CN107446945A - A kind of induced expression of people source polypeptide in pichia yeast - Google Patents
A kind of induced expression of people source polypeptide in pichia yeast Download PDFInfo
- Publication number
- CN107446945A CN107446945A CN201710738272.5A CN201710738272A CN107446945A CN 107446945 A CN107446945 A CN 107446945A CN 201710738272 A CN201710738272 A CN 201710738272A CN 107446945 A CN107446945 A CN 107446945A
- Authority
- CN
- China
- Prior art keywords
- pichia yeast
- rhggf2
- polypeptide
- rhcggf2
- people source
- 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
- 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
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- 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
- C07K14/475—Growth factors; Growth regulators
- C07K14/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The invention provides a kind of induced expression of people source polypeptide in pichia yeast, belong to biomedicine technical field.It solves the technical problems such as induced expression of people's source protein in pichia yeast.A kind of induced expression of people source polypeptide in pichia yeast, the nucleotide construction of the coding prevention of codon optimization and (or) the β of people source polypeptide rhNRG 1 for the treatment of cardiomyopathy is entered into pichia yeast expression vector, then it is transferred to pichia yeast and carries out induced expression, obtains described polypeptide.The present invention has the advantages that the people source polypeptide that can be prevented and (or) treated cardiomyopathy.
Description
Technical field
The invention belongs to biomedicine technical field, is related to a kind of induced expression of people source polypeptide in pichia yeast.
Background technology
Heart failure is also known as heart failure, congestive heart failure etc., is referred to as the epidemic disease of 21st century, is global morbidity
Rate, the death rate and medical expense highest principal disease.Inflammation caused by myocardial infarction, infection, the heart caused by metabolic disorder
The change etc. of structure, function is all to trigger the important risk factor of heart failure.Although the pathogenesis about heart failure has been permitted
More reports, but there is presently no the treatment that very effective medicine is used for heart failure.Angiotensin-Converting (ACE) presses down
Preparation is reduced blood pressure, lightening heart load, there is the effect of certain to heart failure by expanding blood vessel, but therapeutic effect is very limited,
It is also possible to be accompanied by side effect simultaneously.Not only expensive, wound is big as performed the operation for other treatment methods, and donor has very much
Limit.With the aggravation of aging population, the demand of cardiovascular drugs is also stepped up, and the whole world spends in heart failure every year
The fund of disease treatment is about 108,000,000,000 dollars, and market is huge.At home, in terms of heart failure therapy drug development also in rising
The medicine listing of step section, up to now 3 treatment heart failure of only three domestic corporation's exploitations, and biological species medicine is only
1 kind (rhBNP).Therefore, the effective treatment heart failure bio-pharmaceutical of exploitation seems particularly urgent, important.
Nerve modulation element (Neuregulins, NRGs) belongs to the polypeptide nerve growth factor of a kind of regulation growth and differentiation
Son, belong to EGF (EGF) family member, have expression in nervous system, cardiovascular system, mammary gland, intestines and kidney.NRGs
Gene family is made up of NRG-1, NRG-2, NRG-3, NRG-4, and coding produces different NRGs.They all contain similar structure
Domain, including immunoglobulin (Ig) sample cyclic structure, EGF (EGF) spline structure domain, transmembrane region and length are not etc.
Intracellular region domain.It has now been found that NRG-1 is in cardiovascular and cerebrovascular disease, diabetic cardiomyopathy, diabetic nephropathy, obesity
Cardiomyopathy etc. all has certain prevention and treatment effect.Different montages and different promoters due to NRGs mRNA
Enable, the Neuregulin-1 (NRG-1) of cross-cell membrane can produce the different isomer protein of kind more than 30, according to its N-terminal
The difference of sequence, NRG-1 isomers can be divided into 6 classes (I- VI), including acetylcholinergic receptor induced activation body
(acetylcholine receptor inducing activity, ARIA), neural differentiation factor
(neudifferentiation factor, NDF), people Neuregulin (heregulin, HRG), glial growth factor
(glial growth factor, GGF), feel and motor neuron derived factor (sensorimotor-derived
Factor, SMDF) etc..They have the difference in certain tissue specificity and structure.HRG hypotypes contain immune spherical structure
(immunoglobulin, Ig) and EGF-like domains, GGF and GGF2 hypotypes also sequence containing kringle-like, SMDF are sub-
Type but only has EGF-like domains [13,14].Pro-NRG-1 expression of the NRG-1 majorities in the form of cross-film is thin in myocardium endothelium
On after birth, the N-terminal polypeptide that is then in through extracellular protein enzyme hydrolysis outside film, the NRG-1 of maturation is discharged.Shear these Pro-NRG-
1 membrane proteolytic enzyme includes β Amyloid precursor proteins shearing enzyme 1 (β-secretase 1, BACE1) and meltrin (TACE) β
(ADAM19) etc..NRG-1 is one of member of most study in NRGs families, and research shows, NRG-1 can promote breast epithelium
Cell, spongiocyte, neuronal cell and the propagation of myocyte, differentiation and survival.NRG-1 alternative splicings in transcription
And a variety of hypotypes are formed, Type I types include an Ig and ball but Aries domain are immunized, and are the important albumen of heart development；Type
II type includes Kringle regions and immunoglobulin like domain, expresses N-terminal secreting signal peptide；The types of Type III include one
Domain rich in cysteine, is mainly expressed in neuronal cell.But three of the above hypotype all contains EFG domains,
This domain of alternative splicing can form two kinds of variants in transcription, both α and β.Many research discoveries, NRG-1 β hypotype
One of ----GGF2 (refers to GGF2) without specified otherwise, NRG-1 β below, promotes heart development, is given birth in cardiovascular biology and pathology
Aspect of science all plays an important role.Many Research of Animal Model for Study it has been reported that NRG-1 β in ischemic cardiomyopathy, the diabetes heart
Myopathy etc. plays important protective effect, and thinks that NRG-1 β are probably a kind of very promising heart failure therapy medicine
Thing.
In terms of the beta mediated cardiomyopathy protection mechanism researchs of NRG-1, NRG-1 β/ErbB signal paths play important regulating and controlling
Effect.Under compensatory heart failure early stage, myocardium stressed condition, NRG-1 β/ErbB signal paths are activated, and in cardiomyopathy process
And activity reduces under the conditions of metabolic imbalance.Proteolytic enzyme (BACE1, ADAM10) mediates myocardium endothelial cell surface release NRG-
1 β, it interacts with one of myocardial cell surface ErbB3,4 receptor tyrosine kinases, mediates ErbB3/4 or ErbB4/4 dimerization
The formation of body, or mediation ErbB3/2 or ErbB4/2 dimers formation, activate NRG-1 β/ErbB signal paths, as MAPK,
PI3K/Akt etc. influences cells survival, migration, propagation, adhesion and differentiation performance.
In cardiovascular system, NRG-1 β mainly by endocardial endothelial cell and CMEC expression and discharge, and
ErbB4 and ErbB2 expresses in cardiac muscle, and ErbB3 is expressed in the interstitial cell of endocardial cushion.Zoopery proves NRG-1 β/ErbB
System plays a significant role during myocardium bone trabecula and heart development；The NRG-1 β of source of endothelial cells pass through PI3K/Akt
Signal suppresses the release of cromoci and caspase-3 activity and protects oxidative stress and anthracene nucleus medicament to induce cardiac muscle to wither
The cell death of dying property；NRG-1 β cause cardiac muscle insensitive to isoprel by activating myocardium eNOS signals, so as to self
Adjust Acute myocardial stress and chronic heart failure, including diabetes and diabetic cardiomyopathy in chronic myocardial metabolic stress；Also have
Report NRG-1 β can promote appropriate revascularization and arteriogenesis in the damage of scarce inducing heart vessel blood, be advantageous to damage
Recover.
The clinical research for being used to treat heart failure about NRG-1 β has also been carried out, several myocardial infarctions, adriamycin induction cardiac muscle
Disease, diabetic cardiomyopathy and myocarditis results of animal show that NRG-1 β can be obviously improved in heart failure prevention and treatment
Myocardial function, the expression of disease markers is weakened, extend the animal survival cycle.Research report shows that NRG-1 β are used to treat at present
The mechanism of heart failure includes promoting cardiac muscle cell's renewal, protection myocardial cell injury, stable muscle segment institutional framework, remodeling ventricle
Reverse, promote angiogenesis, reduction mitochondria dysfunction, reduce oxidative stress, suppress generation, the myocardium calcium of regulation and control of apoptosis
Ion homeostasis etc..
In summary, NRG-1 beta polypeptides have important answer in terms of heart failure and its related myocardium prevention and treatment of diseases
With value and higher DEVELOPMENT PROSPECT, but the presently used NRG-1 beta polypeptides modes of production are mainly mammalian cell table
Up to Bacillus coli expression two ways, the former production cost is higher, and the latter obtain polypeptide do not possess eucaryote expression institute
Obtain the higher structure and necessary chemical modification (such as glycosylation, nitration modification), particularly Bacillus coli cells wall of polypeptide
Upper endotoxic interference, increase production cost and difficulty for later stage peptide purification.Therefore, a kind of simple, high efficient expression tool of exploitation
The expression system of higher structure and the rhNRG-1 beta polypeptides of chemical modification, and a kind of simple and effective purification process, will be
While keeping polypeptide high activity, production cost is substantially reduced, therefore there is wide exploitation and application prospect.
The content of the invention
The purpose of the present invention is to be directed to above mentioned problem existing for existing technology, there is provided a kind of people source polypeptide is in pichia yeast
In induced expression, the technical problems to be solved by the invention are the people sources for the prevention and treatment cardiomyopathy for how obtaining high activity
Polypeptide.
The purpose of the present invention can be realized by following technical proposal：A kind of induction table of people source polypeptide in pichia yeast
Reach, its feature is, the coding of codon optimization is prevented and treated to the people source polypeptide rhNRG-1 β of cardiomyopathy nucleosides
Acid sequence is built into pichia yeast expression vector, is then transferred to pichia yeast and carries out induced expression, obtains described polypeptide；
RhNRG-1 β include total length people source polypeptide rhGGF2, rhGGF2 part of polypeptide rhcGGF2, pass through HIS pairs
RhGGF2 be marked after polypeptide HIS-rhGGF2 and the polypeptide HIS- after rhcGGF2 is marked by HIS
rhcGGF2；
According to total length people source polypeptide rhGGF2 polypeptid acid sequence and pichia yeast codon-bias, will pass through
The nucleotide sequence of encoding human source polypeptide rhGGF2 after codon optimization is built into Yeast expression carrier, or will pass through password
The nucleotide sequence of encoding human source polypeptide rhGGF2 after son optimization is connected with HIS (6 × his) label, is then transferred to Bi Shi ferment
Mother, screened by positive colony, obtain the high expression bacterial strain of rhGGF2 high expression bacterial strain and HIS-rhGGF2 respectively.
Part of polypeptide rhcGGF2 is according to people source rhGGF2 polypeptid acid sequences, removes rhGGF2 signal peptide sequences, obtains
Obtain the rhcGGF2, then according to pichia yeast codon-bias, the encoding human source polypeptide after codon is optimized
RhcGGF2 nucleotide sequence is built into Yeast expression carrier, or the encoding human source polypeptide after codon is optimized
RhcGGF2 nucleotide sequence is connected with HIS (6 × his) label, is then transferred to pichia yeast, is screened by positive colony, point
Huo get not rhcGGF2 height expression bacterial strain and HIS-rhcGGF2 high expression bacterial strain.
Obviously, any polypeptide interested can be expressed using the above method.
In a kind of induced expression of the above-mentioned people source polypeptide in pichia yeast, the nucleotides sequence of the coding rhGGF2
Row such as Nucleinic acid SEQ ID NO：1, corresponding peptide sequence such as Peptide No.1；The coding HIS-
RhGGF2 nucleotide sequence such as Nucleinic acid SEQ ID NO：2, corresponding peptide sequence such as Peptide No.2；
The nucleotide sequence such as Nucleinic acid SEQ ID NO of the coding rhcGGF2：3, corresponding peptide sequence is such as
Peptide No.3；The nucleotide sequence such as Nucleinic acid SEQ ID NO of the coding HIS-rhcGGF2：4, accordingly
Peptide sequence such as Peptide No.4.
In a kind of induced expression of the above-mentioned people source polypeptide in pichia yeast, the pichia yeast inducible expression carrier
It is pHKY01.Pichia yeast inducible expression carrier can select arbitrary pichia yeast inducible expression carrier, can realize this
Invention, is optimal using pHKY01.
In a kind of induced expression of the above-mentioned people source polypeptide in pichia yeast, the pichia yeast is pichia yeast
GS115.Pichia yeast can also select arbitrary pichia yeast bacterial strain, can realize the present invention.
In a kind of induced expression of the above-mentioned people source polypeptide in pichia yeast, rhGGF2 the and rhcGGF2 polypeptides
In pichia yeast after induced expression, its purification process isolates and purifies the albumen using ion-exchange chromatography.
In a kind of induced expression of the above-mentioned people source polypeptide in pichia yeast, the HIS-rhGGF2 and HIS-
RhcGGF2 polypeptides are in pichia yeast after induced expression, and its purification process is using Ni- nickel affinity purifications column chromatography method purifying institute
State albumen.
The second aspect of the present invention, there is provided the people source polypeptide of one kind prevention and (or) treatment cardiomyopathy, be characterized in, adopt
It is prepared with derivational expression method of the above-mentioned people source polypeptide in pichia yeast.
The third aspect of the present invention, there is provided above-mentioned prevention and (or) the people source polypeptide for the treatment of cardiomyopathy are preparing clinic
Application in medicine.
The beneficial effects of the present invention are：Prevent in the present invention and (or) treat the people source polypeptide of cardiomyopathy in pichia yeast
In derivational expression method be that the nucleotide sequence of the encoding human source polypeptide of codon optimization is built into pichia yeast and lured
Lead in expression vector, be then transferred to pichia yeast and carry out induced expression, prevented and (or) treated the people source polypeptide of cardiomyopathy,
Expression quantity can be of about 100mg/L；People source polypeptide shows through In vitro cell experiment and animal model experiment, can be effectively improved
H2O2The myocardial necrosis that the myocardial cell injury and myocardial ischemia of induction induce.The skillful in design, height can be readily available
The people source polypeptide of the prevention and treatment cardiomyopathy of activity, purifying process is simple, suitable for large-scale promotion application.
Brief description of the drawings
Fig. 1 is plasmid pHKY01-rhGGF2 structure collection of illustrative plates.
Fig. 2 is plasmid pHKY01-rhcGGF2 structure collection of illustrative plates.
Fig. 3 is plasmid pHKY01-HIS-rhGGF2 structure collection of illustrative plates.
Fig. 4 is plasmid pHKY01-HIS-rhcGGF2 structure collection of illustrative plates.
Embodiment
It is the specific embodiment of the present invention and with reference to accompanying drawing below, technical scheme is further described,
But the present invention is not limited to these embodiments.
Embodiment one
Induced expression of a kind of people source rhGGF2 polypeptides in pichia yeast, it is intended to which people source is expressed by pichia yeast
RhGGF2 polypeptides, method are as follows：
According to people source GGF2 protein amino acid sequences and pichia yeast codon-bias, after codon is optimized
Encoding human source rhGGF2 nucleotide sequence is built into Yeast expression carrier pHKY01, or the volume after codon is optimized
Code people source rhGGF2 nucleotide sequence is connected with HIS (6 × his) label, is then transferred to expressive host bacterial strain pichia yeast
GS115, screened by positive colony, obtain high expression bacterial strain GS115-rhGGF2 and high expression bacterial strain GS115-HIS- respectively
rhGGF2；Induced expression in pichia yeast obtains polypeptide rhGGF2, and its purification process is separated pure using ion-exchange chromatography
Change the albumen；Induced expression in pichia yeast obtains polypeptide HIS-rhGGF2, and its purification process is affine pure using Ni- nickel
Change column chromatography method and purify the albumen.
Traditional people source rhGGF2 polypeptides are expressed by zooblast, animal cell expression, purifying and acquisition, its
Expression quantity is small, and the cycle is long, costly, and this programme can not only be obtained similar dynamic by the induced expression in pichia yeast
The expression of thing cell has higher structure and the polypeptide (glycosylation modified etc.) of chemical modification, and pichia yeast genetic engineering bacterium
GS115-rhGGF2 and GS115-HIS-rhGGF2 high expression, high activity, can substantially reduce research and development, production cost, due to
Compared to zooblast, the acquisition of pichia yeast, growing environment, obtain the cycle and all optimize in zooblast, highly this area
Technical staff's promotion and application.
Encode rhGGF2 nucleotide sequence Nucleinic acid SEQ ID NO:1 has more than 70% identical nucleic acid；Compile
Code rhGGF2 nucleotide sequence and Peptide SEQ ID NO:1 amino acid sequence is corresponding；
Encode HIS-rhGGF2 nucleotide sequence Nucleinic acid SEQ ID NO:2 have more than 70% identical core
Acid；Encode HIS-rhGGF2 nucleotide sequence and Peptide SEQ ID NO:2 amino acid sequence is corresponding；
Embodiment two
RhGGF2 is full-length polypeptide, obtains the expression of high activity, can also use part of polypeptide rhGGF2 in pichia yeast
Middle induced expression, its method are as follows：
According to the GENEBANK people source GGF2 protein amino acid sequences shown and its explanation, by the signal of people source GGF2 albumen
Peptide removes, and remaining part of polypeptide is named as rhcGGF2, according to polypeptide rhcGGF2 amino acid sequence and pichia yeast password
Sub- preferences, the nucleotide construction of the encoding human source rhcGGF2 polypeptides after codon optimization enter Yeast expression carrier
In pHKY01, or the nucleotides of the encoding human source rhcGGF2 after codon is optimized and HIS (6 × his,
CATCATCATCATCATCAT) label connects, and is then transferred to pichia yeast GS115, is screened by positive colony, obtains respectively high
Bacterial strain GS115-rhcGGF2 and high expression bacterial strain GS115-HIS-rhcGGF2 are expressed, induced expression is obtained in pichia yeast
RhcGGF2 polypeptides, its purification process isolate and purify the polypeptide using ion-exchange chromatography；Induced expression in pichia yeast
Obtained polypeptide HIS-rhcGGF2, its purification process use Ni- nickel affinity purification column chromatography method purified polypeptides.
Coding rhcGGF2 nucleotide sequence has more than 70% identical core for Nucleinic acid SEQ ID NO.3
Acid, its corresponding amino acid sequence are Peptide SEQ ID NO.3
Coding HIS-rhcGGF2 nucleotide sequence is Nucleinic acid SEQ ID NO.4, its corresponding amino acid
Sequence is Peptide SEQ ID NO.4.
Embodiment three
Polypeptide rhNRG-1 improves H2O2Inducing cardiomyocytes apoptosis
1st, the separation and culture of primary neonatal cardiac myocytes (NRCM)
1) the Sprague-Dawley rats that just birth 1-3 is extremely big are taken, aseptically cut off suckling mouse chest with operating scissors
Chamber, suckling mouse apex of the heart part is cut, be put into D-Hank ' s liquid to the cold and wash three times, then use clean suckling mouse apex of the heart tissue
Scissors shreds (about 1mm3Size)
2) the heart tissue fragment 3-5 of the above is digested under the conditions of 37 DEG C with 0.4% clostridiopetidase A II and 0.5% trypsase
Minute, then digestion supernatant is removed；
3) digestion process of step 2) is repeated, and it is isometric with sterile centrifugation tube, adding to collect supernatant digestive juice
DMEM nutrient solutions (contain 10%FBS)；
4) repeat step 3) 3-5 times, until tissue has digested substantially, then the supernatant of all collections is blown and beaten into cell and hanged
Liquid, collect cell within 5 minutes with 1000 revs/min of centrifugations under normal temperature, add DMEM nutrient solutions (contain 10%FBS) wash,
Cell is collected by centrifugation, repeats 2-3 times.
5) cell suspension is finally inserted into CO2After incubator culture 75-90 minutes, not adherent cell suspension is suctioned out, is moved
Enter in new culture plate, insert CO2Incubator continues culture 24 hours；
6) cardiac muscle cell after being cultivated 24 hours more than changes liquid and removes not adherent cell, hereafter the next day change liquid, micro-
Visible pulsatile heart myocyte under mirror.
2nd, polypeptide rhNRG-1 weakens H2O2Induce the experiment of NRCM apoptosis
1)H2O2Induce the foundation of NRCM Apoptosis Models：In the NRCM of culture, 4 groups are divided into, every group is separately added into final concentration
Respectively 0,100,200,400 μM of H2O2Processing 3 hours, then collects cell, is examined using ANNEXIN V-FITC/PI apoptosis
Test agent box handles cell, then does flow cytometry, as a result shows H2O2It is that cell mortality reaches at final concentration of 200 μM
40% or so, establish H2O2NRCM Apoptosis Models are induced to use final concentration of 200 μM of H2O2, processing time is 3 hours；
2) rhNRG-1 weakens H2O2Induce the experiment of NRCM apoptosis
Point be now 4 each groups by the NRCM of culture, add 0 respectively, 100,200,400ng/mL rhNRG-1 pretreatment cells 30
Minute, then add final concentration of 200 μM of H2O2Processing 3 hours, cell is collected, examined using ANNEXIN V-FITC/PI apoptosis
Test agent box handles cell, then does flow cytometry, as a result shows that the final concentration of 100-400ng/mL of rhNRG-1 can have
Effect reduces H2O2NRCM apoptosis is induced, and 200ng/mL efficiency is best, about reduces 40-50%H2O2Induce NRCM apoptosis.
Example IV
1st, the foundation of male Sprague Dawley rat model of myocardial infarction
1) anaesthetize：The male Sprague Dawley rats of 175-200g weights are chosen, are injected by way of intraperitoneal injection
30mg/kg anaesthetized with pentobarbital rat, to pinch rat toe by the use of hand, rat does not react and is used as suitable level of anesthesia；
2) it is intubated：Fixed rat, rat front tooth is tangled with rope or rubber band, and the holding layback of mouse head is easy to be intubated.With band
The 16G venous detaining needles (cutting off tip) of tube core are gently sent into forward (note through glottis：Not touch glottis surrounding tissue), so
After extract tube core, can with a hair critical point detection intubation whether succeed；
3) setting of lung ventilator：Lung ventilator is arranged to volume controlled pattern, and tidal volume is 1mL/100g body weight, respiratory rate
For 50-65 beats/min, respiratory quotient 1:1, fraction of inspired oxygen 100%.
4) open chest surgery on the left of the progress of the 4th intercostal, is tied arteria coroaria sinistra with 6-0 non-traumatic sewing threads, with observation
Distal myocardium surface is blue at from ligation, and Electrocardiography S-T segment is raised as there is myocardial infarction symptom, is then squeezed
Intrathoracic air is pressed, sutures thoracic cavity, 400,000 units of Penicillin are injected in rat abdominal cavity with pre- aseptic generation.
2nd, rhNRG-1 improves myocardial infarction
Experiment is chosen postoperative one week rat still to survive and is grouped as research object, control group, model group, administration group
(1mg/kg, 3mg/kg, 6mg/kg), administration group is administered by tail vein, is every other day administered once, and blank group injection is without more
The solvent (physiological saline) of peptide medicine, per heart function of weekly check, as a result show, low dose group (1mg/kg) is to heart function
Improve it is limited,.3mg/kg, 6mg/kg dosage group can be obviously improved myocardial function, it was demonstrated that the rhNRG-1 polypeptides of this method production
There is preferable cardiomyopathy therapeutic effect.
Specific embodiment described herein is only to spirit explanation for example of the invention.Technology belonging to the present invention is led
The technical staff in domain can be made various modifications or supplement to described specific embodiment or be replaced using similar mode
Generation, but without departing from the spiritual of the present invention or surmount scope defined in appended claims.
Peptide SEQ ID No.1(rhGGF2)
MRWRRAPRRSGRPGPRAQRPGSAARSSPPLPLLPLLLLLGTAALAPGAAAGNEAAPAGASVCYSSPPSV
GSVQELAQRAAVVIEGKVHPQRRQQGALDRKAAAAAGEAGAWGGDREPPAAGPRALGPPAEEPLLAANGTVPSWPTA
PVPSAGEPGEEAPYLVKVHQVWAVKAGGLKKDSLLTVRLGTWGHPAFPSCGRLKEDSRYIFFMEPDANSTSRAPAAF
RASFPPLETGRNLKKEVSRVLCKRCALPPRLKEMKSQESAAGSKLVLRCETSSEYSSLRFKWFKNGNELNRKNKPQN
IKIQKKPGKSELRINKASLADSGEYMCKVISKLGNDSASANITIVESNATSTSTTGTSHLVKCAEKEKTFCVNGGEC
FMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYSTSTPFLSLPE
Nucleinic acid SEQ ID NO.1(rhGGF2)
ATGAGATGGAGAAGAGCTCCAAGAAGATCTGGTAGACCAGGTCCAAGAGCACAAAGACCAGGTTCTGCC
GCTAGATCTTCTCCACCTTTGCCATTGTTGCCACTATTATTGTTGTTGGGTACAGCTGCATTGGCTCCAGGTGCTGC
CGCTGGCAACGAAGCTGCTCCAGCTGGAGCTTCTGTTTGTTACTCTTCTCCACCATCTGTTGGTTCTGTTCAAGAAT
TGGCTCAAAGGGCTGCTGTTGTTATTGAAGGTAAGGTTCATCCCCAAAGAAGACAACAGGGTGCTTTGGATAGAAAG
GCTGCTGCTGCTGCTGGAGAAGCTGGTGCTTGGGGTGGTGATAGAGAACCACCAGCTGCTGGTCCAAGAGCTTTAGG
TCCACCAGCTGAAGAACCATTGTTGGCTGCTAACGGTACTGTTCCATCTTGGCCAACTGCTCCAGTTCCATCAGCTG
GTGAACCAGGTGAGGAAGCTCCATACTTGGTCAAGGTTCATCAAGTTTGGGCTGTTAAGGCTGGTGGTTTGAAGAAG
GATTCTTTGTTGACTGTTAGGTTGGGTACTTGGGGTCATCCAGCCTTTCCATCTTGTGGTAGGCTGAAGGAGGATTC
TAGATACATTTTTTTCATGGAACCCGATGCTAACTCTACTTCTAGAGCTCCCGCTGCTTTTAGAGCCTCTTTTCCAC
CATTGGAAACTGGCAGAAACTTGAAGAAGGAGGTTTCTAGAGTTTTGTGCAAGAGATGTGCTTTGCCACCAAGATTG
AAGGAAATGAAGTCTCAAGAATCTGCTGCTGGTTCAAAATTGGTTTTGAGGTGCGAGACTTCCAGTGAATACTCCTC
TCTGAGATTCAAGTGGTTTAAGAACGGTAACGAATTGAACCGTAAGAACAAGCCACAAAACATCAAGATTCAGAAGA
AGCCCGGTAAGTCTGAATTGAGAATTAACAAGGCTTCTTTGGCTGACTCTGGTGAGTACATGTGCAAAGTTATCAGT
AAGCTGGGTAACGATTCTGCTTCTGCTAACATTACTATCGTTGAGTCTAACGCAACCTCCACTTCTACTACTGGTAC
CTCTCATTTGGTTAAATGTGCTGAGAAGGAAAAAACTTTCTGTGTTAACGGTGGTGAATGTTTCATGGTTAAGGACT
TGTCTAACCCATCTAGGTACTTGTGCAAGTGTCCAAACGAATTTACAGGTGACAGATGCCAGAACTACGTTATGGCA
TCTTTTTACTCCACTAGTACTCCATTTCTTTCATTGCCTGAA
Peptide SEQ ID No.2(HIS-rhGGF2)
HHHHHHRWRRAPRRSGRPGPRAQRPGSAARSSPPLPLLPLLLLLGTAALAPGAAAGNEAAPAGASVCYS
SPPSVGSVQELAQRAAVVIEGKVHPQRRQQGALDRKAAAAAGEAGAWGGDREPPAAGPRALGPPAEEPLLAANGTVP
SWPTAPVPSAGEPGEEAPYLVKVHQVWAVKAGGLKKDSLLTVRLGTWGHPAFPSCGRLKEDSRYIFFMEPDANSTSR
APAAFRASFPPLETGRNLKKEVSRVLCKRCALPPRLKEMKSQESAAGSKLVLRCETSSEYSSLRFKWFKNGNELNRK
NKPQNIKIQKKPGKSELRINKASLADSGEYMCKVISKLGNDSASANITIVESNATSTSTTGTSHLVKCAEKEKTFCV
NGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYSTSTPFLSLPE
Nucleinic acid SEQ ID NO.2(HIS-rhGGF2)
CATCATCATCATCATCATAGATGGAGAAGAGCTCCAAGAAGATCTGGTAGACCAGGTCCAAGAGCACAA
AGACCAGGTTCTGCCGCTAGATCTTCTCCACCTTTGCCATTGTTGCCACTATTATTGTTGTTGGGTACAGCTGCATT
GGCTCCAGGTGCTGCCGCTGGCAACGAAGCTGCTCCAGCTGGAGCTTCTGTTTGTTACTCTTCTCCACCATCTGTTG
GTTCTGTTCAAGAATTGGCTCAAAGGGCTGCTGTTGTTATTGAAGGTAAGGTTCATCCCCAAAGAAGACAACAGGGT
GCTTTGGATAGAAAGGCTGCTGCTGCTGCTGGAGAAGCTGGTGCTTGGGGTGGTGATAGAGAACCACCAGCTGCTGG
TCCAAGAGCTTTAGGTCCACCAGCTGAAGAACCATTGTTGGCTGCTAACGGTACTGTTCCATCTTGGCCAACTGCTC
CAGTTCCATCAGCTGGTGAACCAGGTGAGGAAGCTCCATACTTGGTCAAGGTTCATCAAGTTTGGGCTGTTAAGGCT
GGTGGTTTGAAGAAGGATTCTTTGTTGACTGTTAGGTTGGGTACTTGGGGTCATCCAGCCTTTCCATCTTGTGGTAG
GCTGAAGGAGGATTCTAGATACATTTTTTTCATGGAACCCGATGCTAACTCTACTTCTAGAGCTCCCGCTGCTTTTA
GAGCCTCTTTTCCACCATTGGAAACTGGCAGAAACTTGAAGAAGGAGGTTTCTAGAGTTTTGTGCAAGAGATGTGCT
TTGCCACCAAGATTGAAGGAAATGAAGTCTCAAGAATCTGCTGCTGGTTCAAAATTGGTTTTGAGGTGCGAGACTTC
CAGTGAATACTCCTCTCTGAGATTCAAGTGGTTTAAGAACGGTAACGAATTGAACCGTAAGAACAAGCCACAAAACA
TCAAGATTCAGAAGAAGCCCGGTAAGTCTGAATTGAGAATTAACAAGGCTTCTTTGGCTGACTCTGGTGAGTACATG
TGCAAAGTTATCAGTAAGCTGGGTAACGATTCTGCTTCTGCTAACATTACTATCGTTGAGTCTAACGCAACCTCCAC
TTCTACTACTGGTACCTCTCATTTGGTTAAATGTGCTGAGAAGGAAAAAACTTTCTGTGTTAACGGTGGTGAATGTT
TCATGGTTAAGGACTTGTCTAACCCATCTAGGTACTTGTGCAAGTGTCCAAACGAATTTACAGGTGACAGATGCCAG
AACTACGTTATGGCATCTTTTTACTCCACTAGTACTCCATTTCTTTCATTGCCTGAA
Peptide SEQ ID No.3(rhcGGF2)
MGNEAAPAGASVCYSSPPSVGSVQELAQRAAVVIEGKVHPQRRQQGALDRKAAAAAGEAGAWGGDREPP
AAGPRALGPPAEEPLLAANGTVPSWPTAPVPSAGEPGEEAPYLVKVHQVWAVKAGGLKKDSLLTVRLGTWGHPAFPS
CGRLKEDSRYIFFMEPDANSTSRAPAAFRASFPPLETGRNLKKEVSRVLCKRCALPPRLKEMKSQESAAGSKLVLRC
ETSSEYSSLRFKWFKNGNELNRKNKPQNIKIQKKPGKSELRINKASLADSGEYMCKVISKLGNDSASANITIVESNA
TSTSTTGTSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYSTSTPFLSLPE
Nucleinic acid SEQ ID NO.3
ATGGGCAACGAAGCTGCTCCAGCTGGTGCCTCTGTTTGTTACTCTTCTCCACCATCTGTTGGTTCTGTT
CAAGAATTGGCTCAAAGAGCTGCTGTTGTTATTGAAGGTAAGGTTCATCCACAACGTAGACAACAAGGTGCTCTGGA
TAGAAAGGCTGCTGCTGCTGCCGGTGAAGCTGGTGCTTGGGGTGGTGATAGAGAACCACCTGCTGCTGGTCCAAGAG
CTTTGGGTCCACCAGCTGAAGAACCATTGTTGGCTGCTAACGGTACTGTTCCATCTTGGCCAACTGCTCCAGTTCCA
TCAGCTGGTGAACCAGGTGAGGAAGCTCCATACCTAGTTAAGGTTCATCAGGTTTGGGCTGTTAAGGCTGGTGGTTT
GAAGAAGGACTCTTTGTTGACTGTTAGATTGGGTACTTGGGGTCATCCAGCTTTTCCATCTTGTGGTCGTTTGAAAG
AAGACTCCAGGTACATTTTTTTCATGGAACCAGATGCTAACTCCACTTCTAGGGCTCCCGCTGCTTTCAGAGCTTCT
TTTCCACCATTGGAAACGGGTAGAAACTTGAAGAAGGAAGTTTCTAGAGTTCTGTGTAAGCGTTGTGCTTTGCCACC
AAGATTGAAGGAAATGAAGTCTCAAGAATCTGCCGCTGGTTCCAAGTTGGTATTGAGATGCGAGACTTCTTCCGAGT
ACAGTTCTTTGAGGTTCAAGTGGTTTAAGAACGGTAACGAATTGAACAGAAAAAACAAGCCCCAAAACATAAAGATT
CAAAAGAAGCCTGGCAAGTCTGAATTGAGGATTAACAAGGCTTCCTTGGCTGATTCTGGTGAATACATGTGTAAGGT
TATTTCCAAGTTGGGTAACGATTCTGCTTCTGCTAACATTACAATTGTTGAGTCCAACGCTACATCTACTTCTACGA
CTGGTACTTCTCACTTGGTTAAGTGCGCTGAAAAGGAAAAGACCTTTTGTGTTAACGGTGGTGAATGTTTTATGGTT
AAGGACTTGTCTAACCCATCTCGTTACTTGTGTAAGTGCCCAAACGAATTCACTGGTGATAGATGTCAAAACTACGT
CATGGCTTCTTTCTACTCTACCTCTACCCCATTTTTGTCTTTGCCAGAA
Peptide SEQ ID No.4(HIS-rhcGGF2)
HHHHHHGNEAAPAGASVCYSSPPSVGSVQELAQRAAVVIEGKVHPQRRQQGALDRKAAAAAGEAGAWGG
DREPPAAGPRALGPPAEEPLLAANGTVPSWPTAPVPSAGEPGEEAPYLVKVHQVWAVKAGGLKKDSLLTVRLGTWGH
PAFPSCGRLKEDSRYIFFMEPDANSTSRAPAAFRASFPPLETGRNLKKEVSRVLCKRCALPPRLKEMKSQESAAGSK
LVLRCETSSEYSSLRFKWFKNGNELNRKNKPQNIKIQKKPGKSELRINKASLADSGEYMCKVISKLGNDSASANITI
VESNATSTSTTGTSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYSTSTPFLSLP
E
Nucleinic acid SEQ ID NO.4(HIS-rhcGGF2)
CATCATCATCATCATCATGGCAACGAAGCTGCTCCAGCTGGTGCCTCTGTTTGTTACTCTTCTCCACCA
TCTGTTGGTTCTGTTCAAGAATTGGCTCAAAGAGCTGCTGTTGTTATTGAAGGTAAGGTTCATCCACAACGTAGACA
ACAAGGTGCTCTGGATAGAAAGGCTGCTGCTGCTGCCGGTGAAGCTGGTGCTTGGGGTGGTGATAGAGAACCACCTG
CTGCTGGTCCAAGAGCTTTGGGTCCACCAGCTGAAGAACCATTGTTGGCTGCTAACGGTACTGTTCCATCTTGGCCA
ACTGCTCCAGTTCCATCAGCTGGTGAACCAGGTGAGGAAGCTCCATACCTAGTTAAGGTTCATCAGGTTTGGGCTGT
TAAGGCTGGTGGTTTGAAGAAGGACTCTTTGTTGACTGTTAGATTGGGTACTTGGGGTCATCCAGCTTTTCCATCTT
GTGGTCGTTTGAAAGAAGACTCCAGGTACATTTTTTTCATGGAACCAGATGCTAACTCCACTTCTAGGGCTCCCGCT
GCTTTCAGAGCTTCTTTTCCACCATTGGAAACGGGTAGAAACTTGAAGAAGGAAGTTTCTAGAGTTCTGTGTAAGCG
TTGTGCTTTGCCACCAAGATTGAAGGAAATGAAGTCTCAAGAATCTGCCGCTGGTTCCAAGTTGGTATTGAGATGCG
AGACTTCTTCCGAGTACAGTTCTTTGAGGTTCAAGTGGTTTAAGAACGGTAACGAATTGAACAGAAAAAACAAGCCC
CAAAACATAAAGATTCAAAAGAAGCCTGGCAAGTCTGAATTGAGGATTAACAAGGCTTCCTTGGCTGATTCTGGTGA
ATACATGTGTAAGGTTATTTCCAAGTTGGGTAACGATTCTGCTTCTGCTAACATTACAATTGTTGAGTCCAACGCTA
CATCTACTTCTACGACTGGTACTTCTCACTTGGTTAAGTGCGCTGAAAAGGAAAAGACCTTTTGTGTTAACGGTGGT
GAATGTTTTATGGTTAAGGACTTGTCTAACCCATCTCGTTACTTGTGTAAGTGCCCAAACGAATTCACTGGTGATAG
ATGTCAAAACTACGTCATGGCTTCTTTCTACTCTACCTCTACCCCATTTTTGTCTTTGCCAGAA
Sequence Nucleinic acid SEQ ID No.1 amplimers
1 ATCTCGAGAAAAGAGAGGCTGAAGCTATGAGATGGAGAAGAGCTCCAAGAAGATCTGGTAG ACCA
39
2 TGGTCTTTGTGCTCTTGGACCTGGTCTACCAGATCTTCTTGGA 43
3 GTCCAAGAGCACAAAGACCAGGTTCTGCCGCTAGATCTTC 40
4 CAACAATGGCAAAGGTGGAGAAGATCTAGCGGCAGAACC 39
5 TCCACCTTTGCCATTGTTGCCACTATTATTGTTGTTGGGTACA 43
6 CCTGGAGCCAATGCAGCTGTACCCAACAACAATAATAGTGG 41
7 GCTGCATTGGCTCCAGGTGCTGCCGCTGGCAACGAA 36
8 AACAAACAGAAGCTCCAGCTGGAGCAGCTTCGTTGCCAGCGGCA 44
9 AGCTGGAGCTTCTGTTTGTTACTCTTCTCCACCATCTGTTG 41
10 TGAGCCAATTCTTGAACAGAACCAACAGATGGTGGAGAAGAGT 43
11 GTTCTGTTCAAGAATTGGCTCAAAGGGCTGCTGTTGTTATTGA 43
12 CTTTGGGGATGAACCTTACCTTCAATAACAACAGCAGCCCTT 42
13 AGGTAAGGTTCATCCCCAAAGAAGACAACAGGGTGCTTTGGA 42
14 GCAGCAGCAGCCTTTCTATCCAAAGCACCCTGTTGTC 37
15 TAGAAAGGCTGCTGCTGCTGCTGGAGAAGCTGGTGC 36
16 GTTCTCTATCACCACCCCAAGCACCAGCTTCTCCAGC 37
17 TTGGGGTGGTGATAGAGAACCACCAGCTGCTGGTCCA 37
18 GCTGGTGGACCTAAAGCTCTTGGACCAGCAGCTGGTG 37
19 AGAGCTTTAGGTCCACCAGCTGAAGAACCATTGTTGGCTG 40
20 CAAGATGGAACAGTACCGTTAGCAGCCAACAATGGTTCTTCA 42
21 CTAACGGTACTGTTCCATCTTGGCCAACTGCTCCAGTTCC 40
22 ACCTGGTTCACCAGCTGATGGAACTGGAGCAGTTGGC 37
23 ATCAGCTGGTGAACCAGGTGAGGAAGCTCCATACTTGGTC 40
24 AGCCCAAACTTGATGAACCTTGACCAAGTATGGAGCTTCCTC 42
25 AAGGTTCATCAAGTTTGGGCTGTTAAGGCTGGTGGTTTGAAG 42
26 CCTAACAGTCAACAAAGAATCCTTCTTCAAACCACCAGCCTTAAC 45
27 AAGGATTCTTTGTTGACTGTTAGGTTGGGTACTTGGGGTCATCC 44
28 CTACCACAAGATGGAAAGGCTGGATGACCCCAAGTACCCAA 41
29 AGCCTTTCCATCTTGTGGTAGGCTGAAGGAGGATTCTAGATACA 44
30 GCATCGGGTTCCATGAAAAAAATGTATCTAGAATCCTCCTTCAGC 45
31 TTTTTTTCATGGAACCCGATGCTAACTCTACTTCTAGAGCTCCCG 45
32 GGAAAAGAGGCTCTAAAAGCAGCGGGAGCTCTAGAAGTAGAGTTA 45
33 CTGCTTTTAGAGCCTCTTTTCCACCATTGGAAACTGGCAGAAA 43
34 ACTCTAGAAACCTCCTTCTTCAAGTTTCTGCCAGTTTCCAATGGT 45
35 CTTGAAGAAGGAGGTTTCTAGAGTTTTGTGCAAGAGATGTGCTTT 45
36 CATTTCCTTCAATCTTGGTGGCAAAGCACATCTCTTGCACAAA 43
37 GCCACCAAGATTGAAGGAAATGAAGTCTCAAGAATCTGCTGCT 43
38 CACCTCAAAACCAATTTTGAACCAGCAGCAGATTCTTGAGACTT 44
39 GGTTCAAAATTGGTTTTGAGGTGCGAGACTTCCAGTGAATACTCC 45
40 TCTTAAACCACTTGAATCTCAGAGAGGAGTATTCACTGGAAGTCTCG 47
41 TCTCTGAGATTCAAGTGGTTTAAGAACGGTAACGAATTGAACCGTA 46
42 TTGATGTTTTGTGGCTTGTTCTTACGGTTCAATTCGTTACCGT 43
43 AGAACAAGCCACAAAACATCAAGATTCAGAAGAAGCCCGGTAA 43
44 GCCTTGTTAATTCTCAATTCAGACTTACCGGGCTTCTTCTGAATC 45
45 GTCTGAATTGAGAATTAACAAGGCTTCTTTGGCTGACTCTGGTG 44
46 TTACTGATAACTTTGCACATGTACTCACCAGAGTCAGCCAAAGAA 45
47 AGTACATGTGCAAAGTTATCAGTAAGCTGGGTAACGATTCTGCTT 45
48 GACTCAACGATAGTAATGTTAGCAGAAGCAGAATCGTTACCCAGC 45
49 CTGCTAACATTACTATCGTTGAGTCTAACGCAACCTCCACTTCTAC 46
50 AACCAAATGAGAGGTACCAGTAGTAGAAGTGGAGGTTGCGTTA 43
51 ACTGGTACCTCTCATTTGGTTAAATGTGCTGAGAAGGAAAAAACT 45
52 TTCACCACCGTTAACACAGAAAGTTTTTTCCTTCTCAGCACATTT 45
53 TTCTGTGTTAACGGTGGTGAATGTTTCATGGTTAAGGACTTGTC 44
54 GCACAAGTACCTAGATGGGTTAGACAAGTCCTTAACCATGAAACA 45
55 TAACCCATCTAGGTACTTGTGCAAGTGTCCAAACGAATTTACAGG 45
56 CGTAGTTCTGGCATCTGTCACCTGTAAATTCGTTTGGACACTT 43
57 TGACAGATGCCAGAACTACGTTATGGCATCTTTTTACTCCACTAG 45
58 ATGCGGCCGCTTCAGGCAATGAAAGAAATGGAGTACTAGTGGAGTAAAAAGATGCCA TAA 60
Sequence Nucleinic acid SEQ ID No.2 amplimers
59 ATCTCGAGAAAAGAGAGGCTGAAGCTCATCATCATCATCATCAT 44
60 CATCATCATCATCATCATAGATGGAGAAGAGCTCCAAGAAGATCTGGTAGACCA 54
2 TGGTCTTTGTGCTCTTGGACCTGGTCTACCAGATCTTCTTGGA 43
3 GTCCAAGAGCACAAAGACCAGGTTCTGCCGCTAGATCTTC 40
4 CAACAATGGCAAAGGTGGAGAAGATCTAGCGGCAGAACC 39
5 TCCACCTTTGCCATTGTTGCCACTATTATTGTTGTTGGGTACA 43
6 CCTGGAGCCAATGCAGCTGTACCCAACAACAATAATAGTGG 41
7 GCTGCATTGGCTCCAGGTGCTGCCGCTGGCAACGAA 36
8 AACAAACAGAAGCTCCAGCTGGAGCAGCTTCGTTGCCAGCGGCA 44
9 AGCTGGAGCTTCTGTTTGTTACTCTTCTCCACCATCTGTTG 41
10 TGAGCCAATTCTTGAACAGAACCAACAGATGGTGGAGAAGAGT 43
11 GTTCTGTTCAAGAATTGGCTCAAAGGGCTGCTGTTGTTATTGA 43
12 CTTTGGGGATGAACCTTACCTTCAATAACAACAGCAGCCCTT 42
13 AGGTAAGGTTCATCCCCAAAGAAGACAACAGGGTGCTTTGGA 42
14 GCAGCAGCAGCCTTTCTATCCAAAGCACCCTGTTGTC 37
15 TAGAAAGGCTGCTGCTGCTGCTGGAGAAGCTGGTGC 36
16 GTTCTCTATCACCACCCCAAGCACCAGCTTCTCCAGC 37
17 TTGGGGTGGTGATAGAGAACCACCAGCTGCTGGTCCA 37
18 GCTGGTGGACCTAAAGCTCTTGGACCAGCAGCTGGTG 37
19 AGAGCTTTAGGTCCACCAGCTGAAGAACCATTGTTGGCTG 40
20 CAAGATGGAACAGTACCGTTAGCAGCCAACAATGGTTCTTCA 42
21 CTAACGGTACTGTTCCATCTTGGCCAACTGCTCCAGTTCC 40
22 ACCTGGTTCACCAGCTGATGGAACTGGAGCAGTTGGC 37
23 ATCAGCTGGTGAACCAGGTGAGGAAGCTCCATACTTGGTC 40
24 AGCCCAAACTTGATGAACCTTGACCAAGTATGGAGCTTCCTC 42
25 AAGGTTCATCAAGTTTGGGCTGTTAAGGCTGGTGGTTTGAAG 42
26 CCTAACAGTCAACAAAGAATCCTTCTTCAAACCACCAGCCTTAAC 45
27 AAGGATTCTTTGTTGACTGTTAGGTTGGGTACTTGGGGTCATCC 44
28 CTACCACAAGATGGAAAGGCTGGATGACCCCAAGTACCCAA 41
29 AGCCTTTCCATCTTGTGGTAGGCTGAAGGAGGATTCTAGATACA 44
30 GCATCGGGTTCCATGAAAAAAATGTATCTAGAATCCTCCTTCAGC 45
31 TTTTTTTCATGGAACCCGATGCTAACTCTACTTCTAGAGCTCCCG 45
32 GGAAAAGAGGCTCTAAAAGCAGCGGGAGCTCTAGAAGTAGAGTTA 45
33 CTGCTTTTAGAGCCTCTTTTCCACCATTGGAAACTGGCAGAAA 43
34 ACTCTAGAAACCTCCTTCTTCAAGTTTCTGCCAGTTTCCAATGGT 45
35 CTTGAAGAAGGAGGTTTCTAGAGTTTTGTGCAAGAGATGTGCTTT 45
36 CATTTCCTTCAATCTTGGTGGCAAAGCACATCTCTTGCACAAA 43
37 GCCACCAAGATTGAAGGAAATGAAGTCTCAAGAATCTGCTGCT 43
38 CACCTCAAAACCAATTTTGAACCAGCAGCAGATTCTTGAGACTT 44
39 GGTTCAAAATTGGTTTTGAGGTGCGAGACTTCCAGTGAATACTCC 45
40 TCTTAAACCACTTGAATCTCAGAGAGGAGTATTCACTGGAAGTCTCG 47
41 TCTCTGAGATTCAAGTGGTTTAAGAACGGTAACGAATTGAACCGTA 46
42 TTGATGTTTTGTGGCTTGTTCTTACGGTTCAATTCGTTACCGT 43
43 AGAACAAGCCACAAAACATCAAGATTCAGAAGAAGCCCGGTAA 43
44 GCCTTGTTAATTCTCAATTCAGACTTACCGGGCTTCTTCTGAATC 45
45 GTCTGAATTGAGAATTAACAAGGCTTCTTTGGCTGACTCTGGTG 44
46 TTACTGATAACTTTGCACATGTACTCACCAGAGTCAGCCAAAGAA 45
47 AGTACATGTGCAAAGTTATCAGTAAGCTGGGTAACGATTCTGCTT 45
48 GACTCAACGATAGTAATGTTAGCAGAAGCAGAATCGTTACCCAGC 45
49 CTGCTAACATTACTATCGTTGAGTCTAACGCAACCTCCACTTCTAC 46
50 AACCAAATGAGAGGTACCAGTAGTAGAAGTGGAGGTTGCGTTA 43
51 ACTGGTACCTCTCATTTGGTTAAATGTGCTGAGAAGGAAAAAACT 45
52 TTCACCACCGTTAACACAGAAAGTTTTTTCCTTCTCAGCACATTT 45
53 TTCTGTGTTAACGGTGGTGAATGTTTCATGGTTAAGGACTTGTC 44
54 GCACAAGTACCTAGATGGGTTAGACAAGTCCTTAACCATGAAACA 45
55 TAACCCATCTAGGTACTTGTGCAAGTGTCCAAACGAATTTACAGG 45
56 CGTAGTTCTGGCATCTGTCACCTGTAAATTCGTTTGGACACTT 43
57 TGACAGATGCCAGAACTACGTTATGGCATCTTTTTACTCCACTAG 45
58 ATGCGGCCGCTTCAGGCAATGAAAGAAATGGAGTACTAGTGGAGTAAAAAGATGCCA TAA 60
Sequence Nucleinic acid SEQ ID No.3 amplimers
61 ATCTCGAGAAAAGAGAGGCTGAAGCTATGGGCAACGAAGCTGC 41
62 CAGAGGCACCAGCTGGAGCAGCTTCGTTGCCCAT 34
63 TCCAGCTGGTGCCTCTGTTTGTTACTCTTCTCCACCATCTGTTGG 45
64 TTTGAGCCAATTCTTGAACAGAACCAACAGATGGTGGAGAAGAG 44
65 TTCTGTTCAAGAATTGGCTCAAAGAGCTGCTGTTGTTATTGAAGG 45
66 TCTACGTTGTGGATGAACCTTACCTTCAATAACAACAGCAGCTC 44
67 TAAGGTTCATCCACAACGTAGACAACAAGGTGCTCTGGATAGA 43
68 CAGCAGCAGCAGCCTTTCTATCCAGAGCACCTTGTTG 37
69 AAGGCTGCTGCTGCTGCCGGTGAAGCTGGTGCT 33
70 GGTTCTCTATCACCACCCCAAGCACCAGCTTCACCGG 37
71 TGGGGTGGTGATAGAGAACCACCTGCTGCTGGTCCAA 37
72 CTGGTGGACCCAAAGCTCTTGGACCAGCAGCAGGT 35
73 GAGCTTTGGGTCCACCAGCTGAAGAACCATTGTTGGCTGCTA 42
74 CCAAGATGGAACAGTACCGTTAGCAGCCAACAATGGTTCTT 41
75 ACGGTACTGTTCCATCTTGGCCAACTGCTCCAGTTCCAT 39
76 CACCTGGTTCACCAGCTGATGGAACTGGAGCAGTTGG 37
77 CAGCTGGTGAACCAGGTGAGGAAGCTCCATACCTAGTTAAG 41
78 ACAGCCCAAACCTGATGAACCTTAACTAGGTATGGAGCTTCCT 43
79 GTTCATCAGGTTTGGGCTGTTAAGGCTGGTGGTTTGAAGAA 41
80 AATCTAACAGTCAACAAAGAGTCCTTCTTCAAACCACCAGCCTTA 45
81 GGACTCTTTGTTGACTGTTAGATTGGGTACTTGGGGTCATCCA 43
82 CGACCACAAGATGGAAAAGCTGGATGACCCCAAGTACCC 39
83 GCTTTTCCATCTTGTGGTCGTTTGAAAGAAGACTCCAGGTACA 43
84 AGCATCTGGTTCCATGAAAAAAATGTACCTGGAGTCTTCTTTCAAA 46
85 TTTTTTTCATGGAACCAGATGCTAACTCCACTTCTAGGGCTCC 43
86 GGAAAAGAAGCTCTGAAAGCAGCGGGAGCCCTAGAAGTGGAGTT 44
87 TGCTTTCAGAGCTTCTTTTCCACCATTGGAAACGGGTAGAAA 42
88 AACTCTAGAAACTTCCTTCTTCAAGTTTCTACCCGTTTCCAATGGT 46
89 CTTGAAGAAGGAAGTTTCTAGAGTTCTGTGTAAGCGTTGTGCTTTG 46
80 TTCATTTCCTTCAATCTTGGTGGCAAAGCACAACGCTTACACAG 44
91 CCACCAAGATTGAAGGAAATGAAGTCTCAAGAATCTGCCGCT 42
92 GCATCTCAATACCAACTTGGAACCAGCGGCAGATTCTTGAGAC 43
93 TTCCAAGTTGGTATTGAGATGCGAGACTTCTTCCGAGTACAGTT 44
94 TCTTAAACCACTTGAACCTCAAAGAACTGTACTCGGAAGAAGTCTC 46
95 CTTTGAGGTTCAAGTGGTTTAAGAACGGTAACGAATTGAACAGAAAA 47
96 ATCTTTATGTTTTGGGGCTTGTTTTTTCTGTTCAATTCGTTACCGT 46
97 AACAAGCCCCAAAACATAAAGATTCAAAAGAAGCCTGGCAAGT 43
98 AGCCTTGTTAATCCTCAATTCAGACTTGCCAGGCTTCTTTTGA 43
99 CTGAATTGAGGATTAACAAGGCTTCCTTGGCTGATTCTGGTGAA 44
100 ACTTGGAAATAACCTTACACATGTATTCACCAGAATCAGCCAAGG 45
101 TACATGTGTAAGGTTATTTCCAAGTTGGGTAACGATTCTGCTTCTG 46
102 TGGACTCAACAATTGTAATGTTAGCAGAAGCAGAATCGTTACCCA 45
103 CTAACATTACAATTGTTGAGTCCAACGCTACATCTACTTCTACGAC 46
104 ACTTAACCAAGTGAGAAGTACCAGTCGTAGAAGTAGATGTAGCGT 45
105 TGGTACTTCTCACTTGGTTAAGTGCGCTGAAAAGGAAAAGACCT 44
106 AAACATTCACCACCGTTAACACAAAAGGTCTTTTCCTTTTCAGCG 45
107 GTGTTAACGGTGGTGAATGTTTTATGGTTAAGGACTTGTCTAACCC 46
108 GCACTTACACAAGTAACGAGATGGGTTAGACAAGTCCTTAACCATA 46
109 ATCTCGTTACTTGTGTAAGTGCCCAAACGAATTCACTGGTGAT 43
110 CCATGACGTAGTTTTGACATCTATCACCAGTGAATTCGTTTGG 43
111 AGATGTCAAAACTACGTCATGGCTTCTTTCTACTCTACCTCTACCC 46
112 ATGCGGCCGCTTCTGGCAAAGACAAAAATGGGGTAGAGGTAGAGTAGAAAGAAG 54
Sequence Nucleinic acid SEQ ID No.4 amplimers
113 ATCTCGAGAAAAGAGAGGCTGAAGCTCATCATCATCATCATCATATGGGCAACGAA GCTGC 59
62 CAGAGGCACCAGCTGGAGCAGCTTCGTTGCCCAT 34
63 TCCAGCTGGTGCCTCTGTTTGTTACTCTTCTCCACCATCTGTTGG 45
64 TTTGAGCCAATTCTTGAACAGAACCAACAGATGGTGGAGAAGAG 44
65 TTCTGTTCAAGAATTGGCTCAAAGAGCTGCTGTTGTTATTGAAGG 45
66 TCTACGTTGTGGATGAACCTTACCTTCAATAACAACAGCAGCTC 44
67 TAAGGTTCATCCACAACGTAGACAACAAGGTGCTCTGGATAGA 43
68 CAGCAGCAGCAGCCTTTCTATCCAGAGCACCTTGTTG 37
69 AAGGCTGCTGCTGCTGCCGGTGAAGCTGGTGCT 33
70 GGTTCTCTATCACCACCCCAAGCACCAGCTTCACCGG 37
71 TGGGGTGGTGATAGAGAACCACCTGCTGCTGGTCCAA 37
72 CTGGTGGACCCAAAGCTCTTGGACCAGCAGCAGGT 35
73 GAGCTTTGGGTCCACCAGCTGAAGAACCATTGTTGGCTGCTA 42
74 CCAAGATGGAACAGTACCGTTAGCAGCCAACAATGGTTCTT 41
75 ACGGTACTGTTCCATCTTGGCCAACTGCTCCAGTTCCAT 39
76 CACCTGGTTCACCAGCTGATGGAACTGGAGCAGTTGG 37
77 CAGCTGGTGAACCAGGTGAGGAAGCTCCATACCTAGTTAAG 41
78 ACAGCCCAAACCTGATGAACCTTAACTAGGTATGGAGCTTCCT 43
79 GTTCATCAGGTTTGGGCTGTTAAGGCTGGTGGTTTGAAGAA 41
80 AATCTAACAGTCAACAAAGAGTCCTTCTTCAAACCACCAGCCTTA 45
81 GGACTCTTTGTTGACTGTTAGATTGGGTACTTGGGGTCATCCA 43
82 CGACCACAAGATGGAAAAGCTGGATGACCCCAAGTACCC 39
83 GCTTTTCCATCTTGTGGTCGTTTGAAAGAAGACTCCAGGTACA 43
84 AGCATCTGGTTCCATGAAAAAAATGTACCTGGAGTCTTCTTTCAAA 46
85 TTTTTTTCATGGAACCAGATGCTAACTCCACTTCTAGGGCTCC 43
86 GGAAAAGAAGCTCTGAAAGCAGCGGGAGCCCTAGAAGTGGAGTT 44
87 TGCTTTCAGAGCTTCTTTTCCACCATTGGAAACGGGTAGAAA 42
88 AACTCTAGAAACTTCCTTCTTCAAGTTTCTACCCGTTTCCAATGGT 46
89 CTTGAAGAAGGAAGTTTCTAGAGTTCTGTGTAAGCGTTGTGCTTTG 46
90 TTCATTTCCTTCAATCTTGGTGGCAAAGCACAACGCTTACACAG 44
91 CCACCAAGATTGAAGGAAATGAAGTCTCAAGAATCTGCCGCT 42
92 GCATCTCAATACCAACTTGGAACCAGCGGCAGATTCTTGAGAC 43
93 TTCCAAGTTGGTATTGAGATGCGAGACTTCTTCCGAGTACAGTT 44
94 TCTTAAACCACTTGAACCTCAAAGAACTGTACTCGGAAGAAGTCTC 46
95 CTTTGAGGTTCAAGTGGTTTAAGAACGGTAACGAATTGAACAGAAAA 47
96 ATCTTTATGTTTTGGGGCTTGTTTTTTCTGTTCAATTCGTTACCGT 46
97 AACAAGCCCCAAAACATAAAGATTCAAAAGAAGCCTGGCAAGT 43
98 AGCCTTGTTAATCCTCAATTCAGACTTGCCAGGCTTCTTTTGA 43
99 CTGAATTGAGGATTAACAAGGCTTCCTTGGCTGATTCTGGTGAA 44
100 ACTTGGAAATAACCTTACACATGTATTCACCAGAATCAGCCAAGG 45
101 TACATGTGTAAGGTTATTTCCAAGTTGGGTAACGATTCTGCTTCTG 46
102 TGGACTCAACAATTGTAATGTTAGCAGAAGCAGAATCGTTACCCA 45
103 CTAACATTACAATTGTTGAGTCCAACGCTACATCTACTTCTACGAC 46
104 ACTTAACCAAGTGAGAAGTACCAGTCGTAGAAGTAGATGTAGCGT 45
105 TGGTACTTCTCACTTGGTTAAGTGCGCTGAAAAGGAAAAGACCT 44
106 AAACATTCACCACCGTTAACACAAAAGGTCTTTTCCTTTTCAGCG 45
107 GTGTTAACGGTGGTGAATGTTTTATGGTTAAGGACTTGTCTAACCC 46
108 GCACTTACACAAGTAACGAGATGGGTTAGACAAGTCCTTAACCATA 46
109 ATCTCGTTACTTGTGTAAGTGCCCAAACGAATTCACTGGTGAT 43
110 CCATGACGTAGTTTTGACATCTATCACCAGTGAATTCGTTTGG 43
111 AGATGTCAAAACTACGTCATGGCTTCTTTCTACTCTACCTCTACCC 46
112 ATGCGGCCGCTTCTGGCAAAGACAAAAATGGGGTAGAGGTAGAGTAGAAAGAAG 54
<110>Hubei University of Science and Technology
<120>A kind of induced expression of people source polypeptide in pichia yeast
Peptide Seq ID No.1 (rhGGF2)
Mrwrraprrsgrpgpraqrpgsaarsspplpllplllllgtaalapgaaagneaapagasvcyssppsvgsvq
elaqraavviegkvhpqrrqqgaldrkaaaaageagawggdreppaagpralgppaeepllaangtvpswptapvps
agepgeeapylvkvhqvwavkagglkkdslltvrlgtwghpafpscgrlkedsryiffmepdanstsrapaafrasf
ppletgrnlkkevsrvlckrcalpprlkemksqesaagsklvlrcetsseysslrfkwfkngnelnrknkpqnikiq
kkpgkselrinkasladsgeymckvisklgndsasanitivesnatststtgtshlvkcaekektfcvnggecfmvk
dlsnpsrylckcpneftgdrcqnyvmasfyststpflslpe
Nucleinic acid Seq ID no.1(rhGGF2)
ATGAGATGGAGAAGAGCTCCAAGAAGATCTGGTAGACCAGGTCCAAGAGCACAAAGACCAGGTTCTGCCGCTA
GATCTTCTCCACCTTTGCCATTGTTGCCACTATTATTGTTGTTGGGTACAGCTGCATTGGCTCCAGGTGCTGCCGCT
GGCAACGAAGCTGCTCCAGCTGGAGCTTCTGTTTGTTACTCTTCTCCACCATCTGTTGGTTCTGTTCAAGAATTGGC
TCAAAGGGCTGCTGTTGTTATTGAAGGTAAGGTTCATCCCCAAAGAAGACAACAGGGTGCTTTGGATAGAAAGGCTG
CTGCTGCTGCTGGAGAAGCTGGTGCTTGGGGTGGTGATAGAGAACCACCAGCTGCTGGTCCAAGAGCTTTAGGTCCA
CCAGCTGAAGAACCATTGTTGGCTGCTAACGGTACTGTTCCATCTTGGCCAACTGCTCCAGTTCCATCAGCTGGTGA
ACCAGGTGAGGAAGCTCCATACTTGGTCAAGGTTCATCAAGTTTGGGCTGTTAAGGCTGGTGGTTTGAAGAAGGATT
CTTTGTTGACTGTTAGGTTGGGTACTTGGGGTCATCCAGCCTTTCCATCTTGTGGTAGGCTGAAGGAGGATTCTAGA
TACATTTTTTTCATGGAACCCGATGCTAACTCTACTTCTAGAGCTCCCGCTGCTTTTAGAGCCTCTTTTCCACCATT
GGAAACTGGCAGAAACTTGAAGAAGGAGGTTTCTAGAGTTTTGTGCAAGAGATGTGCTTTGCCACCAAGATTGAAGG
AAATGAAGTCTCAAGAATCTGCTGCTGGTTCAAAATTGGTTTTGAGGTGCGAGACTTCCAGTGAATACTCCTCTCTG
AGATTCAAGTGGTTTAAGAACGGTAACGAATTGAACCGTAAGAACAAGCCACAAAACATCAAGATTCAGAAGAAGCC
CGGTAAGTCTGAATTGAGAATTAACAAGGCTTCTTTGGCTGACTCTGGTGAGTACATGTGCAAAGTTATCAGTAAGC
TGGGTAACGATTCTGCTTCTGCTAACATTACTATCGTTGAGTCTAACGCAACCTCCACTTCTACTACTGGTACCTCT
CATTTGGTTAAATGTGCTGAGAAGGAAAAAACTTTCTGTGTTAACGGTGGTGAATGTTTCATGGTTAAGGACTTGTC
TAACCCATCTAGGTACTTGTGCAAGTGTCCAAACGAATTTACAGGTGACAGATGCCAGAACTACGTTATGGCATCTT
TTTACTCCACTAGTACTCCATTTCTTTCATTGCCTGAA
Peptide Seq ID No.2 (HIS-rhGGF2)
HHHHHHrwrraprrsgrpgpraqrpgsaarsspplpllplllllgtaalapgaaagneaapagasvcysspps
vgsvqelaqraavviegkvhpqrrqqgaldrkaaaaageagawggdreppaagpralgppaeepllaangtvpswpt
apvpsagepgeeapylvkvhqvwavkagglkkdslltvrlgtwghpafpscgrlkedsryiffmepdanstsrapaa
frasfppletgrnlkkevsrvlckrcalpprlkemksqesaagsklvlrcetsseysslrfkwfkngnelnrknkpq
nikiqkkpgkselrinkasladsgeymckvisklgndsasanitivesnatststtgtshlvkcaekektfcvngge
cfmvkdlsnpsrylckcpneftgdrcqnyvmasfyststpflslpe
Nucleinic acid Seq ID no.2(HIS-rhGGF2)
CATCATCATCATCATCATAGATGGAGAAGAGCTCCAAGAAGATCTGGTAGACCAGGTCCAAGAGCACAAAGAC
CAGGTTCTGCCGCTAGATCTTCTCCACCTTTGCCATTGTTGCCACTATTATTGTTGTTGGGTACAGCTGCATTGGCT
CCAGGTGCTGCCGCTGGCAACGAAGCTGCTCCAGCTGGAGCTTCTGTTTGTTACTCTTCTCCACCATCTGTTGGTTC
TGTTCAAGAATTGGCTCAAAGGGCTGCTGTTGTTATTGAAGGTAAGGTTCATCCCCAAAGAAGACAACAGGGTGCTT
TGGATAGAAAGGCTGCTGCTGCTGCTGGAGAAGCTGGTGCTTGGGGTGGTGATAGAGAACCACCAGCTGCTGGTCCA
AGAGCTTTAGGTCCACCAGCTGAAGAACCATTGTTGGCTGCTAACGGTACTGTTCCATCTTGGCCAACTGCTCCAGT
TCCATCAGCTGGTGAACCAGGTGAGGAAGCTCCATACTTGGTCAAGGTTCATCAAGTTTGGGCTGTTAAGGCTGGTG
GTTTGAAGAAGGATTCTTTGTTGACTGTTAGGTTGGGTACTTGGGGTCATCCAGCCTTTCCATCTTGTGGTAGGCTG
AAGGAGGATTCTAGATACATTTTTTTCATGGAACCCGATGCTAACTCTACTTCTAGAGCTCCCGCTGCTTTTAGAGC
CTCTTTTCCACCATTGGAAACTGGCAGAAACTTGAAGAAGGAGGTTTCTAGAGTTTTGTGCAAGAGATGTGCTTTGC
CACCAAGATTGAAGGAAATGAAGTCTCAAGAATCTGCTGCTGGTTCAAAATTGGTTTTGAGGTGCGAGACTTCCAGT
GAATACTCCTCTCTGAGATTCAAGTGGTTTAAGAACGGTAACGAATTGAACCGTAAGAACAAGCCACAAAACATCAA
GATTCAGAAGAAGCCCGGTAAGTCTGAATTGAGAATTAACAAGGCTTCTTTGGCTGACTCTGGTGAGTACATGTGCA
AAGTTATCAGTAAGCTGGGTAACGATTCTGCTTCTGCTAACATTACTATCGTTGAGTCTAACGCAACCTCCACTTCT
ACTACTGGTACCTCTCATTTGGTTAAATGTGCTGAGAAGGAAAAAACTTTCTGTGTTAACGGTGGTGAATGTTTCAT
GGTTAAGGACTTGTCTAACCCATCTAGGTACTTGTGCAAGTGTCCAAACGAATTTACAGGTGACAGATGCCAGAACT
ACGTTATGGCATCTTTTTACTCCACTAGTACTCCATTTCTTTCATTGCCTGAA
Peptide Seq ID No.3 (rhcGGF2)
Mgneaapagasvcyssppsvgsvqelaqraavviegkvhpqrrqqgaldrkaaaaageagawggdreppaagp
ralgppaeepllaangtvpswptapvpsagepgeeapylvkvhqvwavkagglkkdslltvrlgtwghpafpscgrl
kedsryiffmepdanstsrapaafrasfppletgrnlkkevsrvlckrcalpprlkemksqesaagsklvlrcetss
eysslrfkwfkngnelnrknkpqnikiqkkpgkselrinkasladsgeymckvisklgndsasanitivesnatsts
ttgtshlvkcaekektfcvnggecfmvkdlsnpsrylckcpneftgdrcqnyvmasfyststpflslpe
Nucleinic acid Seq ID no.3
ATGGGCAACGAAGCTGCTCCAGCTGGTGCCTCTGTTTGTTACTCTTCTCCACCATCTGTTGGTTCTGTTCAAG
AATTGGCTCAAAGAGCTGCTGTTGTTATTGAAGGTAAGGTTCATCCACAACGTAGACAACAAGGTGCTCTGGATAGA
AAGGCTGCTGCTGCTGCCGGTGAAGCTGGTGCTTGGGGTGGTGATAGAGAACCACCTGCTGCTGGTCCAAGAGCTTT
GGGTCCACCAGCTGAAGAACCATTGTTGGCTGCTAACGGTACTGTTCCATCTTGGCCAACTGCTCCAGTTCCATCAG
CTGGTGAACCAGGTGAGGAAGCTCCATACCTAGTTAAGGTTCATCAGGTTTGGGCTGTTAAGGCTGGTGGTTTGAAG
AAGGACTCTTTGTTGACTGTTAGATTGGGTACTTGGGGTCATCCAGCTTTTCCATCTTGTGGTCGTTTGAAAGAAGA
CTCCAGGTACATTTTTTTCATGGAACCAGATGCTAACTCCACTTCTAGGGCTCCCGCTGCTTTCAGAGCTTCTTTTC
CACCATTGGAAACGGGTAGAAACTTGAAGAAGGAAGTTTCTAGAGTTCTGTGTAAGCGTTGTGCTTTGCCACCAAGA
TTGAAGGAAATGAAGTCTCAAGAATCTGCCGCTGGTTCCAAGTTGGTATTGAGATGCGAGACTTCTTCCGAGTACAG
TTCTTTGAGGTTCAAGTGGTTTAAGAACGGTAACGAATTGAACAGAAAAAACAAGCCCCAAAACATAAAGATTCAAA
AGAAGCCTGGCAAGTCTGAATTGAGGATTAACAAGGCTTCCTTGGCTGATTCTGGTGAATACATGTGTAAGGTTATT
TCCAAGTTGGGTAACGATTCTGCTTCTGCTAACATTACAATTGTTGAGTCCAACGCTACATCTACTTCTACGACTGG
TACTTCTCACTTGGTTAAGTGCGCTGAAAAGGAAAAGACCTTTTGTGTTAACGGTGGTGAATGTTTTATGGTTAAGG
ACTTGTCTAACCCATCTCGTTACTTGTGTAAGTGCCCAAACGAATTCACTGGTGATAGATGTCAAAACTACGTCATG
GCTTCTTTCTACTCTACCTCTACCCCATTTTTGTCTTTGCCAGAA
Peptide Seq ID No.4 (HIS-rhcGGF2)
hhhhhhgneaapagasvcyssppsvgsvqelaqraavviegkvhpqrrqqgaldrkaaaaageagawggdrep
paagpralgppaeepllaangtvpswptapvpsagepgeeapylvkvhqvwavkagglkkdslltvrlgtwghpafp
scgrlkedsryiffmepdanstsrapaafrasfppletgrnlkkevsrvlckrcalpprlkemksqesaagsklvlr
cetsseysslrfkwfkngnelnrknkpqnikiqkkpgkselrinkasladsgeymckvisklgndsasanitivesn
atststtgtshlvkcaekektfcvnggecfmvkdlsnpsrylckcpneftgdrcqnyvmasfyststpflslpe
Nucleinic acid Seq ID no.4 (HIS-rhcGGF2)
CATCATCATCATCATCATGGCAACGAAGCTGCTCCAGCTGGTGCCTCTGTTTGTTACTCTTCTCCACCATCTG
TTGGTTCTGTTCAAGAATTGGCTCAAAGAGCTGCTGTTGTTATTGAAGGTAAGGTTCATCCACAACGTAGACAACAA
GGTGCTCTGGATAGAAAGGCTGCTGCTGCTGCCGGTGAAGCTGGTGCTTGGGGTGGTGATAGAGAACCACCTGCTGC
TGGTCCAAGAGCTTTGGGTCCACCAGCTGAAGAACCATTGTTGGCTGCTAACGGTACTGTTCCATCTTGGCCAACTG
CTCCAGTTCCATCAGCTGGTGAACCAGGTGAGGAAGCTCCATACCTAGTTAAGGTTCATCAGGTTTGGGCTGTTAAG
GCTGGTGGTTTGAAGAAGGACTCTTTGTTGACTGTTAGATTGGGTACTTGGGGTCATCCAGCTTTTCCATCTTGTGG
TCGTTTGAAAGAAGACTCCAGGTACATTTTTTTCATGGAACCAGATGCTAACTCCACTTCTAGGGCTCCCGCTGCTT
TCAGAGCTTCTTTTCCACCATTGGAAACGGGTAGAAACTTGAAGAAGGAAGTTTCTAGAGTTCTGTGTAAGCGTTGT
GCTTTGCCACCAAGATTGAAGGAAATGAAGTCTCAAGAATCTGCCGCTGGTTCCAAGTTGGTATTGAGATGCGAGAC
TTCTTCCGAGTACAGTTCTTTGAGGTTCAAGTGGTTTAAGAACGGTAACGAATTGAACAGAAAAAACAAGCCCCAAA
ACATAAAGATTCAAAAGAAGCCTGGCAAGTCTGAATTGAGGATTAACAAGGCTTCCTTGGCTGATTCTGGTGAATAC
ATGTGTAAGGTTATTTCCAAGTTGGGTAACGATTCTGCTTCTGCTAACATTACAATTGTTGAGTCCAACGCTACATC
TACTTCTACGACTGGTACTTCTCACTTGGTTAAGTGCGCTGAAAAGGAAAAGACCTTTTGTGTTAACGGTGGTGAAT
GTTTTATGGTTAAGGACTTGTCTAACCCATCTCGTTACTTGTGTAAGTGCCCAAACGAATTCACTGGTGATAGATGT
CAAAACTACGTCATGGCTTCTTTCTACTCTACCTCTACCCCATTTTTGTCTTTGCCAGAA
Sequence Nucleinic acid SEQ ID No.1 amplimers
1 AT CTCGAG AAAAGAGAGGCTGAAGCTATGAGATGGAGAAGAGCTCCAAGAAGATCTGGTAG ACCA 39
2 TGGTCTTTGTGCTCTTGGACCTGGTCTACCAGATCTTCTTGGA 43
3 GTCCAAGAGCACAAAGACCAGGTTCTGCCGCTAGATCTTC 40
4 CAACAATGGCAAAGGTGGAGAAGATCTAGCGGCAGAACC 39
5 TCCACCTTTGCCATTGTTGCCACTATTATTGTTGTTGGGTACA 43
6 CCTGGAGCCAATGCAGCTGTACCCAACAACAATAATAGTGG 41
7 GCTGCATTGGCTCCAGGTGCTGCCGCTGGCAACGAA 36
8 AACAAACAGAAGCTCCAGCTGGAGCAGCTTCGTTGCCAGCGGCA 44
9 AGCTGGAGCTTCTGTTTGTTACTCTTCTCCACCATCTGTTG 41
10 TGAGCCAATTCTTGAACAGAACCAACAGATGGTGGAGAAGAGT 43
11 GTTCTGTTCAAGAATTGGCTCAAAGGGCTGCTGTTGTTATTGA 43
12 CTTTGGGGATGAACCTTACCTTCAATAACAACAGCAGCCCTT 42
13 AGGTAAGGTTCATCCCCAAAGAAGACAACAGGGTGCTTTGGA 42
14 GCAGCAGCAGCCTTTCTATCCAAAGCACCCTGTTGTC 37
15 TAGAAAGGCTGCTGCTGCTGCTGGAGAAGCTGGTGC 36
16 GTTCTCTATCACCACCCCAAGCACCAGCTTCTCCAGC 37
17 TTGGGGTGGTGATAGAGAACCACCAGCTGCTGGTCCA 37
18 GCTGGTGGACCTAAAGCTCTTGGACCAGCAGCTGGTG 37
19 AGAGCTTTAGGTCCACCAGCTGAAGAACCATTGTTGGCTG 40
20 CAAGATGGAACAGTACCGTTAGCAGCCAACAATGGTTCTTCA 42
21 CTAACGGTACTGTTCCATCTTGGCCAACTGCTCCAGTTCC 40
22 ACCTGGTTCACCAGCTGATGGAACTGGAGCAGTTGGC 37
23 ATCAGCTGGTGAACCAGGTGAGGAAGCTCCATACTTGGTC 40
24 AGCCCAAACTTGATGAACCTTGACCAAGTATGGAGCTTCCTC 42
25 AAGGTTCATCAAGTTTGGGCTGTTAAGGCTGGTGGTTTGAAG 42
26 CCTAACAGTCAACAAAGAATCCTTCTTCAAACCACCAGCCTTAAC 45
27 AAGGATTCTTTGTTGACTGTTAGGTTGGGTACTTGGGGTCATCC 44
28 CTACCACAAGATGGAAAGGCTGGATGACCCCAAGTACCCAA 41
29 AGCCTTTCCATCTTGTGGTAGGCTGAAGGAGGATTCTAGATACA 44
30 GCATCGGGTTCCATGAAAAAAATGTATCTAGAATCCTCCTTCAGC 45
31 TTTTTTTCATGGAACCCGATGCTAACTCTACTTCTAGAGCTCCCG 45
32 GGAAAAGAGGCTCTAAAAGCAGCGGGAGCTCTAGAAGTAGAGTTA 45
33 CTGCTTTTAGAGCCTCTTTTCCACCATTGGAAACTGGCAGAAA 43
34 ACTCTAGAAACCTCCTTCTTCAAGTTTCTGCCAGTTTCCAATGGT 45
35 CTTGAAGAAGGAGGTTTCTAGAGTTTTGTGCAAGAGATGTGCTTT 45
36 CATTTCCTTCAATCTTGGTGGCAAAGCACATCTCTTGCACAAA 43
37 GCCACCAAGATTGAAGGAAATGAAGTCTCAAGAATCTGCTGCT 43
38 CACCTCAAAACCAATTTTGAACCAGCAGCAGATTCTTGAGACTT 44
39 GGTTCAAAATTGGTTTTGAGGTGCGAGACTTCCAGTGAATACTCC 45
40 TCTTAAACCACTTGAATCTCAGAGAGGAGTATTCACTGGAAGTCTCG 47
41 TCTCTGAGATTCAAGTGGTTTAAGAACGGTAACGAATTGAACCGTA 46
42 TTGATGTTTTGTGGCTTGTTCTTACGGTTCAATTCGTTACCGT 43
43 AGAACAAGCCACAAAACATCAAGATTCAGAAGAAGCCCGGTAA 43
44 GCCTTGTTAATTCTCAATTCAGACTTACCGGGCTTCTTCTGAATC 45
45 GTCTGAATTGAGAATTAACAAGGCTTCTTTGGCTGACTCTGGTG 44
46 TTACTGATAACTTTGCACATGTACTCACCAGAGTCAGCCAAAGAA 45
47 AGTACATGTGCAAAGTTATCAGTAAGCTGGGTAACGATTCTGCTT 45
48 GACTCAACGATAGTAATGTTAGCAGAAGCAGAATCGTTACCCAGC 45
49 CTGCTAACATTACTATCGTTGAGTCTAACGCAACCTCCACTTCTAC 46
50 AACCAAATGAGAGGTACCAGTAGTAGAAGTGGAGGTTGCGTTA 43
51 ACTGGTACCTCTCATTTGGTTAAATGTGCTGAGAAGGAAAAAACT 45
52 TTCACCACCGTTAACACAGAAAGTTTTTTCCTTCTCAGCACATTT 45
53 TTCTGTGTTAACGGTGGTGAATGTTTCATGGTTAAGGACTTGTC 44
54 GCACAAGTACCTAGATGGGTTAGACAAGTCCTTAACCATGAAACA 45
55 TAACCCATCTAGGTACTTGTGCAAGTGTCCAAACGAATTTACAGG 45
56 CGTAGTTCTGGCATCTGTCACCTGTAAATTCGTTTGGACACTT 43
57 TGACAGATGCCAGAACTACGTTATGGCATCTTTTTACTCCACTAG 45
58 AT GCGGCCGC TTCAGGCAATGAAAGAAATGGAGTACTAGTGGAGTAAAAAGATGCCA TAA 60
Sequence Nucleinic acid SEQ ID No.2 amplimers
59 AT CTCGAG AAAAGAGAGGCTGAAGCTCATCATCATCATCATCAT 44
60 CATCATCATCATCATCATAGATGGAGAAGAGCT CCAAGAAGATCTGGTAGACCA 54
2 TGGTCTTTGTGCTCTTGGACCTGGTCTACCAGATCTTCTTGGA 43
3 GTCCAAGAGCACAAAGACCAGGTTCTGCCGCTAGATCTTC 40
4 CAACAATGGCAAAGGTGGAGAAGATCTAGCGGCAGAACC 39
5 TCCACCTTTGCCATTGTTGCCACTATTATTGTTGTTGGGTACA 43
6 CCTGGAGCCAATGCAGCTGTACCCAACAACAATAATAGTGG 41
7 GCTGCATTGGCTCCAGGTGCTGCCGCTGGCAACGAA 36
8 AACAAACAGAAGCTCCAGCTGGAGCAGCTTCGTTGCCAGCGGCA 44
9 AGCTGGAGCTTCTGTTTGTTACTCTTCTCCACCATCTGTTG 41
10 TGAGCCAATTCTTGAACAGAACCAACAGATGGTGGAGAAGAGT 43
11 GTTCTGTTCAAGAATTGGCTCAAAGGGCTGCTGTTGTTATTGA 43
12 CTTTGGGGATGAACCTTACCTTCAATAACAACAGCAGCCCTT 42
13 AGGTAAGGTTCATCCCCAAAGAAGACAACAGGGTGCTTTGGA 42
14 GCAGCAGCAGCCTTTCTATCCAAAGCACCCTGTTGTC 37
15 TAGAAAGGCTGCTGCTGCTGCTGGAGAAGCTGGTGC 36
16 GTTCTCTATCACCACCCCAAGCACCAGCTTCTCCAGC 37
17 TTGGGGTGGTGATAGAGAACCACCAGCTGCTGGTCCA 37
18 GCTGGTGGACCTAAAGCTCTTGGACCAGCAGCTGGTG 37
19 AGAGCTTTAGGTCCACCAGCTGAAGAACCATTGTTGGCTG 40
20 CAAGATGGAACAGTACCGTTAGCAGCCAACAATGGTTCTTCA 42
21 CTAACGGTACTGTTCCATCTTGGCCAACTGCTCCAGTTCC 40
22 ACCTGGTTCACCAGCTGATGGAACTGGAGCAGTTGGC 37
23 ATCAGCTGGTGAACCAGGTGAGGAAGCTCCATACTTGGTC 40
24 AGCCCAAACTTGATGAACCTTGACCAAGTATGGAGCTTCCTC 42
25 AAGGTTCATCAAGTTTGGGCTGTTAAGGCTGGTGGTTTGAAG 42
26 CCTAACAGTCAACAAAGAATCCTTCTTCAAACCACCAGCCTTAAC 45
27 AAGGATTCTTTGTTGACTGTTAGGTTGGGTACTTGGGGTCATCC 44
28 CTACCACAAGATGGAAAGGCTGGATGACCCCAAGTACCCAA 41
29 AGCCTTTCCATCTTGTGGTAGGCTGAAGGAGGATTCTAGATACA 44
30 GCATCGGGTTCCATGAAAAAAATGTATCTAGAATCCTCCTTCAGC 45
31 TTTTTTTCATGGAACCCGATGCTAACTCTACTTCTAGAGCTCCCG 45
32 GGAAAAGAGGCTCTAAAAGCAGCGGGAGCTCTAGAAGTAGAGTTA 45
33 CTGCTTTTAGAGCCTCTTTTCCACCATTGGAAACTGGCAGAAA 43
34 ACTCTAGAAACCTCCTTCTTCAAGTTTCTGCCAGTTTCCAATGGT 45
35 CTTGAAGAAGGAGGTTTCTAGAGTTTTGTGCAAGAGATGTGCTTT 45
36 CATTTCCTTCAATCTTGGTGGCAAAGCACATCTCTTGCACAAA 43
37 GCCACCAAGATTGAAGGAAATGAAGTCTCAAGAATCTGCTGCT 43
38 CACCTCAAAACCAATTTTGAACCAGCAGCAGATTCTTGAGACTT 44
39 GGTTCAAAATTGGTTTTGAGGTGCGAGACTTCCAGTGAATACTCC 45
40 TCTTAAACCACTTGAATCTCAGAGAGGAGTATTCACTGGAAGTCTCG 47
41 TCTCTGAGATTCAAGTGGTTTAAGAACGGTAACGAATTGAACCGTA 46
42 TTGATGTTTTGTGGCTTGTTCTTACGGTTCAATTCGTTACCGT 43
43 AGAACAAGCCACAAAACATCAAGATTCAGAAGAAGCCCGGTAA 43
44 GCCTTGTTAATTCTCAATTCAGACTTACCGGGCTTCTTCTGAATC 45
45 GTCTGAATTGAGAATTAACAAGGCTTCTTTGGCTGACTCTGGTG 44
46 TTACTGATAACTTTGCACATGTACTCACCAGAGTCAGCCAAAGAA 45
47 AGTACATGTGCAAAGTTATCAGTAAGCTGGGTAACGATTCTGCTT 45
48 GACTCAACGATAGTAATGTTAGCAGAAGCAGAATCGTTACCCAGC 45
49 CTGCTAACATTACTATCGTTGAGTCTAACGCAACCTCCACTTCTAC 46
50 AACCAAATGAGAGGTACCAGTAGTAGAAGTGGAGGTTGCGTTA 43
51 ACTGGTACCTCTCATTTGGTTAAATGTGCTGAGAAGGAAAAAACT 45
52 TTCACCACCGTTAACACAGAAAGTTTTTTCCTTCTCAGCACATTT 45
53 TTCTGTGTTAACGGTGGTGAATGTTTCATGGTTAAGGACTTGTC 44
54 GCACAAGTACCTAGATGGGTTAGACAAGTCCTTAACCATGAAACA 45
55 TAACCCATCTAGGTACTTGTGCAAGTGTCCAAACGAATTTACAGG 45
56 CGTAGTTCTGGCATCTGTCACCTGTAAATTCGTTTGGACACTT 43
57 TGACAGATGCCAGAACTACGTTATGGCATCTTTTTACTCCACTAG 45
58 AT GCGGCCGC TTCAGGCAATGAAAGAAATGGAGTACTAGTGGAGTAAAAAGATGCCA TAA 60
Sequence Nucleinic acid SEQ ID No.3 amplimers
61 AT CTCGAG AAAAGAGAGGCTGAAGCTATGGGCAACGAAGCTGC 41 62
CAGAGGCACCAGCTGGAGCAGCTTCGTTGCCCAT 34 63
TCCAGCTGGTGCCTCTGTTTGTTACTCTTCTCCACCATCTGTTGG 45 64
TTTGAGCCAATTCTTGAACAGAACCAACAGATGGTGGAGAAGAG 44 65
TTCTGTTCAAGAATTGGCTCAAAGAGCTGCTGTTGTTATTGAAGG 45 66
TCTACGTTGTGGATGAACCTTACCTTCAATAACAACAGCAGCTC 44 67
TAAGGTTCATCCACAACGTAGACAACAAGGTGCTCTGGATAGA 43 68
CAGCAGCAGCAGCCTTTCTATCCAGAGCACCTTGTTG 37 69
AAGGCTGCTGCTGCTGCCGGTGAAGCTGGTGCT 33 70
GGTTCTCTATCACCACCCCAAGCACCAGCTTCACCGG 37 71
TGGGGTGGTGATAGAGAACCACCTGCTGCTGGTCCAA 37 72
CTGGTGGACCCAAAGCTCTTGGACCAGCAGCAGGT 35 73
GAGCTTTGGGTCCACCAGCTGAAGAACCATTGTTGGCTGCTA 42 74
CCAAGATGGAACAGTACCGTTAGCAGCCAACAATGGTTCTT 41 75
ACGGTACTGTTCCATCTTGGCCAACTGCTCCAGTTCCAT 39 76
CACCTGGTTCACCAGCTGATGGAACTGGAGCAGTTGG 37 77
CAGCTGGTGAACCAGGTGAGGAAGCTCCATACCTAGTTAAG 41 78
ACAGCCCAAACCTGATGAACCTTAACTAGGTATGGAGCTTCCT 43 79
GTTCATCAGGTTTGGGCTGTTAAGGCTGGTGGTTTGAAGAA 41 80
AATCTAACAGTCAACAAAGAGTCCTTCTTCAAACCACCAGCCTTA 45 81
GGACTCTTTGTTGACTGTTAGATTGGGTACTTGGGGTCATCCA 43 82
CGACCACAAGATGGAAAAGCTGGATGACCCCAAGTACCC 39 83
GCTTTTCCATCTTGTGGTCGTTTGAAAGAAGACTCCAGGTACA 43 84
AGCATCTGGTTCCATGAAAAAAATGTACCTGGAGTCTTCTTTCAAA 46 85
TTTTTTTCATGGAACCAGATGCTAACTCCACTTCTAGGGCTCC 43 86
GGAAAAGAAGCTCTGAAAGCAGCGGGAGCCCTAGAAGTGGAGTT 44 87
TGCTTTCAGAGCTTCTTTTCCACCATTGGAAACGGGTAGAAA 42 88
AACTCTAGAAACTTCCTTCTTCAAGTTTCTACCCGTTTCCAATGGT 46
89 CTTGAAGAAGGAAGTTTCTAGAGTTCTGTGTAAGCGTTGTGCTTTG 46
80 TTCATTTCCTTCAATCTTGGTGGCAAAGCACAACGCTTACACAG 44 91
CCACCAAGATTGAAGGAAATGAAGTCTCAAGAATCTGCCGCT 42 92
GCATCTCAATACCAACTTGGAACCAGCGGCAGATTCTTGAGAC 43 93
TTCCAAGTTGGTATTGAGATGCGAGACTTCTTCCGAGTACAGTT 44 94
TCTTAAACCACTTGAACCTCAAAGAACTGTACTCGGAAGAAGTCTC 46 95
CTTTGAGGTTCAAGTGGTTTAAGAACGGTAACGAATTGAACAGAAAA 47 96
ATCTTTATGTTTTGGGGCTTGTTTTTTCTGTTCAATTCGTTACCGT 46 97
AACAAGCCCCAAAACATAAAGATTCAAAAGAAGCCTGGCAAGT 43 98
AGCCTTGTTAATCCTCAATTCAGACTTGCCAGGCTTCTTTTGA 43 99
CTGAATTGAGGATTAACAAGGCTTCCTTGGCTGATTCTGGTGAA 44 100
ACTTGGAAATAACCTTACACATGTATTCACCAGAATCAGCCAAGG 45 101
TACATGTGTAAGGTTATTTCCAAGTTGGGTAACGATTCTGCTTCTG 46 102
TGGACTCAACAATTGTAATGTTAGCAGAAGCAGAATCGTTACCCA 45 103
CTAACATTACAATTGTTGAGTCCAACGCTACATCTACTTCTACGAC 46 104
ACTTAACCAAGTGAGAAGTACCAGTCGTAGAAGTAGATGTAGCGT 45 105
TGGTACTTCTCACTTGGTTAAGTGCGCTGAAAAGGAAAAGACCT 44 106
AAACATTCACCACCGTTAACACAAAAGGTCTTTTCCTTTTCAGCG 45 107
GTGTTAACGGTGGTGAATGTTTTATGGTTAAGGACTTGTCTAACCC 46 108
GCACTTACACAAGTAACGAGATGGGTTAGACAAGTCCTTAACCATA 46 109
ATCTCGTTACTTGTGTAAGTGCCCAAACGAATTCACTGGTGAT 43 110
CCATGACGTAGTTTTGACATCTATCACCAGTGAATTCGTTTGG 43 111
AGATGTCAAAACTACGTCATGGCTTCTTTCTACTCTACCTCTACCC 46 112
AT GCGGCCGC TTCTGGCAAAGACAAAAATGGGGTAGAGGTAGAGTAGAA AGAAG 54
Sequence Nucleinic acid SEQ ID No.4 amplimers
113 AT CTCGAG AAAAGAGAGGCTGAAGCTCATCATCATCATCATCATATGGGCAACGAA GCTGC 59
62 CAGAGGCACCAGCTGGAGCAGCTTCGTTGCCCAT 34 63
TCCAGCTGGTGCCTCTGTTTGTTACTCTTCTCCACCATCTGTTGG 45 64
TTTGAGCCAATTCTTGAACAGAACCAACAGATGGTGGAGAAGAG 44 65
TTCTGTTCAAGAATTGGCTCAAAGAGCTGCTGTTGTTATTGAAGG 45 66
TCTACGTTGTGGATGAACCTTACCTTCAATAACAACAGCAGCTC 44 67
TAAGGTTCATCCACAACGTAGACAACAAGGTGCTCTGGATAGA 43 68
CAGCAGCAGCAGCCTTTCTATCCAGAGCACCTTGTTG 37 69
AAGGCTGCTGCTGCTGCCGGTGAAGCTGGTGCT 33 70
GGTTCTCTATCACCACCCCAAGCACCAGCTTCACCGG 37 71
TGGGGTGGTGATAGAGAACCACCTGCTGCTGGTCCAA 37 72
CTGGTGGACCCAAAGCTCTTGGACCAGCAGCAGGT 35 73
GAGCTTTGGGTCCACCAGCTGAAGAACCATTGTTGGCTGCTA 42 74
CCAAGATGGAACAGTACCGTTAGCAGCCAACAATGGTTCTT 41 75
ACGGTACTGTTCCATCTTGGCCAACTGCTCCAGTTCCAT 39 76
CACCTGGTTCACCAGCTGATGGAACTGGAGCAGTTGG 37 77
CAGCTGGTGAACCAGGTGAGGAAGCTCCATACCTAGTTAAG 41 78
ACAGCCCAAACCTGATGAACCTTAACTAGGTATGGAGCTTCCT 43 79
GTTCATCAGGTTTGGGCTGTTAAGGCTGGTGGTTTGAAGAA 41 80
AATCTAACAGTCAACAAAGAGTCCTTCTTCAAACCACCAGCCTTA 45 81
GGACTCTTTGTTGACTGTTAGATTGGGTACTTGGGGTCATCCA 43 82
CGACCACAAGATGGAAAAGCTGGATGACCCCAAGTACCC 39 83
GCTTTTCCATCTTGTGGTCGTTTGAAAGAAGACTCCAGGTACA 43 84
AGCATCTGGTTCCATGAAAAAAATGTACCTGGAGTCTTCTTTCAAA 46 85
TTTTTTTCATGGAACCAGATGCTAACTCCACTTCTAGGGCTCC 43 86
GGAAAAGAAGCTCTGAAAGCAGCGGGAGCCCTAGAAGTGGAGTT 44 87
TGCTTTCAGAGCTTCTTTTCCACCATTGGAAACGGGTAGAAA 42 88
AACTCTAGAAACTTCCTTCTTCAAGTTTCTACCCGTTTCCAATGGT 46
89 CTTGAAGAAGGAAGTTTCTAGAGTTCTGTGTAAGCGTTGTGCTTTG 46
90 TTCATTTCCTTCAATCTTGGTGGCAAAGCACAACGCTTACACAG 44 91
CCACCAAGATTGAAGGAAATGAAGTCTCAAGAATCTGCCGCT 42 92
GCATCTCAATACCAACTTGGAACCAGCGGCAGATTCTTGAGAC 43 93
TTCCAAGTTGGTATTGAGATGCGAGACTTCTTCCGAGTACAGTT 44 94
TCTTAAACCACTTGAACCTCAAAGAACTGTACTCGGAAGAAGTCTC 46 95
CTTTGAGGTTCAAGTGGTTTAAGAACGGTAACGAATTGAACAGAAAA 47 96
ATCTTTATGTTTTGGGGCTTGTTTTTTCTGTTCAATTCGTTACCGT 46 97
AACAAGCCCCAAAACATAAAGATTCAAAAGAAGCCTGGCAAGT 43 98
AGCCTTGTTAATCCTCAATTCAGACTTGCCAGGCTTCTTTTGA 43 99
CTGAATTGAGGATTAACAAGGCTTCCTTGGCTGATTCTGGTGAA 44 100
ACTTGGAAATAACCTTACACATGTATTCACCAGAATCAGCCAAGG 45 101
TACATGTGTAAGGTTATTTCCAAGTTGGGTAACGATTCTGCTTCTG 46 102
TGGACTCAACAATTGTAATGTTAGCAGAAGCAGAATCGTTACCCA 45 103
CTAACATTACAATTGTTGAGTCCAACGCTACATCTACTTCTACGAC 46 104
ACTTAACCAAGTGAGAAGTACCAGTCGTAGAAGTAGATGTAGCGT 45 105
TGGTACTTCTCACTTGGTTAAGTGCGCTGAAAAGGAAAAGACCT 44 106
AAACATTCACCACCGTTAACACAAAAGGTCTTTTCCTTTTCAGCG 45 107
GTGTTAACGGTGGTGAATGTTTTATGGTTAAGGACTTGTCTAACCC 46 108
GCACTTACACAAGTAACGAGATGGGTTAGACAAGTCCTTAACCATA 46 109
ATCTCGTTACTTGTGTAAGTGCCCAAACGAATTCACTGGTGAT 43 110
CCATGACGTAGTTTTGACATCTATCACCAGTGAATTCGTTTGG 43 111
AGATGTCAAAACTACGTCATGGCTTCTTTCTACTCTACCTCTACCC 46 112
AT GCGGCCGC TTCTGGCAAAGACAAAAATGGGGTAGAGGTAGAGTAGAA AGAAG 54
Claims (6)
1. a kind of induced expression of people source polypeptide in pichia yeast, its feature are, the coding that codon is optimized is pre-
Anti- and treatment cardiomyopathy people source polypeptide rhNRG-1 β nucleotide construction enters pichia yeast expression vector, is then transferred to Bi Shi ferment
Mother carries out induced expression, obtains described polypeptide；
RhNRG-1 β include total length people source polypeptide rhGGF2, rhGGF2 part of polypeptide rhcGGF2, rhGGF2 are entered by HIS
Polypeptide HIS-rhGGF2 after the line flag and polypeptide HIS-rhcGGF2 after rhcGGF2 is marked by HIS；
It is according to total length people source polypeptide rhGGF2 amino acid sequence and pichia yeast codon-bias, codon is excellent
The nucleotide construction of encoding human source polypeptide rhGGF2 after change enters in Yeast expression carrier, or the volume after codon is optimized
Code people source polypeptide rhGGF2 nucleotides is connected with HIS (6 × his) label, is then transferred to pichia yeast, is sieved by positive colony
Choosing, the high expression bacterial strain of rhGGF2 high expression bacterial strain and HIS-rhGGF2 is obtained respectively.
Part of polypeptide rhcGGF2 is according to people source rhGGF2 polypeptid acid sequences, removes rhGGF2 signal peptide sequences, obtains institute
RhcGGF2 is stated, then according to pichia yeast codon-bias, the encoding human source polypeptide after codon is optimized
RhcGGF2 nucleotide construction enters in Yeast expression carrier, or the encoding human source polypeptide rhcGGF2 after codon is optimized
Nucleotides be connected with HIS (6 × his) label, be then transferred to pichia yeast, screened by positive colony, obtained respectively
The high expression bacterial strain of rhcGGF2 high expression bacterial strain and HIS-rhcGGF2.
A kind of 2. induced expression of people source polypeptide in pichia yeast according to claim 1, it is characterised in that the coding
RhGGF2 nucleotide sequence such as Nucleinic acid SEQ ID NO：1, corresponding peptide sequence such as Peptide No.1；
The nucleotide sequence such as Nucleinic acid SEQ ID NO of the coding HIS-rhGGF2：2, corresponding peptide sequence is such as
Peptide No.2；The nucleotide sequence such as Nucleinic acid SEQ ID NO of the coding rhcGGF2：3, it is corresponding more
Peptide sequence such as Peptide No.3；The nucleotide sequence such as Nucleinic acid SEQ ID of the coding HIS-rhcGGF2
NO：4, corresponding peptide sequence such as Peptide No.4.
3. induced expression of a kind of people source polypeptide according to claim 1 or claim 2 in pichia yeast, it is characterised in that described
Pichia yeast inducible expression carrier is pHKY01.Pichia yeast inducible expression carrier can select arbitrary pichia yeast induction table
Up to carrier, the present invention can be realized, is optimal using pHKY01.
4. induced expression of a kind of people source polypeptide according to claim 1 or claim 2 in pichia yeast, it is characterised in that described
Pichia yeast is pichia yeast GS115.Pichia yeast can also select arbitrary pichia yeast bacterial strain, can realize this hair
It is bright.
5. induced expression of a kind of people source polypeptide according to claim 1 or claim 2 in pichia yeast, it is characterised in that described
In pichia yeast after induced expression, its purification process is isolated and purified rhGGF2 and rhcGGF2 polypeptides using ion-exchange chromatography
The albumen.
6. induced expression of a kind of people source polypeptide according to claim 1 or claim 2 in pichia yeast, it is characterised in that described
For HIS-rhGGF2 and HIS-rhcGGF2 polypeptides in pichia yeast after induced expression, its purification process uses Ni- nickel affinity purifications
Column chromatography method purifies the albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710738272.5A CN107446945A (en) | 2017-08-25 | 2017-08-25 | A kind of induced expression of people source polypeptide in pichia yeast |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710738272.5A CN107446945A (en) | 2017-08-25 | 2017-08-25 | A kind of induced expression of people source polypeptide in pichia yeast |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107446945A true CN107446945A (en) | 2017-12-08 |
Family
ID=60493224
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710738272.5A Pending CN107446945A (en) | 2017-08-25 | 2017-08-25 | A kind of induced expression of people source polypeptide in pichia yeast |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107446945A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101336251A (en) * | 2005-12-02 | 2008-12-31 | 上海泽生科技开发有限公司 | Neuregulin variants and methods of screening and using thereof |
| US20140086897A1 (en) * | 2012-09-26 | 2014-03-27 | Morehouse School Of Medicine | Chimeric neuregulins and method of making and use thereof |
| CN106636175A (en) * | 2016-09-30 | 2017-05-10 | 湖北科技学院 | Induced expression method of cell injury repair protein in pichia pastoris and purification method and application of cell injury repair protein |
-
2017
- 2017-08-25 CN CN201710738272.5A patent/CN107446945A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101336251A (en) * | 2005-12-02 | 2008-12-31 | 上海泽生科技开发有限公司 | Neuregulin variants and methods of screening and using thereof |
| US20140086897A1 (en) * | 2012-09-26 | 2014-03-27 | Morehouse School Of Medicine | Chimeric neuregulins and method of making and use thereof |
| CN106636175A (en) * | 2016-09-30 | 2017-05-10 | 湖北科技学院 | Induced expression method of cell injury repair protein in pichia pastoris and purification method and application of cell injury repair protein |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| TW211585B (en) | ||
| CN104548137B (en) | A kind of medical composition and its use of the inhibitor containing lncRNA | |
| CN107019794A (en) | Agent is applied for treating or preventing the neuregulin of heart failure or the therapeutic of its subsequence | |
| CN101134105B (en) | Pharmaceutical composition containing recombination human pancreatic kininogenase for treating and/or preventing cerebral infarction | |
| CN108379275B (en) | Lysophosphatidic acid, lysophosphatidic acid receptor 3, and use of lysophosphatidic acid receptor 3 agonist | |
| CN105732789A (en) | Haemadipsa sylvestris analgesic peptide mh2620 and gene and application thereof | |
| CN103215225A (en) | Construction method of glial cell line-derived neurotrophic factor gene-modified embryo neural stem cell | |
| CN107778362A (en) | It is a kind of to be used to suppress polypeptide of OSCC migration and invasive ability and application thereof | |
| CN112851791B (en) | Novel FGF analogue for resisting metabolic disorder and application thereof | |
| CN107385033B (en) | PiRNA-5938 and application of antisense nucleic acid thereof in diagnosis and treatment of ischemic heart diseases | |
| CN108410893A (en) | A kind of tumour cell specific response expression vector started by NF- κ B and its expression product and application | |
| CN105194660B (en) | Ubiquitin specific proteinase 18（USP18）Function and application in myocardial hypertrophy is treated | |
| CN102921021B (en) | MiRNA-361 (micro-ribonucleic acid-361) and application of antisense nucleotide thereof | |
| CN107446945A (en) | A kind of induced expression of people source polypeptide in pichia yeast | |
| CN1978466B (en) | Transduction peptide-human brain-derived neurotrophic factor fusion protein and its use | |
| CN101897953B (en) | Non-invasive high-penetrability epidermal growth factor and application thereof | |
| CN110437311A (en) | A kind of polypeptide and its application | |
| CN102266570A (en) | New application of miRNA-484, pharmaceutical composition containing miRNA-484 and use thereof | |
| CN104524599A (en) | Antisense nucleotide MiRNA-532 containing pharmaceutical composition and application thereof | |
| CN108187029A (en) | Leukocytic immunity globulin sample receptor subfamily B member 4 is in the application for preparing prevention, alleviating and/or treat myocardial hypertrophy drug | |
| CN106636175A (en) | Induced expression method of cell injury repair protein in pichia pastoris and purification method and application of cell injury repair protein | |
| CN114432332A (en) | Application of circUTRN in preparation of medicine for treating heart failure, recombinant vector and medicine for treating heart failure | |
| CN101444629B (en) | Non-injection gene therapy medicine and medicine box thereof | |
| CN105315359A (en) | Deletion type recombinant human mesencephalic astrocyte-derived neurotrophic factor | |
| CN109839508A (en) | Application of the Rbm24-S181 site phosphorylation as stress class disease and the marker of related cardiac conditions medication |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20171208 |