CN103743910B - CTBS albumen is as the application of target spot in screening sperm ovum binding inhibitor of sperm ovum binding acceptor - Google Patents
CTBS albumen is as the application of target spot in screening sperm ovum binding inhibitor of sperm ovum binding acceptor Download PDFInfo
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- CN103743910B CN103743910B CN201310739152.9A CN201310739152A CN103743910B CN 103743910 B CN103743910 B CN 103743910B CN 201310739152 A CN201310739152 A CN 201310739152A CN 103743910 B CN103743910 B CN 103743910B
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Abstract
The present invention relates to the target spot application in screening sperm ovum binding inhibitor of a kind of CTBS albumen as sperm ovum binding acceptor, CTBS is distinctive sperm ovum binding acceptor on rodent sperm, CTBS albumen only has specific expressed on the sperm of rodent, the sperm of people does not express this albumen, and CTBS antibody capable suppresses the sperm ovum binding of mouse.The sperm ovum binding inhibitor screened is Dequalinium Chloride, its half suppression constant < 100nM.The present invention adopts Dequalinium Chloride to use as the birth control agents of muroid, effectively can control the sperm ovum binding of rodent, reach and control the population quantity of muroid, realizing can control in rodent pests object without the need to agricultural chemicals, and its natural enemy eats this medicine but can not produce harm.
Description
Technical field
The present invention relates to the target spot application in screening sperm ovum binding inhibitor of a kind of CTBS albumen as sperm ovum binding acceptor.
Background technology
Dequalinium Chloride (Dequaliniumchloride, CAS#522-51-0), use as cancer therapy drug as a kind of pkc inhibitor, its IC50 is 7-18 μM, it is also a kind of selectivity apamin responsive type potassium-channel blocker simultaneously, and its IC50 is 1.1 μMs
[1,2,3,4].In recent years, Dequalinium Chloride is used for the antibacterials (medicine is called " dequaline chloride ") of non-antibiotic class as a kind of cationic surfactant.Recently, find that Dequalinium Chloride also can suppress the activity of microbe-derived chitin hydrolytic enzyme on nanomolar range
[5].Dequalinium Chloride is very micro-to the toxicity of human body, does not affect the reproduction of the mankind, has spread effect to eye and skin.To the lethal quantity of mouse be: oral 150-1000mg/kg body weight, hypodermic injection 70mg/kg body weight, intravenous injection 1.9mg/kg body weight; Be 500mg/kg body weight (MSDS) to the oral lethal dose of rabbit and dog.
And two-N-acetylchitobiose enzymes, namely Di-N-acetylchitobiase (being called for short CTBS) is the one of chitin hydrolytic enzyme, and in mammal, the liver only in rodent and mankind's body has expression
[6,7,8].
The reproduction isolation of species is a kind of general spontaneous phenomenons, and on the carbohydrates on ovum oolemma and sperm, the interaction of protein is one of mechanism wherein.Have report to show, oligosaccharides LewisX can be incorporated into mouse sperm, and can suppress the in vitro fertilization of mouse specifically, the fertility isolation of pointing out the albumen its sperm identifying LewisX may take part in mouse
[9], but be only a kind of conjecture.
The plague of rats refers to that muroid produces to agricultural etc. the harm caused.Muroid belongs to Mammalia (Mam-malia) grinding tooth day (Rodentia) animal, has kind more than 1600.Often, the pregnancy period is short, and farrowing rate is high in muroid breeding, and sexal maturity is fast, and quantity can sharply increase in a short time; Its adaptability is very strong, except Antarctica, can survive on the ground all over the world, underground, tree, in water, no matter Plain, high mountain, forest, grassland so that desert area has its trace, often huge disaster is led to productions such as agriculturals.
Therefore be badly in need of providing a kind of medicine that can control muroid fertility, especially suppress the inhibitor of the sperm ovum binding of muroid, and this inhibitor can suppress the plague of rats in the wild and its natural enemy eats harmless, without the need to adopting agricultural chemicals, the plague of rats being suppressed.
List of references of the present invention is as follows:
ManettaA，etal.GynecolOncol，1993，50(1)，38-44。
CastleNA，etal.EurJPharmacol，1993，236(2)，201-207。
DunnPM，etal.EurJPharmacol，1994，252(2)，189-194。
RotenbergSA，etal.JBiolChem，1998，273(4)，2390-2395。
PantoomS，etal，J.Biol.Chem.2011，286∶24312-24323。
AronsonNNJr，etal，ArchofBiochemBiophy,1989，272(2)，290-300。
LiuB，etal，Glycobiology,1999，9(6)，589-593。
ParkC，etal，JBiolChem2005，280，37204-37216。
KerrCL，etal，BiolReprod，2004，71(3)，770-777。
RaoFV,etal，ChemBio，2005，12，973-980。
ShurBD，etal，JBiolChem1988,263，17706-17714。
Summary of the invention
The object of the present invention is to provide a kind of CTBS albumen as the application of target spot in screening sperm ovum binding inhibitor of sperm ovum binding acceptor, and screen a kind of Dequalinium Chloride inhibitor that can suppress the sperm ovum binding of muroid, this inhibitor can be used for the population quantity controlling muroid, thus reach without the need to adopting agricultural chemicals can the effect of control in rodent pests, and the natural enemy of muroid to eat this medicine harmless.
To achieve these goals, the present invention adopts following technical scheme:
A kind of CTBS albumen is as the application of target spot in screening sperm ovum binding inhibitor of sperm ovum binding acceptor.
Above-mentioned CTBS is two-N-acetylchitobiose enzymes, i.e. Di-N-acetylchitobiase, referred to as CTBS.
We find that CTBS is distinctive sperm ovum binding acceptor on rodent sperm after deliberation, and CTBS only expresses on the sperm of rodent, and CTBS antibody can suppress the inseminatio externalis of mouse, and does not express this albumen on the sperm of people.
The inhibitor Dequalinium Chloride obtained through screening can suppress the in vitro fertilization of mouse effectively by suppressing the sperm ovum binding of mouse, and very micro-to the toxic action of human body.Find after deliberation, described Dequalinium Chloride is receptor protein CTBS inhibitor specific expressed on sperm in the sperm ovum binding of described muroid, CTBS is requisite albumen in the sperm ovum binding of muroid, therefore the birth control agents of Dequalinium Chloride as muroid is used, and Dequalinium Chloride can suppress the in vitro fertilization of mouse, the affinity that described inhibitor Dequalinium Chloride is combined with CTBS is strong, suppress the activity of the sperm ovum binding of muroid high, experiment shows that it suppresses half of sperm ovum binding to suppress constant < 100nM; Further, its half suppression constant is 50nM.
Therefore, Dequalinium Chloride, as the birth control agents of muroid, may be used for the population quantity controlling muroid, thus reaches the object of control in rodent pests.
The present invention adopts CTBS albumen as the Sites Screening sperm ovum binding inhibitor of sperm ovum binding acceptor, Dequalinium Chloride through screening acquisition effectively can control the sperm ovum binding of rodent, reach the population quantity controlling muroid, realizing can control in rodent pests object without the need to agricultural chemicals, and its natural enemy eats this medicine but can not produce harm.
Accompanying drawing explanation
Fig. 1 is the suppression figure of Dequalinium Chloride to mouse in vitro fertilization.
Fig. 2 A is the qualification thinking figure of LewisX acceptor.
Fig. 2 B is the mouse sperm albumen figure utilizing LewisX probe to find.
Fig. 2 C is the mass spectrophotometry collection of illustrative plates of albumen.
Fig. 3 is the picture that the antibody suppression LewisX of CTBS is attached to mouse sperm.
Fig. 4 is the binding site of LewisX on mouse sperm and the CTBS positioning result picture at mouse sperm.
Fig. 5 A be restructuring people source CTBS and Mouse Eggs oolemma there is interactional schematic diagram.
Fig. 5 B be restructuring people source CTBS and Mouse Eggs oolemma there is interactional light field and fluorescence photo.
Fig. 5 C is RFP or the RFP-CTBS Western blot analysis that the latex particle of CTBS antibody is enriched to.
Fig. 6 is the inductive effect figure of CTBS Antibody on Mouse sperm acrosome reaction.
Fig. 7 is the figure in vitro fertilization of the CTBS antibody suppression mouse of variable concentrations.
Fig. 8 is the western blot analysis figure of mouse, rat and human spermatogoa lysate.
Embodiment
By particular specific embodiment, embodiments of the present invention are described below, person skilled in the art scholar the content disclosed by this instructions can understand other advantages of the present invention and effect easily.
Embodiment 1:
Through experiment, we find that CTBS expresses on the sperm of rodent, and CTBS antibody can suppress the inseminatio externalis of mouse, and do not express this albumen on the sperm of people.Its experimental technique is as follows:
(1) discovery of CTBS on mouse sperm:
Utilize lewis oligosaccharide X (i.e. LewisX, is abbreviated as LeX) probe identification to arrive CTBS on mouse sperm, and prove that CTBS can be incorporated into the ovum oolemma of mouse.
(1) first identify LewisX acceptor, its identity process as shown in Figure 2 A, comprises the identity process of the agglutinoid albumen that can identify LewisX and endogenic Identification of Fusion Protein flow process of modifying containing LewisX.Its principle is that bovine serum albumin(BSA) (BSA) finishing LewisX is made Neoglycoproteins LeX-BSA, and this Neoglycoproteins is modified further upper UV-crosslinked reagent [6-(biotin acylamino)-2-(p-azido benzoyl is amino) hexanoyl is amino] ethyl-1,3 '-dithiopropionic acid sulfosuccinimide ester (Sulfo-NHS-2-(6-[biotinamido]-2-(p-azidobenzamido)-hexanoamido) ethyl-1,3 '-dithiopropionate, is called for short Sulfo-SBED) as LewisX probe.This probe forms LewisX probe-receptor complex through UV-crosslinked after mouse sperm, utilization can identify that four rib lotus agglutinins (Lotustetragonolobusagglutinin the is called for short LTA) resin of LewisX carrys out the LewisX probe-receptor complex in separation and purification spermatoblast lysate.Due to the albumen may modified containing endogenous LewisX in mouse sperm, be provided with the endogenic Identification of Fusion Protein flow process containing LewisX modification and distinguish this albuminoid (control group) in contrast.In general, namely the differential protein that two flow processs identify is likely the receptor protein of LewisX.
(2) albumen on the mouse sperm utilizing LewisX probe to find:
LewisX probe is utilized to find mouse sperm albumen, as shown in Figure 2 B.Differential band 1,2,3,4 in Fig. 2 B is taken off, for the Mass Spectrometric Identification of albumen, mass spectrophotometry figure as that shown in fig. 2 c, by analysis, likely as shown in table 1 with the albumen identity of carbohydrates effect.
Table 1
Note: the first hurdle in table 1 is nucleocytoplasmic ratio, the second hurdle is peptide sequence, and third column is protein identity.
(3) antibody capable of CTBS suppresses LewisX fluorescence probe to be attached to mouse sperm:
LewisX fluorescence probe LeX-BSA-Alexa430 adds fluorophor Alexa430 by the Neoglycoproteins LeX-BSA of LewisX and is prepared from.As shown in Figure 3, this probe can be incorporated into mouse epididymis afterbody take out mature sperm on (positive control).Antibody is the rabbit polyclonal antibody prepared by the CTBS recombinant protein in people source, purchased from Wuhan Sanying Bio-Technology Co., Ltd..
Find to only have the antibody capable of CTBS to suppress LeX-BSA-Alexa430 to be attached to mouse sperm after deliberation, as shown in Figure 3.Further, a kind of weak chitinase inhibitors oxpentifylline (Pentoxifylline is called for short PTX) also can suppress this combination
[10], nonspecific combination that the space steric effect eliminating antibody itself causes suppresses.
(4) binding site of LewisX on mouse sperm is consistent in the location of mouse sperm with CTBS:
LewisX fluorescence probe LeX-BSA-Alexa430 adds fluorophor Alexa430 by the Neoglycoproteins LeX-BSA of LewisX and is prepared from.This probe can be incorporated on the mature sperm of mouse epididymis afterbody taking-up, as shown in Fig. 4 illustration, consistent with previous report in conjunction with situation
[9].Antibody is the rabbit polyclonal antibody prepared by the CTBS recombinant protein in people source, purchased from Wuhan Sanying Bio-Technology Co., Ltd..By Immunofluorescent localization, we find that the binding site of LeX-BSA-Alexa430 on mouse sperm is consistent with the CTBS protein localization of mouse sperm, as shown in Figure 4.
(5) interaction that people source CTBS (i.e. rCTBS) of recombinating and Mouse Eggs oolemma (ZP) occur:
A. experimental considerations as shown in Figure 5A, is fixed on latex particle by red fluorescent protein (RFP) antibody, will be expressed in the restructuring RFP-CTBS protein enrichment of 293 cells on latex particle, and be added in the nutrient culture media of Mouse Eggs.If CTBS can identify the carbohydrates on Mouse Eggs oolemma, by the latex particle of sclerozone red fluorescence on mouse zona pellucida.
B. Fig. 5 B is light field (left side) and red fluorescence (right side) photo.Found that the latex particle being only enriched to RFP cannot be attached to (as shown in Fig. 5 B lastrow) on the ovum oolemma of mouse; And can be incorporated into (as shown in Fig. 5 B middle row) on the ovum oolemma of mouse with the latex particle of RFP-CTBS, CTBS antibody then can hinder this combination (as shown in Fig. 5 B next line).The fluorescence photo on right side confirms above-mentioned observation.
C. Fig. 5 C is western blot, and graphical results shows, the latex particle being combined with RFP antibody can enrichment RFP or RFP-CTBS.Wherein white arrow refers to RFP, and black arrow refers to RFP-CTBS.
The people source CTBS that the above results demonstrates restructuring can interact with Mouse Eggs oolemma.
(2) CTBS take part in the fertilization process of mouse.
(1) inductive effect of CTBS Antibody on Mouse sperm acrosome reaction:
Adopt the acrosome reaction of the CTBS antibody induction mouse sperm of variable concentrations between 0-30ug/ml, as shown in Figure 6, result proves that CTBS antibody can the acrosome reaction of inducing mouse sperm, and the feature that this receptor protein relevant with sperm ovum binding on the mouse sperm that prior art is reported shows is consistent
[11].
(2) CTBS antibody suppression mouse in vitro fertilization of variable concentrations:
Adopt the in vitro fertilization of the CTBS antibody suppression mouse of variable concentrations, as shown in Figure 7, result proves that CTBS antibody capable well suppresses the in vitro fertilization of mouse, shows that the CTBS on mouse sperm is really relevant with the sperm ovum binding of mouse further.
(3) western blot analysis of the sperm lysate of mouse, rat and people.
Carry out western blot analysis to the sperm lysate of mouse, rat and people, the applied sample amount of sperm lysate is mouse 50ug, rat 60ug, people 60ug.Antibody is the rabbit polyclonal antibody prepared by the CTBS recombinant protein in people source, purchased from Wuhan Sanying Bio-Technology Co., Ltd.; The applied sample amount of human cell line Hela cell pyrolysis liquid is 10ug; Use Alpha one tubulin (α-tubulin) as the reference of albumen applied sample amount.Result as shown in Figure 8, shows that the CTBS in CTBS antibody on human source has very high sensitivity, further demonstrates CTBS and do not express on human spermatogoa, and only on the sperm of rodent, have expression.
Embodiment 2: Dequalinium Chloride is as the inhibitors experiment suppressing mouse sperm ovum binding
Using Dequalinium Chloride as the inhibitor suppressing mouse sperm ovum binding, adopt each dosage experiments mouse eggs > 50 of CTBS variable concentrations, repeat 3 times, each amount ovum > 150, its experimental result as shown in Figure 1, its half suppression constant, at 50nM, shows that Dequalinium Chloride can well suppress the in vitro fertilization of mouse.
To sum up embodiment 1 and all results described in embodiment 2 show, CTBS is receptor protein specific expressed on rodent sperm, and it take part in the physiology course of sperm ovum binding, and CTBS antibody capable suppresses the in vitro fertilization of mouse.Dequalinium Chloride can well suppress the in vitro fertilization of mouse as the birth control agents of muroid, and experiment shows that its half suppression constant is at 50nM.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any person skilled in the art scholar all without prejudice under spirit of the present invention and category, can modify above-described embodiment or changes.Therefore, such as have in art usually know the knowledgeable do not depart from complete under disclosed spirit and technological thought all equivalence modify or change, must be contained by claim of the present invention.
Claims (4)
1.CTBS albumen is as the application of target spot in screening sperm ovum binding inhibitor of rodent sperm ovum binding acceptor.
2. CTBS albumen as claimed in claim 1 is as the application of target spot in screening sperm ovum binding inhibitor of rodent sperm ovum binding acceptor, it is characterized in that, CTBS is distinctive sperm ovum binding acceptor on rodent sperm, CTBS albumen only has specific expressed on the sperm of rodent, the sperm of people does not express this albumen, and CTBS antibody capable suppresses the sperm ovum binding of mouse.
3. CTBS albumen as claimed in claim 1 or 2 is as the application of target spot in screening sperm ovum binding inhibitor of rodent sperm ovum binding acceptor, and it is characterized in that, the sperm ovum binding inhibitor screened is Dequalinium Chloride; Described Dequalinium Chloride is the inhibitor of receptor protein CTBS specific expressed on sperm in the sperm ovum binding of muroid.
4. CTBS albumen as claimed in claim 3 is as the application of target spot in screening sperm ovum binding inhibitor of rodent sperm ovum binding acceptor, it is characterized in that, described Dequalinium Chloride suppresses half of muroid sperm ovum binding to suppress constant <100nM.
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Citations (3)
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| CN1136778A (en) * | 1993-12-10 | 1996-11-27 | 查珀尔希尔北卡罗来纳大学 | Sperm antigen corresponding to sperm zone binding protein autoantigenic epitope |
| CN1643148A (en) * | 2002-02-14 | 2005-07-20 | 独立行政法人科学技术振兴机构 | Mouse spermatogenesis genes, human male sterility-associated genes and diagnostic system using the same |
| CN101086809A (en) * | 2006-06-05 | 2007-12-12 | 中国医学科学院基础医学研究所 | An animal model for studying sperm generation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| DK2537942T3 (en) * | 2004-05-21 | 2015-12-14 | Trustees Of The University Of Arkansas System Board Of | Use of genudtryksprofilering to predict survival in cancer patients |
| AU2006238733A1 (en) * | 2005-04-16 | 2006-10-26 | Axordia Limited | Cytotrophoblast stem cell |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1136778A (en) * | 1993-12-10 | 1996-11-27 | 查珀尔希尔北卡罗来纳大学 | Sperm antigen corresponding to sperm zone binding protein autoantigenic epitope |
| CN1643148A (en) * | 2002-02-14 | 2005-07-20 | 独立行政法人科学技术振兴机构 | Mouse spermatogenesis genes, human male sterility-associated genes and diagnostic system using the same |
| CN101086809A (en) * | 2006-06-05 | 2007-12-12 | 中国医学科学院基础医学研究所 | An animal model for studying sperm generation |
Non-Patent Citations (2)
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| 卵巢移植对雄性受体鼠精子及生殖能力的影响;彭南妮等;《湖南农业大学学报（自然科学版）》;20100228;第36卷(第1期);73-76 * |
| 精卵结合分子机制的研究进展;周密;《中国男科学杂志》;20071231;第21卷(第12期);67-71 * |
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