CN107033247A - Compound, protein and the preparation method of the two - Google Patents
Compound, protein and the preparation method of the two Download PDFInfo
- Publication number
- CN107033247A CN107033247A CN201710256655.9A CN201710256655A CN107033247A CN 107033247 A CN107033247 A CN 107033247A CN 201710256655 A CN201710256655 A CN 201710256655A CN 107033247 A CN107033247 A CN 107033247A
- Authority
- CN
- China
- Prior art keywords
- protein
- compound
- preparation
- peptide
- present
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Endocrinology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a kind of compound, protein and the preparation method of the two, it is related to the technical field of biological medicine.The complex stabilities that the present invention is provided are strong, on the basis of long-acting function of blood sugar reduction is reached using glucagon-like peptide 1, realization can be administered by oral mode, be that the further exploitation of diabetes related drugs is laid a good foundation, and be worth follow-up continuation research and development；The preparation method for the protein that the present invention is provided, physics deoxidation is carried out under conditions of 75 85 DEG C, the purity of protein can be greatly improved；The protein that the present invention is provided, make use of the preparation method of the protein of the invention provided to prepare, purity reaches more than 80%.Also, the protein purification method provided by the present invention can remove the heat toxin albumen and nucleic acid substances that saccharomycetes to make fermentation is introduced, and be that protein offers convenience as the security and quality research in the application later stage of pharmaceutical preparation.
Description
Technical field
The present invention relates to biomedicine technical field, more particularly, to a kind of compound, protein and the preparation side of the two
Method.
Background technology
Diabetes (Diabetes mellitus, DM) are the metabolic diseases being characterized with the relative or absolute deficiency of insulin
Disease, insulin is relative or definitely deficiency triggers hyperglycaemia, and then cause three major nutrient metabolic disorder, and final influence patient is just
Normal physiological function and cause complication.Global diabetic is growing day by day, it is contemplated that the year two thousand thirty is up to 4.39 hundred million.
Glucagon-like-peptide-1 be one containing 37 amino acid, the peptide of response can be made to food intake, be by
The secretion of intestines L- cells.It has been found that glucagon-like-peptide-1 can stimulate insulin secretion (insulinotropic activity), so as to draw
Absorption and serum level of glucose of the cell to glucose is played to decline.However, glucagon-like-peptide-1 activity is very low, and serum
Half-life period is only 3-5 minutes.At present, studied and used fusion protein technology to extend glucagon-like-peptide-1 in vivo
Remaining time, but need daily in drug administration by injection before meals, it is inconvenient in Clinical practice.
Therefore, develop it is a kind of can be orally, reach the stabilization of long-acting function of blood sugar reduction using glucagon-like-peptide-1
Protein complex be increasingly becoming the focus of research.
Simultaneously there is provided a kind of high-purity, the protein of stable compound can be formed with glucagon-like-peptide-1
It is particularly important.
In view of this, it is special to propose the present invention.
The content of the invention
The present invention first purpose be to provide a kind of compound, with alleviate present in prior art utilize the high blood of pancreas
The sugared hypoglycemic of element sample peptide -1 can not be realized orally, and the short technical problem of timeliness.Second object of the present invention is to provide a kind of
The preparation method of compound.Third object of the present invention is to provide a kind of preparation method of protein, to alleviate existing skill
Purification process present in art purifies the low technical problem of obtained lipidated protein.Fourth object of the present invention is to provide
A kind of protein, to alleviate the technical problem that lipidated protein present in prior art is low.
A kind of compound that the present invention is provided, including protein and the plain peptide -1 of pancreas hyperglycaemia sample.
Further, the protein is following (1), (2) or (3)：
(1) protein being made up of the amino acid sequence in sequence table shown in SEQ ID NO.1；
(2) protein being made up of the amino acid sequence in sequence table shown in SEQ ID NO.2；
(3) the taking by one or several amino acid residues by SEQ ID NO.1 or SEQ ID NO.2 amino acid sequence
Generation and/or missing and/or addition are by its derivative protein.
Present invention also offers a kind of preparation method of above-mentioned compound, comprise the following steps：
Step (a)：By the protein and the plain peptide -1 of the pancreas hyperglycaemia sample according to molar ratio 1:8-4:1 is dissolved in mops
In buffer solution, mixed liquor is obtained, and adjust pH to 1.7-10.8；
Step (b)：By the mixed liquor after 25-35 DEG C is incubated, fully shaking；
Step (c)：Mixed liquor through concussion is subjected to excusing from death ripple processing；
Step (d)：Refrigeration cooling is stayed overnight.
Further, the time being incubated in the step (b) is 10-30min, and the time of concussion is 3-10min.
Further, the time of ultrasonication is 1-5min in the step (d), and the temperature of the refrigeration is 4 DEG C.
Present invention also offers the preparation method of above-mentioned protein, including：
The pH to 2.0-4.0 of yeast fermentation broth is adjusted, is placed after 8-12h, 75-85 DEG C is heated to, physics deoxidation is carried out, from
The heart simultaneously collects precipitation.
Further, the yeast fermentation broth is Pichia yeast fermentation broths.
Further, the method for the physics deoxidation is to enter bubbling inert gas in the Pichia yeast fermentation broths
Portion.
Further, the time of the physics deoxidation is 2-6h.
In addition, present invention also offers a kind of protein, being prepared using above-mentioned preparation method.
The complex stabilities that the present invention is provided are strong, and long-acting function of blood sugar reduction is being reached using glucagon-like-peptide-1
On the basis of, realization can be administered by oral mode, be that the further exploitation of diabetes related drugs is laid a good foundation, and be worth
Follow-up continuation research and development；The preparation method for the protein that the present invention is provided, carries out physics deoxidation, energy under conditions of 75-85 DEG C
Enough greatly improve the purity of protein；The protein that the present invention is provided, make use of the preparation method of the protein of the invention provided
Prepare, purity reaches more than 80%.Also, the protein purification method provided by the present invention can remove saccharomycete and send out
Heat toxin albumen and nucleic acid substances that ferment is introduced, are security and quality research band of the protein as the application later stage of pharmaceutical preparation
To facilitate.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art
The accompanying drawing used required in embodiment or description of the prior art is briefly described, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
The liquid chromatography results figure for the protein that Figure 1A provides for the present invention；
Figure 1B is the liquid chromatography results figure of the plain peptide -1 of pancreas hyperglycaemia sample；
Fig. 1 C are superimposed for the protein liquid chromatogram figure that provides of the present invention and glucagon-like-peptide-1 liquid chromatogram
Figure；
The plain peptide -1 of protein and pancreas hyperglycaemia sample that Fig. 1 D provide for the present invention is 1 in molar ratio：Compound is formed when 1
Liquid chromatography results figure；
The plain peptide -1 of protein and pancreas hyperglycaemia sample that Fig. 1 E provide for the present invention is 4 in molar ratio:Compound is formed when 1
Liquid chromatography results figure；
The plain peptide -1 of protein and pancreas hyperglycaemia sample that Fig. 1 F provide for the present invention is 8 in molar ratio:Compound is formed when 1
Liquid chromatography results figure；
Fig. 2A is isolated rat islet cells plasma insulin levels result figure；
Fig. 2 B are isolated rat islet cells cAMP accumulating level result figures；
The Regulation of blood glucose result figure for the compound that Fig. 3 A provide for single to invention；
Fig. 3 B are the sugar tolerance experimental result picture of the compound repeatedly provided to invention；
Insulin stimulating result of the test figures of Fig. 4 A for the compound of the invention provided in diabetic mice body；
Determination of blood glucose level result figure after the compound that Fig. 4 B provide for animal pattern single to invention；
Fig. 5 A are the TEM electron-microscope scanning result figures of 100nM compound；
Fig. 5 B are the TEM electron-microscope scanning result figures of 10 μM of compound；
Fig. 5 C are the TEM electron-microscope scanning result figures of 1mM compound；
Fig. 5 D determine the compound nanosphere diameter test result figure that the present invention is provided for DLS；
Fig. 6 simulates calculating figure for the Composite Results that the present invention is provided；
Fig. 7 is is titrated into the result figure of film test to the compound that provides of the present invention under different pH condition；
Fig. 8 A are the INS-1 cell lines of normal incubation medium culture；
Fig. 8 B are the islet tissue after oral 42 days of the compound that provides of the present invention；
Fig. 9 handled for the protein that the preparation method that provides of the present invention is prepared through zinc sulfate after electrophoresis result figure.
Embodiment
In order to illustrate more clearly of the present invention, with reference to preferred embodiment, the present invention is described further.Ability
Field technique personnel should be appreciated that following specifically described content is illustrative and be not restrictive, and this should not be limited with this
The protection domain of invention.Unreceipted actual conditions person in embodiment, the condition advised according to normal condition or manufacturer is carried out.Institute
It is the conventional products that can be obtained by commercially available purchase with reagent or the unreceipted production firm person of instrument.
Traditional protein purification, the general method purified using direct ultra-filtration or liquid phase, the effect on yield and initial purity
It is undesirable, raw material are not only wasted, and add the energy and cost of labor.
The present invention is in the prior art, there is provided one kind for protein yield and the undesirable technical problem of initial purity effect
The preparation method of protein, including：
The pH to 2.0-4.0 of yeast fermentation broth is adjusted, is placed after 8-12h, 75-85 DEG C is heated to, physics deoxidation is carried out, from
The heart simultaneously collects precipitation.
Wherein, pH value for example can be, but be not limited to 2.0,3.0 or 4.0；Standing time for example can be, but be not limited to
8h, 9h, 10h, 11h or 12h；Heating-up temperature for example can be, but be not limited to 75 DEG C, 80 DEG C or 85 DEG C.
In the present invention, protein is following (1), (2) or (3)：
(1)QQCTTGQLQCCESTSTANDPATSELLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCD
SSPIGGLVSIGCVPVNV(SEQ ID NO.1)；
(2)QQCTTGQLQCCESTSTANDPATSKLLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCD
SSPIGGLVSIGCVPVNV(SEQ ID NO.2)；
(3) the taking by one or several amino acid residues by SEQ ID NO.1 or SEQ ID NO.2 amino acid sequence
Generation and/or missing and/or add by its derivative protein,
It for example can be, but be not limited to：
QQCTTGQLQCCKSTSTANDPATSELLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIG
CVPVNV (SEQ ID NO.3) or
QQCTTGQLQCCKSTSTANDPATSKLLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIG
CVPVNV(SEQ ID NO.4)。
In the present invention, yeast fermentation broth is Pichia yeast fermentation broths.
In the present invention, the method for physics deoxidation is to enter bubbling inert gas in the Pichia yeast fermentation broths
Portion.
Wherein, inert gas can be nitrogen or argon gas, preferably nitrogen.
In another embodiment, the method for physics deoxidation is that reduced vacuum is evacuated deoxidation.
In the present invention, the time of physics deoxidation is 2-6h.
Wherein, the time of physics deoxidation for example can be, but be not limited to 2h, 3h, 4h, 5h or 6h.
In addition, the protein precipitation that the method for producing protein provided by the present invention is prepared, can also use sulfuric acid
Zinc carries out single treatment, further the whole purity of lifting protein.
Wherein, sulfuric acid zinc concentration is 1.3M.
The preparation method for the protein that the present invention is provided, carries out physics deoxidation under conditions of 75-85 DEG C, by this behaviour
Make, have compared to traditional direct ultra-filtration and liquid phase purifying method on yield and initial purity and be significantly improved, conventional method
In completion ultrafiltration or liquid phase, purity of protein is only 10% after purification, and the albumen that the hot bubbling method described using the present invention is obtained
Purity, after zinc sulfate (1.3M) precipitation process once, can make the whole purity of protein up to more than 80%>90%,
Meet the requirement as complex formulation.Also, the protein purification method provided by the present invention can remove saccharomycete and send out
Heat toxin albumen and nucleic acid substances that ferment is introduced, are security and quality research band of the protein as the application later stage of pharmaceutical preparation
To facilitate.
Present invention also offers a kind of protein, apply above-mentioned preparation method and prepare.
Present invention also offers a kind of compound, compound includes above-mentioned protein and the plain peptide -1 of pancreas hyperglycaemia sample.
In addition, present invention also offers the preparation method of above-mentioned compound, comprising the following steps：
Step (a)：By protein and the plain peptide -1 of pancreas hyperglycaemia sample according to molar ratio 1:8-4:1 is dissolved in mops buffer solutions
In, mixed liquor is obtained, and adjust pH to 1.7-10.8；
Step (b)：By mixed liquor after 25-35 DEG C is incubated, fully shaking；
Step (c)：Mixed liquor through concussion is subjected to excusing from death ripple processing；
Step (d)：Refrigeration cooling is stayed overnight.
Wherein, in step (a), the molar ratio of protein and the plain peptide -1 of pancreas hyperglycaemia sample for example can be, but not limit
In 1:8,1:5,1:3,1:1,2:1 or 4:1；The pH of adjustment for example can be, but be not limited to 1.7,3.5,6.08,6.8 or 10.8.
Alternatively, also can be by protein and the plain peptide -1 of pancreas hyperglycaemia sample according to molar ratio 1:8-4:1 is molten
In physiological saline, mixed liquor is obtained.
Wherein, in step (b), the temperature being incubated for example can be, but be not limited to 25 DEG C, 30 DEG C or 35 DEG C；Enter
The time that row is incubated for example can be, but be not limited to 10min, 20min or 30min；The time of concussion for example can be, but not limit
In 3min, 5min or 10min.
Wherein, in step (d), the time of ultrasonication for example can be, but be not limited to 1min, 3min or 5min；
The temperature of refrigeration is 4 DEG C.
The compound that the preparation method provided using the present invention is prepared, stability is strong, is utilizing glucagon
Peptide -1 reaches on the basis of long-acting function of blood sugar reduction that realization can be administered by oral mode, be diabetes related drugs
Further exploitation is laid a good foundation, and is worth follow-up continuation research and development.
The method of the present invention can be carried out in traditional commercial equipment or device, and those of ordinary skill in the art can root
According to the device used in the condition designed, designed of the inventive method.Agents useful for same is commercially available, can also be made by oneself.
Technical scheme is described further below in conjunction with embodiment and comparative example.
Embodiment 1pH values
A kind of preparation method of protein, including：
Embodiment 1-1
Adjust the pH to 3.0 of Pichia yeast fermentation broths；
Embodiment 1-2
Adjust the pH to 2.0 of Pichia yeast fermentation broths；
Embodiment 1-3
Adjust the pH to 4.0 of Pichia yeast fermentation broths；
Embodiment 1-4
Adjust the pH to 3.0 of Pichia yeast fermentation broths；
Comparative example 1-1
Adjust the pH to 1.0 of Pichia yeast fermentation broths；
Comparative example 1-2
Adjust the pH to 5.0 of Pichia yeast fermentation broths；
Above-described embodiment 1-1 to 1-4 and comparative example 1-1 and 1-2 yeast fermentation broth are placed after 10h, 80 DEG C are heated to,
Nitrogen bubble is entered simultaneously and is centrifuged after the inside of Pichia yeast fermentation broths, 4h and collects precipitation.Wherein embodiment 1-4 will be precipitated
Single treatment is carried out with 1.3M zinc sulfate.
In order to examine the different pH value of Pichia yeast fermentation broths to the yield of protein and the influence of purity, apply respectively
The preparation method that embodiment 1-1 to 1-4 and comparative example 1-1 and 1-2 are provided prepares protein, and application western-blot inspection
Survey means are detected to the yield and purity of the protein obtained by preparation, the results are shown in Table 1.
The different pH value of the Pichia yeast fermentation broths of table 1 is to the yield of protein and the influence of purity
| Yield (%) | Purity (%) | |
| Embodiment 1-1 | 46 | 88 |
| Embodiment 1-2 | 38 | 82 |
| Embodiment 1-3 | 41 | 83 |
| Embodiment 1-4 | 39 | 95 |
| Comparative example 1-1 | 19 | 55 |
| Comparative example 1-2 | 22 | 68 |
As can be seen from Table 1, the preparation method that Application Example 1-1 to 1-4 is provided prepares the yield and purity of protein
Increased significantly compared with prior art.When the pH value of Pichia yeast fermentation broths is 3.0, and it will precipitate with 1.3M zinc sulfate
When carrying out single treatment, yield and purity highest, are preferable ph.
The standing time of embodiment 2
A kind of preparation method of protein, including：The pH of Pichia yeast fermentation broths is adjusted to 3.0,
Embodiment 2-1
Place 8h；
Embodiment 2-2
Place 9h；
Embodiment 2-3
Place 11h；
Embodiment 2-4
Place 12h；.
Comparative example 2-1
Place 5h；
Comparative example 2-2
Place 15h；
Above-described embodiment 2-1 to 2-3 and comparative example 2-1 and 2-2 were placed after the corresponding time, 80 DEG C are heated to, simultaneously will
Nitrogen bubble, which enters, centrifuges and collects precipitation after the inside of Pichia yeast fermentation broths, 4h.
In order to examine different standing times to the yield of protein and the influence of purity, difference Application Example 1-1,2-
1 to 2-4 and the preparation method that provides of comparative example 2-1 and 2-2 prepare protein, and application western-blot detection means pair
The yield and purity of protein obtained by preparation are detected, the results are shown in Table 2.
The different standing time of table 2 is to the yield of protein and the influence of purity
As can be seen from Table 2, the preparation method that Application Example 2-1 to 2-4 is provided prepares the yield and purity of protein
Increased significantly compared with prior art.When the time of placement is 10h, yield and purity highest, are preferred standing time.
The heating-up temperature of embodiment 3
A kind of preparation method of protein, including：The pH to 3.0 of Pichia yeast fermentation broths is adjusted, 10h is placed.
Embodiment 3-1
75 DEG C are heated to, while nitrogen bubble to be entered to the inside of Pichia yeast fermentation broths.
Embodiment 3-2
85 DEG C are heated to, while nitrogen bubble to be entered to the inside of Pichia yeast fermentation broths.
Comparative example 3-1
70 DEG C are heated to, while nitrogen bubble to be entered to the inside of Pichia yeast fermentation broths.
Comparative example 3-2
90 DEG C are heated to, while nitrogen bubble to be entered to the inside of Pichia yeast fermentation broths.
Centrifuge and collect after above-described embodiment 3-1 and 3-2, the zymotic fluid of comparative example 3-1 and 3-2 processing, nitrogen bubble 4h
Precipitation.
In order to examine different heating-up temperatures to the yield of protein and the influence of purity, difference Application Example 1-1,3-
The preparation method that 1 and 3-2 and comparative example 3-1 and 3-2 are provided prepares protein, and application western-blot detection means pair
The yield and purity of protein obtained by preparation are detected, the results are shown in Table 3.
The different heating-up temperature of table 3 is to the yield of protein and the influence of purity
As can be seen from Table 3, the preparation method that Application Example 3-1 and 3-2 is provided prepares the yield and purity of protein
Increased significantly compared with prior art.When heating-up temperature is 80 DEG C, yield and purity highest, are preferred heating-up temperature.
The time of the physics deoxidation of embodiment 4
A kind of preparation method of protein, including：The pH to 3.0 of Pichia yeast fermentation broths is adjusted, is placed after 10h, plus
Heat is to 80 DEG C.
Embodiment 4-1
Nitrogen bubble is entered simultaneously and is centrifuged after the inside of Pichia yeast fermentation broths, 2h and collects precipitation.
Embodiment 4-2
Nitrogen bubble is entered simultaneously and is centrifuged after the inside of Pichia yeast fermentation broths, 3h and collects precipitation.
Embodiment 4-3
Nitrogen bubble is entered simultaneously and is centrifuged after the inside of Pichia yeast fermentation broths, 5h and collects precipitation.
Embodiment 4-4
Nitrogen bubble is entered simultaneously and is centrifuged after the inside of Pichia yeast fermentation broths, 6h and collects precipitation.
Comparative example 4-1
Nitrogen bubble is entered simultaneously and is centrifuged after the inside of Pichia yeast fermentation broths, 1h and collects precipitation.
Comparative example 4-2
Nitrogen bubble is entered simultaneously and is centrifuged after the inside of Pichia yeast fermentation broths, 7h and collects precipitation.
In order to examine the different time of physics deoxidation to the yield of protein and the influence of purity, difference Application Example 1-
1st, the preparation method that 4-1 to 4-4 and comparative example 4-1 and 4-2 are provided prepares protein, and application western-blot detection hand
Section is detected to the yield and purity of the protein obtained by preparation, the results are shown in Table 4.
The different time of the physics deoxidation of table 4 is to the yield of protein and the influence of purity
As can be seen from Table 4, the preparation method that Application Example 4-1 to 4-4 is provided prepares the yield and purity of protein
Increased significantly compared with prior art.When the time of physics deoxidation is 4h, yield and purity highest, are preferred physics deoxidation
Time.
When the pH of regulation Pichia yeast fermentation broths is 3.0, standing time it can be seen from above example and comparative example
For 10h, heating-up temperature is 80 DEG C, while nitrogen bubble to be entered to the inside 4h of Pichia yeast fermentation broths, the albumen prepared
Matter yield and purity highest, are preferred preparation condition.
The protein prepared using optimization protein preparation condition, is prepared compound with the plain peptide -1 of pancreas hyperglycaemia sample
Thing.
The molar ratio of the protein of embodiment 5 and the plain peptide -1 of pancreas hyperglycaemia sample
A kind of preparation method of protein and the plain compound of peptide -1 of pancreas hyperglycaemia sample, including：
Embodiment 5-1
By protein and the plain peptide -1 of pancreas hyperglycaemia sample according to molar ratio 1:3 are dissolved in mops buffer solutions.
Embodiment 5-2
By protein and the plain peptide -1 of pancreas hyperglycaemia sample according to molar ratio 1:5 are dissolved in mops buffer solutions.
Embodiment 5-3
By protein and the plain peptide -1 of pancreas hyperglycaemia sample according to molar ratio 1:8 are dissolved in mops buffer solutions.
Embodiment 5-4
By protein and the plain peptide -1 of pancreas hyperglycaemia sample according to molar ratio 1:1 is dissolved in mops buffer solutions.
Embodiment 5-5
By protein and the plain peptide -1 of pancreas hyperglycaemia sample according to molar ratio 2:1 is dissolved in mops buffer solutions.
Embodiment 5-6
By protein and the plain peptide -1 of pancreas hyperglycaemia sample according to molar ratio 4:1 is dissolved in mops buffer solutions.
Comparative example 5-1
By protein and the plain peptide -1 of pancreas hyperglycaemia sample according to molar ratio 1:10 are dissolved in mops buffer solutions.
Comparative example 5-2
By protein and the plain peptide -1 of pancreas hyperglycaemia sample according to molar ratio 10:1 is dissolved in mops buffer solutions.
After above-described embodiment 5-1 to 5-6 and comparative example 5-1 and 5-2 mixed liquor adjustment pH to 6.08, in 30 DEG C of progress
20min, fully shaking 5min are incubated, afterwards after ultrasonication 2min, cooling is refrigerated in 4 DEG C and stays overnight.
In order to verify the different mol ratio example of the plain peptide -1 of protein and pancreas hyperglycaemia sample to the steady of the compound for preparing
Qualitative and envelop rate influence, the preparation method that Application Example 5-1 to 5-6 and comparative example 5-1 and 5-2 are provided respectively prepares multiple
Compound, the stability of the compound obtained by preparing is detected as liquid phase method, and application dialysis detects the compound obtained by preparing
Envelop rate, testing result is shown in Table 5.
Influence of the molar ratio of the protein of table 5 and the plain peptide -1 of pancreas hyperglycaemia sample to compound
| Stability (%) | Envelop rate (%) | |
| Embodiment 5-1 | 88 | 91 |
| Embodiment 5-2 | 77 | 84 |
| Embodiment 5-3 | 83 | 88 |
| Embodiment 5-4 | 81 | 88 |
| Embodiment 5-5 | 76 | 86 |
| Embodiment 5-6 | 75 | 79 |
| Comparative example 5-1 | 25 | 15 |
| Comparative example 5-2 | 24 | 19 |
As can be seen from Table 5, the preparation method that Application Example 5-1 to 5-6 is provided prepares the stability and bag of compound
Envelope rate is in higher level.When the molar ratio of protein and the plain peptide -1 of pancreas hyperglycaemia sample is 1:When 3, stability and envelop rate
Highest, is preferred molar ratio example.
The incubation temperature of embodiment 6
A kind of preparation method of protein and the plain compound of peptide -1 of pancreas hyperglycaemia sample, including：By protein and pancreas hyperglycaemia
Sample element peptide -1 is according to molar ratio 1:3 are dissolved in mops buffer solutions, obtain mixed liquor.
Embodiment 6-1
The mixed liquor that embodiment 6 is obtained is incubated in 25 DEG C.
Embodiment 6-2
The mixed liquor that embodiment 6 is obtained is incubated in 35 DEG C.
Comparative example 6-1
The mixed liquor that embodiment 6 is obtained is incubated in 20 DEG C.
Comparative example 6-2
The mixed liquor that embodiment 6 is obtained is incubated in 40 DEG C.
It is after 6.08, to be incubated 20min by above-described embodiment 6-1,6-2 and comparative example 6-1 and 6-2 mixed liquor adjustment pH,
Fully shaking 5min, afterwards after ultrasonication 2min, refrigerates cooling in 4 DEG C and stays overnight.
In order to verify that different incubation temperatures, to the stability of compound prepared and the influence of envelop rate, are applied respectively
The preparation method that embodiment 5-1,6-1 and 6-2 and comparative example 6-1 and 6-2 are provided prepares compound, is provided by embodiment 5
The stability and envelop rate of compound obtained by method detection preparation, testing result are shown in Table 6.
The different incubation temperatures of table 6 are to the stability of compound and the influence of envelop rate
| Stability (%) | Envelop rate (%) | |
| Embodiment 5-1 | 88 | 91 |
| Embodiment 6-1 | 79 | 86 |
| Embodiment 6-2 | 80 | 86 |
| Comparative example 6-1 | 24 | 43 |
| Comparative example 6-2 | 31 | 51 |
As can be seen from Table 6, the preparation method that Application Example 6-1 and 6-2 is provided prepares the stability and bag of compound
Envelope rate is in higher level.When incubation temperature is 30 DEG C, stability and envelop rate highest, are preferred incubation temperature.
The incubation time of embodiment 7
A kind of preparation method of protein and the plain compound of peptide -1 of pancreas hyperglycaemia sample, including：By protein and pancreas hyperglycaemia
Sample element peptide -1 is according to molar ratio 1:3 are dissolved in mops buffer solutions, obtain mixed liquor, adjust the pH to 6.08 of mixed liquor, will be mixed
Liquid is closed to be incubated in 30 DEG C.
Embodiment 7-1
The mixed liquor that embodiment 7 is obtained is incubated 10min.
Embodiment 7-2
The mixed liquor that embodiment 7 is obtained is incubated 30min.
Comparative example 7-1
The mixed liquor that embodiment 7 is obtained is incubated 5min.
Comparative example 7-2
The mixed liquor that embodiment 7 is obtained is incubated 40min.
By above-described embodiment 7-1 and 7-2 and comparative example 7-1 and 7-2 mixed liquor fully shaking 5min, afterwards at ultrasonic wave
Manage after 2min, refrigerating cooling in 4 DEG C stays overnight.
In order to verify that different incubation times, to the stability of compound prepared and the influence of envelop rate, are applied respectively
The preparation method that embodiment 5-1,7-1 and 7-2 and comparative example 7-1 and 7-2 are provided prepares compound, is provided by embodiment 5
The stability and envelop rate of compound obtained by method detection preparation, testing result are shown in Table 7.
The different incubation times of table 7 are to the stability of compound and the influence of envelop rate
| Stability (%) | Envelop rate (%) | |
| Embodiment 5-1 | 88 | 91 |
| Embodiment 7-1 | 81 | 90 |
| Embodiment 7-2 | 85 | 85 |
| Comparative example 7-1 | 34 | 41 |
| Comparative example 7-2 | 33 | 44 |
As can be seen from Table 7, the preparation method that Application Example 7-1 and 7-2 is provided prepares the stability and bag of compound
Envelope rate is in higher level.When incubation time is 20min, stability and envelop rate highest, are preferred incubation time.
Embodiment 8 is incubated pH value
A kind of preparation method of protein and the plain compound of peptide -1 of pancreas hyperglycaemia sample, including：By protein and pancreas hyperglycaemia
Sample element peptide -1 is according to molar ratio 1:3 are dissolved in mops buffer solutions, obtain mixed liquor.
Embodiment 8-1
The mixed liquor that embodiment 8 is obtained adjusts pH to 1.7.
Embodiment 8-2
The mixed liquor that embodiment 8 is obtained adjusts pH to 3.5.
Embodiment 8-3
The mixed liquor that embodiment 8 is obtained adjusts pH to 6.8.
Embodiment 8-4
The mixed liquor that embodiment 8 is obtained adjusts pH to 10.8.
Comparative example 8-1
The mixed liquor that embodiment 8 is obtained adjusts pH to 1.2.
Comparative example 8-2
The mixed liquor that embodiment 8 is obtained adjusts pH to 11.5.
Above-described embodiment 8-1 to 8-4 and comparative example 8-1 and 8-2 mixed liquor are subjected to incubation 20min in 30 DEG C.Fully
5min is shaken, afterwards after ultrasonication 2min, cooling is refrigerated in 4 DEG C and stays overnight.
In order to verify that different incubation times, to the stability of compound prepared and the influence of envelop rate, are applied respectively
Embodiment 5-1,8-1 to 8-4 and comparative example 8-1 and the 8-2 preparation method provided prepares compound, is provided by embodiment 5
The stability and envelop rate of compound obtained by method detection preparation, testing result are shown in Table 8.
The different pH that are incubated of table 8 are to the stability of compound and the influence of envelop rate
Meanwhile, also carry out influence of the different pH value for compound formation nanometer ball particle, specific test operation is：Point
Using the experiment of titration film forming not under conditions of pH is 1.7,3.5,6.08,6.8 and 10.8, it was demonstrated that compound is in pH=6.08
It is with good nanosphere form, as a result as shown in Figure 7.
The preparation method that Application Example 8-1 to 8-4 is provided it can be seen from table 8 and Fig. 7 prepares the stability of compound
Higher level is in envelop rate.When it is 6.08 to be incubated pH, stability and envelop rate highest, are preferred incubation time.
In order to further illustrate beneficial effects of the present invention, with reference to above-mentioned optimum condition, i.e., with embodiment 1-4,2-1,3-1
Protein is prepared with the 4-1 each conditions provided, the electrophoresis result of the protein prepared is as shown in figure 9, wherein M is albumen
Maker, lane 1 is protein finished product, and lane 2 is Pichia yeast fermentation broths.And with gained protein and pancreas hyperglycaemia
The optimum condition that sample the element Application Example of peptide -1 5-1 and embodiment 6-1 are provided prepares compound, and passes through following experiment detection system
The indices of standby obtained compound.
1. liquid chromatogram
Using liquid-phase chromatography method, the formation of the compound prepared using optimum condition and optimal compound are detected
Loading ratio.As a result as shown in Figure 1A, 1B, 1C, 1D, 1E and 1F.Wherein, when the delay of protein is can be seen that from Figure 1A
Between be 3.3min；As can be seen that the holdup time of the plain peptide -1 of pancreas hyperglycaemia sample is 5.2min from Figure 1B；Can from Fig. 1 D
Go out, with the plain peptide -1 of pancreas hyperglycaemia sample：The high-visible compounds of 18.5min in the raising of the mol ratio of protein, liquid chromatogram
Formation；As can be seen that in protein from Fig. 1 E and 1F：The molar ratio of pancreas hyperglycaemia sample element peptide -1 is 4:1 (Fig. 1 E) or
8:When 1 (Fig. 1 F), the plain peptide -1 of final pancreas hyperglycaemia sample can form compound by protein encapsulation completely.
2. Determination of Physiological Activity
The physiologically active evaluation criterion of protein and the plain compound of peptide -1 of pancreas hyperglycaemia sample includes：Insulin stimulating secretes work(
Result and Regulation of blood glucose function that energy, cAMP are largely accumulated.The application is commented in strict accordance with the plain analog of peptide -1 of hyperglycaemia sample
Valency method has carried out above-mentioned evaluation to compound.In addition, the oral compound provided to the present invention of animal is provided
Hypoglycemic evaluation method.Total result is confirmed：Compound has orally available, long-acting function of blood sugar reduction, be worth it is follow-up after
Continuous exploitation.
2.1 isolated tests confirm that the insulin stimulating secretion of compound and cAMP stimulate accumulation function
Insulin stimulating is secreted：Isolated rat islet cells is taken, is divided into control group and compound group, every group sets 3 repetitions.
Cellular control unit is incubated in physiological saline；The cell culture of compound group after 48h, adds 55 in 15mmol/L glucose solutions
The μ L of μ g/L compounds 20 continue to cultivate 8 hours.Two groups of islet cells are collected, being separately added into 15mmol/L glucose solutions stimulates training
Support 1h.Nutrient solution is collected, the level of insulin secretion stimulated with its basis of measured by radioimmunoassay and glucose.As a result figure is seen
2A。
CAMP stimulates accumulation function：Isolated rat islet cells is taken, is divided into control group, the pancreas hyperglycaemia sample element group of peptide -1 and multiple
Compound group, every group sets 3 repetitions.Cellular control unit is incubated in physiological saline；Pancreas hyperglycaemia sample element the cell culture of the group of peptide -1 in
In 15mmol/L glucose solutions after 48h, the plain solution of peptide -1 of 55 μ g/L pancreas hyperglycaemia samples is added, continues to cultivate 8h；Compound group
Cell culture after 48h, adds the μ L of 55 μ g/L compounds 20 and continues to cultivate 8 hours in 15mmol/L glucose solutions.Collect each
Group islet cells, being separately added into 15mmol/L glucose solutions stimulates culture 1h.Collect nutrient solution, with measured by radioimmunoassay its
The cAMP accumulating functions that basis and glucose are stimulated.As a result Fig. 2 B are seen.
Wherein, Fig. 2A is plasma insulin levels result figure.Plasma insulin levels use radio-immunity reagent in experiment
Box (EIA- Insulin Kits) is operated.It can be seen that compared to control group (▼), compound group () has obvious
Insulin stimulating function；Fig. 2 B are cAMP accumulating level result figures, it can be seen that compared to control group (zero) and pancreas
The hyperglycaemia sample element group of peptide -1 (●), compound group () substantially increases cAMP accumulating amount.It follows that what the present invention was provided
Compound, with the active function stimulated insulin secretion with cAMP, and is significantly better than in stability and simple to use the high blood of pancreas
Sugar-like element peptide -1.
The compound function of blood sugar reduction of 2.2 normal mouses
From Kunming mouse, 20 ± 2g of body weight carries out sugar tolerance research.
Single is tested to compound：Mouse is divided into the plain group of peptide -1 of pancreas hyperglycaemia sample and compound group, every group 3, if 3
Repeat.Compound group mouse gives physiological saline (the 500 μ g/ of the compound provided dissolved with the present invention by the method for gavage
Kg), the plain group of peptide -1 mouse of pancreas hyperglycaemia sample gives the physiological saline dissolved with the plain peptide -1 of pancreas hyperglycaemia sample by the method for gavage
Feeding is mixed with the feed (3.5g/kg) of sugar, 30min-120h areal surveys at once after (500 μ g/kg), compound group mouse stomach
Mouse blood sugar value.As a result as shown in Figure 3A.
Repeatedly give compound experiment：Mouse is divided into the plain group of peptide -1 of pancreas hyperglycaemia sample and compound group, every group 3, if 3
Repeat.The feeding of compound group mouse is mixed with the feed (500 μ g/kg) for the compound that the present invention is provided, pancreas hyperglycaemia sample element peptide -1
Group mouse feeding is mixed with the feed (500 μ g/kg) of the plain peptide -1 of pancreas hyperglycaemia sample, and the feeding of sugar is mixed with to feeding after compound 30min
Expect (3.5g/kg) that daily same time measurement blood glucose continues 35 days.As a result as shown in Figure 3 B (● be compound group, is pancreas
Hyperglycaemia sample element peptide -1 group).
Regulation of blood glucose result from Fig. 3 A single to compound, after single oral is to compound, compound group is small
The blood glucose of mouse is in 24 hours still in normal range (NR)；It is multiple from Fig. 3 B sugar tolerance experimental result for repeatedly giving compound
After the experiment in 35 days of compound group mouse, internal Saccharification blood gp declines 1.09 ± 0.06%.
It follows that the compound that the present invention is provided has stable long-acting function of blood sugar reduction.
The compound function of blood sugar reduction of 2.3 diabetic mices
Select the Spontaneous Diabetic mouse db/db of monogenic inheritance, body weight：20 ± 2g, is divided into pancreas hyperglycaemia by mouse
The sample element group of peptide -1 and compound group, every group 6, if 3 repetitions.Compound group mouse daily feeding be mixed with the present invention provide answer
The feed (500 μ g/kg) of compound, the pancreas hyperglycaemia sample element group of peptide -1 mouse feeding daily is mixed with the feed of the plain peptide -1 of pancreas hyperglycaemia sample
(500 μ g/kg), measures blood sugar level and insulin level daily.As a result as illustrated in figures 4 a and 4b (● be compound group, zero is
Pancreas hyperglycaemia sample element peptide -1 group).
Wherein, Fig. 4 A are that insulin stimulating of the compound of the invention provided in diabetic mice body is tested, as a result table
Bright, compound increases the blood glucose sensitiveness of diabetic mice；Blood after the compound that Fig. 4 B provide for animal pattern single to invention
Sugar level is determined, and as a result shows compound single to maintaining good Regulation of blood glucose ability after compound in 20 hours.
3. the Senile Mouse of compound
Senile Mouse for compound is carried out using transmission electron microscope (TEM) and dynamic light scattering (DLS),
As a result as shown in Fig. 5 A, 5B, 5C and 5D.
As shown in Figure 5A, the TEM electron-microscope scanning figures of the compound of low concentration (100nM) show compound into spherical point of nanometer
Cloth；Fig. 5 B are the TEM electron-microscope scanning figures of 10 μM of compound, and Fig. 5 C are the TEM electron-microscope scanning figures of 1mM compound, by Fig. 5 B
Understood with 5C, after the concentration rise of compound, these single nanospheres of TEM scanning graph discoveries also have the work(mutually assembled
Can, so as to form one layer of stable nanometer spherical network structure；As shown in Figure 5 D, nanosphere diameter test is determined using DLS
As a result, it was demonstrated that the size of compound is 80nm.
As a result show, by controlling the self assembly condition of compound, can finally form homogeneous, stable nano particle knot
Structure.
The application has carried out result simulation calculating to compound simultaneously, as a result confirms protein and pancreas that the present invention is provided
Hyperglycaemia sample element peptide -1 can form nanometer spherical compound, as shown in Figure 6.
4. the islet cells of compound recovers function
Because the plain peptide -1 of pancreas hyperglycaemia sample itself has the function of suppressing Intra-islet Apoptosis, and by this function come extensive
The islet cells being damaged again.Therefore, the application is by evaluating the volume of islet cells come the pancreas islet recovery function of evaluating combined thing.
Fig. 8 A are the INS-1 cell lines of normal incubation medium culture, it can be seen that pancreas islet atrophy does not have caused by the height sugar processing of early stage
It is restored because low sugar condition is returned to, Fig. 8 B are the islet tissues after oral 42 days of compound.As a result this hair is shown
The compound of bright offer can substantially recover islet damage caused by high sugar.
5. the toxicity of compound
The application has carried out the anxious poison and long term toxication of mouse, as a result shows that compound does not possess blood and physiological-toxicity.
5.1 body weight and overview
SC213 male mices are divided into blank group, solvent group, low dose group, middle dose group and high dose group, every group 30
Only, if 3 repetitions.Each group mouse is through subcutaneously giving compound (low dose group：5mg/kg body weight；Middle dose group：15mg/kg body weight；
High dose group：50mg/kg body weight) after 30 days, the body weight of each group mouse is measured, 8 are the results are shown in Table.
The SC213 mouse of table 8 subcutaneously give 30 days long term toxicity body weight tables of compound
As seen from the results in Table 8, SC213 mouse subcutaneously to the influence after the compound that provides of the present invention one month to body weight not
Substantially, each group the weight of animals increasess slowly to compound mid-term compared with blank control group ratio, and the later stage recovers；The drinking water amount of intake with
The change of body weight is varied from.
5.2 organ index results
SC213 male mices are divided into blank group, solvent group, low dose group, middle dose group and high dose group, every group 20
Only, if 3 repetitions.Each group mouse is after subcutaneously giving compound 30 days, by determining mouse weight and organ weights acquisition each group
The long term toxicity organ index of mouse, as a result as shown in table 9.
The SC213 mouse of table 9 subcutaneously give 30 days long term toxicity organ index tables of compound
As seen from the results in Table 9, SC213 mouse subcutaneously show to organ index result after the compound that provides of the present invention one month
Show, the thymus index to mouse is in dose-dependent increase, and high dose group has statistical significance with blank control group ratio.
5.3 biochemistry detection
SC213 male mices are divided into blank group, solvent group, low dose group, middle dose group and high dose group, every group 8,
If 3 repetitions.Each group mouse is after subcutaneously giving compound 30 days, and the long term toxicity blood for determining each group mouse using biochemical instruments is given birth to
Change index, as a result as shown in Table 10 and Table 11.
The SC213 mouse of table 10 subcutaneously determine table to 30 days long term toxicity blood biochemicals of compound
The SC213 mouse of table 11 subcutaneously determine continued to 30 days long term toxicity blood biochemicals of compound
From table 10 and the result of table 11, SC213 mouse subcutaneously to compound after one month blood parameters point out nothing
Substantially with dose-dependent toxicity；The blood glucose value of each group mouse decreases with the increase of dosage, there is certain dosage correlation.
5.4 peripheral blood cell counts
SC213 male mices are divided into blank group, solvent group, low dose group, middle dose group and high dose group, every group 7,
If 3 repetitions.Each group mouse determines the long term toxicity blood of each group mouse using automatic biochemical analyzer after subcutaneously giving compound 30 days
The liquid index of correlation, as a result as shown in table 12 to table 16.
The SC213 mouse of table 12 subcutaneously determine table to 30 days long term toxicity hematologies of compound
The SC213 mouse of table 13 subcutaneously determine continued -1 to 30 days long term toxicity hematologies of compound
The SC213 mouse of table 14 subcutaneously determine continued -2 to 30 days long term toxicity hematologies of compound
The SC213 mouse of table 15 subcutaneously determine continued -3 to 30 days long term toxicity hematologies of compound
The SC213 mouse of table 16 subcutaneously determine continued -4 to 30 days long term toxicity hematologies of compound
Note：* the P compared with blank control group is represented<0.05, with significant difference, * * represent to compare P with blank control group<
0.01, with pole significant difference.
From the result of table 12 to table 16, SC213 mouse subcutaneously to compound after one month hematological indices point out nothing
Significantly with dose-dependent toxicity.
Therefore, the protein of preparation method preparation and the stable composite of glucagon-like-peptide-1 are provided using the present invention
Property it is strong, on the basis of long-acting function of blood sugar reduction is reached using glucagon-like-peptide-1, realization can pass through oral mode
Administration, is that the further exploitation of diabetes related drugs is laid a good foundation, and is worth follow-up continuation research and development.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
The present invention is described in detail with reference to foregoing embodiments for pipe, it will be understood by those within the art that：Its according to
The technical scheme described in foregoing embodiments can so be modified, or which part or all technical characteristic are entered
Row equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology
The scope of scheme.
SEQUENCE LISTING
<110>Tianjin bio tech ltd handed down through generations
<120>Compound, protein and the preparation method of the two
<160> 4
<210> 1
<211> 83
<212> PRT
<213>Pichia yeast
<400> 1
Gln Gln Cys Thr Thr Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr
1 5 10 15
Ala Asn Asp Pro Ala Thr Ser Glu Leu Leu Gly Leu Ile Gly Val Val
20 25 30
Ile Ser Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser
35 40 45
Val Ile Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys
50 55 60
Cys Asp Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro
65 70 75 80
Val Asn Val
<210> 2
<211> 83
<212> PRT
<213>Pichia yeast
<400> 2
Gln Gln Cys Thr Thr Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr
1 5 10 15
Ala Asn Asp Pro Ala Thr Ser Lys Leu Leu Gly Leu Ile Gly Val Val
20 25 30
Ile Ser Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser
35 40 45
Val Ile Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys
50 55 60
Cys Asp Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro
65 70 75 80
Val Asn Val
<210> 3
<211> 83
<212> PRT
<213>Pichia yeast
<400> 3
Gln Gln Cys Thr Thr Gly Gln Leu Gln Cys Cys Lys Ser Thr Ser Thr
1 5 10 15
Ala Asn Asp Pro Ala Thr Ser Glu Leu Leu Gly Leu Ile Gly Val Val
20 25 30
Ile Ser Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser
35 40 45
Val Ile Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys
50 55 60
Cys Asp Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro
65 70 75 80
Val Asn Val
<210> 4
<211> 83
<212> PRT
<213>Pichia yeast
<400> 4
Gln Gln Cys Thr Thr Gly Gln Leu Gln Cys Cys Lys Ser Thr Ser Thr
1 5 10 15
Ala Asn Asp Pro Ala Thr Ser Lys Leu Leu Gly Leu Ile Gly Val Val
20 25 30
Ile Ser Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser
35 40 45
Val Ile Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys
50 55 60
Cys Asp Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro
65 70 75 80
Val Asn Val
Claims (10)
1. a kind of compound, it is characterised in that the compound includes protein and the plain peptide -1 of pancreas hyperglycaemia sample.
2. compound according to claim 1, it is characterised in that the protein is following (1), (2) or (3)：
(1) protein being made up of the amino acid sequence in sequence table shown in SEQ ID NO.1；
(2) protein being made up of the amino acid sequence in sequence table shown in SEQ ID NO.2；
(3) SEQ ID NO.1 or SEQ ID NO.2 amino acid sequence is passed through to the substitution of one or several amino acid residues
And/or missing and/or addition are by its derivative protein.
3. the preparation method of compound as claimed in claim 1 or 2, it is characterised in that the preparation method includes following step
Suddenly：
Step (a)：By the protein and the plain peptide -1 of the pancreas hyperglycaemia sample according to molar ratio 1:8-4:1 is dissolved in mops bufferings
In liquid, mixed liquor is obtained, and adjust pH to 1.7-10.8；
Step (b)：By the mixed liquor after 25-35 DEG C is incubated, fully shaking；
Step (c)：Mixed liquor through concussion is subjected to excusing from death ripple processing；
Step (d)：Refrigeration cooling is stayed overnight.
4. preparation method according to claim 3, it is characterised in that the time being incubated in the step (b) is 10-
30min, the time of concussion is 3-10min.
5. preparation method according to claim 3, it is characterised in that the time of ultrasonication is in the step (d)
1-5min, the temperature of the refrigeration is 4 DEG C.
6. the preparation method of protein as claimed in claim 2, it is characterised in that the preparation method includes：
The pH to 2.0-4.0 of yeast fermentation broth is adjusted, is placed after 8-12h, 75-85 DEG C is heated to, physics deoxidation is carried out, centrifugation is simultaneously
Collect precipitation.
7. preparation method according to claim 6, it is characterised in that the yeast fermentation broth ferments for Pichia yeast
Liquid.
8. preparation method according to claim 7, it is characterised in that the method for the physics deoxidation is to rouse inert gas
Steep the inside into the Pichia yeast fermentation broths.
9. preparation method according to claim 8, it is characterised in that the time of the physics deoxidation is 2-6h.
10. a kind of protein, it is characterised in that the preparation method described in application claim any one of 6-9 is prepared.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710256655.9A CN107033247A (en) | 2017-04-18 | 2017-04-18 | Compound, protein and the preparation method of the two |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710256655.9A CN107033247A (en) | 2017-04-18 | 2017-04-18 | Compound, protein and the preparation method of the two |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107033247A true CN107033247A (en) | 2017-08-11 |
Family
ID=59535163
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710256655.9A Pending CN107033247A (en) | 2017-04-18 | 2017-04-18 | Compound, protein and the preparation method of the two |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107033247A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108187060A (en) * | 2018-01-11 | 2018-06-22 | 王�琦 | Pharmaceutical carrier, pharmaceutical preparation and preparation method |
| CN113527506A (en) * | 2020-04-15 | 2021-10-22 | 博锐生物科技有限公司 | Fusion protein and its application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103980353A (en) * | 2014-05-21 | 2014-08-13 | 乔明强 | Hydrophobin and medicinal preparation containing same |
| CN104117220A (en) * | 2014-07-04 | 2014-10-29 | 南开大学 | Foam distillation purification method |
| CN104945514A (en) * | 2015-07-17 | 2015-09-30 | 李瑛� | Glucagon-like peptide-1 fusion protein, preparing method thereof and medicine combination thereof |
| CN106892978A (en) * | 2017-04-18 | 2017-06-27 | 天津世传生物科技有限公司 | Compound, protein and the preparation method of the two |
-
2017
- 2017-04-18 CN CN201710256655.9A patent/CN107033247A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103980353A (en) * | 2014-05-21 | 2014-08-13 | 乔明强 | Hydrophobin and medicinal preparation containing same |
| CN104117220A (en) * | 2014-07-04 | 2014-10-29 | 南开大学 | Foam distillation purification method |
| CN104945514A (en) * | 2015-07-17 | 2015-09-30 | 李瑛� | Glucagon-like peptide-1 fusion protein, preparing method thereof and medicine combination thereof |
| CN106892978A (en) * | 2017-04-18 | 2017-06-27 | 天津世传生物科技有限公司 | Compound, protein and the preparation method of the two |
Non-Patent Citations (3)
| Title |
|---|
| BAOLONG NIU等: "Heterologous expression and characterization of the hydrophobin HFBI in Pichia pastoris and evaluation of its contribution to the food industry", 《AMINO ACIDS》 * |
| 王振宇等: "《天然产物分离技术》", 31 January 2012, 中国轻工业出版社 * |
| 赵立强等: "疏水蛋白rHGFI与rHFBI的发泡能力研究", 《南开大学学报（自然科学版）》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108187060A (en) * | 2018-01-11 | 2018-06-22 | 王�琦 | Pharmaceutical carrier, pharmaceutical preparation and preparation method |
| CN108187060B (en) * | 2018-01-11 | 2021-03-09 | 王�琦 | Drug carrier, drug preparation and preparation method |
| CN113527506A (en) * | 2020-04-15 | 2021-10-22 | 博锐生物科技有限公司 | Fusion protein and its application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Frank et al. | A direct in vitro demonstration of insulin binding to isolated brain microvessels | |
| AU2015262214B2 (en) | Composition for treating or preventing metabolic disease, containing, as active ingredient, extracellular vesicles derived from Akkermansia muciniphila bacteria | |
| Bales et al. | The clinical and biochemical features of variegate porphyria: an analysis of 300 cases studied at Groote Schuur Hospital, Cape Town | |
| CN106879413A (en) | A kind of cultivation of selenium-rich blue or green money willow, extracts active ingredients and application | |
| CN101905022A (en) | Stable medicinal composition containing artemisia pollen allergen and preparation method thereof | |
| CN110150669A (en) | A kind of probiotic composition and its application suitable for patients with diabetes mellitus | |
| CN105367664B (en) | Activate GLP-1 receptor and the preparation of the fusion protein of the difunctional effect of Amylin receptor and application thereof | |
| Besser et al. | Immunoreactive corticotrophin levels in adrenocortical insufficiency | |
| JP3960393B2 (en) | Health composition | |
| CN107033247A (en) | Compound, protein and the preparation method of the two | |
| CN104257696B (en) | A kind of steady sugar yeast bacterium powder of hypoglycemic and its preparation method and application | |
| CN101525387B (en) | Recombined long-acting glucagons peptide analogue and preparation method thereof | |
| CN100381167C (en) | Medicine for treating neurasthenia | |
| Graffe et al. | Abnormal increase in urinary aquaporin-2 excretion in response to hypertonic saline in essential hypertension | |
| CN106892978A (en) | Compound, protein and the preparation method of the two | |
| CN103344764A (en) | Reagent, method and kit for detection of biological activity of glucagon-like peptide-1 (GLP-1) | |
| CN111905097B (en) | Application of an oral hypoglycemic peptide in preparing medicine for treating or preventing diabetes complicated with cardiovascular disease | |
| Maurer | The Therapy of Diabetes: Insulin therapy does not always prevent the serious complications of diabetes; current research is directed at more closely reproducing the functioning of a healthy pancreas | |
| CN106608915A (en) | GLP-1(7-37) polypeptide analog | |
| CN118546271A (en) | A kind of roxburghii polysaccharide CLP2-2 and its preparation method and use | |
| CN101716332B (en) | Use of phycocyanin for preventing and treating diabetes mellitus | |
| CN103344773A (en) | Reagent, method and kit for detection of biological activity of glucagon-like peptide-1 (GLP-1) | |
| CN113930397B (en) | Construction and culture method for yeast cell mitochondria transplanted breast tumor cells | |
| Espiner et al. | Aldosterone regulation in primary aldosteronism: influence of salt balance, posture and ACTH | |
| CN109136151A (en) | A kind of composite plant Lemonal and its application with reduction blood glucose function |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180629 Address after: 300380 A2-2 District, No. 2 Xinghua two branch road, Xiqing economic and Technological Development Zone, Tianjin Applicant after: Tianjin Bray Biotechnology Co., Ltd. Address before: 300000 Tianjin Binhai New Area Economic and Technological Development Zone Nangang industrial zone comprehensive service area office block D block two story 213-03 room. Applicant before: Tianjin Shichuan Biological Technology Co., Ltd |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170811 |
|
| RJ01 | Rejection of invention patent application after publication |