CN106636205A - Method for efficiently expressing polypeptide PA1b (Pea Albumin 1b) - Google Patents
Method for efficiently expressing polypeptide PA1b (Pea Albumin 1b) Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
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- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
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- Molecular Biology (AREA)
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- Microbiology (AREA)
- Plant Pathology (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a method for efficiently expressing a polypeptide PA1b (Pea Albumin 1b). The method comprises the following steps: (1) synthesizing a target gene PA1b; (2) connecting the target gene PA1b and a vector; (3) transforming a recombinant plasmid PUSCN1-PA1b; (4) carrying out enzyme digestion and sequencing identification on the recombinant plasmid; (5) carrying out transfection on 293F (human embryonic kidney suspension cells) cells. The method provided by the invention has the main essentials that the PA1b is successfully expressed by adopting human-derived mammalian cells for the first time, the quality of expressed protein is stable and products are uniform; the purity reaches 98 percent; the polypeptide PA1b protein can be applied to production of drugs and foods for treating diabetes mellitus; the production cost is lower than that of insulin and the polypeptide PA1b is more convenient than the insulin; the polypeptide PA1b can be developed into an oral administration type and has no toxic or side effect; the polypeptide PA1b does not express a blood glucose lowering effect when a blood glucose level is lower than a physiological level.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to a kind of method of high efficient expression polypeptide PA1b and by upper
State applications of the polypeptide PA1b of method preparation in treatment diabetes.
Background technology
Pea albumin 1b (pea albumin lb, PAlb) is the single-stranded peptide containing 37 amino acid, and molecular weight is 3,
742Da, the amino acid of the 3rd, 7,15,20,22 and 32 is cysteine, and by 3 pairs of disulfide bond cystine knot die body is constituted.Should
Peptide participates in growing the regulation with metabolism, its binding mode and insulin and IGF in animal body in plant
Effect is similar.It can again can be with reference to animal insulin and insulin with reference to 7S Bg albumen, 7S Bg albumen in soya seeds
Like growth factor, therefore PA1b also known as leginsulin Leginsulin.
Diabetes are a kind of metabolic syndromes with hyperglycaemia as characteristic feature.According to WH02016 annual reports, global diabetes
Oneself reaches 4.22 hundred million to patient, and oneself reaches 1.3 hundred million to diabetes mellitus in China people, accounts for the 9.4 of population.Estimate according to WHO, 2005-2015
Year, China is for economic loss caused by diabetes and related cardiovascular disease up to 557,700,000,000 dollars.Although the treatment of diabetes
Global great attention is obtained with the pre- root of fangji, but omnibus survey in recent years shows, during either European and American developed countries still develop
Country, blood glucose control situation allows of no optimist.
At present the medicine of diabetes is concentrated mainly on insulin, sulfonylurea drugs, GLP-1 receptor stimulating agents, DDP-W
Inhibitor, melbine class, thiazolidinediones etc..Regrettably these medicines all have the shortcomings that obvious, such as diformazan
Biguanides is easily caused lacticemia, and thiazolidinediones has the side effects such as hepatotoxicity wind agitation and bone loss, and sulfonylurea drugs are long
Phase takes and easily cause hypoglycemia, and long-term taking DDP-W inhibitor easily produces cough, insulin then have can not orally, make
With birth defects such as inconveniences.
Recently, NMR researchs display, leginsulin and insulin, Bg albumen has with insulin receptor on space structure
Similarity.Whether so PA1b there is Insulin-Like to adjust function carbohydrate metabolismCause the interest of researcher.Specifically,
This plant polypeptide is found in animal gastrointestinal tract simultaneously to be present, and whether it is new similar incretin polypeptide hormone,
The problem for also making numerous researchers interested.CN105944103A is had found the Soybean Peptide with function of polysaccharide
Hypoglycemic effect is not only substantially increased after PAlb and other hypoglycemics and/or lipid-loweringing composition compounding, dosage is reduced, and is compounded
Medicine has the function of protection islet cells；Leginsulin (PAlb) gene cloning, expression and fusion protein purification,《Central China
University of Science and Technology's master thesis》, Du Wen, 2005, for the first time PA1b is prepared with method of gene recombination, it is the work(for furtheing investigate PAIb
Can, exploitation " insulin type " oral hypoglycemic drug provides the foundation.
But the method also existing defects of existing PA1b protein expressions, its expression efficiency is low, and purity is not high, quality is unstable
It is fixed, therefore, developing the method for high efficiency stable expression Soybean Peptide PA1b becomes the focus of research.
The content of the invention
It is an object of the invention to provide a kind of method of high efficient expression polypeptide PA1b, to solve the table existing for prior art
Up to the problem of inefficiency, the method for the present invention has good, safe expression stability, low cost and easily extensive life
Produce；In addition, the polypeptide PA1b prepared by this method achieves good effect in treatment diabetes are applied to.
To solve the above problems, the technical solution used in the present invention is：
A kind of method of high efficient expression polypeptide PA1b, comprises the steps：
(1) synthesis of genes of interest PA1b：PA1b gene orders are searched, upstream and downstream primer is designed；And it is literary with pea cDNA
Storehouse is template, enters performing PCR amplification with upstream primer, downstream primer and obtains PA1b genes of interest fragments；
(2) connection of PA1b genes and carrier：By genes of interest fragment PA1b and carrier PUSCN1 respectively with restricted
Enzyme cutting BamHI and XhoI enzyme carries out double digestion, then by T4 ligases by genetic fragment PA1b and carrier after digestion
PUSCN1 is attached acquisition recombinant plasmid PUSCN1-PA1b；
(3) conversion of recombinant plasmid PUSCN1-PA1b：The recombinant plasmid PUSCN1-PA1b that step (2) is obtained is using warm
The mode of sharp conversion is imported to is cultivated in bacillus coli DH 5 alpha competent cell, and recombinant plasmid PUSCN1- is extracted afterwards
PA1b；
(4) digestion, the sequencing identification of recombinant plasmid：The recombinant plasmid BamHI and XhoI digestions that will be extracted, 37 DEG C of digestions
Overnight, digestion result is detected with 1% agarose gel electrophoresis；Digestion result is correct, plasmid is sent into sequencing mechanism and is identified
To determine purpose fragment of the fragment for having connected for needed for；
(5) transfection of 293F (human embryo kidney (HEK) suspension cell) cell：After the qualification result of step (4) passes through, will be described heavy
Group plasmid PUSCN1-PA1b imports to 293F (human embryo kidney (HEK) suspension cell) cell and is transfected, and after transfection terminates, cell continues to train
Foster 7 days, afterwards 6000RPM is centrifuged 10min and collects supernatant, and nickel ion post affinity purification PA1b albumen, its purity reaches 98%,
It is identified to be polypeptide PA1b.
The condition of PCR amplification is：95 DEG C of 5min denaturations, 94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 50s, 40 circulations,
72 DEG C of extension 10min.
Cell density is adjusted to 2.5 × 106/mL before transfection in the step (5).
The transfection is to carry out under the following conditions：Cell：Plasmid：Ratio=1 of PEI:3:9, the time of transfection is 7
My god.
The qualification process of albumen is in the step (5)：Pvdf membrane is proceeded to by albumen is wet, then incubation one resists, and closes it
It is incubated two again afterwards to resist, the horseradish peroxidase reaction resisted using ECL Reagent and two, generation launch wavelength is 428nm
Light wave, this light Jing X-ray photosensitive records get off, and as a result show that expressing protein is polypeptide PA1b.
In addition, the present invention is also claimed the polypeptide PA1b and polypeptide PA1b that are prepared by said method in treatment
Application in diabetes.
The main inventive of the present invention is characterized by first using people source mammalian cell successful expression PA1b, mammal
Cell expression system can provide posttranslational modification closest to native state, the meeting during protein expression for recombinant protein
Protein folding and the polymerization of close native protein are formed, possesses space structure necessary to activated protein and modification.By lactation
The exogenous proteins that reprocessing modification is produced after zooblast translation, outclass in terms of activity prokaryotic expression system and yeast,
The eukaryotic expression systems such as insect cell, closer to native protein.
The present invention technique effect be：(1) expression of PA1b albumen is carried out using people source mammalian cell, due to its people
Source relation is near, protein modified accurate, with very high transfection efficiency；(2) method of the present invention expression is high and be secretion table
Reach, expression may be up to 1-3g/L；(3) present invention adopts autonomous improved carrier PUSCN1, and on autonomous expression vector
Addition secreting signal peptide or label, with the secreting, expressing for promoting destination protein purifying is easy to, and on the one hand increased WPRE elements (soil
Dial mouse hepatitis posttranscriptional modification controlling element), enhance the expression of foreign gene；Secondly, N-terminal increased the signal of the IL2 of people
Peptide (its amino acid sequence-MYRMQLLSCI ALSLALVTNS, base sequence
ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGT)；(4) express
Albumen quality stablize, product is homogeneous；(5) people source mammalian cell tolerance shearing force is strong, can be mass-produced；(6)293F
Cell is human archeocyte, not the pathogenic carcinogen of synthesis secretion, high safety；(7) can be mass-produced, low cost；(8) this
Bright polypeptide PA1b albumen can be applied in the medicine and food for manufacturing treatment diabetes, and its cost of manufacture is lower than insulin, and
And it is more convenient than insulin, oral type can be developed into, have no toxic side effect, less than physiological level it is not play hypoglycemic in blood sugar level
Effect.
Description of the drawings
Fig. 1 is the PA1b protein sequence figures for determining；
Fig. 2 is the hum pattern of carrier PUSCN1；
Specific embodiment
Technical scheme is further elaborated with reference to embodiment：
Embodiment 1
A kind of method of high efficient expression polypeptide PA1b, comprises the steps：
(1) synthesis of genes of interest PA1b：PA1b gene orders are searched, upstream and downstream primer is designed；And it is literary with pea cDNA
Storehouse is template, enters performing PCR amplification with upstream primer, downstream primer and obtains PA1b genes of interest fragments；The upstream primer is upper
Trip primer is 5-CGCGGATCCGCAAGCTGC-3, and its restriction enzyme site is BamHI；Downstream primer is
5-CCGCTCGAGTTCCAGATGGATG-3, its restriction enzyme site is XhoI；The condition of PCR amplification is：95℃
5min denaturations, 94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 50s, 40 circulations, 72 DEG C of extension 10min；PCR reaction system be：10
The μ l of × Buffer 1, synthesis PA1b gene 1 μ l, the μ l of upstream primer 0.5, the μ L of downstream primer 0.5, the μ l of high-fidelity PFU enzyme 0.5,
The μ l of dNTP 1, the μ l of distilled water 5.5.
(2) connection of PA1b genes and carrier：By genes of interest fragment PA1b and carrier PUSCN1 respectively with restricted
Enzyme cutting BambI and XhoI enzyme carries out double digestion, then by ligase by genetic fragment PA1b after digestion and carrier PUSCN1
It is attached acquisition recombinant plasmid PUSCN1-PA1b；
(3) conversion of recombinant plasmid PUSCN1-PA1b：The recombinant plasmid PUSCN1-PA1b that step (2) is obtained is using warm
The mode of sharp conversion is imported to is cultivated in bacillus coli DH 5 alpha competent cell, and recombinant plasmid PUSCN1- is extracted afterwards
PA1b；
(4) digestion, the sequencing identification of recombinant plasmid：The recombinant plasmid BamHI and XhoI digestions that will be extracted, 37 DEG C of digestions
Overnight, digestion result is detected with 1% agarose gel electrophoresis；Digestion result is correct, plasmid is sent into sequencing mechanism and is identified
To determine purpose fragment of the fragment for having connected for needed for；
(5) after the qualification result in step (4) passes through, the recombinant plasmid PUSCN1-PA1b is imported into 293F (people's embryos
Kidney suspension cell) cell transfected, and supernatant is collected in centrifugation after transfection terminates, nickel ion post affinity purification PA1b albumen, and its is pure
Degree reaches 98%, identified as polypeptide PA1b；Cell density is adjusted to 2.5 × 106/mL before transfection；The transfection is as follows
Under the conditions of carry out：Cell：Plasmid：Ratio=1 of PEI:3:9, the time of transfection is 7 days；In addition, the qualification process of albumen is：
Pvdf membrane is proceeded to by albumen is wet, then incubation one resists, be incubated two after closing again and resist, resisted using ECL Reagent and two
Horseradish peroxidase reacts, and produces light wave of the launch wavelength for 428nm, and this light Jing X-ray photosensitive records get off, as a result show
Expressing protein is polypeptide PA1b.
(6) detection of polypeptide active：Add BXPC-3 (people original position pancreas adenocarcinoma cell) cell suspension, density on 24 orifice plates
About 1 × 105/ hole, the μ l/ holes of cell suspension 500；5%CO2,37 DEG C of incubation 24h；Renew the growth medium of fresh dosing, by 0,
10th, 25,50 μM of concentration add 5%CO2 in cell culture fluid, 37 DEG C of incubation 30min；To each hole add 1 μ g/m L LPS, 5%
CO2,37 DEG C of incubation 24h；Using the nitric oxide detection kit in the green skies, step reference reagent box specification：Detection is different
The NO content differences of concentration PA1b cultured cells BXPC-3, polypeptide PA1b is active for identification, the difference when PA1b concentration reaches 50 μM
Significantly.
Embodiment 2
Effects of the measuring polypeptide PA1b in animal diabetes model：
(1) foundation of animal model and packet
Diabetes B rat model builds：2 monthly age SD rats are taken, high glucose and high fat forage feed is given 1 month, then abdomen
Chamber injection 20mg/kg alloxans (STZ), continues high lipid food and feeds, and monitors its change of blood sugar.
(2) animal packet：Measure on the basis of soybean active polypeptide containing 98%Palb, median dose is 5 times of datum quantity polypeptides
Albumen, high dose is 30 times of datum quantity polypeptide proteins.After modeling success, model mouse is divided into (low dose of positive controls, datum quantity
Amount) Palb groups, median dose Palb group, high dose Palb groups, tea polysaccharide group, Tea Polyphenols group and melbine group.
(3) drug therapy：Each experimental group gives respectively corresponding medicine and is treated：Low dose group gavage gives 5mg/
Kg/d purity is the soybean active polypeptide of 98%Palb, and middle dose group gavage gives 25mg/kg/d purity for the big of 98%Palb
Beans active peptides, high dose group gavage gives the soybean active polypeptide that 100mg/kg/d purity is 98%Palb, and tea polysaccharide group is filled
Stomach gives 15mg/kg/d99% tea polysaccharides, and Tea Polyphenols group gavage gives the Tea Polyphenols of 15mg/kg/d 95%, melbine group gavage
Give 100mg/kg/d melbine.
(4) effect assessment：Before administration, administration after 1 week, administration after 2 weeks, administration after 3 weeks and be administered after 4 weeks inspection
Survey each experimental group rat blood sugar value.The results are shown in Table 1.
Its test result such as table 1 below：
As can be seen from Table 1, various dose group soybean active polypeptide drugs can in various degree reduce the blood sugar of model mouse,
Wherein high dose soybean active polypeptide group is the most obvious.
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, the present invention is not limited to,
Although being described in detail to the present invention with reference to the foregoing embodiments, for a person skilled in the art, it still may be used
To modify to the technical scheme described in foregoing embodiments, or equivalent is carried out to which part technical characteristic.
All any modification, equivalent substitution and improvements within the spirit and principles in the present invention, made etc., should be included in the present invention's
Within protection domain.
Claims (8)
1. a kind of method of high efficient expression polypeptide PA1b, it is characterised in that comprise the steps：
(1) synthesis of genes of interest PA1b：PA1b gene orders are searched, upstream and downstream primer is designed；And be with Pea cDNA library
Template, enters performing PCR amplification and obtains PA1b genes of interest fragments with upstream primer, downstream primer；
(2) connection of PA1b genes and carrier：Genes of interest fragment PA1b and carrier PUSCN1 are used into respectively restriction enzyme
BamHI and XhoI enzymes carry out double digestion, are then entered genetic fragment PA1b after digestion and carrier PUSCN1 by T4 ligases
Row connection obtains recombinant plasmid PUSCN1-PA1b；
(3) conversion of recombinant plasmid PUSCN1-PA1b：The recombinant plasmid PUSCN1-PA1b that step (2) is obtained is turned using heat shock
The mode of change is imported to is cultivated in bacillus coli DH 5 alpha competent cell, and recombinant plasmid PUSCN1-PA1b is extracted afterwards；
(4) digestion, the sequencing identification of recombinant plasmid：The recombinant plasmid BamHI and XhoI digestions that will be extracted, 37 DEG C of digestions
At night, with 1% agarose gel electrophoresis digestion result is detected；Digestion result is correct, by plasmid be sent to sequencing mechanism identified with
It is determined that purpose fragment of the fragment for having connected for needed for；
(5) transfection of 293F (human embryo kidney (HEK) suspension cell) cell：After the qualification result of step (4) passes through, by the restructuring matter
Grain PUSCN1-PA1b imports to 293F (human embryo kidney (HEK) suspension cell) cell and is transfected, and after transfection terminates, cell continues to cultivate 7
My god, afterwards 6000RPM is centrifuged 10min and collects supernatant, and nickel ion post affinity purification PA1b albumen, its purity reaches 98%, Jing
Identification is polypeptide PA1b.
2. the method for high efficient expression polypeptide PA1b according to claim 1, it is characterised in that：The upstream primer is 5-
CGCGGATCCGCAAGCTGC-3, its restriction enzyme site is BamHI；Downstream primer is 5-CCGCTCGAGTTCCAGATGGATG-3,
Its restriction enzyme site is XhoI.
3. the method for high efficient expression polypeptide PA1b according to claim 1, it is characterised in that：The condition of PCR amplification is：
95 DEG C of 5min denaturations, 94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 50s, 40 circulations, 72 DEG C of extension 10min.
4. the method for high efficient expression polypeptide PA1b according to claim 1, it is characterised in that：In the step (5) before transfection
Cell density is adjusted to 2.5 × 106/mL.
5. the method for high efficient expression polypeptide PA1b according to claim 1, it is characterised in that：The transfection is in following condition
Under carry out：Cell：Plasmid：Ratio=1 of PEI:3:9, the time of transfection is 7 days.
6. the method for high efficient expression polypeptide PA1b according to claim 1, it is characterised in that：Albumen in the step (5)
Qualification process is：Pvdf membrane is proceeded to by albumen is wet, then incubation one resists, be incubated two after closing again and resist, using ECL Reagent
Horseradish peroxidase reaction on anti-with two, produces light wave of the launch wavelength for 428nm, under this light Jing X-ray photosensitive records
Come, as a result show that expressing protein is polypeptide PA1b.
7. the polypeptide PA1b that a kind of method of the high efficient expression polypeptide PA1b according to any one of claim 1-6 is prepared.
8. polypeptide PA1b described in a kind of claim 7 is preparing treatment diabetes medicament or the application in food.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101033465A (en) * | 2007-01-26 | 2007-09-12 | 华南理工大学 | Pea albumin subunit PA1b recombination albumen and its preparing method and application |
| CN101082046A (en) * | 2007-01-26 | 2007-12-05 | 华南理工大学 | Heterogenetic expression method for recombined pea albumin subfraction PA1b gene |
| CN105944103A (en) * | 2016-06-15 | 2016-09-21 | 武汉格睿特科技有限公司 | Oral compound natural glucose-lowering medicine |
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2017
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101033465A (en) * | 2007-01-26 | 2007-09-12 | 华南理工大学 | Pea albumin subunit PA1b recombination albumen and its preparing method and application |
| CN101082046A (en) * | 2007-01-26 | 2007-12-05 | 华南理工大学 | Heterogenetic expression method for recombined pea albumin subfraction PA1b gene |
| CN105944103A (en) * | 2016-06-15 | 2016-09-21 | 武汉格睿特科技有限公司 | Oral compound natural glucose-lowering medicine |
Non-Patent Citations (3)
| Title |
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| 李国坤等: "哺乳动物细胞表达系统研究进展", 《中国生物工程杂志》 * |
| 柏家林等: "哺乳动物细胞高效表达系统研究进展", 《西北民族大学学报（自然科学报）》 * |
| 王登等: "用于重组蛋白表达的哺乳动物细胞系的研究进展", 《药物生物技术》 * |
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