g, disodium-hydrogen in per 100ml extract solutions
1.19g, sodium dihydrogen phosphate dihydrate 0.25g and Sodium azide 0.05g；
(4) digestion fluorescent microsphere：The acrosome zyme extract 100uL obtained in aspiration step (3), adds 100uLPB bufferings
Solution, adds and contains 5*104The solution of individual acrosin Substrate fluorescence microballoon, makes digestion system for 250uL, 24 degrees Celsius of incubations
After 1 hour, terminator terminating reaction 1-2min is added；The formula of wherein PB cushioning liquid is：0.1M NaH2PO4, 0.5M
NaCl, pH value is 7-8, and the buffer solution for having configured is with 0.22 micron of membrane filtration；The terminator be going containing benzenecarboximidamide from
The sub- aqueous solution, 17.46g benzenecarboximidamides are contained in every 100ml terminate liquids；The acrosin Substrate fluorescence microballoon includes a diameter of micron order
Microballoon, on the microballoon coupling have and fluorescence molecule can be had by the polypeptide of acrosin digestion or protein, polypeptide or protein labeling
Group, the fluorescence molecule group fluorescence launched that is excited on flow cytometer after light irradiation can be examined by flow cytometer
Survey；The material of the microballoon is polystyrene (PS), polymethyl methacrylate (PMMA), silica (SiO2), PLA
Or polylactic-co-glycolic acid polymer (PLGA) (PLA)；Polypeptide refers to contain the arginine or bad ammonia that can be recognized by acrosin
The polypeptide of acid, protein refers to contain the arginine that can be recognized by acrosin or the protein of lysine；
(5) flow cytometer detection：Mixed liquor after endonuclease reaction in step (4) is detected on flow cytometer；With BD
(flow cytometer of C6, Calibur or other models can be also used as a example by the C5 flow cytometers of company), detection parameter is
In slow patterns, the intensity dot plots according to SSC-A and FSC-A carry out circle door R1 (see Figure of description 1), collect 5000 in R1
It is individual, by this 5000 average fluorescent strengths of point output valve as a result, the scope of the FSC of fluorescent microsphere is shown by Fig. 1
It is 0.40-0.76 (* 106) and SSC scope be 0.11-0.50 (* 106), R1 refers in Fig. 1 institute in rectangle black box
The region for showing；Fig. 2 is the front and rear peak fluorescence intensity on flow cytometer of fluorescent microsphere digestion, and fluorescent microsphere is illustrated by Fig. 2
Before and after digestion, its fluorescence values changes, and fluorescence peak figure is subjected to displacement, and figure shows the fluorescence peak reduction of microballoon after digestion, peak
Figure offsets to the left.
Mean Fluorescence Mean and the CV value of microballoon is as shown in the table before and after digestion,
| # | Sample | Gate | Count | %R1 | Mean X | CVX |
| 1 | After digestion | R1 | 5.101 | 100.00% | 10.090 | 18.98% |
| 2 | Before digestion | 5.078 | 100.00% | 14.505 | 19.46% |
Before and after by upper table explanation digestion, the parameter of fluorescent microsphere, wherein Mean values are most meaningful, represent digestion effect, with
Mean values 14505 compare before digestion, and the fluorescent value after digestion is 10090, and fluorescent value is decayed, and expression there occurs endonuclease reaction.
(6) enzyme activity is calculated：According to the fluorescent value obtained on flow cytometer, fluorescence decay rate is calculated, decayed according to enzyme activity
Rate standard curve y=0.0001X2+ 0.0355X+67.324, calculates the enzyme activity for obtaining acrosin, and wherein y represents acrosin enzyme activity
Property, unit is μ IU, and X represents fluorescence decay rate.
The computing formula of above-mentioned fluorescence decay rate is (fluorescent value-control group fluorescent value after digestion)/control group fluorescent value *
100%, the drawing process of standard curve is：Use registered listing acrosin immue quantitative detection reagent box (producer be Shenzhen China
Health biomedical engineering Co., Ltd, 20 person-portions of model/box, equipment registration number is：Guangdong food medicine prison tool (standard) word 2,012 the
No. 2400314) certain density sperm sample measures are obtained with a series of enzyme activity values as ordinate, simultaneous equal sperm sample
Digestion fluorescent microsphere is carried out using this patent methods described, a series of attenuation rate values being calculated as abscissa, by two sides
A series of data point simulation corresponding to method obtains standard curve y=0.0001X2+ 0.0355X+67.324, wherein y are represented
Acrosin enzymatic activity, unit is pIU, and X represents fluorescence decay rate.
Described above not limitation of the present invention, the present invention is also not limited to the example above.The art it is common
In essential scope of the invention, change, remodeling, addition or the replacement made should also belong to protection of the invention to technical staff
Scope.
Claims (10)
1. a kind of rapid extracting method of acrosin, it is characterised in that comprise the following steps：
(1) 7.5*10 will be contained6The seminal fluid of individual sperm number is added in centrifuge tube, and 2min is centrifuged in 10000rpm, sucks centrifugation
The refining in pipe partly, retains lower floor's Sperm pellets afterwards；
(2) 200uL rupture of membranes liquid is added in the centrifuge tube containing Sperm pellets, after Sperm pellets is rolled into a ball even suspension, in
After 7000rpm centrifugations 2min, remove supernatant, be subsequently adding 400uL extract solutions, make sperm in 37 DEG C of suspension 10min, then at
After 12000rpm centrifugations 3min, supernatant is collected, that is, obtain acrosome enzyme extract, wherein phosphorous acid dihydride in per 100ml rupture of membranes liquid
Sodium 1.2g, Triton-100 0.2-0.4ml and Sodium azide 0.05g；1g containing BSA in per 100ml extract solutions, sodium chloride 0.35g,
Disodium-hydrogen 1.19g, sodium dihydrogen phosphate dihydrate 0.25g and Sodium azide 0.05g.
2. a kind of rapid extracting method of acrosin according to claim 1, it is characterised in that described rupture of membranes liquid
Compound method it is as follows：Precise sodium dihydrogen phosphate 1.2g, Sodium azide 0.05g and 0.2-0.4ml Triton-100 are dissolved in 80
In milliliter deionized water, after adjusting pH to 4-6 with 1N HCl, 100 milliliters are settled to deionized water, with 0.22 micron of filter membrane
Rupture of membranes liquid is obtained after filtering.
3. a kind of rapid extracting method of acrosin according to claim 1, it is characterised in that described extract solution
Compound method it is as follows：Precise bovine serum albumin(BSA) (BSA) 1g, sodium chloride 0.35g, disodium-hydrogen 1.19g, two water phosphorus
Acid dihydride sodium 0.25g and Sodium azide 0.05g, is dissolved in 80 milliliter deionized waters, adjusts pH to 7.0-8.0 with 1N NaOH, so
100 milliliters are settled to deionized water afterwards, extract solution is obtained with after 0.22 micron of membrane filtration.
4. a kind of flow cytometer detection method of sperm acrosin activities in assessing, it is characterised in that comprise the following steps：
(1) sample is collected：By fresh collection, complete sperm sample is liquefied after Spermatozoon counter is counted, take 7.5*106It is individual
The seminal fluid of sperm number, 2min is centrifuged in 10000rpm, is inhaled with pipette tips and is abandoned supernatant, leaves sperm sample precipitation stand-by；
(2) cell lysis：200uL rupture of membranes liquid is added in being precipitated to the spermatoblast obtained in step (1), is blown and beaten up and down with pipette tips
Cell, makes spermatoblast fully resuspended, (is otherwise pushed up influence no more than 5min after re-suspension liquid 7000rpm is centrifuged into 2-4min
Body enzymatic activity), supernatant is sucked with pipette tips, leave precipitation stand-by；
(3) acrosin is dissolved：400uL extract solutions are added in being rolled into a ball to the sedimentation cell in step (2), is inhaled up and down with pipette tips and beaten, filled
Divide and mix precipitation, after being incubated 10min on 37 degrees Celsius of metal baths, 12000rpm centrifugation 2min, the body that Aspirate supernatant is
Zyme extract；
(4) digestion fluorescent microsphere：The acrosome zyme extract 100uL obtained in aspiration step (3), adds 100uLPB cushioning liquid,
Add and contain 5*104The solution of individual acrosin Substrate fluorescence microballoon, makes digestion system for 250uL, and 24 degrees Celsius are incubated 1 hour
Afterwards, terminator terminating reaction 1-2min is added；
(5) flow cytometer detection：Mixed liquor after endonuclease reaction in step (4) is detected on flow cytometer；
(6) enzyme activity is calculated：According to the fluorescent value obtained on flow cytometer, fluorescence decay rate is calculated, according to enzyme activity attenuation rate mark
Directrix curve y=0.0001X2+ 0.0355X+67.324, calculates the enzyme activity for obtaining acrosin, and wherein y represents acrosin enzymatic activity,
Unit represents fluorescence decay rate for μ IU, X.
5. the flow cytometer detection method of sperm acrosin activities in assessing according to claim 4, it is characterised in that described in step (2)
Rupture of membranes formula of liquid be：Phosphoric acid sodium dihydrogen 1.2g, Triton-100 0.2-0.4ml and Sodium azide in per 100ml rupture of membranes liquid
0.05g。
6. the flow cytometer detection method of sperm acrosin activities in assessing according to claim 4, it is characterised in that described in step (3)
Extract recipe be：1g containing BSA, sodium chloride 0.35g, disodium-hydrogen 1.19g, phosphate dihydrate two in per 100ml extract solutions
Hydrogen sodium 0.25g and Sodium azide 0.05g.
7. the flow cytometer detection method of sperm acrosin activities in assessing according to claim 4, it is characterised in that described in step (4)
The formula of PB cushioning liquid is：0.1M NaH2PO4, 0.5M NaCl, pH value is 7-8, and the buffer solution for having configured is with 0.22 micron
Membrane filtration.
8. the flow cytometer detection method of sperm acrosin activities in assessing according to claim 4, it is characterised in that described in step (4)
Terminator be the deionized water solution containing benzenecarboximidamide, per 100ml terminate liquids in contain 17.46g benzenecarboximidamides.
9. the flow cytometer detection method of sperm acrosin activities in assessing according to claim 4, it is characterised in that：Institute in step (4)
The acrosin Substrate fluorescence microballoon stated includes a diameter of micron-sized microballoon, and have can be by acrosin digestion for coupling on described microballoon
Polypeptide or protein, described polypeptide or protein labeling have fluorescence molecule group, and described fluorescence molecule group is in streaming
The fluorescence launched after the light irradiation that is excited on cell instrument can be detected by flow cytometer.
10. the flow cytometer detection method of sperm acrosin activities in assessing according to claim 9, it is characterised in that：Described microballoon
Material be polystyrene, polymethyl methacrylate, silica, PLA or polylactic-co-glycolic acid polymer；It is described
Polypeptide refer to that, containing the arginine that can be recognized by acrosin or the polypeptide of lysine, described protein refers to containing can
The arginine or the protein of lysine recognized by acrosin.
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| CN109609483A (en) * | 2019-02-15 | 2019-04-12 | 深圳华康生物医学工程有限公司 | A kind of extracting method of acrosin |
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