CN102827285A - Mouse-specific anti-fertility polypeptide - Google Patents
Mouse-specific anti-fertility polypeptide Download PDFInfo
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- CN102827285A CN102827285A CN2011101624511A CN201110162451A CN102827285A CN 102827285 A CN102827285 A CN 102827285A CN 2011101624511 A CN2011101624511 A CN 2011101624511A CN 201110162451 A CN201110162451 A CN 201110162451A CN 102827285 A CN102827285 A CN 102827285A
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Abstract
The invention discloses six immune anti-fertility polypeptide segments, all of which have excellent immunogenicity. The six immune anti-fertility polypeptide segments can stimulate the organisms to generate specific antibodies and induce the anti-fertility function. According to the fertility comparison of antigen polypeptide immunized mice and the immunized mice in a control group (KLH (keyhole limpet homocyanin) control group), immunization by the six polypeptide antigens mixed group can significantly reduce the farrowing rate of the male and the female mice, wherein litter sizes in the male and the female mice is respectively reduced by 62.8% and 50%. The athletic ability and activity of sperms are significantly reduced in the HE6 polypeptide group or the mixed group immunized male mice. The polypeptide with high-efficiency anti-fertility effect establishes the foundation for research of the mouse-specific immune anti-fertility vaccines, and can be used for application of various anti-fertility preparations.
Description
Technical field:
The present invention relates to the polypeptide of one group of mouse specific anti fertility, particularly relate to the immune antifertility polypeptide that comprises mouse specificity epididymal proteins and sperm protein.
Background technology:
According to The World Health Organization's statistics, there is mouse in the whole world more than 17,000,000,000 at present, and there is mouse in China more than 4,000,000,000, and agricultural, forestry, livestock industry and human beings'health have been caused serious harm.According to Food and Argriculture OrganizationFAO (FAO) report, the whole world is equivalent to 25 impoverished nation's gross national incomes because the plague of rats causes grain loss to can reach the 15%-20% of Yield, the annual grain ration of enough 1.5 hundred million people.Be broken on the annual nearly ten million mu of forest of China and 1,000,000,000 mu of grasslands and be desert or waste continent, loss surpasses 3,000,000,000 Renminbi.In addition, mouse can be propagated multiple infecting both domestic animals and human communicable disease, carries to such an extent that can make that the people is morbific just to have 57 kinds surplus 200 in kind of the pathogenic agent.Therefore, research low toxicity special mouse killing technology harmless, not pollution of ecological environment and medicine are to have very important significance.
It is main killing mouse at present with chemical deratization, but chemical deratization exists problems such as people and animals' toxic side effect and muroid resistance, and because the muroid prolificacy is extremely strong, often kills soon, and the recovery of muroid quantity is also fast.In recent years, the structure that Australian scientist is successful the proteic mouse pox virus of expression mouse ovum zona pellucida ZP3, successfully suppressed the fertility prolificacy of muroid.But because the ZP3 albumen of mouse and people's ZP3 albumen height homology, promptly target does not possess specificity, thereby has caused the public to this mouse pox virus worry of the security of life-time service in the open air, has limited promoting the use of of it.
Therefore, the specific antifertility target of research mouse has very important significance.As the specific antifertility target of mouse, the most important condition is that target proteins must be the important reproduction GAP-associated protein GAP of function; Next is to have the mouse specificity; Be at last immune antifertility target can with antibody direct interaction, preferably membranin or secretory protein.
In addition, the vaccine prerequisite of impelling body to produce high titre antibody is that vaccine has strong immunogenicity.Modern protective immunity theory thinks that effectively protective immunity comes from the reasonable combination and the collocation of one group of epi-position.Proteantigen is not through its complete molecule performance function, but embodies its specificity through its epi-position.With regard to a certain proteantigen, just contain and the closely-related epi-position structure of immunity identification, produce immune response.Because there is cross reaction in the immunoreation that many natural antigens cause; Can not well satisfy the clinical practice needs; Therefore, in order to improve the specificity of proteantigen, just must on epitope levels, make a choice; Keep and improve the specific polypeptide fragment, to obtain better vaccine targets molecule.
Summary of the invention:
The object of the invention is to disclose one group of immune antifertility polypeptide that comprises mouse specificity epididymal proteins and sperm protein.
The objective of the invention is to realize through such technical scheme:
One group of polypeptide with antifertility, comprise any a plurality of balanced mix among the SEQ ID:1-6 shown in aminoacid sequence.
A kind of pharmaceutical prepn of antifertility, comprise among the above-mentioned SEQ ID 1-6 described a plurality of balanced mix arbitrarily shown in aminoacid sequence as activeconstituents.
A kind of preparation that kills mouse, comprise arbitrary described a plurality of balanced mix among the above-mentioned SEQ ID 1-6 shown in aminoacid sequence as activeconstituents.
A kind of pharmaceutical composition with antifertility action comprises described polypeptide and pharmaceutically acceptable carrier and vehicle.
A kind of specificity sperm protein Sp38 immunity antifertility polypeptide that derives from mouse, its aminoacid sequence is: SEQ ID:1 and SEQ ID:2.
A kind of specificity epididymal proteins HE6 immunity antifertility polypeptide that derives from mouse, its aminoacid sequence is: SEQ ID:3 and SEQ ID:4.
A kind of specificity sperm protein Slc26a8 immunity antifertility polypeptide that derives from mouse, its aminoacid sequence is: SEQ ID:5 and SEQ ID:6.
A kind of polypeptide with antifertility, its aminoacid sequence is: 6 polypeptide are processed with balanced mix among the SEQ ID:1-6.
A kind of albumen of antifertility is made up of with keyhole limpet hemocyanin (KLH) coupling respectively said polypeptide.
Polypeptide described in the above-mentioned SEQ ID 1-6 is during as activeconstituents, and the polypeptide total content is 100 μ g in the unit formulation, and " every " of unit formulation pointed injection agent mixes by equivalent when many polypeptide described in the SEQ ID 1-6 mix.
6 polypeptide fragments of the present invention have good immunogenicity, can stimulate body to produce specific antibody, induce antifertility action.Find that from the fertility contrast of antigenic peptide immune mouse and control group (KLH control group) immune mouse the immunity of 6 polypeptide antigen combined group can make male and farrowing rate female mice obviously descends, litter size descends 62.8% and 50% respectively.HE6 polypeptide group or combined group immunity male mice can significantly reduce the motor capacity and the vigor of sperm.Polypeptide with efficient antifertility effect shown in the present is that the research of mouse specific immune antifertility vaccine is laid a good foundation.
Antigen of the present invention is different with natural antigen on antigenic characteristic; But immunoreation specificity that causes and immune effect and natural antigen are as good as; It is little or do not have the advantage epi-position of cross reactivity to study to screen cross reactivity targetedly, and the result shows that 6 kinds of mouse specific anti fertility polypeptide that the applicant screens have good immune antifertility effect and have very optimistic application prospect.The immune antifertility polypeptide of six kinds of mouse specificity epididymal proteins and sperm protein has significant antigenicity, and immune antifertility effect is obvious, and has no side effect.
Following experimental example and embodiment further specify but are not limited to the present invention.
The applicant combines sufficient literature survey to find through comparative genomics and bioinformatics technique; Epididymis HE6 receptor protein, sperm Sp38 and Slc26a8 albumen, they are membranin, knock out the back male mice and all are sterile phenotype; All can be used as the potential target of immune antifertility; These three albumen of while are participated in different processes such as the heavily absorption of seminal fluid, the combination of smart ovum and sperm motility growth respectively, so they also possibly have synergy because the position that distributes is different.
Because this synthetic polypeptide molecular weight is too little, can not induce the generation immunoreation in animal body, therefore polypeptide and carrier proteins just can be carried out immunity after crosslinked, can significantly increase immunogenicity of antigens.(Keyhole limpet hemocyanin KLH) carries out crosslinkedly, is because it is a kind ofly self just can cause immunoreactive macromolecular carrier albumen, and immunogenicity is superior to other carrier proteins such as BSA to select keyhole limpet hemocyanin.Therefore, the present invention all uses KLH and polypeptide fragment to carry out coupling.
The screening of experimental example 1 antifertility peptide sequence
Both has the mouse Idiotype in order to screen; Has good antigenic polypeptide fragment again; The present invention has used Protean analysis kit in online polypeptide analysis instrument (http://peptideselect.invitrogen.com/peptide) and the DNAStar analysis software of Invitrogen company, and the protein sequence of people/mouse epididymal proteins HE6, sperm protein Sp38 and Slc26a8 is analyzed.Specificity is good according to choosing, antigenicity, wetting ability strong, avoid being positioned at the principle of helical region structural polypeptide; Finally choosing Sp38 proteic 4-19 position and 40-54 amino acids sequence, Slc26a8 proteic 2-20 position and 36-51 amino acids sequence and HE6 proteic 3-14 position and 35-50 amino acids sequence studies; The result of Protean analysis kit analysis peptide sequence sees Fig. 1 in the DNAStar analysis software, chooses the sequence results of polypeptide and sees table 1.
Amino acid sequence information in the table 1 antifertility polypeptide of the present invention
The antifertility research of experimental example 2 polypeptide immune mice serum antibody of the present invention
Reagent and preparation method thereof:
Encapsulate diluent composition and preparation: form the yellow soda ash of 1.5g and the NaHCO of 2.9g by 0.05mol/L yellow soda ash-sodium bicarbonate buffer liquid
3After the mixing, add bi-distilled water, transfer to pH9.6 to 1000mL.
Confining liquid is formed and preparation: 5%BSA-PBS solution, and BSA5g adds PBS (pH7.4) 100mL and obtains phosphate buffered saline buffer (PBS):
A liquid: 0.2mol/L biphosphate sodium water solution, the preparation method is NaH
2PO
4, H
2O 27.6g is dissolved in bi-distilled water 1000mL.
B liquid: the 0.2mol/L Sodium phosphate, dibasic aqueous solution, the preparation method is Na
2HPO
47H
2O 53.6g, Na
2HPO
412H
2O71.6g or Na
2HPO
42H
2O 35.6g is dissolved in bi-distilled water 1000mL.
Sample diluent: PBS, the 0.01mol/L phosphate buffered saline, the preparation method: PBA liquid 19mL mixes with PBB liquid 81mL, adds NaCl 18.5g, adds bi-distilled water to 1000mL.
Washings: PBST, pH7.4, the preparation method: PBS liquid 1000mL adds Tween200.5mL, transfers to pH7.4.
Substrate solution: OPD-H
2O
2Source: OPD (O-Phenylene Diamine<yan Suanyan>) (Sigma company, USA)
A liquid: the 0.1mol/L citric acid solution, the preparation method: Hydrocerol A 19.2g adds bi-distilled water to 1000mL.
B liquid: 0.2mol/LNaH
2PO
4Solution, preparation method: Na
2HPO
412H
2O 71.7g adds bi-distilled water to 1000mL.
Stop buffer: 2mol/LH
2SO
4Solution, preparation method: after in bi-distilled water 600mL, slowly dripping vitriol oil 100mL and continuous the stirring, add bi-distilled water to 900mL.
The goat anti-mouse IgG of horseradish peroxidase-labeled (Cell Signaling company, USA).
Fu Shi Freund's complete adjuvant and freund 's incomplete adjuvant (Sigma company, USA).
Bovine serum albumin (BSA) (Sigma company, USA).
Key instrument:
ELIASA (Bio-Rad company, USA)
96 holes gather the third ethene elisa plate (Costar company, USA)
Electric heating constant temperature hydro-thermal case (Shanghai one permanent Science and Technology Ltd., China)
Experimental technique:
Laboratory animal divides into groups: 50 of healthy Balb/c mouse (Beijing Military Medical Science Institute Experimental Animal Center provides), female and male each 25,6～8 ages in week; 10～16g is divided into 10 groups at random, mixes with 2 described in the SEQ ID 1-6 or many polypeptide and uses; The using dosage of every polypeptide mixes by equivalent, and each total dose of using is 100 μ g/, divides into groups respectively; Every group 5, by following identical method and dosage immune mouse.6 peptide species are used for the echelon design of immunity and see table 2.The echelon design of table 2 polypeptide immune mouse of the present invention
Immune programme for children: first immunisation, polypeptide is mixed with Fu Shi Freund's complete adjuvant equal-volume by 100 μ g//inferior dosage, fully emulsified, make immunogen, divide be expelled to Balb/c mouse peritoneum undertissue position at 2; At interval 2 weeks, booster immunization mixes by 100 μ g//inferior dosage polypeptide with the freund 's incomplete adjuvant equal-volume, fully emulsified, makes immunogen, divides be expelled to Balb/c mouse peritoneum undertissue position at 2; Again 2 weeks of interval, in kind repeat booster immunization 1 time.Immunity finishes back 14 days, will accept mice immunized tail vein and get blood, collects its serum.
The collection of sample with separate: with the venesection of immune mouse tail, separation of serum is preserved subsequent use in-20 ℃ of refrigerators.
Indirect enzyme-linked immunosorbent (ELISA) method detects antibody: adopt the standard indirect elisa method to measure antibody titers in the serum, measure every treated animal specific antibody titre respectively.Coating buffer, diluent and washings, ELIAS secondary antibody are goat anti-mouse igg, and atural object is OPD; Using 96 hole elisa plate envelope antigen concentration is 10 μ g/mL, and every hole 100 μ l put 37 ℃ of constant water bath box after 4 hours, discarded liquid in the hole; With 5%BSA-PBS sealase mark reacting hole, 37 ℃ of constant water bath box are after 40 minutes, and washing hydroful hole is washed each 3 minutes 3 times.Add each hole according to the serum ratio of 10 times of serial dilutions, every hole 100 μ l, 1 hour after scouring of 37 ℃ of constant water bath box 3 times; Add ELIAS secondary antibody, 37 ℃ of constant water bath box 40 minutes add substrate OPD, and every hole 100 μ l put 37 ℃ and covered the light coupling reaction 20 minutes, and termination reaction in 20 minutes, in 492nm mensuration absorbance A value, is made negative control with normal mouse serum with ELIASA.The result is with the average OD value representation in two multiple holes, and sample OD value to be measured/negative control OD value＞2.1 are judged to be the positive.Immunity is female sees Fig. 2 and Fig. 3 with male mice Serum Antibody Detection result.
Detected result shows: the antibody titer that produces to polypeptide Sp38-1, HE6-1 and Slc26a8-2 in the polypeptide group immunity male mice serum is all higher; Antibody titer to Slc26a8-1 in the combined group immunity male mice serum is the highest, and all decreases to the antibody titer of Sp38-1, HE6-1 and Slc26a8-2; All produced all higher antibody to 6 peptide species in the polypeptide immune female mice serum.
The antifertility effect of experimental example 3 polypeptide immune mouse of the present invention
Main agents:
Fu Shi Freund's complete adjuvant and freund 's incomplete adjuvant (Sigma company, USA).
Experimental technique:
Laboratory animal divides into groups: 50 of healthy Balb/c mouse (Beijing Military Medical Science Institute Experimental Animal Center provides), female and male each 25,6～8 ages in week; 10～16g is divided into 10 groups at random, and the polypeptide described in the SEQ ID 1-6 mixes when using; Carry out sample according to the echelon design table 2 in the experimental example 2 and divide into groups, the using dosage of every polypeptide mixes by equivalent, and each total dose of using is that 100 μ g/ only divide into groups respectively; Every group 5, by following identical method and dosage immune mouse.
Immune programme for children: first immunisation, polypeptide is mixed with Fu Shi Freund's complete adjuvant equal-volume by 100 μ g//inferior dosage, fully emulsified, make immunogen, divide be expelled to Balb/c mouse peritoneum undertissue position at 2; At interval 2 weeks, booster immunization mixes by 100 μ g//inferior dosage polypeptide with the freund 's incomplete adjuvant equal-volume, fully emulsified, makes immunogen, divides be expelled to Balb/c mouse peritoneum undertissue position at 2; Again 2 weeks of interval, in kind repeat booster immunization 1 time.Immunity finishes the back to be observed 14 days, again immune mouse and normal healthy mouse was mated raising according to 1: 1 ratio.Mate after 14 days and divide cage, divide cage after 4 days, observe the mouse condition of production morning every day, and the statistics litter size, the result sees table 3.
The antifertility effect detection result of table 3 polypeptide immune male mice of the present invention
Detected result shows: the immune separately influence (the P value is all greater than 0.05) that the fertility of male mouse is not had significance of any one group of polypeptide among Sp38, HE6 and the Slc26a8; And the litter size of combined group immune mouse obviously reduces; Litter size is 32.6% of a control group; The P value is less than 0.02, and each polypeptide of prompting combined group possibly produce synergy, has disturbed normal growing process.Though HE6-1 and HE6-2 from the HE6 polypeptide do not have statistical significance (the P value is 0.15) to the influence that male mouse gives birth to, sterile with the male mouse that can cause 20% after its immunity, litter size is 62.8% of a control group.Combined group immunity female mice has immune antifertility action (the P value is less than 0.05), can make the litter size of female mouse reduce by 50%, and prompting Sp38 and Slc26a8 also have synergy.The immunity of Sp38 and Slc26a8 polypeptide group can make litter size reduce 33% and 25% respectively, and the P value is all greater than 0.05 (being respectively 0.28 and 0.14).
The pathology of experimental example 5 polypeptide immune mouse epididymis tissues of the present invention detect antifertility research
Main agents:
4% Paraformaldehyde 96 (Beijing Suo Laibao Science and Technology Ltd., China).
Experimental technique:
Laboratory animal divides into groups: 50 of healthy Balb/c mouse (Beijing Military Medical Science Institute Experimental Animal Center provides), female and male each 25,6～8 ages in week; 10～16g is divided into 10 groups at random, and the polypeptide described in the SEQ ID 1-6 mixes when using; Carry out sample according to the echelon design table 2 in the experimental example 2 and divide into groups, the using dosage of every polypeptide mixes by equivalent, and each total dose of using is that 100 μ g/ only divide into groups respectively; Every group 5, by following identical method and dosage immune mouse.
Immune programme for children: first immunisation, polypeptide is mixed with Fu Shi Freund's complete adjuvant equal-volume by 100 μ g//inferior dosage, fully emulsified, make immunogen, divide be expelled to Balb/c mouse peritoneum undertissue position at 2; At interval 2 weeks, booster immunization mixes by 100 μ g//inferior dosage polypeptide with the freund 's incomplete adjuvant equal-volume, fully emulsified, makes immunogen, divides be expelled to Balb/c mouse peritoneum undertissue position at 2; Again 2 weeks of interval, in kind repeat booster immunization 1 time.Immunity finishes the back to be observed 14 days, again immune mouse and normal healthy mouse was mated raising according to 1: 1 ratio.Mate after 14 days and divide cage, the dissection mouse is got the epididymis tissue and utilizes 4% Paraformaldehyde 96 to fix, and detects the pathological change whether antibody that produces behind the immune male mouse can cause male mouse reproductive system, and the result sees Fig. 4.
Detected result shows: the hybrid injection group (dividing into groups 4) and the tube chamber of HE6 polypeptide group mice immunized epididymis head and afterbody diminish, and tubule is thinning to be dredged; The intraluminal sperm of afterbody also reduces.Considerable change does not all take place in Sp38 and Slc26a8 polypeptide group mice immunized epididymis head and afterbody.
Experimental example 6 polypeptide immunes of the present invention are to the influence of mouse to sperm motility and vigor
Experimental technique:
Laboratory animal divides into groups: 50 of healthy Balb/c mouse (Beijing Military Medical Science Institute Experimental Animal Center provides), female and male each 25,6～8 ages in week; 10～16g is divided into 10 groups at random, and the polypeptide described in the SEQ ID 1-6 mixes when using; Carry out sample according to the echelon design table 2 in the experimental example 2 and divide into groups, the using dosage of every polypeptide mixes by equivalent, and each total dose of using is that 100 μ g/ only divide into groups respectively; Every group 5, by following identical method and dosage immune mouse.
Immune programme for children: first immunisation, polypeptide is mixed with Fu Shi Freund's complete adjuvant equal-volume by 100 μ g//inferior dosage, fully emulsified, make immunogen, divide be expelled to Balb/c mouse peritoneum undertissue position at 2; At interval 2 weeks, booster immunization mixes by 100 μ g//inferior dosage polypeptide with the freund 's incomplete adjuvant equal-volume, fully emulsified, makes immunogen, divides be expelled to Balb/c mouse peritoneum undertissue position at 2; Again 2 weeks of interval, in kind repeat booster immunization 1 time.Immunity finishes the back to be observed 14 days, again immune mouse and normal healthy mouse was mated raising according to 1: 1 ratio.Mate after 14 days and divide cage, detect motility of sperm, motor capacity and the sperm count of the male mice of polypeptide immune.The result sees table 4.
Table 4 polypeptide immune of the present invention is to the influence result of mouse sperm motion with vigor
Detected result shows: the immunity of HE6 polypeptide group can significantly suppress the motion and the vigor of sperm, and sperm count also decreases.The sperm motility of hybrid injection group and vigor also significantly reduce, and Sp38 and the immunity of Slc26 polypeptide group are little to sperm motility and effect of vigor, show that HE6 polypeptide group plays an important role in the immune antifertility of hybrid injection group.
Description of drawings:
Fig. 1: the Protean analysis kit is analyzed the result of peptide sequence in the DNAStar analysis software
Fig. 2: polypeptide ELISA of the present invention detects male mouse-anti serum antibody titer
Fig. 3: polypeptide ELISA of the present invention detects female mouse mouse-anti serum antibody titer
Fig. 4: the pathology detected result of polypeptide immune mouse epididymis tissue of the present invention
Following embodiment all can realize the effect of above-mentioned experimental example.
Embodiment 1
To the mouse-anti fertility peptide sequence of screening on full-automatic polypeptide synthetic instrument by fixing carboxyl to the aminoterminal synthetic back acquisition bullion that successively decreases; Dissolve laggard horizontal high voltage liquid chromatography (HPLC) analysis through 30% second eyeball again; Calculate main peak area and collection; The synthetic polypeptide of purifying after freezing vacuum is drained, mass spectrum is identified.Synthetic trust Beijing health of peptide sequence is century bio tech ltd completion.
Carrier proteins is selected keyhole limpet hemocyanin (Keyhole limpet hemocyanin; KLH); With bifunctional reagent maleic acid amides phenylformic acid-N-succinate (MBS) connection method KLH and synthetic peptide SEQ ID 1-2 balanced mix are used, carry out coupling: get KLH 5mg (0.11 μ mol contains Methionin 2.2 μ mol); Be dissolved in 0.75mL coupling buffer 1 (the 50mmol/L borate buffer solution, pH8.5); 3mgMBS (11 μ mol) is dissolved in 75 μ l diformamides (DMF).Divide MBS solution 3 times and add KLH solution, rotation mixing, room temperature effect 30 minutes.After centrifugal rapidly coupling buffer 2 (0.1mol/L phosphoric acid salt, 0.15mol/LNaCL, 0.01mol/LNa are used in reaction mixture (about 0.8mL) adding in advance
2EDTA, pH=7.0) equilibrated PD-10 post.With coupling buffer 2 wash-outs, collect elutriant (being MBS-KLH solution).The synthetic polypeptide of dissolving 1.5mg adds MBS-KLH damping fluid 0.56mL in 0.15mL coupling buffer 2, and rotation mixes under the room temperature; Acting on 2 hours postposition spends the night for 4 ℃; Phosphate buffered saline buffer equilibrated PD-10 post is used in reaction mixture (0.7mL) adding in advance, and the phosphoric acid buffer wash-out is collected effluent.KLH, MBS-KLH and conjugate are carried out SDS-PAGE (polyacrylamide gel electrophoresis) identify coupling efficiency,,, process injection according to ordinary method with the albumen that obtains, after be used for female mouse of the present invention or the fertility of male mouse-anti is used.
Embodiment 2
To the mouse-anti fertility peptide sequence of screening on full-automatic polypeptide synthetic instrument by fixing carboxyl to the aminoterminal synthetic back acquisition bullion that successively decreases; Dissolve laggard horizontal high voltage liquid chromatography (HPLC) analysis through 30% second eyeball again; Calculate main peak area and collection; The synthetic polypeptide of purifying after freezing vacuum is drained, mass spectrum is identified.Synthetic trust Beijing health of peptide sequence is century bio tech ltd completion.
Carrier proteins is selected keyhole limpet hemocyanin (Keyhole limpet hemocyanin; KLH); With bifunctional reagent maleic acid amides phenylformic acid-N-succinate (MBS) connection method 2 polypeptide balanced mix described in KLH and the SEQ ID 3-4 are used, carried out coupling: get KLH 5mg (0.11 μ mol contains Methionin 2.2 μ mol); Be dissolved in 0.75mL coupling buffer 1 (the 50mmol/L borate buffer solution, pH8.5); 3mgMBS (11 μ mol) is dissolved in 75 μ l diformamides (DMF).Divide MBS solution 3 times and add KLH solution, rotation mixing, room temperature effect 30 minutes.After centrifugal rapidly coupling buffer 2 (0.1mol/L phosphoric acid salt, 0.15mol/L NaCL, 0.01mol/LNa are used in reaction mixture (about 0.8mL) adding in advance
2EDTA, pH=7.0) equilibrated PD-10 post.With coupling buffer 2 wash-outs, collect elutriant (being MBS-KLH solution).The synthetic polypeptide of dissolving 1.5mg adds MBS-KLH damping fluid 0.56mL in 0.15mL coupling buffer 2, and rotation mixes under the room temperature; Acting on 2 hours postposition spends the night for 4 ℃; Phosphate buffered saline buffer equilibrated PD-10 post is used in reaction mixture (0.7mL) adding in advance, and the phosphoric acid buffer wash-out is collected effluent.KLH, MBS-KLH and conjugate are carried out SDS-PAGE (polyacrylamide gel electrophoresis) identify coupling efficiency,,, process tablet according to ordinary method with the albumen that obtains, after be used for female mouse of the present invention or the fertility of male mouse-anti is used.
Embodiment 3
To the mouse-anti fertility peptide sequence of screening on full-automatic polypeptide synthetic instrument by fixing carboxyl to the aminoterminal synthetic back acquisition bullion that successively decreases; Dissolve laggard horizontal high voltage liquid chromatography (HPLC) analysis through 30% second eyeball again; Calculate main peak area and collection; The synthetic polypeptide of purifying after freezing vacuum is drained, mass spectrum is identified.Synthetic trust Beijing health of peptide sequence is century bio tech ltd completion.
Carrier proteins is selected keyhole limpet hemocyanin (Keyhole limpet hemocyanin; KLH); With bifunctional reagent maleic acid amides phenylformic acid-N-succinate (MBS) connection method 2 polypeptide balanced mix described in KLH and the SEQ ID 5-6 are used, carried out coupling: get KLH 5mg (0.11 μ mol contains Methionin 2.2 μ mol); Be dissolved in 0.75mL coupling buffer 1 (the 50mmol/L borate buffer solution, pH8.5); 3mgMBS (11 μ mol) is dissolved in 75 μ l diformamides (DMF).Divide MBS solution 3 times and add KLH solution, rotation mixing, room temperature effect 30 minutes.After centrifugal rapidly coupling buffer 2 (0.1mol/L phosphoric acid salt, 0.15mol/L NaCL, 0.01mol/LNa are used in reaction mixture (about 0.8mL) adding in advance
2EDTA, pH=7.0) equilibrated PD-10 post.With coupling buffer 2 wash-outs, collect elutriant (being MBS-KLH solution).The synthetic polypeptide of dissolving 1.5mg adds MBS-KLH damping fluid 0.56mL in 0.15mL coupling buffer 2, and rotation mixes under the room temperature; Acting on 2 hours postposition spends the night for 4 ℃; Phosphate buffered saline buffer equilibrated PD-10 post is used in reaction mixture (0.7mL) adding in advance, and the phosphoric acid buffer wash-out is collected effluent.KLH, MBS-KLH and conjugate are carried out SDS-PAGE (polyacrylamide gel electrophoresis) identify coupling efficiency,,, process film according to ordinary method with the albumen that obtains, after be used for female mouse of the present invention or the fertility of male mouse-anti is used.
Embodiment 4
To the mouse-anti fertility peptide sequence of screening on full-automatic polypeptide synthetic instrument by fixing carboxyl to the aminoterminal synthetic back acquisition bullion that successively decreases; Dissolve laggard horizontal high voltage liquid chromatography (HPLC) analysis through 30% second eyeball again; Calculate main peak area and collection; The synthetic polypeptide of purifying after freezing vacuum is drained, mass spectrum is identified.Synthetic trust Beijing health of peptide sequence is century bio tech ltd completion.
Carrier proteins is selected keyhole limpet hemocyanin (Keyhole limpet hemocyanin; KLH); With bifunctional reagent maleic acid amides phenylformic acid-N-succinate (MBS) connection method 6 polypeptide balanced mix described in KLH and the SEQ ID 1-6 are used, carried out coupling: get KLH 5mg (0.11 μ mol contains Methionin 2.2 μ mol); Be dissolved in 0.75mL coupling buffer 1 (the 50mmol/L borate buffer solution, pH8.5); 3mgMBS (11 μ mol) is dissolved in 75 μ l diformamides (DMF).Divide MBS solution 3 times and add KLH solution, rotation mixing, room temperature effect 30 minutes.After centrifugal rapidly coupling buffer 2 (0.1mol/L phosphoric acid salt, 0.15mol/L NaCL, 0.01mol/LNa are used in reaction mixture (about 0.8mL) adding in advance
2EDTA, pH=7.0) equilibrated PD-10 post.With coupling buffer 2 wash-outs, collect elutriant (being MBS-KLH solution).The synthetic polypeptide of dissolving 1.5mg adds MBS-KLH damping fluid 0.56mL in 0.15mL coupling buffer 2, and rotation mixes under the room temperature; Acting on 2 hours postposition spends the night for 4 ℃; Phosphate buffered saline buffer equilibrated PD-10 post is used in reaction mixture (0.7mL) adding in advance, and the phosphoric acid buffer wash-out is collected effluent.KLH, MBS-KLH and conjugate are carried out SDS-PAGE (polyacrylamide gel electrophoresis) identify coupling efficiency,,, process injection according to ordinary method with the albumen that obtains, after be used for female mouse of the present invention or the fertility of male mouse-anti is used.
Claims (12)
1. the polypeptide of an antifertility is characterized in that comprising that any a plurality of aminoacid sequence among the SEQ ID:1-6 is according to balanced mix.
2. antifertility polypeptide as claimed in claim 1 is characterized in that SEQ ID:1 and SEQ ID:2 process with balanced mix.
3. antifertility polypeptide as claimed in claim 1 is characterized in that SEQ ID:3 and SEQ ID:4 process with balanced mix.
4. antifertility polypeptide as claimed in claim 1 is characterized in that SEQ ID:5 and SEQ ID:6 process with balanced mix.
5. antifertility polypeptide as claimed in claim 1 is characterized in that SEQ ID:1-6 processes with balanced mix.
6. the albumen of an antifertility is characterized in that being made up of the polypeptide and keyhole limpet hemocyanin (KLH) coupling of many balanced mix of SEQ ID 1-6.
7. antifertility albumen as claimed in claim 6 is characterized in that SEQ ID:1 and SEQ ID:2 process with balanced mix.
8. antifertility albumen as claimed in claim 6 is characterized in that SEQ ID:3 and SEQ ID:4 process with balanced mix.
9. antifertility albumen as claimed in claim 6 is characterized in that SEQ ID:5 and SEQ ID:6 process with balanced mix.
10. antifertility albumen as claimed in claim 6 is characterized in that SEQ ID:1-6 processes with balanced mix.
11., it is characterized in that the pharmaceutical composition that said polypeptide and pharmaceutically acceptable carrier and vehicle are formed like the described antifertility polypeptide of claim 1-5.
12., it is characterized in that the pharmaceutical composition that said albumen and pharmaceutically acceptable carrier and vehicle are formed like the described antifertility albumen of claim 6-10.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110162451.1A CN102827285B (en) | 2011-06-16 | 2011-06-16 | Mouse-specific anti-fertility polypeptide |
Applications Claiming Priority (1)
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| CN115554403A (en) * | 2022-08-16 | 2023-01-03 | 山东大学 | Application of steroid hormone DHEA as receptor ADGRG2 agonist ligand |
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| US20020006656A1 (en) * | 1999-12-23 | 2002-01-17 | Holloway James L. | Zcys5: a member of the cystatin superfamily |
| CN101906163A (en) * | 2009-06-05 | 2010-12-08 | 上海交通大学医学院 | Immunocontraceptive synthetic peptide and immunocontraceptive chimeric peptide and application thereof |
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| US20020006656A1 (en) * | 1999-12-23 | 2002-01-17 | Holloway James L. | Zcys5: a member of the cystatin superfamily |
| CN101906163A (en) * | 2009-06-05 | 2010-12-08 | 上海交通大学医学院 | Immunocontraceptive synthetic peptide and immunocontraceptive chimeric peptide and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115554403A (en) * | 2022-08-16 | 2023-01-03 | 山东大学 | Application of steroid hormone DHEA as receptor ADGRG2 agonist ligand |
| CN115554403B (en) * | 2022-08-16 | 2024-03-08 | 山东大学 | Use of the steroid hormone DHEA as receptor ADGRG2 agonist ligand |
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