CN108060195A - A kind of method of -1 albumen of fermenting and producing Gluca Gen sample peptide - Google Patents
A kind of method of -1 albumen of fermenting and producing Gluca Gen sample peptide Download PDFInfo
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- CN108060195A CN108060195A CN201711487690.8A CN201711487690A CN108060195A CN 108060195 A CN108060195 A CN 108060195A CN 201711487690 A CN201711487690 A CN 201711487690A CN 108060195 A CN108060195 A CN 108060195A
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- 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
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
The invention discloses a kind of methods of 1 albumen of fermenting and producing Gluca Gen sample peptide, which is characterized in that comprises the following steps：The genetic engineering bacterium of nucleotide sequence containing coding 1 albumen of Gluca Gen sample peptide is cultivated with hypophosphate culture medium, control cultivation stage pH value and dissolved oxygen amount, after fermentation, centrifugation removal thalline obtains 1 albumen of Gluca Gen sample peptide by purifying supernatant.The present invention method can high-purity, stably excreting expression GLP 1, simplify separation purifying technique.
Description
Technical field
The invention belongs to technical field of polypeptide, and in particular to a kind of -1 (GLP- of fermenting and producing Gluca Gen sample peptide
1) method of albumen.
Background technology
Global diabetes morbidity is in the rapid growth phase, according to International Diabetes Federation (IDF) newest announcement
7th edition diabetes data show, global diabetic's number 4.15 hundred million in 2015, illness rate 8.8%.Global patient of diabetes
Sick rate is in rapid increase trend, it is contemplated that the year two thousand forty diabetic's number will be up to 6.42 hundred million, and illness rate will rise to 10.4%.China
Diabetic's number 2015 up to 1.096 hundred million, ranks the first in the world, it is contemplated that will be up to 1.507 to the year two thousand forty diabetes mellitus in China patient number
Hundred million.Diabetes often cause severe complication, such as the traditional complication of vascular lesion, retinopathy, nephrosis and peripheral neuropathy.
Glucagon-like-peptide-1 (GLP-1) is the hormone secreted by intestinal wall L- cells, and effect includes stimulating glucose
Dependence insulin secretion promotes biological insulin synthesis, reduces blood glucose, protect B cell, improves the disease of diabetes B
Shape.In addition, finding that it can also promote the multiplication and nerve to occur of nerve cell in recent years, inhibit nerve cell apoptosis.
It has been reported that at present using gene engineering expression GLP-1 based on expressing fusion protein, technique is needed to large intestine bar
Bacterium is crushed, and the purifying later stage needs, using cleavage, to purify again after cutting, and whether cleavage has an impact albumen, into
This costliness greatly limits the industrialization of GLP-1.Therefore, a kind of method of production GLP-1 more efficient and at low cost is sought
It is significantly.
The content of the invention
It is an object of the invention to provide a kind of methods of -1 albumen of fermenting and producing Gluca Gen sample peptide, can
High-purity, steadily secreting, expressing GLP-1.
The technical solution used in the present invention is：
A kind of method of -1 albumen of fermenting and producing Gluca Gen sample peptide, comprises the following steps：Coding will be contained
The genetic engineering bacterium of the nucleotide sequence of -1 albumen of Gluca Gen sample peptide is cultivated with hypophosphate culture medium, control training
Stage pH value and dissolved oxygen amount are supported, after fermentation, centrifugation removal thalline obtains the restructuring pancreas hyperglycaemia by purifying supernatant
Plain -1 albumen of sample peptide.
The method according to the invention, the nucleotide sequence such as SEQ ID of -1 albumen of coding Gluca Gen sample peptide
NO:Shown in 1.
The method according to the invention, the genetic engineering bacterium are included in expression vector phoA, the expression vector phoA and inserted
Entering has the nucleotide sequence of -1 albumen of coding Gluca Gen sample peptide.Preferably, periplasmic secretion signal peptide for pelB,
One kind in phoA, ompA, ompF, ompT, lamB, SPA, StII, MalE, DsbA, TorA or HlyA.Preferably, expression carries
Also contain STII signal peptide sequences in body phoA.PhoA is escherichia coli alkaline phosphatase PhoA gene promoters, can be utilized
Low-phosphorous induced expression.On the contrary, some other promoters are mostly induced by IPTG, insoluble inclusion body is easily generated, simultaneously
Derivant IPTG is costly.
The method according to the invention, the genetic engineering bacterium are Escherichia coli YK537.
The method according to the invention, the construction step of the genetic engineering bacterium are as follows：
The nucleotide sequence of -1 gene of step 1. synthesis Gluca Gen sample peptide introduces NheI enzymes at 5 ' ends of the gene
Enzyme site and 3 ' end introduce BamHI restriction enzyme sites；
Step 2. construction of expression vector：Handle the gene and table of step 1 synthesis respectively with NheI and BamHI double digestions
Up to carrier phoA, 37 DEG C of digestions 2 are recycled corresponding segment T4DNA ligases and are stayed overnight in 16 DEG C of connections when small；Connection product turns
Change into Escherichia coli, positive colony is selected on the LB tablets containing l00ug/mL ampicillins, carried so as to build expression
Body；
The expression vector that the step 2 is built is transformed into Escherichia coli YK537 by step 3..
Preferably, the primer that the nucleotide sequence is synthesized in the step 1 is as follows：
Primer sequence GLP-1-U：GGTGCTAGCGCTCATGCTGAAGGGACCTTTACC；
Primer sequence GLP-1-D：GGTGGATCCTCATCCTCGGCCTTTCACCAG.
The method according to the invention, the composition for stating hypophosphate culture medium are 10g containing peptone in every 1000ml culture mediums,
Yeast extract 2.5g, MgSO4·7H2The TrisHCl 50ml of O 0.5g, 1mol/L pH 8.0, phenol red 2ml, glucose
10g。
Preferably, hypophosphate medium pH is 7.0-8.0, and more preferable pH is 7.0-7.4.
In the hypophosphate culture medium, carbon source and nitrogen source can be added in due course.
Preferably, in the hypophosphate culture medium, the carbon source added includes glycerine, glucose, molasses.More preferably
Ground, the carbon source added are glycerine.
Preferably, in the hypophosphate culture medium, the nitrogen source added includes organic nitrogen source and/or inorganic nitrogen-sourced；
Wherein organic nitrogen source includes peptone, yeast extract (yeast extract or yeast extract)；It is inorganic nitrogen-sourced including ammonium hydroxide or ammonium salt.
It is furthermore preferred that in the hypophosphate culture medium, the nitrogen source added is ammonium hydroxide, it can provide suitable nitrogen source, simultaneously
Play the role of that pH value can be adjusted.
The present invention will encode the nucleotide sequence insertion coli alkaline of -1 (GLP-1) albumen of Gluca Gen sample peptide
In phosphatase expression vector phoA, structure obtains the GLP-1 colibacillus engineerings of efficient stably excreting expression.Through high density
Fermented and cultured, the high high adjustment induction of phosphorus concentration, GLP-1 are secreted into soluble form in culture supernatant, and expression is high, and has
There is natural activity, not only avoid and slightly propose destruction of the process to protein, and improve purifying yield, reduce cost.It is logical
Easy, quick purification process is crossed, that is, obtains the GLP-1 sterlings of high quality.
Description of the drawings
Fig. 1 is the plasmid construction schematic diagram of expression vector phoA-GLP-1.
It is the qualification result of GLP-1 in Fig. 2, wherein, A is culture supernatant electrophoretogram：1st, 2,3 be supernatant after induction；B is point
Sub- gel exclusion chromatography figure：1st, 2 be eluting peak.
Fig. 3 is the result of efficient liquid phase chromatographic analysis purity of protein.
Fig. 4 is Mass Spectrometer Method figure.
Table 1 is the experimental result using GLP-1 treatment diabetes rats.
Specific embodiment
The present invention is expressed, which contains phoA and open by in-depth study extensively with preferred carrier phoA
Mover can start the expression of destination protein, while band on carrier in the phosphoric acid salt culture medium (low-phosphorous culture medium) containing low concentration
There is the signal peptide of secreting, expressing, contribute to correct folding of the GLP-1 eukaryotic genes in protokaryon, and be secreted into soluble form
In culture supernatant, so as to simplify separation purifying technique.
With reference to specific embodiment, the present invention is further explained, but not limited to this.
Embodiment l
GLP-1 expresses the structure of engineering bacteria
1. target gene synthesizes
According to the natural acid sequence of known GLP-1, under conditions of amino acid sequence is not changed, GLP-1 is designed
Coded sequence primer.
Primer sequence GLP-1-U：GGTGCTAGCGCTCATGCTGAAGGGACCTTTACC(SEQ ID NO.2)；
Primer sequence GLP-1-D：GGTGGATCCTCATCCTCGGCCTTTCACCAG(SEQ ID NO.3).
The full length sequence of GLP-1 is obtained by bridging PCR method.Obtained GLP-1 sequences such as SEQ ID NO：1 institute
Show.
The nucleotide sequence of artificial synthesized restructuring GLP-1 genes introduces NheI restriction enzyme sites and 3 ' at 5 ' ends of the gene
End introduces BamHI restriction enzyme sites.
2. the structure of expression plasmid phoA-GLP-1 and conversion host strain
Inventor is expressed, which contributes to GLP-1 by in-depth study extensively with preferred carrier phoA
Correct folding of the eukaryotic gene in protokaryon, and secreted with soluble form in culture supernatant, simplify separation purifying technique.
The phoA expression vectors (expression vector contains phoA secreting signal peptides) that expression plasmid preserves for this laboratory, place
Main bacterium is Escherichia coli YK537.
Gene GLP-1 and expression vector phoA are handled respectively with NheI and BamHI double digestions, and 37 DEG C of digestions 2 are recycled when small
Corresponding segment is stayed overnight with T4DNA ligases in 16 DEG C of connections.Connection product conversion is containing ammonia benzyl mould into Escherichia coli
Positive colony is selected on the LB tablets of plain (l00ug/mL).The plasmid construction schematic diagram of expression vector phoA-GLP-1 is shown in Fig. 1.
The expression vector phoA-GLP-1 built is routinely operated and is transformed into Escherichia coli YK537.
Embodiment 2
The research and purifying of engineering bacteria high density fermentation
The genetic engineering bacterium that embodiment 1 obtains is inoculated into the 250ml conical flasks of the culture mediums of LB containing 50mI, cultivated
Overnight.Next day, by the bacterium solution being incubated overnight by 1：100 ratio is inoculated in low-phosphorous culture medium.Treat that OD600 reaches 6~8, by 1：
100 (low-phosphorous culture mediums) suitably add carbon source (such as glycerine, glucose, molasses, preferably glycerine), (nitrogen source added includes nitrogen source
Organic nitrogen source and/or inorganic nitrogen-sourced；Wherein organic nitrogen source includes peptone, yeast extract (yeast extract or yeast extract)；
It is inorganic nitrogen-sourced including ammonium hydroxide or ammonium salt；Most preferably ammonium hydroxide), phosphate buffer adjusted to pH7.2~7.4, DO>50%th, temperature
30~32 DEG C, induction 12h terminate.
After fermentation expression GLP-1, thalline is removed with centrifugation, purifying obtains fermented liquid supernatant.
The composition of above-mentioned hypophosphate culture medium is 10g containing peptone, yeast extract 2.5g in every 1000ml culture mediums,
MgSO4·7H2The TrisHCl 50ml, phenol red 2ml, glucose 10g of O 0.5g, 1mol/L pH 8.0.
After adjusting fermentation supernatant pH to 3.5, loading to SP-Sepharose FF chromatographic columns, 0~1mol/L NaCl are linearly terraced
Degree elution, collects eluting peak, is freezed with after RP-C18 column desalinations, using the Tricine-SDS-PAGE methods improved to GLP-
1 is identified, then, using coomassie brilliant blue staining, the results are shown in Figure 2, in the purposeful bands of 3.5KD.Using efficient liquid phase
Chromatography detects protein concentration, as shown in figure 3, its purity can reach 96.7%.Then, as shown in figure 4, destination protein passes through matter
Spectrum analysis determines its molecular size range, and the results show obtains destination protein.
Embodiment 3
Determination of biological activity
Using the activity of diabetes B rat model detection GLP-1,30 rats are purchased from Nanfang Medical Univ experimental animal
Center, 10 are Normal group, with normal mice grain feeding.After another 20 only with high glucose and high fat diet one month, once
The method of low dosage STZ is injected intraperitoneally, detects within the 2nd day its blood glucose and then thinks modeling success higher than 16.7mM.Then by diabetes
Group is randomly divided into two groups, and one group is treated using GLP-1, and one group is not treated.As shown in the following Table 1, GLP-1 can be substantially reduced glycosuria
Sick rat blood sugar concentration (p<0.01).
The blood sugar concentration of the different group rats of table 1
**p<0.01, compared with pre-treatment.
Sequence table
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<120>A kind of method of -1 albumen of fermenting and producing Gluca Gen sample peptide
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gaattcattg cttggctggt gaaaggccga gga 93
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Claims (10)
- A kind of 1. method of -1 albumen of fermenting and producing Gluca Gen sample peptide, which is characterized in that comprise the following steps：It will contain The genetic engineering bacterium for having the nucleotide sequence of -1 albumen of coding Gluca Gen sample peptide is cultivated with hypophosphate culture medium, Cultivation stage pH value and dissolved oxygen amount are controlled, after fermentation, centrifugation removal thalline obtains the restructuring pancreas by purifying supernatant - 1 albumen of glucagon-like peptide.
- 2. the according to the method described in claim 1, it is characterized in that, core of -1 albumen of coding Gluca Gen sample peptide Acid sequence such as SEQ ID NO:Shown in 1.
- It is 3. described according to the method described in claim 1, it is characterized in that, the genetic engineering bacterium includes expression vector phoA Nucleotide sequence inserted with -1 albumen of coding Gluca Gen sample peptide in expression vector phoA.
- 4. according to the method described in claim 3, it is characterized in that, also contain STII signal peptide sequences in the expression vector phoA Row.
- 5. according to the method described in claim 1, it is characterized in that, the genetic engineering bacterium is Escherichia coli YK537.
- 6. according to the method described in claim 1, it is characterized in that, the construction step of the genetic engineering bacterium is as follows：The nucleotide sequence of -1 gene of step 1. synthesis Gluca Gen sample peptide introduces NheI digestions position at 5 ' ends of the gene It puts and introduces BamHI restriction enzyme sites at 3 ' ends；Step 2. construction of expression vector：Gene and the expression load that the step 1 synthesizes are handled respectively with NheI and BamHI double digestions Body phoA, 37 DEG C of digestions 2 are recycled corresponding segment T4DNA ligases and are stayed overnight in 16 DEG C of connections when small；Connection product is transformed into Enter Escherichia coli, positive colony is selected on the LB tablets containing 100ug/mL ampicillins, so as to construction of expression vector；The expression vector that the step 2 is built is transformed into Escherichia coli YK537 by step 3..
- 7. according to the method described in claim 6, it is characterized in that, the primer of the nucleotide sequence is synthesized in the step 1 such as Under：Primer sequence GLP-1-U：GGTGCTAGCGCTCATGCTGAAGGGACCTTTACC；Primer sequence GLP-1-D：GGTGGATCCTCATCCTCGGCCTTTCACCAG.
- 8. according to the method described in claim 1, it is characterized in that, the composition of the hypophosphate culture medium is to be trained per 1000ml Support 10g containing peptone, yeast extract 2.5g, MgSO in base4·7H2The TrisHCl 50ml of O 0.5g, 1mol/L pH 8.0, Phenol red 2ml, glucose 10g.
- 9. the method according to claim 1 or 8, which is characterized in that the hypophosphate medium pH is 7.0-8.0.
- 10. the method according to claim 1 or 8, which is characterized in that the hypophosphate medium pH is 7.0-7.4.
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Cited By (5)
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|---|---|---|---|---|
| CN109868281A (en) * | 2019-03-06 | 2019-06-11 | 江苏大学附属医院 | A method of Gluca Gen sample peptide -1 is expressed using bacillus subtilis |
| CN111763679A (en) * | 2019-05-22 | 2020-10-13 | 杭州杰迪生物科技有限公司 | Preparation method of glucagon-like peptide-1 analogue, coding gene, expression vector and host cell |
| CN112831516A (en) * | 2021-02-08 | 2021-05-25 | 青岛海华莱康生物医药技术有限公司 | Recombinant bacterium for expressing GLP-1-like factor and application thereof |
| CN113474361A (en) * | 2019-02-24 | 2021-10-01 | 昂科斯密斯生物技术私人有限公司 | Method for continuous production of recombinant GLP-1 peptides from bacteria |
| CN114774496A (en) * | 2022-06-21 | 2022-07-22 | 北京惠之衡生物科技有限公司 | Method for preparing GLP-1 analogue through high-density fermentation |
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| CN114774496B (en) * | 2022-06-21 | 2022-10-04 | 北京惠之衡生物科技有限公司 | Method for preparing GLP-1 analogue through high-density fermentation |
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Application publication date: 20180522 |
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