JPH05506639A - Regulation of milk secretion - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Regulation of milk secretion Background of the invention The present invention provides protein freshly isolated from cow's milk; protein or its relative to regulate milk secretion in lactating animals ANT1 bodies.

Description of prior art The rate of milk secretion by a lactating animal is regulated by the frequency of milk removal.

That is, an animal's milk supply is replaced by an animal's offspring or offspring. has a mechanism that works to adapt to the requirements of farmers' milk regimes. Ru. Part of this regulation is due to galactinization during suckling and withdrawal. This is achieved by the release of galactopoietic hormones. Ru.

However, the Haunch Research Institute on Milk-producing Goats (Scott A study by Rand workers showed that other factors were involved. .

This is at the local level (local 1 level), that is, at the individual level of the breast. It is an inhibitor that reduces the secretion of milk in each lymph gland.

This inhibitor is a whey protein with a molecular weight of 1O-30KDa. Goat's milk fraction containing tin(w)Iey proteins) i, lk fraction), and this molecular weight range is the Nominal size of filter used in ultra filtration (nominal 5 It has already been shown that it is determined by The effect is shown in vitro (i It has been demonstrated both in vitro and in vivo.

This in vitro technology (Biochemistry of C. J. Wide et al., J, 242.28 5-288.1987) is a study of rabbit mammary glands with and without milk fraxilan. Cultivating flat pieces and demonstrating inhibition of lactose and casein synthesis In addition, G. M. Stewart et al. J. Endocrinology 1 18, R1-R3, 1988), in vivo technology (C, J, Wi de etc., Quar Journal of Experimental Ph. ysilogY 73.391-397.1988), this milk milk Laccidine was introduced into goat milk via a test canal. injected into the lymph glands. Temporary administration (dosse)-dependent decrease in milk yield observed.

Determine whether a suppressant is present in the cow and, if so, use a chemical or The problem remained of purifying it sufficiently to identify it by biological synthesis.

Lord & Hada Gorin! The inhibitory activity is 1O-30KDa fraction of whey (whe3') from cow's milk. This fraction was analyzed by ion-exchange FPLC (Fast Pr (Otein Liquid Chromatography) This inhibitory activity can be separated into two peaks. It was found to be mainly concentrated in special peaks.

There are various ways to identify the proteins of the invention or to vary the degree of reliability. Ru. One presently preferred specification inhibits milk secretion by lactating cows, and The proteins present in the two important peaks of M2 (shown in Figure 1). At that time, the nominal value of milk whey protein (nomina 1) 10-30 The KDa fraction is a single fraction with a pendant -CH2N(CHs)s” group. Monodisperse Hydroph The separation (r esolve). The particles are 10 ± 0.5 μm, especially 1 monokyu -(M.

no Q)-column, 10mM imidazole buffer y-(imidazo 1 ebuffer), pH 7.0 and sodium chloride elution gradient (elution gradient).

Peak 2A protein as determined by gel filtration chromatography The molecular weight is approximately 7KDa. This peak 2A protein is superose (Su cross-linked agarose gel with a particle size of 10 ± 2 μm, such as by gel filtration chromatography on agaro, se gel , when further extracted, the majority of the components (the ones that appear second) are , consisting of inhibitory proteins. Furthermore, peak 2A (or anion exchange chromatography Its equivalent in which the whey is not fractionated before the feed) is a droopy tertiary grade (-N ゝHR4)- and a single having a quaternary (-N+R,) amine group (R represents an organic group) Dispersed hydrophilic polymer particles (the particle size is 10 ± 0.5 μm) Containing chromatography columns (especially “Mono” P columns) 10mM imidazole, PH6,5 and 6. 5-4. Generates a PH gradient of 0 (using an amphoteric buffer of pH 4,0 to Protein is the second f! It is present at the important peak (“2A, 2”).

Any combination of the inhibitory activity of this protein and one or more of the above components ination) is also sufficient to identify the protein and therefore The applicant may wish to re-determine one of them or some aspect of one of them. and are considered insufficient for their identification above, while the remaining configurations are If the Leaving no doubt as to Lotin's identity. Exactly which configuration makes the most sense and Reliability is in any case a matter of judgment and depends on the applicant's current situation. This is a preferred limitation given to reflect current judgment. For that purpose, here Proteins defined by other combinations of the configurations listed are encompassed by the invention. It may be thought that

A property useful for limiting the protein is its isoelectric point (isoelectr point). ic point) PI. determined in a polyacrylamide gel tube Sometimes peak 2A gives a range (7)P[ of 4.8-5.0, while peak 2A A.

2 is an isoelectric fore swing. giving a PI of 4,9-5.0 as determined by

The protein can be either glycosidated or non-glycosidated. It can be in lykoxidized form.

Antibodies against the protein can be polyclonal, monoclonal or engineered. However, it is within the scope of the present invention.

Where national patent laws allow, milk yield control for cows or other animals is an inhibitor that reduces or an antibody that at least partially inhibits the activity of this inhibitor is within scope ii1 of the invention.

Kumen Ω! Sato A Seho Figure 1 shows the 1O-30KDa fraction by anion-exchange chromatography. shows the separation of the components.

Figures 2 and 3 show the anion exchange chromatography of Figure 1 by gel filtration chromatography. 2 shows further separation of the two peaks in the graph.

Figure 4 shows anion-exchange chromatography using chromatofocusing. Further separation of one peak obtained from

beautiful The protein of the invention is present in the milk of cows, possibly in a glycooxidized form. . The effect of glycosylation is due to the simple transfer of proteins to appropriate cells within the mammary lymph glands. It is believed that this is because of the grant. for that , the protein is localized in the lymph glands in non-glycosylated form (Io ca! ly) is expected to be granted.

Concerning goat milk using whey protein 1O-30KDa fraction Inhibition of lactose and casein synthesis in mammary gland transplant cultures It has been shown that the effect is dose-dependent. Furthermore, the graft is an inhibitor exposed to the containing fraction, washed, and in fresh medium in the absence of inhibitors. The ability to synthesize lactose and casein is restored when the cells are re-cultured.

In vivo, the treatment of broth on mammary lymph glands is Reduces yield within hours and recovers full yield 24-26 hours after one treatment do. However, changes in milking frequency - and hence autocrine When (autOcrine) control is extended over several weeks, the synthetic key Effects on capacity, i.e. differentiation of secretory cells due to autocrine inhibitors There was a degree of (differentiation). Information on mammary gland cell activity The effect of period is on cell-surface hormone receptors for proteins. Milking three times the daily milking of a lactating goat for 64 weeks with changes in the number of Increases cell activity and the number of protein receptors in cells, while increasing cell activity for 21 weeks. A decrease in milking efficiency that extends beyond the secretion Reduces cell mutations and protein receptor numbers. These long-term effects, and also Rapid II! caused by the autocrine inhibitor of the present invention! ! + is the endocrine (e ndocrine) depending primarily on changes in the sensitivity of individual lymph glands to regulation. There is. For cows, the same effect can be achieved with the protein of the invention obtained from cow's milk. There is reason to believe that it will be possible to prove that

Antibodies are raised against the proteins of the invention. In the usual way, e.g. Reclonal anti-whal, mouse monoclonal antibody, cow-mouse hybrid mono Clonal antibodies, etc. have been raised, and by the methods currently used, engineering Antibodies, etc. that have been tested are listed. Causes a reduction in the effectiveness of natural inhibitors Active immunization methods are used to increase the lactate rate. be caught. However, milk quotas are often not met. There will be a need to reduce the lactic acid rate in order to There, the inhibitor itself My body is controlled. Usual carriers and adjuvants known in vaccination is used.

The present invention is applicable to any animal that is sensitive to the inhibitors identified herein.

When a 1O-30KDa goat's milk fraction was injected into the mammary lymph glands of a rabbit, Females have been found to be successful in reducing milk accumulation and proper yeast activity. Bovine pore suppressants are also likely to be effective in several other types of milk-producing animals. breast lymph For internal injection into the gland, 1 to 50 μg, especially 5 to 20 μg of inhibitor. A range of dosages appears to be effective and may be repeated as needed, e.g. daily. and can be reduced when administered over a long period of time. Wear.

The protein of the present invention can be obtained from cow's milk by the method described in the Examples or a modified method thereof. can be obtained from It is produced by long dialysis against water and freeze-drying. Recovered in pure form from the eluent (using a suitable membrane that retains the protein, e.g. (with a typical molecular weight cut-off of about 6 KDa). However, it It is expected that it will be synthesized by lotin synthesis or DNA method.

The following examples illustrate the invention.

X delivery This example describes the preparation and properties of the inhibitor of the present invention.

1 Frag John's Milk is obtained from Holstein (Frjesian) cows in the morning milking. , degreased by centrifugation (2500 g, 15 °C, 20 min) and washed with glass wool. filtered through the filter. Skimmed milk was centrifuged at M (80,000 g, 15°C, 2 hours). ), casein micelles (casein mlcelles) pellets and A clean supernatant containing A-protein was obtained. The whey fraction is Dialysis was performed for 24 hours against distilled water.

The whey fraction has a molecular weight of 30.000 Daltons (Dalton, Da ) with a nominal cut-off value of Ultra filtration was performed using a filter that This 30,000 Da The filtrate filtered using a filter was filtered using a filter equipped with a 10.0OODa filter. It was concentrated by filter filtration. Its 10.000-30.0OODa fraction is extensively dialyzed against water, sterilized by filter sterilization, and its and concentrated by freeze-drying for anion exchange chromatography.

2, - Whey protein anion Romato Rough - Part 1O-30KDa Whey fluxin 3 is produced using the 'FPLC' chromatography system (Pharm "MonoQ HR10/10" anion exchange using acia resolved on the column. The whey fraction is used to concentrate the original milk four times. was dissolved in 10 mM imidazole and its pH was adjusted to 7.0. nine Before chromatography, the sample and buffer (degassed) are +1 Filtered through a 2 μm filter. 1 ml of 4 times concentrated whey flax each section was separated. Its flow rate (f’low rate) is 4.0ml /minute. A sodium chloride elution gradient was used.

Fractions containing protein peaks eluted from the column were added to distilled water. and then extensively dialyzed, freeze-dried, and used in the next step. It was previously stored at -20°C.

Figure 1 shows the elution of proteins from a chromatography column. left hand The protein concentration (as light absorption at 280 nm) on the ordinate is the concentration on the abscissa. Plotted against the cumulative volume of the eluator. the plot is shown as a line . The right hand ordinate is the sodium chloride gradient IF used in the eluent (O to 1°OM ) is scaled to indicate. The dashed line is a plot of sodium chloride concentration. Ru.

The peak is the void that contains material that is not bound to the column. voIume) wVo and in the first elution order, those peaks Whey proteins that were not ultrafiltered were chromatographed using the same method. A system that relates them to what they get when they receive They are numbered far apart.

The sono numbering is 1.2A, 2B13.4.5.6A, 6B, 6c, 7 and 8. (The last two peaks are for whey that was not ultrafiltered. although they are not interrelated).

3. Fraction ex 1ant Bioassay approx. 1cm', weight 0.　 Small pieces of breast tissue of 5-0.7 mg of parenchyma were cultured as explants. The explant is , R, Dils and Ai, Horsis (1, A, FOR syth) is Mentos in Enzymology (Methods in En z　Molo　) Eng. 2, 724-742 (1981), New Sealant was manufactured from breast tissue of white rabbits in the third trimester. This explant is cultured in a specified manner. Medium [Medium 199; Gibuco Eu1ope Ltd, ) (Baseley, UK) J, each containing 30 explants. Hold the explant in place so that it is in contact with the medium but is not completely immersed in the medium. Cultured on stainless steel. Insulin (5 μg/ml L cortisol (1 00ng/ml) and prolactin (lμg/m+). explant The pieces were incubated in this medium under an air/COx (19:1 v/v) atmosphere for 42 hours. The cells were cultured for 24 hours, and the medium was replenished after 24 hours. At this point, explant groups (3-4 groups/treatment (2000) were transferred to fresh medium containing the hormones and one of the milk fractions under test. Ta. The milk fraxilan tested in this experiment was not ultrafiltered (see 1 above). O-30KDa fraction), anion exchange chromatography as described above. It was obtained from milk whey, which was fractionated by graphics.

Dissolve these in 10mM Hepes (pH 7,4) at twice the concentration in raw milk. In addition to the volume of the double concentration medium, add 100% of the raw milk concentration of the normal concentration medium. I did it. A control culture containing only a dilution of milk fraxilan was included in each experiment. Breasts lactose and cold during an additional 6 hours of incubation in the presence or absence of lactose The average rate of in-synthesis was determined in this medium by [[J-140]glucose (U-uniformly labeled ;0゜18mC1/mmo 1) and L-[4,5-'H]Cffisine (2, It was measured by adding 22 mC1/mmol, respectively. 6 hour period At the end of the period, the explants and culture medium were separated and stored frozen in liquid nitrogen.

The explants were incubated at 4°C in 5mM ethylene glycol-bis-(2-aminoethyl ether) N, N, N', N'-tetraacetic acid (EGTA) and 2mM 10mM Tris/HCI containing phenylmethanesulfonyl fluoride (p H7,0) in 1.0 ml with a glass/PTFE homogenizer. arc and subsequent 30 s of ultrasonic treatment [Contes Ultrasonic Cell Di Slabter (Kontes Ultrasonic cell disrupt) or), 30% maximum power] for 5 minutes at 10.000g A particle-free supernatant was produced by centrifugation. Particles of 3H-labeled casein produced by precipitation at its isoelectric point from a supernatant containing no , C, J, Wild (C, J, Wi Ide) etc. , 151.519-532 (1984). Mid-gel electrophoresis was performed. By Coomassie brilliant blue staining, Make the band corresponding to casein polypeptide visible, extract it, varnish it, and make it visible. , Russell (S, M, Ru5sell) et al. , 714, 34-45 (1982), [3H] radioactivity was measured. Established. Explant homogenate and ethanol/diethyl ether (3:1, V/V ) to selectively precipitate [14C]lactose from the culture medium using Lee, Kuhn (N, J, Kuhn) and A, White (A, Wh ite) , Biochim, Jay, 14th, ? ? -84 (1975) Radioactivity of L sediment It was measured. By measuring the [+4(:) radioactivity after extracting the non-culture medium] Therefore, the carry-over of glucose from the culture medium (+ 4 () carry-thr The results were corrected for (typically <O, OS%). milk fraction The addition did not affect the secretion distribution between the extracellular space of the explant and the medium. .

The amount of radioactive substances (casein and lactose) is that of explants without milk fraction added. expressed as a percentage of the amount produced by The results are shown in Table 1. Number in parentheses The numbers are the number of experiments performed for various peaks.

Peak No., lactose synthesis, casein synthesis A vs. history +7)% and -011 series − Not added (control) 100 100 1 96, 6 ± 3.7 (5) 90.6 ± 3.5 (5) 2 (=2A’+2B) 65.8 ± 7.5 (6) books 63.1 ± 14.2 (6) zero 3 94, 6 ± 4. 6 (5) 86.7 ± 12.2 (5) 4 104, 0 ± 6.0 (5) 146. 4 ± 22.8 (5) 5 102, 1 ± 1.7 (4) 94.3 ± 21.3 (5) 7 85, 3 ± 9.8 (4) 91-6 ± 19.11 (5) 8 90, 4 ± 4. 8 (3) 94.4 ± 6.1 (5) 9 110, 9 ± 5.6 (3) 90.3 ± 10.8 (4) 10 102.0 (2) 120.3 (2) Main exam vs. vs. Check; pro, 05 (paired test) From the table, peak 2 is lactose synthesis, A major determinant of milk yield and more active than other peaks in inhibiting casein synthesis It is obvious that.

Next, repeat the above experiment using fractions from peak 2A and peak 2B. The results are shown in Table 2 below.

Table 2 Peak No., Lactose Synthesis, Casein Synthesis, Control Ω, Lff Control, 9 No addition (control) 100 100 2 A 71.4±9.1 519±5.92 B 80.9±9.6 80. 8±6.6 Results are mean values of 3 experiments±s, e, m,.

Although both fractions are inhibitory to some extent, the inhibitor component between peaks 2A and 2B It is thought that some of these effects may be due to the lack of clear separation of Ru.

4 Peak gel ', chromatography As described above, 1O-30KDa filter Gel filtration of each of peaks 2A and 2B produced from the rFPRCJ clone Matography system and “Sucrose 12HR10/30J column (Pharmacia) It was carried out using “Sucrose 12” has a particle size of 10 ± 2 μm and 2xlO’D It is a highly crosslinked agarose with an exclusion limit of a. Filter the buffer before use. (0.2μm filter), degassed, 50mM T containing 100mM KCI It was ris/HCI. Sample (routinely 1-10 μl in a maximum volume of 200 μl) g) was dissolved in the same buffer and filtered (0.2 μm filter) before use.

The column was loaded with a molecular weight standard in the molecular weight range 200.000-12.400 (Sigma MW- GF-200 kit), aprotinin (molecular weight 6.500), and bovine α-lactoa calibra precin B using rubumin (molecular weight 14.200). tion)t,ta. Create a Calipre silane curve of logarithm [molecular weight] versus V, /v0. In this case ■. - Empty volume, ■, = elution amount of each protein. vo is dex 6 Figure 2 and 6 measured using Tranblue (Sigma: approximately molecular weight 2,0OOKDa) 3 is the result of gel filtration, at 280 nm on the ordinate plotted against the elution amount on the abscissa. shows protein absorption. From Figure 2, peak 2 is the 3 types of peaks and the first main peak. It is seen that it is decomposed into a shoulder downstream of the .

The m, w, 7 and 4KDa components are located at the first major peak and its shoulder, respectively. assigned. The 7KDa component is clearly the main component of peak 2A. Pi -2B is resolved into three types of peaks and a shoulder downstream of the main peak, m, w, 7 and 4KDa components are assigned to the shoulder and main peak, respectively. It will be done. The molecular weights of the main components were found to be approximately 7KDa and 4KDa. (SD Attempts to measure m, w by S-PAGE yielded unusual results: high molecular weight aggregates. body is formed). The surprisingly low molecular weight is probably due to ultra filtration. clogging of the nominal 10,000 Dalton filter, which allows for the consonance of small molecules. This seems to be explained by the following.

5 Points of whey protein °゛ Ta. For this award, Be Etch, O'Farrell (P, H, O' Fa r rel l) is 2ei, A&*Atl-, 250, 4007-4021 (1975) i: As stated, range 4. 0-9. A that produces a linear slope of 0 Inforin mixture (4% v/v pH range 5-8; 1% v/v p H range 11!3. 5-10. Manufacture 4% acrylamide gel using BioRad) did. The sample (25ttg of Beak 2 protein) was mixed with 9.5M urea, 2% (w/ v) NP40, 1.6% (v/v) pH 5-8 ampholine and 0. 4% (v /v) pH 3,5-10 dissolved in a solution containing ampholine. anode solution and The cathode solutions were 10mM H3PO4 and 20mM NaOH, respectively.

Electrophoresis was carried out at 300v for 18 hours, then at 400v for 4 hours.

Extrude the gel and first add 25% (V/V) isopropanol/10% (V/V) vinegar. in acid, then 5% (w/v) TCA 15% (w/v) sulfosalicylic acid/1% ( V/V) fixed in methanol and containing 0.1% (W/V) Coomassie Blue. Stained with 5% (v/v) isopropanol and 710% (v/v) acetic acid. No bleaching Performed in sopropatol/acetic acid.

Two bands were obtained. The large band had an isoelectric point of 4.84; the small band had an isoelectric point of 4°9 Concentrate on the isoelectric point of 2.

6. Chromatofocusing of peak 2A due to chromatofocusin is Separate proteins based on their isoelectric point (pI). Pharmacia "Thing" Decomposition by P HR5/2 OJ column reduces pI by only 0.02 pH units. is a decomposition in which molecules with different values are separated. “Mono PJ is based on mono beads It is a weak anion exchanger, i.e. various tertiary (-N'' HR2) and fourth ( -N”Rs) Monodisperse hydrophilic polymer particles introduced with amine groups (10 ±0.5 μm diameter). "Mono P" has buffering capacity and the amount of charge it has changes with pH. Therefore, its ionic capacity also changes with pH. chroma For force-shinging, the column is equilibrated with the starting buffer and a large amount is added. elute with another buffer and gradually adjust the solution to a lower pH. A pH gradient is created on the column. Coupled to column at starting pi ( The proteins eluted at different points in the pH gradient depending on their pI.

"Pre-clean the mono PJ column with 1 ml of IM NaOH, and The solution was equilibrated in pH 6.5. Once the pH of the column eluate is 6.5 Once you have returned the sample, anion as described in Section 2 of this example. Ultra-filtered bovine hojii protein obtained by exchange chromatography, 50 μg of Ark 2 was applied. Place the column in distilled water with “Polybuffer” 74 (pH Eluted with a 1/10 v/v dilution of 4,0). Polybuffer (Pharmacia) contains a number of ampholytic buffers of different pKa. Buffer to 0. 2μm fill It was filtered through a filter. Flow of 0.5 ml/min until the pH of the eluate is 4.5. The elution was carried out with the amount. The protein eluted from the column was measured at 280 nm. Detection was done by monitoring the pH of the effluent.

As shown in FIG. 4, peak 2A is three major peaks (2A, 1.2A, 2.2A.

6) and 3 small peaks (2A, 3.2A, 4.2A, 5). these The relative abundance of the components changes with separation. but , qualitatively reproducible with respect to the relative positions of the peaks. pH 5.0 to 4.9 The second large peak eluting at 2A, 2 is a peak mainly containing the inhibitor. Yes (See Sexitan 7 # below).

7 Chromatofocused peak bioassay A was performed as described in Section 3. The samples tested in this experiment were As mentioned in Section 5, the chromatophoretic force of ion exchange peak 2A I fractionated it. These were added to 10mM Hepes (pH 7,4) to concentrate the raw milk. Add to the volume of the 2x concentration medium and add 1/2 of the raw milk concentration of the normal concentration medium. 00%. A control culture containing only a dilution of Milk Fraxin 3 was added to each experiment. added to. The average rates of lactose and casein synthesis were determined as previously described (section 3) Measured.

The amount of radioactivity is expressed as a percentage of the amount produced by the explant without the addition of the milk fraction. It was expressed as The results are shown in Table 3 below. The results are the average value range s when 3 experiments were conducted. , e, m.

beard Dan2 IN Dan - Lactose Synthesis, Casein Synthesis" Contrast Ω and Go vs. Shin Ω Not added (control) 100 100 2A, 1 142 (1) 107 (1) 2A, 2 66, El±11.1 (3) 67, 0 ± 19.5 (3) 2A, 6 101.5 (2) 102.9 (2) From the table above, it can be seen that among the fractions tested, peak 2 and only peak 2 were inhibitory. I understand.

Gel filtration of peak 2A, 2 romato roughy peak 2A, 2 7) as mentioned in section 4 regarding the anion exchange peak. LC chromatography system and Superose 12HR 10/30” column It was carried out using LUMACIA). Peak 2A.

2 eluted as a single protein-containing peak. Other peaks represent detectable protein. It contained no fins and had a low molecular weight (<I KD a). Peak 2A, 2 minutes The molecular weight was calculated to be approximately 7.0 KDa, which was determined in Section 4 for the anion. This was the molecular weight calculated for exchange peak 2A.

9. Romantofukasshin Phi 2A, 2, ``Pharmacia's It was carried out using a PhastGe 1 J electrophoresis system. this method [Fast Gel IEFJ was used. “Fastgel rEFJ medium is [Pharma A highly prized polyacrylate containing Pharmalyte J carrier amphoteric electrolyte It is a lylamide gel. "Pharmalite" is used during electrophoresis, in this case the pH range 4 to 6.5, creating a stable, linear pH gradient in the gel.

Under an electric field, proteins are essentially unhindered by the porous gel and their p ■ Move to a point on the pH gradient corresponding to (isoelectric point).

Sample of peak 2A, 2 (section 6) extensively dialyzed against distilled water. Apply 1 μg to one hole of the gel, and apply the "Pharmacia PL Calibration Kit ” protein was applied to the holes on both sides of the sample hole. Proty generated after electrophoresis The band was made visible by Coomassie blue staining. Peak 2A, 2 is p A single protiin band corresponding to 14.9-5.0 was generated.

8 distance (/f)l) % “Polybuffer 74” pH 4 international search report lIIItnnl1MalA$ltsl1ml&PCT/GO9070174 3 International search report :21=”″, :Z:::q−==View Yu’l’:2==dm l*m m7% 7++y, /2W@#m 8m m mom m ($Fsle+tl alllm II + M wH11m1d@Iw 1mmmd m m pot-1---m m-W-11miaaH axis 9 No. 9, page 1 [Phase] Int, C1,' Identification symbol Office serial number 0 clearer Beaker, Malcolm Igiritsuno (- 0 shots clear Wild, Colin James England Z Sri Lanka Air K.F.4KEX, Alloway, A.C. 13 Sri Lanka Air K.F.4N.F., Alloway, Raihi

Claims (8)

[Claims] 1. Inhibits the secretion of milk by lactating cows and reduces whey protein in the milk. The nominal 10-30 KDa fraction of the -CH2N(CH3)3+ Particles of unit-dispersed hydrohylic polymer having groups (the particle size is 10 to 0.5 μm) anion exchange column (10mM imidazole buffer, PH7 ．． 0 and using a sodium chloride elution gradient). protein present in the peak (“2A”). 2. molecular weight (determined by gel filtration chromatography) of approximately 7 KDa. The protein according to claim 1. 3. etc. within the range of 4.8-4.95 determined in a tube of polyacrylamide gel. The protein according to claim 1 or 2, which has an electric point. 4. Peak 2A indicates the drooping tertiary (-N+HR2) and quaternary (-N+R3) amino acids. Particles of a monodisverse hydrohylic polymer having an organic group (R represents an organic group) chromatographic focus column containing particles whose particle size is 10 ± 0.5 μm (10mM imidazole, pH 6.5 and to generate a pH gradient of 6.5-4.5) When further separated (using a pH 4.0 buffer), the second important The protein according to claim 1, 2 or 3, which is present in a peak. 5. Claim 1, Claim 2, Claim 3 or Claim 4 in non-glycosylated form Proteins listed. 6. An antibody against the protein according to any one of claims 1 to 5. 7. The protein according to any one of claims 1 to 5, which regulates milk secretion in animals. proteins or their general equivalents, or antibodies against such proteins. . 8. The protein according to claim 7, wherein the animal is a cow.