CN102816244A - Fusion protein of exendin-4 peptide and human serum albumin (HSA) and preparation method thereof - Google Patents
Fusion protein of exendin-4 peptide and human serum albumin (HSA) and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a fusion protein of exendin-4 peptide and human serum albumin (HSA) and a preparation method thereof, belonging to the technical field of long-term recombination fusion protein medicine. The invention relates to a preparation method and a product of fusion protein of insulin secretion promoting peptide (Exendin-4) and human serum albumin (HSA). The fusion protein is characterized in that a genetic engineering method is adopted to directly fuse a single Exndin-4 peptide and HAS and without connecting peptide at the middle part to form stable and long half-life period Exendin-4-HAS fusion protein, and is applied in a treatment of non-insulin-dependent diabetes and obesity.
Description
Technical field
Fusion rotein of a kind of Exendin-4 peptide and human serum albumin (HSA) and preparation method thereof belongs to long-acting recombination fusion rotein technical field of pharmaceuticals.
The present invention is through directly merging single Exendin-4 polypeptide and human serum albumin; In the drug effect that keeps Exendin-4, make its transformation period in vivo obtain to prolong; Improve bioavailability and therapeutic value; Alleviate patient's treatment burden, good prospects for application is arranged at pharmaceutical field.The present invention relates to treat the preparation of non-insulin-dependent type ii diabetes depot drug product.
Background technology
Mellitus are the healthy chronic diseases of a kind of serious threat the people of the world, and number of the infected is in continuous increase, and whole world diabetic subject will reach 4.35 hundred million to the year two thousand thirty.The sickness rate of the mellitus of China is up to 9.7%, and the diabetics in the whole nation will become mellitus first big country in the world, and the development of mellitus be the trend of becoming younger above one hundred million.Therefore, research and develop the significant and urgency of effective treating diabetes and control device.
Mellitus are divided into insulin-dependent type i diabetes and non-insulin-dependent type ii diabetes, and type ii diabetes accounts for more than 90% of total patient's number.The II mellitus are because a variety of causes causes the hypofunction of patient's pancreatic beta cell, due to the excretory insufficient insulin, or the insulin receptor dysfunction can not normally utilize Regular Insulin, causes carbohydrate metabolism disturbance, blood sugar increasing.Take various treatment meanss, recover islet function, increasing insulin secretion or improving its utilising efficiency is one of main means of treating at present type ii diabetes.
Exendin-4 (claiming insulin secretion accelerating peptide again); Be from the Gila monster saliva that is grown in Southwestern United Stares and Mexico desert, to find to contain 39 amino acid whose small peptides, the hyperglycemic-glycogenolytic factor of regulating and controlling blood sugar similar peptide GLP-1-1 has 53% homology in its aminoacid sequence and the human blood, is the similar peptide acceptor of hyperglycemic-glycogenolytic factor (Glucogan like Peptide 1R; GLP-1R) agonist; With the GLP-1 functional similarity, have directly hypoglycemicly, promote the effect of pancreatic secretion Regular Insulin; Can also the absorption of food be slowed down through slowing down GI wriggling, thereby reduce the peak that the glucose in the food occurs in blood.Because by dipeptidyl peptidase-IV (DPP-IV) degraded, Exendin-4 does not have higher more stable BA, become Remedies for diabetes exploitation focus.
The fugitive Exendin-4 peptide (having another name called Byetta) that commercialization is used for the type ii diabetes treatment has obtained the drugs approved by FDA listing, becomes a kind of new drug of treatment type ii diabetes.Under physiological condition; Byetta (Exendin-4) is similar with the GLP-1 effect; Depend on blood sugar concentration what stimulate insulin secretion as the time spent, can hypoglycemia not take place, avoided the shortcoming of Regular Insulin because of using dosage improper meeting causing hypoglycemic reaction because using excessive.Yet because the Exendin-4 transformation period in vivo is 2-3 hour, the patient still needs to inject every day the curative effect that could obtain expection for twice, and not only expense is high, and is also more loaded down with trivial details.
In recent years; The exploitation of the long-acting curative of mellitus all is devoted in the home and abroad; Adopt gene engineering method that the Fc section of Exendin-4 and Tegeline (IgG) is merged like Lilly company; The method that Canada ConjuChem company adopts artificial lotus root to join combines Exendin-4 to produce CJC-1134-PC external with human serum albumin, all possibly effectively prolong the Exendin-4 transformation period in vivo, improves its result of treatment.But external Exendin-4 and HSA are carried out the production cost height that chemical lotus root joins, usefulness is low.
HSA is a high molecular weight protein that molecular weight is 66KD of human body, has the various biological function, is the carrier proteins of many biotic factors; Because of it can not be by glomerular filtration; The transformation period reaches 14-21 days in the blood, is normally used for the carrier of medicine, with the transformation period of prolong drug.The present invention adopts genetic engineering technique to make up the expression of recombinant yeast plasmid of the fusion rotein that can express Exendin-4 and human serum albumin; Exendin-4 and HSA directly merge in the expression product; Can increase the stability of Exendin-4; Prolong it in the intravital transformation period of people, improve the treatment of diabetes effect.The yeast external secretion expression system that makes up can prepare this fusion rotein in enormous quantities, has reduced production cost.
Summary of the invention
The present invention provides fusion rotein depot drug product of a kind of insulin secretion accelerating peptide (Exendin-4) and human serum albumin (HSA) and preparation method thereof.The present invention also aims to develop one type of hypoglycemic drug, on the basis of the physiological property that keeps Exendin-4, prolong its transformation period, make it to become long-acting treatment diabetes medicament of new generation with high stability, low spinoff.
Technical scheme of the present invention: the fusion rotein Exendin-4-HSA of a kind of Exendin-4 and human serum albumin HSA; Contain 2 peptide zones; Its 1 district is an Exendin-4 polypeptide, and its 2 district is human serum albumin (HSA), and the terminal N-with the 2nd district through its C-in 1 district is terminal directly to be connected; The centre does not add any connection peptides, and its structural formula is: Exendin-4-HSA.
1 district Exendin-4 polypeptid acid sequence and sequence SEQ ID NO:1 maintain at least 90% homology.
2 district's polypeptide are selected from the mature peptide of human serum albumin HSA, and its aminoacid sequence and sequence SEQ ID NO:2 maintain at least 90% homology.
Described Exendin-4-HSA fusion rotein, it is nucleotide sequence coded to be SEQ ID NO:3.
Described Exendin-4-HSA fusion rotein, its aminoacid sequence are SEQ ID NO:7.
Described Exendin-4-HSA fusion rotein, its recombinant nucleotide sequence are inserted in a kind of expression vector and cell host system.
The preparation method of described Exendin-4-HSA fusion rotein comprises and transcribing, translation, albumen sepn and purifying, and authentication step.
The application of said Exendin-4-HSA fusion rotein is characterized in that making blood glucose level mammals normalizing, is used to prepare non-insulin-dependent type ii diabetes and obese person's treatment preparation.Administering mode is selected injection for use, and is oral, and in the nose, respiratory tract is inhaled clothes.
In order to improve the medication effect, the Exendin-4-HSA fusion rotein can be processed different dosage forms, includes but not limited to use the formed salt form of various soda acids.
In order to improve the medication effect, the Exendin-4-HSA fusion rotein also can use with other drug jointly.
The present invention adopts genetic engineering technique, preparation Exendin-4-HSA fusion rotein long-acting protein medicament, and its long-acting scheme comprises: the one, contain the Exendin-4 polypeptide in this fusion rotein; The 2nd, this fusion rotein contains the human serum albumin HSA peptide section the long half-lift of in human blood, having.Through biochemistry and cell and experimentation on animals checking, the Exendin-4-HSA fusion rotein has the transformation period in better stability and the body than Exendin-4 polypeptide drugs.
Exendin-4 polypeptid acid sequence among the present invention is to be 39 amino acid, maintains at least 90% homology with sequence SEQ ID NO:1, and preferred homology is 95%.Substituting of multiple amino acids site wherein can be arranged, and purpose is to make this bioactive peptide more stable, and activity is higher.
HSA aminoacid sequence among the present invention and sequence SEQ ID NO:2 maintain at least 90% homology, and preferred homology is 95%.Substituting of multiple amino acids site wherein can be arranged, and purpose is to make this bioactive peptide more stable, and activity is higher.
The present invention correctly folds in order to make the fusion rotein that obtains to express, and can carry out external secretion, and the N-end of Exendin-4-HSA fusion rotein links to each other with the signal peptide sequence of HSA.Particularly, when carrying out the structure of yeast recombinant expression plasmid, the Exendin-4-HSA fusion gene is inserted into the downstream of HSA signal peptide gene among the expression plasmid pWX530, and makes both read the frame endomixis.
The preferred plasmid of the present invention is pWX530, is adapted at carrying out in the Yeast engineering bacteria albumen external secretion and expresses.The upper reaches of this plasmid MCS have the gene order of coding HSA protein signal peptide, and its nucleotides sequence is classified as: atg aag tgg gta acc ttt att tcc ctt ctt ttt ctc ttt agc tcg gct tat tcc agg ggt gtg ttt cgt cga.This signal peptide gene can carry out the frame endomixis with the exogenous genetic fragment that inserts.But the fusion rotein among the present invention also can be selected to utilize other plasmid to express, and these expression plasmids include, but are not limited to protokaryon, eukaryotic expression system plasmid commonly used.
Particularly, this expression plasmid contains suitable promotor, in order to the control Expression of Fusion Protein.These promotors include, but are not limited to the promotor PRB1 of the following stated, and preferred promotor is PRB1.
The present invention makes the Exendin-4-HSA fusion rotein of expression not have amino-acid residue beyond any the fusion rotein, and in that 3 of HSA gene ' end adds terminator codon, fusion rotein is translated can be with any composition of going up on the expression vector in the process.Complete Exendin-4-HSA antigen-4 fusion protein gene sequence such as SEQ ID NO:6.
The present invention provides construction process and the engineering bacteria construction process that makes up above-mentioned preferred Exendin-4-HSA Expression of Fusion Protein plasmid.(Fig. 1)
At first the clone obtains a reorganization polymerized nucleoside acid sequence, and this sequence contains the nucleotide sequence district of coding Exendin-4 peptide zone and the nucleotide sequence district of coding HSA peptide zone.The aminoacid sequence of coding Exendin-4 peptide zone and sequence SEQ ID NO:1 maintain at least 90% homology.The aminoacid sequence of coding HSA peptide zone and sequence SEQ ID NO:2 maintain at least 90% homology.
Particularly, through round pcr, reverse transcription obtains the nucleotide sequence of coding HSA peptide zone from the RNA in people's tire liver source, and makes 3 of HSA mature peptide gene ' end have the restriction enzyme site of Nhe1.The nucleotide sequence of coding Exendin-4 peptide zone obtains the Exendin-4 dna fragmentation through synthetic, and has the StuI restriction enzyme site at its 5 ' end, and makes 5 ' terminal sequence of its 3 ' end band top HSA mature peptide gene.Synthetic Exendin-4 gene fragment is mixed with HSA mature peptide gene fragment; Adopt overlapping PCR method to prepare Exendin-4-HSA antigen-4 fusion protein gene fragment; 5 of Exendin-4-HSA antigen-4 fusion protein gene ' end has the StuI restriction enzyme site, and 3 ' end has the restriction enzyme site of Nhe1.Exendin-4-HSA antigen-4 fusion protein gene fragment is carried out the TA clone, preparation recombinant plasmid Exendin-4-HSA/pEGM-T.
With Stu1, Nhe1 recombinant plasmid Exendin-4-HSA/pEGM-T is carried out double digestion, preparation Exendin-4-HSA gene fragment.With method expression plasmid of yeast pWX530 is carried out double digestion, preparation linearization plasmid pWX530 dna fragmentation.With Exendin-4-HSA gene fragment and the linearization plasmid pWX530 dna fragmentation mixed of 3-5 ︰ 1 in molar ratio, connect with the T4 dna ligase, make up expression of recombinant yeast plasmid EX-HSA/pWX530.EX-HSA is the abbreviation of Exendin-4-HSA.
The recombinant plasmid EX-HSA/pWX530 that the present invention expresses the Exendin-4-HSA fusion rotein can express in multiple yeast; Its preferred yeast is a yeast saccharomyces cerevisiae, and its preferred strain is CICIM Y0600 (Southern Yangtze University's China's colleges and universities' industrial microorganism resource and information center provide).
The present invention provides a kind of engineering bacteria large scale fermentation to express the method for Exendin-4-HSA fusion rotein.
Single yeast (CICIM Y0600) colony inoculation that particularly, will contain recombinant expression plasmid EX-HSA/pWX530 is to selective medium SD [0.67%YNB, 0.609% disodium hydrogen phosphate dodecahydrate (Na
2HPO
412H
2O), 5.04% Citric acid (C
6H
8O
7), 2% sucrose] in, 30 ℃ of joltings are spent the night.Calculate and obtain OD
600The total amount of=0.4 required overnight culture.Centrifugal collection bacterial sediment, and with selective medium SD suspension, and change and plant fermention medium YPD (1% yeast powder; 2% peptone, 2% sucrose) in, 48h is cultivated in 30 ℃ of joltings; Centrifugal collection fermented supernatant fluid ,-80 ℃ of preservations are used for the analysis and the purifying of expressing fusion protein.
Expression of Fusion Protein can detect through the biochemical means of general albumen, including, but not limited to: SDS-PAGE, Western blotting etc.
The present invention also provides a kind of separation purification method of Exendin-4-HSA fusion rotein.
Particularly, this separation purification method has utilized the characteristics of the isoelectric pH about 4.7 of Exendin-4-HSA, selects the cation-exchange chromatography post, uses SP Sepharose FF separating effect best, and target protein matter elution peak is the highest, contains assorted minimum.Selecting SP Seharose FF is filler, has successfully caught the Exendin-4-HSA fused protein in the fermented liquid.The Exendin-4-HSA fused protein of catching passes through hydrophobic type chromatography column Butyl Sepharose FF again, and anion-exchange chromatography DEAE Sepharose FF carries out polishing purification, and the purity of target protein can reach more than 98%.
The present invention also provides a kind of evaluation Exendin-4-HSA fusion rotein bioactive method.
The present invention also provides the method that the transformation period is identified in a kind of Exendin-4-HSA of mensuration fusion rotein vitro stability and the body.Particularly, detection method is measured the active shelf-life (shelf life) and the intravital active transformation period of animal of fusion rotein.Animal includes, but are not limited to mouse, dog, and monkey, and human.
Exendin-4-HSA fusion rotein of the present invention can be used to comprise the treatment of symptoms such as mellitus.Administering mode can be injection, and is oral, and in the nose, respiratory tract is inhaled clothes etc.
In order to improve the medication effect, Exendin-4-HSA fusion rotein of the present invention also can be processed different dosage forms, includes but not limited to use the formed salt form of various soda acids.
In order to improve the medication effect, Exendin-4-HSA fusion rotein of the present invention also can use with other drug jointly.
Beneficial effect of the present invention: the present invention adopts recombinant gene; Exendin-4 polypeptide and human serum albumin are directly merged; Produce insulin secretion accelerating peptide fusion rotein Exendin-4-HSA; The centre does not add any connection peptides, on the basis of the pharmacological property that keeps Exendin-4, prolongs its transformation period in vivo, comprises that at pharmaceutical field type ii diabetes and obesity treatment will have good prospects for application.
Description of drawings
Fig. 1 Exendin-4-HSA fusion rotein construction of recombinant expression plasmid figure.
Fig. 2 DNA electrophorogram (1% agarose); Left side A is the pcr amplification product condition diagram; Right B is DNA restricted enzyme cutting analysis figure.
Fig. 3 10%SDS-PAGE electrophorogram and Western bloting be figure as a result; A left side is A protein fermentation expression product S DS-PAGE (a 10%) electrophorogram; The right side is B Western bloting figure as a result.
Fig. 4 purifying Exendin-4-HSA fusion rotein SDS-PAGE (10%) electrophorogram; 1, protein molecular weight mark, 2, Exendin-4-HSA fusion rotein purified product.
Fig. 5 mouse carbohydrate tolerance test blood sugar concentration graphic representation.
Fig. 6 plasma insulin determination figure.
The long-effect active of Fig. 7 Exendin-4-HSA fusion rotein is observed figure.
Various variation diagrams after Fig. 8 diabetes model mouse BSK db/db administration in 1-11 days; A, 1-11 days change of blood sugar of diabetes model mouse BSK db/db; B, 1-11 days body weight of diabetes model mouse BSK db/db change; C, the diabetes model mouse BSK db/db variation of ingesting in 1-11 days; D, 1-11 days drinking-water of diabetes model mouse BSK db/db change.
Plasma Concentration-time plot that Fig. 9 cynomolgus monkey single-dose records; A, cynomolgus monkey single subcutaneous administration 250 μ gkg
-1The time Plasma Concentration-time curve of recording; B, cynomolgus monkey single subcutaneous administration 500 μ gkg
-1The time Plasma Concentration-time curve of recording; C, cynomolgus monkey single subcutaneous administration 750 μ gkg
-1The time Plasma Concentration-time curve of recording; D, cynomolgus monkey single intravenously administrable 500 μ gkg
-1The time Plasma Concentration-time curve of recording.
Embodiment
The following embodiment that provides can explain content of the present invention in more detail.But content of the present invention is not limited to the content that these embodiment set forth.
Embodiment 1: the human albumin gene clone
Adopt RT-PCR to prepare people HSA mature peptide gene, design of primers is following:
HSA1:5′-GATGCACACA AGAGTGAGGT TGCTCA-3′，
HSA2:5′-AAGCTAGCTT ATAAGCCTAA GGCAGCTTG ACTTGC-3′。
Get people's tire hepatic tissue, prepare total RNA with the Trizol method.RT-PCR amplification: in the PCR pipe of a 0.2mL; Add 2 * RT PCR damping fluid, 25 μ L, total RNA 3 μ L (50ng), primer HSA1 0.5 μ L (25 μ M), HSA2 0.5 μ L (25 μ M); RT-Phusion mixed enzyme 1 μ L adds DEPC water to final volume 50 μ L.PCR condition: 45 ℃ of 30min; 94 ℃ of sex change 2min then; Again by 94 ℃, 30sec; 55 ℃, 1min, 72 ℃, 2min, 30 circulations altogether.At last, 72 ℃ of insulation 5min.The PCR product is carried out agarose gel electrophoresis observe amplification and product size.Adopt glue absorption method purifying HSA gene fragment then; Concrete grammar be on sepharose behind the electrophoresis, downcut molecular weight in the 1750bp left and right sides DNA band; PCR product glue with Promega company reclaims test kit purifying HSA gene DNA fragment again, and method is pressed process specifications.
Embodiment 2:Exendin-4 gene is synthetic
The Exendin-4 gene adopts the artificial synthesis preparation.Sequence by SEQ ID NO:5 is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's Exendin-4 gene, and makes the restriction enzyme site of its 5 ' terminal band StuI.
The structure and the clone of embodiment 3:Exendin-4-HSA fusion gene
This experiment adopts overlapping PCR method Exendin-4 and HSA gene to be merged directly to constitute the Exendin-4-HSA fusion gene be example, and the method for synthesizing fusion gene is described.Concrete grammar is following: under the condition that does not add primer and polysaccharase, mix each composition according to following system.
5 * phusion HF buffer, 20 μ L, dNTPs (10 mM) 2 μ L, Exendin-4 dna fragmentation 2 μ L, HSA DNA fragment 2 μ L, ddH
2O 69 μ L, TV 95 μ L.
99 ℃ of sex change 10 min put the room temperature naturally cooling then.Add Phusion high-fidelity enzyme 0.5 μ L, 72 ℃ of insulation 15min, and then add primer
Ex1: (5 '-TA
AGGCCTCACGGTGAAGGTACTTTCACTT-3 ', Stu1) (50 μ M) and
HSA2: (5 '-AAGCTAGCTTATAAGCCTAAGGCAGCTTGACTTGC-3 ', Nhe1) (50 μ M) each 2 μ L, Phusion high-fidelity enzyme 0.5 μ L, carry out conventional PCR amplification:
98 ℃ of preparatory sex change 30 sec, 98 ℃ of sex change 10 sec, 72 ℃ of annealing 30 sec carry out 35 circulations.At last at 72 ℃ of insulation 7min.Amplified production through 1% agarose gel electrophoresis analysis, is observed amplified production situation (Fig. 2 A).With PCR product purification test kit purifying fusion gene Exendin-4-HSA DNA fragment, working method is pressed the test kit specification sheets.
The TA clone of fusion gene: purifying fusion gene product in the PCR reaction system that only contains deoxidation Adenosine triphosphate purine, is carried out 5 ' end and adds " A " tail under the effect of Taq DNA polysaccharase.Reaction system is following: 10 * Taq polymerase buffer, 10 μ L, MgCl
2(2.5 mM) 10 μ L, dATP (2 mM) 10 μ L, the PCR product 60 μ L of purifying, sterilization de-ionized H
2O 9 μ L, Taq DNA polysaccharase 1 μ L, TV 80 μ L.72 ℃ of reaction 30 min on the PCR instrument.The Exendin-4-HSA gene fragment that adds " A " tail with PCR product purification test kit purifying.With Exendin-4-HSA fragment and TA clone carrier pGEM-T mixed according to molecule mole number 3-5 ︰ 1, under the effect of T4 DNA ligase enzyme, connect, 16 ℃ of water-baths connect spends the night, and makes up the recombinant plasmid EX-HSA/pGEM-T that contains fusion gene.Connect the product electricity and be transformed into competence E.coli DH5, coat on the LB flat board that contains 100 μ g/mL penbritins (AMP), 40 μ L X-gal (20 mg/mL) 37 ℃ of overnight cultures.The single bacterium colony of growing on the picking AMP flat board carries out the accuracy that DNA restricted enzyme cutting analysis (Fig. 2 B) and dna sequencing are confirmed the fusion gene sequence.
The structure of embodiment 4:Exendin-4-HSA expression plasmid
Below be example with the Exendin-4-HSA fusion gene, the structure of expressing vector plasmid is described.It is the expression vector plasmid that pWX530 is adopted in this experiment, and its preparation method is molecular biology method commonly used.The bacterial classification that promptly contains expression vector through cultivation extracts and obtains this plasmid.-20 ℃ of preservations are for use behind the preparation plasmid.
Specific as follows, carry out Stu1 and Nhe1 double digestion to expressing vector plasmid pWX530 earlier.Actual conditions is following.Expression vector plasmid pWX530 10 μ L; Stu1 1 μ L, Nhe1 1 μ L, 10 * T4 DNA ligase damping fluid, 5 μ L; DdH
2O 33 μ L, TV are 50 μ L.37 ℃ of thermostat water bath internal reactions 3 hours reclaim linearizing pWX530 DNA through agarose gel electrophoresis.Exendin-4-HSA/pGEM-T DNA makes same double digestion, and reclaiming molecular weight through agarose gel electrophoresis is the Exendin-4-HSA gene fragment of 1891bp.
The Exendin-4-HSA DNA that reclaims is connected construction of fusion protein expression plasmid EX-HSA/pWX530 with the expression plasmid pWX530DNA of above-mentioned double digestion.Linked system is generally 10 μ L, and the mol ratio of the pWX530 plasmid vector of double digestion and double digestion Exendin-4-HSA DNA is 1 ︰ 2-10,10 * T4 DNA ligase damping fluid, 1 μ L, and adding sterilized water to TV is 10 μ L.16 ℃ of thermostat water bath internal reactions of ligation 16 hours.Connect product transformed into escherichia coli DH5 α competent cell.The evaluation of positive colony is to select positive colony through the LB flat board that contains Ampicilin, the extracting plasmid, and carry out the accuracy that dna sequencing is confirmed the fusion gene sequence.
The structure of embodiment 5:Exendin-4-HSA expressing fusion protein engineering bacteria
This experiment utilizes electric method for transformation and Exendin-4-HSA fusion gene to be example, and the method that Exendin-4-HSA expressing fusion protein engineering bacteria makes up is described.Concrete grammar is following.
At first select the bacterial clone that contains EX-HSA/pWX530 expression vector plasmid, extract the expression vector plasmid.Then, the method for electricity consumption conversion changes plasmid among the yeast saccharomyces cerevisiae INVSc1 over to.The DNA process for extracting is undertaken by Promega company DNA purification kit operation instructions.
Electricity method for transformation transformed saccharomyces cerevisiae is specific as follows.
Carry out the preparation of thalline earlier.Picking yeast list bacterium colony is seeded in the 50mL triangular flask that contains 5mL YPD substratum 30 ℃, 250-300r/min overnight cultures.The culture of getting 100-500 μ L then is seeded to the 2L triangle that contains the 500mL fresh culture and shakes in the bottle, and 28 ~ 30 ℃, 250-300 r/min overnight cultures are to OD
600Reach 1-1.5.Again with cell culture in 4 ℃, centrifugal 5 min of 1500g are with the ddH of the ice precooling of 500 mL
2O is resuspended with bacterial sediment.After centrifugal, use the ddH of the ice precooling of 250mL again
2O is resuspended with bacterial sediment.Centrifugal again, with the Sorbitol Solution USP of 1 mol of the ice precooling of 20mL that bacterial sediment is resuspended.Centrifugal again, with the Sorbitol Solution USP of the 1mol/L of the ice precooling of 1mL that bacterial sediment is resuspended, its final volume is about 1.5mL.
Transform with electric-shocking method then.Specific as follows.
5 ~ 20 μ g EX-HSA/ pWX530 expression plasmid DNA are dissolved in 5 ~ 10 μ L TE solution, and with the above-mentioned competence thalline mixing of 80 μ L, the electricity that goes to the precooling of 0.2 cm ice transforms in the cup.Electricity is transformed cup ice bath 5 min.According to corresponding electric conversion instrument, parameters such as the voltage that employing is optimized, electric current, electric capacity shock by electricity then.After electric shock finished, the Sorbitol Solution USP that adds the precooling of 1 mL ice went to the thalline mixing in the EP pipe of 1.5 mL, and the thalline suspension is coated on the SD selectivity flat board, per 200 ~ 600 μ L coating one flat plate.Flat board is placed 30 ℃ of cultivations, occur until single bacterium colony.This experiment recommends shock parameters to be: voltage 1.5kV; Electric capacity 25 μ F; Resistance 200 Ω.The electric shock time is 4～10 msec.
Embodiment 6:Exendin-4-HSA expressing fusion protein and purifying
With EX-HSA/pWX530 plasmid electricity transformed yeast, to select fuller bacterium colony through SD screening culture medium flat board and be connected to enlarged culturing in the SD screening culture medium, thalli growth is connected to fermentation expression fused protein in the YPD substratum behind finite concentration.Fermented liquid is got supernatant and is carried out the protein expression product band (Fig. 3 A) that SDS-PAGE analyzes demonstration 70 kDa through centrifugal, and Western bloting test shows to have specificity to combine (Fig. 3 B) with Anti-Exendin-4 antibody.
Fermented supernatant fluid is crossed SP Sepharose FF post; The Exendin-4-HSA fused protein of catching; Pass through hydrophobic type chromatography column Butyl Sepharose FF again; Anion-exchange chromatography DEAE Sepharose FF separate fine purifying obtains purity and can reach the Exendin-4-HSA (Fig. 4) more than 98%.
Embodiment 7: the anti-sugar test of mouse
The C57BL/6 mouse, male and female half and half, fasting 18h, IP injection Exendin-4 (10 μ g/kg) or Exendin-4-HSA (expression and purification), Exendin-4-HSA (1mg/kg).Injectable dextrose monohydrate behind the 1h (1.5mg/kg, IP), behind the injectable dextrose monohydrate respectively at 10,20,30,60, the 120min tail vein blood, measure glucose content in the blood sample with blood glucose meter, observe the above-mentioned influence of treating test product to mouse blood sugar.The result shows that administration can reduce the fasting plasma glucose peak concentration effectively, makes blood sugar concentration curve tend to be steady (Fig. 5) simultaneously.
The promoting insulin secretion of embodiment 8:Exendin-4-HSA fusion rotein
Use the carbohydrate tolerance test model; C57BL/6J injected in mice Exendin-4-HSA fusion rotein has two dose groups of 200 μ g/kg and 1mg/kg, behind the administration 1.0h; Abdominal injection glucose 1.5mg/g; When 20min, get tail vein, adopt enzyme linked immunological adsorption method (ELISA, an anti-Anti-insulin antibody D6C4; Match two anti-Anti-insulin biotinlated antibody D3E7, by ABC Antibody.com supply) Regular Insulin in the mice serum is detected, observe Exendin-4-HSA and whether can promote insulin secretion effectively.Compare with control group (injecting normal saline), the insulin content of Exendin-4-HSA fusion rotein group all significantly raises, and with Exendin-4 similar effect (Fig. 6) is arranged.This shows that injection Exendin-4-HSA fusion rotein can promote insulin secretion effectively.
The long-effect active of embodiment 9:Exendin-4-HSA fusion rotein is observed
Give injected in mice Exendin-4-HSA (1.0mg/kg; IP); Then in 1,24,48,72,96 hour injectable dextrose monohydrate (IP); Behind the injectable dextrose monohydrate 0,10,20,30,60,120min time point blood sampling, measure glucose content in the blood sample with blood glucose meter, observe Exendin-4-HSA in of the influence of different injectable dextrose monohydrate time points to mouse blood sugar.HSA compares with negative control, the obvious lowering blood glucose peak value of Exendin-4-HSA, and the blood sugar concentration fluctuation is less, shows obvious lowering blood glucose effect, and has long-acting characteristic (Fig. 7).
Embodiment 10:Exendin-4-HSA fusion rotein is in the observation of diabetes model mouse drug effect
Diabetes model mouse BSK db/db is characterized by hyperglycemia (19-32 mmol/L, blood sugar are 2-3 times of normal mice), fat (the 40-56 gram/only; Body weight be normal mice 2-3 doubly), this experiment pass through injected in mice Exendin-4-HSA (1.0mg/kg, IP), per two days once; Totally 12 days, 30min after the observation administration, 60 min; 2h, 24h, 48h blood glucose value; 1,3,5,7,9,11 days drinking-water, diet, changes of weight.HSA compares with the negative control medicine, and Exendin-4-HSA obviously reduces diabetes model mouse BSK db/db glycemic peaks, has the effect of remarkable lowering blood glucose concentration, and has long-acting characteristic, and the result sees Fig. 8 A; Obviously reduce diabetes model mouse BSK db/db body weight, ingest and the amount of drinking water result sees Fig. 8 B, 8C, 8D.
Pharmacokinetics in the body of embodiment 11:Exendin-4-HSA fusion rotein
Adopt single dose intravenous injection Exendin-4-HSA fusion rotein 500 μ g/kg or subcutaneous injection 250 μ g/kg, 500 μ g/kg, basic, normal, high three dose groups of 750 μ g/kg to cynomolgus monkey.The pharmacokinetic parameter that the analysis of two chamber models (weight=1/C2) obtains shows that during intravenous injection Exendin-4-HSA fusion rotein (500 μ g/kg), the elimination transformation period is approximately 140.6 hours.Basic, normal, high three dose groups of subcutaneous injection Exendin-4-HSA fusion rotein are eliminated the transformation period and were respectively 98.8,100.1,102.8 hours, do not have between group significant difference (P＞0.05) (Fig. 9 A, 9B, 9C).Subcutaneous injection Tmax is 12.5-27.3h (Fig. 9 D).Dosage and AUC and Cmax are linear dependence.Clearance rate CLz/F is 1.0mL/h/kg.Bioavailability is 82.1%.Exendin-4-HSA fusion rotein biological activity assay result shows that the Exendin-4-HSA fusion rotein pharmaceutical activity of subcutaneous injection 500 μ g/kg is held time and reaches 72 ~ 96 hours in the blood sample of cynomolgus monkey.
<160> 7
<210> SEQ ID NO: 1
<211> 39
<212> PRT
<213> Exendin-4
<400> 1
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
5 10 15
Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly
20 25 30
Pro Ser Ser Gly Ala Pro Pro Pro Ser
35 39
<210> SEQ ID NO: 2
<211> 585
<212> PRT
<213> HSA
<400> 2
Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly
5 10 15
Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr
20 25 30
Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu
35 40 45
Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu
50 55 60
Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys
65 70 75
Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys
80 85 90
Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His
95 100 105
Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val
110 115 120
Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu
125 130 135
Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr
140 145 150
Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe
155 160 165
Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro
170 175 180
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys
185 190 195
Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala
200 205 210
Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys
215 220 225
Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys
230 235 240
Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp
245 250 255
Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser
260 265 270
Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu
275 280 285
Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala
290 295 300
Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val
305 310 315
Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe
320 325 330
Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu
335 340 345
Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys
350 355 360
Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp
365 370 375
Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln
380 385 390
Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn
395 400 405
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr
410 415 420
Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser
425 430 435
Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu
440 445 450
Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu
455 460 465
Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser
470 475 480
Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu
485 490 495
Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His
500 505 510
Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys
515 520 525
Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr
530 535 540
Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val
545 550 555
Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu
560 565 570
Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu
575 580 585
<210> SEQ ID NO: 3
<211> 117
<212> DNA
<213> Exendin-4
<400> 3
cacggtgaag gtactttcac ttctgatttg tctaagcaaa tggaagaaga agctgttaga 60
ttgttcattg aatggttgaa gaacggtggt ccatcttctg gtgctccacc accatct 117
<210> SEQ ID NO: 4
<211> 1758
<212> DNA
<213> HSA
<400> 4
gatgcacaca agagtgaggt tgctcatcga tttaaagatt tgggagaaga aaatttcaaa 60
gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120
aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtcagctgaa 180
aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240
cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300
tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360
gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420
gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480
tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540
aagctcgatg aacttcggga tgaagggaag gcttcgtctg ccaaacagag actcaagtgt 600
gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660
cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720
gtccacacgg aatgctgcca tggagatctg cttgaatgtg ctgatgacag ggcggacctt 780
gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840
aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900
gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960
gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020
tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080
tgtgccgctg cagatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140
gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200
tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260
ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gcagcaaatg ttgtaaacat 1320
cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380
tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440
ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg atgaaacata cgttcccaaa 1500
gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560
agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620
aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680
gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740
gctgccttag gcttataa 1758
<210> SEQ ID NO: 5
<211> 150
<212> DNA
<213> Exendin-4
<400> 5
taaggcctca cggtgaaggt actttcactt ctgatttgtc taagcaaatg gaagaagaag 60
ctgttagatt gttcattgaa tggttgaaga acggtggtcc atcttctggt gctccaccac 120
catct
gatgc acacaagagt gaggttgctc 150
<210> SEQ ID NO: 6
<211> 1891
<212> DNA
<213> Exendin-4-HSA
<400> 6
taaggcctca cggtgaaggt actttcactt ctgatttgtc taagcaaatg gaagaagaag 60
ctgttagatt gttcattgaa tggttgaaga acggtggtcc atcttctggt gctccaccac 120
catctgatgc acacaagagt gaggttgctc atcgatttaa agatttggga gaagaaaatt 180
tcaaagcctt ggtgttgatt gcctttgctc agtatcttca gcagtgtcca tttgaagatc 240
atgtaaaatt agtgaatgaa gtaactgaat ttgcaaaaac atgtgttgct gatgagtcag 300
ctgaaaattg tgacaaatca cttcataccc tttttggaga caaattatgc acagttgcaa 360
ctcttcgtga aacctatggt gaaatggctg actgctgtgc aaaacaagaa cctgagagaa 420
atgaatgctt cttgcaacac aaagatgaca acccaaacct cccccgattg gtgagaccag 480
aggttgatgt gatgtgcact gcttttcatg acaatgaaga gacatttttg aaaaaatact 540
tatatgaaat tgccagaaga catccttact tttatgcccc ggaactcctt ttctttgcta 600
aaaggtataa agctgctttt acagaatgtt gccaagctgc tgataaagct gcctgcctgt 660
tgccaaagct cgatgaactt cgggatgaag ggaaggcttc gtctgccaaa cagagactca 720
agtgtgccag tctccaaaaa tttggagaaa gagctttcaa agcatgggca gtagctcgcc 780
tgagccagag atttcccaaa gctgagtttg cagaagtttc caagttagtg acagatctta 840
ccaaagtcca cacggaatgc tgccatggag atctgcttga atgtgctgat gacagggcgg 900
accttgccaa gtatatctgt gaaaatcaag attcgatctc cagtaaactg aaggaatgct 960
gtgaaaaacc tctgttggaa aaatcccact gcattgccga agtggaaaat gatgagatgc 1020
ctgctgactt gccttcatta gctgctgatt ttgttgaaag taaggatgtt tgcaaaaact 1080
atgctgaggc aaaggatgtc ttcctgggca tgtttttgta tgaatatgca agaaggcatc 1140
ctgattactc tgtcgtgctg ctgctgagac ttgccaagac atatgaaacc actctagaga 1200
agtgctgtgc cgctgcagat cctcatgaat gctatgccaa agtgttcgat gaatttaaac 1260
ctcttgtgga agagcctcag aatttaatca aacaaaattg tgagcttttt gagcagcttg 1320
gagagtacaa attccagaat gcgctattag ttcgttacac caagaaagta ccccaagtgt 1380
caactccaac tcttgtagag gtctcaagaa acctaggaaa agtgggcagc aaatgttgta 1440
aacatcctga agcaaaaaga atgccctgtg cagaagacta tctatccgtg gtcctgaacc 1500
agttatgtgt gttgcatgag aaaacgccag taagtgacag agtcaccaaa tgctgcacag 1560
aatccttggt gaacaggcga ccatgctttt cagctctgga agtcgatgaa acatacgttc 1620
ccaaagagtt taatgctgaa acattcacct tccatgcaga tatatgcaca ctttctgaga 1680
aggagagaca aatcaagaaa caaactgcac ttgttgagct cgtgaaacac aagcccaagg 1740
caacaaaaga gcaactgaaa gctgttatgg atgatttcgc agcttttgta gagaagtgct 1800
gcaaggctga cgataaggag acctgctttg ccgaggaggg taaaaaactt gttgctgcaa 1860
gtcaagctgc cttaggctta taagctagct t 1891
<210> SEQ ID NO: 7
<211> 624
<212> PRT
<213> Exendin-4-HSA
<400> 7
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
5 10 15
Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly
20 25 30
Pro Ser Ser Gly Ala Pro Pro Pro Ser Asp Ala His Lys Ser Glu
35 40 45
Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala
50 55 60
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe
65 70 75
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys
80 85 90
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu
95 100 105
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg
110 115 120
Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
125 130 135
Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
140 145 150
Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala
155 160 165
Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu
170 175 180
Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe
185 190 195
Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala
200 205 210
Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg
215 220 225
Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala
230 235 240
Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val
245 250 255
Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val
260 265 270
Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys
275 280 285
His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala
290 295 300
Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys
305 310 315
Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala
320 325 330
Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala
335 340 345
Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu
350 355 360
Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
365 370 375
Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys
380 385 390
Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro
395 400 405
His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val
410 415 420
Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu
425 430 435
Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr
440 445 450
Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val
455 460 465
Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro
470 475 480
Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val
485 490 495
Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp
500 505 510
Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro
515 520 525
Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu
530 535 540
Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu
545 550 555
Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu
560 565 570
Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala
575 580 585
Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala
590 595 600
Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
605 610 615
Ala Ala Ser Gln Ala Ala Leu Gly Leu ***
620 624
Claims (6)
1. the fusion rotein Exendin-4-HSA of Exendin-4 peptide and human serum albumin HSA; It is characterized in that this fusion rotein contains 2 peptide zones; Its 1 district is an Exendin-4 polypeptide, and its 2 district is a human serum albumin HSA, and the terminal N-with the 2nd district through its C-in 1 district is terminal directly to be connected; The centre does not add any connection peptides, and its structural formula is: Exendin-4-HSA;
1 district Exendin-4 polypeptid acid sequence and sequence SEQ ID NO:1 maintain at least 90% homology;
2 district's polypeptide are selected from the mature peptide of human serum albumin HSA, and its aminoacid sequence and sequence SEQ ID NO:2 maintain at least 90% homology;
Described Exendin-4-HSA fusion rotein, it is nucleotide sequence coded to be SEQ ID NO:3;
Described Exendin-4-HSA fusion rotein, its aminoacid sequence are SEQ ID NO:7;
Described Exendin-4-HSA fusion rotein, its recombinant nucleotide sequence are inserted in a kind of expression vector and cell host system.
2. the preparation method of the said Exendin-4-HSA fusion rotein of claim 1 is characterized in that comprising and transcribes, translation, albumen sepn and purifying, and authentication step; Step is:
Through round pcr, reverse transcription obtains the nucleotide sequence of coding HSA peptide zone from the RNA in people's tire liver source, and makes 3 of HSA mature peptide gene ' end have the restriction enzyme site of Nhe1; The nucleotide sequence of coding Exendin-4 peptide zone obtains the Exendin-4 dna fragmentation through synthetic, and has the StuI restriction enzyme site at its 5 ' end, and makes 5 ' terminal sequence of its 3 ' end band top HSA mature peptide gene; Synthetic Exendin-4 gene fragment is mixed with HSA mature peptide gene fragment; Adopt overlapping PCR method to prepare Exendin-4-HSA antigen-4 fusion protein gene fragment; 5 of Exendin-4-HSA antigen-4 fusion protein gene ' end has the StuI restriction enzyme site, and 3 ' end has the restriction enzyme site of Nhe1; Exendin-4-HSA antigen-4 fusion protein gene fragment is carried out the TA clone, preparation recombinant plasmid Exendin-4-HSA/pEGM-T;
With Stu1, Nhe1 recombinant plasmid Exendin-4-HSA/pEGM-T is carried out double digestion, preparation Exendin-4-HSA gene fragment;
With method expression plasmid of yeast pWX530 is carried out double digestion, preparation linearization plasmid pWX530 dna fragmentation; With Exendin-4-HSA gene fragment and the linearization plasmid pWX530 dna fragmentation mixed of 3-5 ︰ 1 in molar ratio, connect with the T4 dna ligase, make up expression of recombinant yeast plasmid EX-HSA/pWX530; EX-HSA is the abbreviation of Exendin-4-HSA;
Recombinant plasmid EX-HSA/pWX530 can express in multiple yeast, and its preferred yeast is a yeast saccharomyces cerevisiae, and its preferred strain is CICIM Y0600.
3. the fermentation expression method of the said Exendin-4-HSA fusion rotein of claim 1; The single yeast CICIM Y0600 colony inoculation that it is characterized in that containing recombinant expression plasmid EX-HSA/pWX530 is in selective medium SD; 30 ℃ of joltings are spent the night, and calculate to obtain OD
600The total amount of=0.4 required overnight culture; Centrifugal collection bacterial sediment, and with selective medium SD suspension, and change and plant in fermention medium YPD, 48h are cultivated in 30 ℃ of joltings, centrifugal collection fermented supernatant fluid, and-80 ℃ of preservations are used for the analysis and the purifying of expressing fusion protein;
Selective medium SD is: 0.67%YNB, 0.609% disodium hydrogen phosphate dodecahydrate, 5.04% Citric acid C
6H
8O
7, the solution of 2% sucrose;
Fermention medium YPD is 1% yeast powder, 2% peptone, the solution of 2% sucrose.
4. the application of the said Exendin-4-HSA fusion rotein of claim 1 is characterized in that being used to prepare non-insulin-dependent type ii diabetes and obese person's treatment preparation; Administering mode is selected injection for use, and is oral, and in the nose, respiratory tract is inhaled clothes.
5. according to the application of the said Exendin-4-HSA fusion rotein of claim 4, it is characterized in that the Exendin-4-HSA fusion rotein can be processed different dosage forms in order to improve the medication effect, include but not limited to use the formed salt form of various soda acids.
6. according to the application of the said Exendin-4-HSA fusion rotein of claim 4, it is characterized in that the Exendin-4-HSA fusion rotein also can use with other drug jointly in order to improve the medication effect.
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| CN103204944A (en) * | 2013-03-26 | 2013-07-17 | 江苏健德生物药业有限公司 | Long-acting immune fusion protein for treating diabetes mellitus |
| CN103613670A (en) * | 2013-11-21 | 2014-03-05 | 中国人民解放军军事医学科学院生物工程研究所 | HSA (Human Serum Albumin) long-acting fusion protein and rapid assembly, separation and purification method thereof |
| US9670261B2 (en) | 2012-12-21 | 2017-06-06 | Sanofi | Functionalized exendin-4 derivatives |
| US9694053B2 (en) | 2013-12-13 | 2017-07-04 | Sanofi | Dual GLP-1/glucagon receptor agonists |
| US9750788B2 (en) | 2013-12-13 | 2017-09-05 | Sanofi | Non-acylated exendin-4 peptide analogues |
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| US9758561B2 (en) | 2014-04-07 | 2017-09-12 | Sanofi | Dual GLP-1/glucagon receptor agonists derived from exendin-4 |
| US9771406B2 (en) | 2014-04-07 | 2017-09-26 | Sanofi | Peptidic dual GLP-1/glucagon receptor agonists derived from exendin-4 |
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| US9932381B2 (en) | 2014-06-18 | 2018-04-03 | Sanofi | Exendin-4 derivatives as selective glucagon receptor agonists |
| US9982029B2 (en) | 2015-07-10 | 2018-05-29 | Sanofi | Exendin-4 derivatives as selective peptidic dual GLP-1/glucagon receptor agonists |
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