CN102807612B - Polypeptide fragment and preparation method and application thereof - Google Patents
Polypeptide fragment and preparation method and application thereof Download PDFInfo
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- CN102807612B CN102807612B CN201210271177.6A CN201210271177A CN102807612B CN 102807612 B CN102807612 B CN 102807612B CN 201210271177 A CN201210271177 A CN 201210271177A CN 102807612 B CN102807612 B CN 102807612B
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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 the preparation method of aforementioned polypeptides fragment simultaneously.
Background technology
Phosphotidylethanolabinding binding protein (PEBP) be a class can with the albumen of phosphatidylethanolamine (PE) combination, all members of this family are contained the conservative region of being combined with PE, PEBP family kind wide material sources, comprise the odorant binding protein (OBP) of fruit bat sense of touch, the antigen A g16 of the immune serum identification after the fluke infection of dish tailfiber, the factor CEN that suppresses CDC25 sudden change in yeast, in mammiferous in-vivo tissue enchylema.PEBP family member function is extensive, relates to opposing apoptosis, and film forms, the physiological function of the each side such as the maturation of sperm.
Diabetes are diseases of the easy overheap of glucose in a kind of blood.The external another name to it is " reticent killer " (Silent Killer), particularly " adult diabetes mellitus ".It is high especially that more than 40 years old middle-aged peoples catches rate, and in Japan, in 40 years old above population, sickness rate accounts for 10%, in the middle of ten people, just has a diabetic subject, once suffer from " diabetes ", will reduce 10 years more than life-span, and contingent complication spreads all over whole body.The medicine of primary treatment diabetes is all medicine and the direct supplementation with insulin that promotes insulin secretion at present, but these medicine effects are bad or can produce insulin resistance.So it is always underway to find the medicine of new treatment diabetes.
Summary of the invention
The invention provides a kind of polypeptide fragment, find afterwards that after testing it is the fragment after phosphotidylethanolabinding binding protein blocks, amino-acid residue by phosphotidylethanolabinding binding protein 93-124 position forms, and its aminoacid sequence is as follows: KGNDISSGTVLSDYVGSGPPKGTGLHRYVWLV.
The present invention also provides polypeptide as above approved modified polypeptide in biological medicine simultaneously.
The invention provides the preparation method of aforementioned polypeptides, comprise the following steps: (1), immerse in boiling water after will chitling road cleaning and soak 3～7 minutes, after immersion completes, taking-up is freezing and mince, then after enteron aisle being leached with acetic acid, filter to obtain leach liquor under 0～10 ° of C condition, with hydrochloric acid, the extract wash-out in leach liquor suction filtration are obtained to heat-resisting intestines polypeptide;
(2), heat-resisting intestines polypeptide is dissolved in the water, add antioxidant and sufficient isopropanol solution, the centrifugal precipitation of removing after sufficient standing under room temperature, and then add enough temperature be the aqueous isopropanol of-25 ℃～-15 ℃ and under-25 ℃～-15 ℃ conditions after sufficient standing suction filtration remove precipitation, the pH value of gained filtrate is adjusted to 5.8～6.1, under-25 ℃～-15 ℃ conditions, after sufficient standing, suction filtration obtains white precipitate again, after white precipitate is fully washed, makes thick peptide;
(3), the thick peptide making in step (2) is dissolved in acetum and makes peptide solution, peptide solution is crossed to column chromatography for separation and obtain isolate, choose the portion in isolate with stimulating pancreas excreting insulin function, its freeze-drying is obtained to crude extract;
(4), crude extract be dissolved in ammonium bicarbonate soln and stir, centrifugal rear removal precipitates to obtain supernatant liquor, gained supernatant liquor is crossed to column chromatography and adopt the ammonium bicarbonate soln that pH value is 8.0, concentration is 0.02mol/L to wash, the washes freeze-drying obtaining after washing, again by washes after twice high performance liquid chromatography separation, isolate freeze-drying is made to the polypeptide of aminoacid sequence described in claim 1.
In step (1), the concentration of acetic acid is 0.5mol/L, and the concentration of hydrochloric acid is 0.2mol/L.
In step (2), antioxidant is thiodiglycol, and addition is 1.2 milliliters; Washing is carried out at twice, and aqueous isopropanol is selected in washing for the first time, and diethyl ether solution is selected in washing for the second time, after having washed for the second time, white precipitate is put into vacuum environment ether is evaporated in a vacuum.
In twice high performance liquid chromatography separation in described step (4), the trifluoroacetic acid aqueous solution that separated eluant used is 0.1% for the first time, the acetonitrile solution of the trifluoroacetic acid that separated eluant used is 0.1% for the second time.
The present invention also provides the purposes of aforementioned polypeptides and pharmacy acceptable salt thereof simultaneously, for the manufacture of the medicine of stimulating pancreas excreting insulin.
Polypeptide provided by the invention with and in biological medicine approved modified polypeptide, effective stimulating pancreas emiocytosis Regular Insulin, adopt maximum height sugar to compare with current treatment diabetes medicament, the effect of polypeptide provided by the invention is more obvious.
Accompanying drawing explanation
Fig. 1 is the thick peptide chromatographic separation result figure in the embodiment of the present invention 1;
Fig. 2 is high performance liquid chromatography separating resulting figure for the second time in the embodiment of the present invention 1;
Fig. 3 is the aminoacid sequence figure of polypeptide provided by the invention;
Fig. 4 is the experimental result picture of pancreas irritant test provided by the invention.
Embodiment
Below in conjunction with specific embodiment, the present invention is done to detailed specific description.
In the present embodiment, adopt following preparation process:
(1), get 30 kilograms of fresh chitling, after chitling road is cleaned, immerse in boiling water and soak 4 minutes, after immersion completes, taking-up is freezing and mince, then after enteron aisle being leached with the acetic acid of 0.5mol/L, filter to obtain leach liquor under 5 ° of C conditions, then with the hydrochloric acid of 0.2mol/L, the extract wash-out in leach liquor suction filtration are obtained to approximately 30 grams of heat-resisting intestines polypeptide, the productive rate of heat-resisting intestines polypeptide is about 0.01% of chitling weight in wet base.
(2), 30 grams of heat-resisting intestines polypeptide are dissolved in 0.24L water, add 1.2 milliliters of antioxidant thiodiglycols and 1.08L aqueous isopropanol, the centrifugal precipitation of removing after standing 2 hours under room temperature, and then add 1.32L temperature for the aqueous isopropanol of-20 ℃ and under-20 ℃ of conditions after standing 24 hours suction filtration remove precipitation, the pH value of gained filtrate is adjusted to 6 with hydrochloric acid, under-20 ℃ of conditions, after sufficient standing, suction filtration obtains white precipitate again, white precipitate is washed at twice, aqueous isopropanol is selected in washing for the first time, diethyl ether solution is selected in washing for the second time, after having washed for the second time, white precipitate being put into vacuum environment evaporates ether to make 6.5 grams of thick peptides in a vacuum.
(3), 6.5 grams of the thick peptides making in step (2) are dissolved in the acetum of 300mL0.2mol/L and make peptide solution, peptide solution is crossed to column chromatography for separation and obtain isolate, choose the portion in isolate with stimulating pancreas excreting insulin function, its freeze-drying is obtained to crude extract, as shown in Figure 1, the portion of choosing institute's mark in Fig. 1 is target compound.
(4), crude extract be dissolved in the ammonium bicarbonate soln of 81.5mL0.01mol/L and stir 20 minutes, centrifugal rear removal precipitates to obtain supernatant liquor, gained supernatant liquor is crossed to column chromatography and adopt the ammonium bicarbonate soln that pH value is 8.0, concentration is 0.02mol/L to wash, the washes freeze-drying obtaining after washing, again by washes after twice high performance liquid chromatography separation, in twice described high performance liquid chromatography separation, the trifluoroacetic acid aqueous solution that separated eluant used is 0.1% for the first time; The acetonitrile solution of the trifluoroacetic acid that separated eluant used is 0.1% for the second time, collects gained precipitation after separated for the second time and freeze-drying makes the polypeptide of aminoacid sequence described in claim 1, and as shown in Figure 2, target compound is polypeptide provided by the present invention.
To the polypeptide of making in above-mentioned steps through SDS-PAGE glue purification.Carry out amino acid sequencing and do peptide sequence analysis with 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) there is very large similarity.Therefrom known, the polypeptide in the present invention is the polypeptide fragment that phosphotidylethanolabinding binding protein the 93rd to the 124th amino-acid residue forms.
The polypeptide performance making in the present invention is studied, and test is carried out the impact of the insulin secretion of glucose induction at isolated rat pancreas islet perfusion system.Male SD rat (200～250 grams of body weight) takes out pancreas after anesthesia, then uses collagenase digesting.Above-mentioned pancreas spends the night by RPMI 1640 culture medium culturing at 37 ℃, first group of glucose that is added with 3.3mM in described substratum, in second group, be added with the glucose of 16.7mM, be added with the glucose of 16.7mM and the phosphotidylethanolabinding binding protein 93-124 of 10nM in the 3rd group.With radio immunoassay, determine the amount of insulin that three pancreas discharge.As shown in Figure 4, phosphotidylethanolabinding binding protein 93-124 has the effect of obvious insulin secretion accelerating to measurement result as can be seen here.
Embodiment 2
In the present embodiment, adopt following preparation process:
(1), get 20 kilograms of fresh chitling, after chitling road is cleaned, immerse in boiling water and soak 6 minutes, after immersion completes, taking-up is freezing and mince, then after enteron aisle being leached with the acetic acid of 0.5mol/L, filter to obtain leach liquor under 8 ° of C conditions, then with the hydrochloric acid of 0.2mol/L, the extract wash-out in leach liquor suction filtration are obtained to approximately 19 grams of heat-resisting intestines polypeptide, the productive rate of heat-resisting intestines polypeptide is about 0.01% of chitling weight in wet base.
(2), 19 grams of heat-resisting intestines polypeptide are dissolved in 0.15L water, add 1 milliliter of antioxidant thiodiglycol and 0.7L aqueous isopropanol, the centrifugal precipitation of removing after standing 2 hours under room temperature, and then add 0.89L temperature for the aqueous isopropanol of-23 ℃ and under-20 ℃ of conditions after standing 24 hours suction filtration remove precipitation, the pH value of gained filtrate is adjusted to 6 with hydrochloric acid, under-18 ℃ of conditions, after sufficient standing, suction filtration obtains white precipitate again, white precipitate is washed at twice, aqueous isopropanol is selected in washing for the first time, diethyl ether solution is selected in washing for the second time, after having washed for the second time, white precipitate being put into vacuum environment evaporates ether to make 4.3 grams of thick peptides in a vacuum.
(3), 4.3 grams of the thick peptides making in step (2) are dissolved in the acetum of 200mL0.2mol/L and make peptide solution, peptide solution is crossed to column chromatography for separation and obtain isolate, choose the portion in isolate with stimulating pancreas excreting insulin function, its freeze-drying is obtained to crude extract.
(4), crude extract be dissolved in the ammonium bicarbonate soln of 54mL0.01mol/L and stir 20 minutes, centrifugal rear removal precipitates to obtain supernatant liquor, gained supernatant liquor is crossed to column chromatography and adopt the ammonium bicarbonate soln that pH value is 8.0, concentration is 0.02mol/L to wash, the washes freeze-drying obtaining after washing, again by washes after twice high performance liquid chromatography separation, in twice described high performance liquid chromatography separation, the trifluoroacetic acid aqueous solution that separated eluant used is 0.1% for the first time; The acetonitrile solution of the trifluoroacetic acid that separated eluant used is 0.1% for the second time, collects gained precipitation after separated for the second time and freeze-drying makes the polypeptide of aminoacid sequence described in claim 1.
To the polypeptide of making in above-mentioned steps through SDS-PAGE glue purification.Carry out amino acid sequencing
With Edman edman degradation Edman, do peptide sequence analysis.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) there is very large similarity.Therefrom known, the polypeptide in the present invention is the polypeptide fragment that phosphotidylethanolabinding binding protein the 93rd to the 124th amino-acid residue forms.
Performance to the polypeptide making in the present embodiment is studied, and its result is identical with embodiment 1.
Claims (2)
1. a peptide species, its aminoacid sequence is as follows:
KGNDISSGTVLSDYVGSGPPKGTGLHRYVWLV。
2. the purposes of polypeptide in the medicine of preparing stimulating pancreas excreting insulin described in claim 1.
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