CN102807612A - Polypeptide fragment and preparation method and application thereof - Google Patents
Polypeptide fragment and preparation method and application thereof Download PDFInfo
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- CN102807612A CN102807612A CN2012102711776A CN201210271177A CN102807612A CN 102807612 A CN102807612 A CN 102807612A CN 2012102711776 A CN2012102711776 A CN 2012102711776A CN 201210271177 A CN201210271177 A CN 201210271177A CN 102807612 A CN102807612 A CN 102807612A
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- 229920001184 polypeptide Polymers 0.000 title claims abstract description 53
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 53
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 239000012634 fragment Substances 0.000 title abstract description 9
- 238000013467 fragmentation Methods 0.000 title 1
- 238000006062 fragmentation reaction Methods 0.000 title 1
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims abstract description 24
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- 230000008014 freezing Effects 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 24
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- 238000005406 washing Methods 0.000 claims description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 18
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 16
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 16
- 239000002244 precipitate Substances 0.000 claims description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
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- YODZTKMDCQEPHD-UHFFFAOYSA-N thiodiglycol Chemical group OCCSCCO YODZTKMDCQEPHD-UHFFFAOYSA-N 0.000 claims description 3
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- 239000000284 extract Substances 0.000 claims description 2
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- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 abstract description 4
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides polypeptide and a preparation method and application thereof. The polypeptide is a fragment in which phosphatidyl ethanolamine combined with protein is cut off and is composed of amino acid residues from the 93rd to 124th, and the amino acid sequence of the polypeptide is shown as follow: KGNDISSGTVLSDYVGSGPPKGTGLHRYVWLV. The invention further provides the preparation method of the polypeptide. The preparation method includes firstly extracting heatproof intestinal polypeptide from chitterlings, extracting crude peptide from the heatproof intestinal polypeptide then, dissolving the crude peptide into acetum to prepare peptide solution, isolating the peptide solution to obtain isolate by column chromatography, selecting a part having a function of stimulating pancreas to secrete insulin from the isolate, freezing and drying the part to obtain crude extracts, and purifying the crude extracts to obtain the polypeptide. Besides, the invention provides application of the polypeptide. The application includes that the polypeptide is acceptable to salt pharmaceutically and used for manufacturing drugs capable of stimulating the pancreas to secrete insulin.
Description
Technical field
The present invention relates to the fragment after a kind of phosphotidylethanolabinding binding protein brachymemma, this polypeptide fragment has the effect that stimulates excreting insulin, and the present invention also provides aforementioned polypeptides segmental preparation method simultaneously.
Background technology
Phosphotidylethanolabinding binding protein (PEBP) is one type of ability and phosphatidylethanolamine (PE) bonded albumen; All members of this family are contained and PE bonded conservative region; PEBP family kind wide material sources; The antigen A g16 that comprises the immune serum identification after the tactile odorant binding protein(OBP) of fruit bat, the dish tailfiber fluke infection suppresses the factor CEN of CDC25 sudden change, in the mammiferous in-vivo tissue enchylema in the yeast.PEBP family member function is extensive, relates to the opposing apoptosis, and film forms, the physiological function of each side such as the maturation of sperm.
Mellitus are diseases of the easy overheap of glucose in a kind of blood.External another name to it is " reticent killer " (Silent Killer), particularly " adult diabetes mellitus ".It is high especially that middle-aged people more than 40 years old catches rate, and in Japan, sickness rate accounts for 10% in the population more than 40 years old, and a diabetic subject is promptly just arranged in the middle of ten people, in case suffer from " mellitus ", will reduce 10 years more than life-span, and contingent complication spreads all over whole body.The medicine of present main treatment mellitus all is the medicine and direct supplementation with insulin that promotes insulin secretion, but these medicine effects are bad or can produce insulin resistance.So it is underway always to seek the medicine of new treatment mellitus.
Summary of the invention
The invention provides a kind of polypeptide fragment; Fragment after it blocks for phosphotidylethanolabinding binding protein through discovery after the detection; Amino-acid residue by phosphotidylethanolabinding binding protein 93-124 position is formed, and its aminoacid sequence is following: KGNDISSGTVLSDYVGSGPPKGTGLHRYVWLV.
The present invention also provides aforesaid polypeptide approved through modified polypeptides on biological medicine simultaneously.
The invention provides the preparation method of aforementioned polypeptides; May further comprise the steps: (1), the back is cleaned in the chitling road immersed in the boiling water and soaked 3～7 minutes; It is freezing and mince soak to accomplish the back taking-up; Under 0～10 ° of C condition, enteron aisle is leached after-filtration then and get leach liquor, extract wash-out in the leach liquor and suction filtration are got heat-resisting intestines polypeptide with hydrochloric acid with acetic acid;
(2), heat-resisting intestines polypeptide is dissolved in the water; Add inhibitor and sufficient isopropanol solution; The centrifugal deposition of removing behind the sufficient standing under the room temperature, and then add the capacity temperature be-25 ℃～-15 ℃ aqueous isopropanol and behind sufficient standing under-25 ℃～-15 ℃ conditions suction filtration remove deposition, the pH value that gained is filtrated is adjusted to 5.8～6.1; Suction filtration gets white precipitate behind sufficient standing under-25 ℃～-15 ℃ conditions again, with promptly making thick peptide behind the white precipitate thorough washing;
(3), the thick peptide that makes in the step (2) be dissolved in make peptide solution in the acetum, peptide solution is crossed column chromatography for separation obtains isolate, choose the portion that has stimulating pancreas excreting insulin function in the isolate, its freeze-drying is promptly got crude extract;
(4), be dissolved in the ammonium bicarbonate soln crude extract and stirring; Centrifugal back remove precipitate supernatant; The gained supernatant is crossed column chromatography and adopted the ammonium bicarbonate soln that pH value is 8.0, concentration is 0.02mol/L to wash; The washes freeze-drying that obtains after the washing after washes being separated through twice performance liquid chromatography, promptly makes the isolate freeze-drying polypeptide of the said aminoacid sequence of claim 1 again.
The concentration of acetic acid is 0.5mol/L in the step (1), and the concentration of hydrochloric acid is 0.2mol/L.
Inhibitor is a thiodiglycol in the step (2), and addition is 1.2 milliliters; Washing is carried out at twice, and aqueous isopropanol is selected in washing for the first time for use, and diethyl ether solution is selected in washing for the second time for use, after washing is accomplished for the second time white precipitate is put into vacuum environment ether is evaporated in a vacuum.
During twice performance liquid chromatography in the said step (4) separates, separate used eluant for the first time and be 0.1% trifluoroacetic acid aqueous solution, separate used eluant for the second time and be the acetonitrile solution of 0.1% trifluoroacetic acid.
The present invention also provides the purposes of aforementioned polypeptides and pharmacy acceptable salt thereof simultaneously, is used to make the medicine of stimulating pancreas excreting insulin.
Polypeptide provided by the invention with and approved through modified polypeptides on biological medicine; Effective stimulating pancreas emiocytosis Regular Insulin; Compare with adopting maximum high sugar in the present treatment diabetes medicament, the effect of polypeptide provided by the invention is more obvious.
Description of drawings
Fig. 1 is the thick peptide chromatographic separation figure as a result in the embodiment of the invention 1;
Fig. 2 is performance liquid chromatography separating resulting figure for the second time in the embodiment of the invention 1;
Fig. 3 is amino acid sequence of polypeptide figure provided by the invention;
Fig. 4 is the experimental result picture of pancreas irritant test provided by the invention.
Embodiment
The present invention is done bright in detail specifically below in conjunction with specific embodiment.
Adopt following preparation process in the present embodiment:
(1), gets 30 kilograms of fresh chitling; With soaking 4 minutes in the clean back immersion in the chitling road boiling water; It is freezing and mince soak to accomplish the back taking-up; Under 5 ° of C conditions, with the acetic acid of 0.5mol/L enteron aisle is leached after-filtration then and get leach liquor, the hydrochloric acid with 0.2mol/L gets about 30 grams of heat-resisting intestines polypeptide to extract wash-out in the leach liquor and suction filtration then, and the productive rate of heat-resisting intestines polypeptide is about 0.01% of chitling weight in wet base.
(2), the heat-resisting intestines polypeptide of 30 grams is dissolved in the 0.24L water; Add 1.2 milliliters of inhibitor thiodiglycols and 1.08L aqueous isopropanol; Leave standstill the centrifugal deposition of removing after 2 hours under the room temperature; And then add the 1.32L temperature for-20 ℃ aqueous isopropanol and after leaving standstill 24 hours under-20 ℃ of conditions suction filtration remove deposition, the pH value of gained filtrating is adjusted to 6 with hydrochloric acid, suction filtration gets white precipitate behind sufficient standing under-20 ℃ of conditions again; White precipitate is washed at twice; Aqueous isopropanol is selected in washing for the first time for use, and diethyl ether solution is selected in washing for the second time for use, after washing is accomplished for the second time white precipitate is put into vacuum environment and ether is evaporated in a vacuum promptly make thick peptide 6.5 grams.
(3), the thick peptide that makes in the step (2) 6.5 gram is dissolved in the acetum of 300mL0.2mol/L makes peptide solution; Peptide solution is crossed column chromatography for separation obtain isolate; Choose the portion that has stimulating pancreas excreting insulin function in the isolate; Its freeze-drying is promptly got crude extract, and as shown in Figure 1, the portion of choosing institute's mark among Fig. 1 is a target compound.
(4), be dissolved in crude extract in the ammonium bicarbonate soln of 81.5mL0.01mol/L and stirred 20 minutes; Centrifugal back remove precipitate supernatant; The gained supernatant is crossed column chromatography and adopted the ammonium bicarbonate soln that pH value is 8.0, concentration is 0.02mol/L to wash, and the washes freeze-drying that obtains after the washing is after washes being separated through twice performance liquid chromatography again; During described twice performance liquid chromatography separates, separate used eluant for the first time and be 0.1% trifluoroacetic acid aqueous solution; Separate for the second time used eluant and be the acetonitrile solution of 0.1% trifluoroacetic acid, with separating for the second time that back gained deposition is collected and freeze-drying promptly makes the polypeptide of the said aminoacid sequence of claim 1, as shown in Figure 2, target compound is polypeptide provided by the present invention.
To the polypeptide made in the above-mentioned steps through the SDS-PAGE glue purification.Carry out amino acid sequencing and do peptide sequence analysis with the Edman edman degradation Edman.Applied Biosystems 477A instrument coupling 120 A analyzer, Milligen 6600, Waters 440 analysers carry out amino acid sequence analysis.
Sequencing result is as shown in Figure 3:
Polypeptide: 1 KGNDISSGTVLSDYVGSGPPKGTGLHRYVWLV 32
With phosphotidylethanolabinding binding protein (sequence is:
90 VNMKGNDISSGTVLSDYVGSGPPKGTGLHRYVWLVYEQ 127) has very big similarity.Can know that therefrom the polypeptide among the present invention is the polypeptide fragment that phosphotidylethanolabinding binding protein the 93rd to the 124th amino-acid residue is formed.
Polypeptide performance to making among the present invention is studied, and test is carried out the influence of the insulin secretion of glucose induction at isolated rat pancreas islet perfusion system.Male SD rat (body weight 200～250 grams) takes out pancreas after anesthesia, use collagenase digesting then.Above-mentioned pancreas spends the night with RPMI 1640 culture medium culturing down at 37 ℃; First group of glucose that is added with 3.3mM in the described substratum; Be added with the glucose of 16.7mM in second group, be added with the glucose of 16.7mM and the phosphotidylethanolabinding binding protein 93-124 of 10nM in the 3rd group.Confirm three amount of insulin that pancreas discharged with radio immunoassay.Mensuration result is as shown in Figure 4, this shows that phosphotidylethanolabinding binding protein 93-124 has the effect of tangible insulin secretion accelerating.
Embodiment 2
Adopt following preparation process in the present embodiment:
(1), gets 20 kilograms of fresh chitling; With soaking 6 minutes in the clean back immersion in the chitling road boiling water; It is freezing and mince soak to accomplish the back taking-up; Under 8 ° of C conditions, with the acetic acid of 0.5mol/L enteron aisle is leached after-filtration then and get leach liquor, the hydrochloric acid with 0.2mol/L gets about 19 grams of heat-resisting intestines polypeptide to extract wash-out in the leach liquor and suction filtration then, and the productive rate of heat-resisting intestines polypeptide is about 0.01% of chitling weight in wet base.
(2), the heat-resisting intestines polypeptide of 19 grams is dissolved in the 0.15L water; Add 1 milliliter of inhibitor thiodiglycol and 0.7L aqueous isopropanol; Leave standstill the centrifugal deposition of removing after 2 hours under the room temperature; And then add the 0.89L temperature for-23 ℃ aqueous isopropanol and after leaving standstill 24 hours under-20 ℃ of conditions suction filtration remove deposition, the pH value of gained filtrating is adjusted to 6 with hydrochloric acid, suction filtration gets white precipitate behind sufficient standing under-18 ℃ of conditions again; White precipitate is washed at twice; Aqueous isopropanol is selected in washing for the first time for use, and diethyl ether solution is selected in washing for the second time for use, after washing is accomplished for the second time white precipitate is put into vacuum environment and ether is evaporated in a vacuum promptly make thick peptide 4.3 grams.
(3), the thick peptide that makes in the step (2) 4.3 gram is dissolved in the acetum of 200mL0.2mol/L makes peptide solution; Peptide solution is crossed column chromatography for separation obtain isolate; Choose the portion that has stimulating pancreas excreting insulin function in the isolate, its freeze-drying is promptly got crude extract.
(4), be dissolved in crude extract in the ammonium bicarbonate soln of 54mL0.01mol/L and stirred 20 minutes; Centrifugal back remove precipitate supernatant; The gained supernatant is crossed column chromatography and adopted the ammonium bicarbonate soln that pH value is 8.0, concentration is 0.02mol/L to wash, and the washes freeze-drying that obtains after the washing is after washes being separated through twice performance liquid chromatography again; During described twice performance liquid chromatography separates, separate used eluant for the first time and be 0.1% trifluoroacetic acid aqueous solution; Separate for the second time used eluant and be the acetonitrile solution of 0.1% trifluoroacetic acid, with separating for the second time that back gained deposition is collected and freeze-drying promptly makes the polypeptide of the said aminoacid sequence of claim 1.
To the polypeptide made in the above-mentioned steps through the SDS-PAGE glue purification.Carry out amino acid sequencing
Do peptide sequence analysis with the Edman edman degradation Edman.Applied Biosystems 477A instrument coupling 120 A analyzer, Milligen 6600, Waters 440 analysers carry out amino acid sequence analysis.
Sequencing result is as shown in Figure 3:
Polypeptide: 1 KGNDISSGTVLSDYVGSGPPKGTGLHRYVWLV 32
With phosphotidylethanolabinding binding protein (sequence is:
90 VNMKGNDISSGTVLSDYVGSGPPKGTGLHRYVWLVYEQ 127) has very big similarity.Can know that therefrom the polypeptide among the present invention is the polypeptide fragment that phosphotidylethanolabinding binding protein the 93rd to the 124th amino-acid residue is formed.
Performance to the polypeptide that makes in the present embodiment is studied, and its result is identical with embodiment 1.
Claims (8)
1. a peptide species, its aminoacid sequence is following:
KGNDISSGTVLSDYVGSGPPKGTGLHRYVWLV。
2. polypeptide as claimed in claim 1 is approved through modified polypeptides on biological medicine, and its aminoacid sequence is following: KGNDISSGTVLSDYVGSGPPKGTGLHRYVWLV.
3. the preparation method of polypeptide as claimed in claim 1 is characterized in that adopting following steps:
(1), with soaking 3～7 minutes in the clean back immersion in the chitling road boiling water; It is freezing and mince soak to accomplish the back taking-up; Under 0～10 ° of C condition, enteron aisle is leached after-filtration then and get leach liquor, extract wash-out in the leach liquor and suction filtration are got heat-resisting intestines polypeptide with hydrochloric acid with acetic acid;
(2), heat-resisting intestines polypeptide is dissolved in the water; Add inhibitor and sufficient isopropanol solution; The centrifugal deposition of removing behind the sufficient standing under the room temperature, and then add the capacity temperature be-25 ℃～-15 ℃ aqueous isopropanol and behind sufficient standing under-25 ℃～-15 ℃ conditions suction filtration remove deposition, the pH value that gained is filtrated is adjusted to 5.8～6.1; Suction filtration gets white precipitate behind sufficient standing under-25 ℃～-15 ℃ conditions again, with promptly making thick peptide behind the white precipitate thorough washing;
(3), the thick peptide that makes in the step (2) be dissolved in make peptide solution in the acetum, peptide solution is crossed column chromatography for separation obtains isolate, choose the portion that has stimulating pancreas excreting insulin function in the isolate, its freeze-drying is promptly got crude extract;
(4), be dissolved in the ammonium bicarbonate soln crude extract and stirring; Centrifugal back remove precipitate supernatant; The gained supernatant is crossed column chromatography and adopted the ammonium bicarbonate soln that pH value is 8.0, concentration is 0.02mol/L to wash; The washes freeze-drying that obtains after the washing after washes being separated through twice performance liquid chromatography, promptly makes the isolate freeze-drying polypeptide of the said aminoacid sequence of claim 1 again.
4. preparation method according to claim 3 is characterized in that: the concentration of acetic acid is 0.5mol/L in the step (1), and the concentration of hydrochloric acid is 0.2mol/L.
5. preparation method according to claim 3 is characterized in that: inhibitor is a thiodiglycol in the step (2), and addition is 1.2 milliliters; Washing is carried out at twice, and aqueous isopropanol is selected in washing for the first time for use, and diethyl ether solution is selected in washing for the second time for use, after washing is accomplished for the second time white precipitate is put into vacuum environment ether is evaporated in a vacuum.
6. preparation method according to claim 3; It is characterized in that: during twice performance liquid chromatography in the said step (4) separates; Separate for the first time used eluant and be 0.1% trifluoroacetic acid aqueous solution, separate used eluant for the second time and be the acetonitrile solution of 0.1% trifluoroacetic acid.
7. the purposes of the said polypeptide of claim 1 is characterized in that: the medicine that is used to make the stimulating pancreas excreting insulin.
8. the said polypeptide of claim 2 approved purposes through modified polypeptides on biological medicine is characterized in that: the medicine that is used to make the stimulating pancreas excreting insulin.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103571803A (en) * | 2013-10-11 | 2014-02-12 | 中南民族大学 | Polypeptide segment, and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1477117A (en) * | 2002-08-19 | 2004-02-25 | 浙江大学免疫学研究所 | Novel human phosphotidylethanolamine binding protein, its coding sequence and application |
| CN1792218A (en) * | 2005-12-31 | 2006-06-28 | 浙江大学 | Intestine nutrition peptide for piglings |
| CN101812126A (en) * | 2009-02-25 | 2010-08-25 | 中国人民解放军第二军医大学 | Novel HLA-A2 restrictive epitope polypeptide coming from hPEBP4 protein and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1477117A (en) * | 2002-08-19 | 2004-02-25 | 浙江大学免疫学研究所 | Novel human phosphotidylethanolamine binding protein, its coding sequence and application |
| CN1792218A (en) * | 2005-12-31 | 2006-06-28 | 浙江大学 | Intestine nutrition peptide for piglings |
| CN101812126A (en) * | 2009-02-25 | 2010-08-25 | 中国人民解放军第二军医大学 | Novel HLA-A2 restrictive epitope polypeptide coming from hPEBP4 protein and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103571803A (en) * | 2013-10-11 | 2014-02-12 | 中南民族大学 | Polypeptide segment, and preparation method and application thereof |
| CN103571803B (en) * | 2013-10-11 | 2018-05-11 | 中南民族大学 | VIII 52-69 of cytochrome c oxidase and preparation method thereof and function |
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| CN102807612B (en) | 2014-04-09 |
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