CN101875700A - Method for improving bioactivity of exendin fusion protein - Google Patents

Abstract

The invention discloses a method for improving the bioactivity of an exendin fusion protein, which belongs to the technical field of biopharmaceuticals. In the method, an Exendin-4 and GLP-1 are connected in series first and then are connected with human serum albumin HSA or human immunoglobulin Fc to form the fusion protein, so as to increase the bioactivity of the exendin fusion protein. The fusion protein is expressed as: (Exendin-4-GLP-1)n-HSA, (GLP-1-Exendin-4)n-HSA, (Exendin-4-GLP-1)n-Fc or (GLP-1-Exendin-1)n-Fc, wherein the value of n ranges from 1 to 5. The fusion protein prepared by the method has higher bioactivity than a fusion protein Exendin-4-HSA or a GLP-1-HSA protein. The fusion protein of the invention can be used for preparing medicaments for treating non-insulin-dependent diabetes, various symptoms of the non-insulin-dependent diabetes and obesity.

Description

The bioactive method of a kind of increase insulin secretion accelerating peptide fusion rotein

Technical field

The invention provides the bioactive method of a kind of increase insulin secretion accelerating peptide fusion rotein, relate to the preparation of the depot drug product for the treatment of non-insulin-dependent diabetes mellitus (NIDDM) and obesity, good prospects for application is arranged, belong to the biological medicine technology field at pharmaceutical field.

Background technology

1, diabetes

Along with the change of remarkable decline of modern humans's labour intensity and food habits, the sickness rate of diabetes rises rapidly, and according to the WHO recent statistics, there is diabetic subject 1.25 hundred million in the whole world, wherein is type ii diabetes more than 90%.Diabetes are divided into two kinds of type i diabetes and type ii diabetes usually.Type i diabetes accounts for 10% of diabetic sum, often betides children and teenager, and the cause of disease is the cell thoroughly damage that pancreas produces Regular Insulin in patient's body, thereby has lost the function that produces Regular Insulin fully.The patient need rely on the exogenous insulin survival, in case end insulinize then life-threatening.Type ii diabetes accounts for 90% of diabetic sum, and the age of onset majority is after 35 years old.Type ii diabetes is certain mechanism of non insulin dependent diabetes, does not still have final conclusion at present, but infers the influence may be subjected to heredity and intemperance of taking food, shortage motion, obesity, chemicals etc., the unbalance disease of a kind of internal secretion of Xing Chenging gradually.Main root is impaired in the β of pancreas islet cell, causes hypoinsulinism; Or pancreas islet acceptor obstacle and can not normally utilize Regular Insulin.Along with the fast development of Chinese national economy, living standards of the people improve constantly, and the rising of onset diabetes rate and patient's the phenomenon that becomes younger is more and more obvious.At the end of the seventies in last century, the morbidity of domestic diabetes is less than 1% also, and has reached at present more than 3%, and statistics in 2007 is more than existing 40,000,000 people of Chinese diabetic subject; According to the prediction of WHO latest data, will increase by 4 times to Chinese diabetics in 2010, and the development of diabetic population just is being tending towards becoming younger.Therefore preventing and treating diabetes has been a hot job too impatient to wait.

2, obesity

Obesity, particularly upper body obesity are the central modal nutrition disorders of crowd of overnutrition in the world.Studies confirm that in a large number, lose weight and to reduce the danger of chronic disease greatly, described chronic disease such as diabetes, hypertension, hyperlipidemia, coronary heart disease and flesh bone disease.Losing weight is objectives of many chronic disease treatments.Help slimming method all can not allow the people satisfied fully at present.Some obesity patient can lose weight by adjustment behavior painstakingly, as changing diet and increasing motion; By these methods can't slimming patient then may be owing to some inherited genetic factors, and they cause that appetite increases, and causes the metabolic tendency of preference high lipid food or steatogenesis.Therefore, be badly in need of helping slimming novel method and composition (as medicine) to replenish old method.

3, insulin secretion accelerating peptide-1

Insulin secretion accelerating peptide-1, also claim glucagon-like sample peptide-1 (Glucogan Like Peptide 1, abbreviation GLP-1) being one contains 37 amino acid whose small peptides, the mature peptide of GLP-1 is made up of 7-36 amino acid, has the function that promotes insulin secretion under the higher situation of blood sugar concentration.Therefore GLP-1 can not produce the hypoglycemia side effect when regulating hyperglycemia.Again can depress appetite because of GLP-1, slow down gastric secretion and stomach emptying (Nauck, 1993; Gutniak et al, 1992), so also can be with its medicine as management of body weight.But GLP-1 is degraded rapidly in human body, and the transformation period in serum has only 1-2 minute, therefore can't bring into play its biological function.Studies show that the derivative of some GLP-1 and analogue have better biological activity and stability.For example, the Exenatide/Exendin-4 of Amylin company is 2-3 hour the intravital transformation period the people, injects 2 every day and can improve plasma glucose levels, and body weight is also obviously reduced after 24 weeks simultaneously.But Exenatide/Exendin-4 also needs every day injects twice, still very inconvenient.The NN2211 (Liraglutide) of N Nordisk company is GLP-1 and connects 16-carbon fatty acid chain, this NN2211 in vivo with the plasma albumin combination, cause to surpass 10 hours plasma half-life, can realize injecting every day 1 time.

4, the plain secretion of long lasting insulinotropic peptide fusion protein matter medicine

By fusion, can produce the plain secretion of long lasting insulinotropic peptide fusion protein matter medicine with insulin secretion accelerating peptide and human serum albumin (HSA) or immunoglobulin Fc.Human serum albumin is difficult for seeing through renal glomerulus under the normal physiological state, transformation period in serum reaches 14-21 days, utilize genetic engineering technique, the protein molecular of human serum albumin and biologically active is carried out recombinant expressed fusion rotein, become an important means of the long-acting protein drug of exploitation.The Albugon of HGS company is the fusion rotein of GLP-1 and human serum albumin, and it can reach 3 days in the intravital transformation period of monkey.And for example Lilly company adopts gene engineering method that the Fc of GLP-1 and immunoglobulin (Ig) (IgG) is merged; Exendin-4 and human serum albumin that Canada ConjuChem company develops

Produce Exendin-4-HSA (CJC-1134-PC) in external combination.Though these fusion roteins can possess the pharmacological property that promotes the insulin secretion peptide, and prolong its transformation period in vivo, the biological activity of these fusion roteins only is the 1/10--1/40 (waiting mole number to compare) of nonfused monomer Exendin-4 or GLP-1.Adopt gene recombination technology among the present invention, to merge with the Fc of human serum albumin or immunoglobulin (Ig) (IgG) behind Exendin-4 and the GLP-1 series combination, on the basis of the pharmacological property that keeps Exendin-4, prolong its transformation period in vivo, has fusion rotein than the Fc of single Exendin-4 or GLP-1 and human serum albumin or immunoglobulin (Ig) (IgG) simultaneously (as Exendin-4-HSA, GLP-1-HSA, Exendin-4-Fc, GLP-1-Fc) have higher biological activity, comprise that at pharmaceutical field type ii diabetes and obesity treatment will have good prospects for application.

Summary of the invention

The objective of the invention is to develop a class and have highly active insulin secretion accelerating peptide fusion rotein, make it to become the medicine of a new generation's treatment diabetes and obesity.For achieving the above object, the invention provides a kind of insulin secretion accelerating peptide fusion rotein and preparation method thereof, increase the biological activity of this fusion rotein.

Technical scheme of the present invention: a kind of insulin secretion accelerating peptide fusion rotein, adopt Exendin-4 and GLP-1 series connection back to be connected to form fusion rotein with HSA or human normal immunoglobulin Fc, to increase the biological activity of insulin secretion accelerating peptide fusion rotein; This fusion rotein contains 2 peptide zones, wherein the first peptide zone sequence is made up of Exendin-4-GLP-1 or GLP-1-Exendin-4 tandem sequence, series system is C-terminal connection of terminal N-with Exendin-4 of C-end with N-end or the GLP-1 of GLP-1 of Exendin-4, above-mentioned tandem sequence can repeat n time successively with same direction, and repeat number n is 1-5; Preferential repeat number is 1.

Above-mentioned (Exendin-4-GLP-1) tandem sequence amino acid has the aminoacid sequence shown in the SEQ ID NO:1 or with this sequence 90% above homology is arranged; Described (GLP-1-Exendin-4) tandem sequence amino acid has the aminoacid sequence shown in the SEQ ID NO:2 or with this sequence 90% above homology is arranged.

Second peptide zone is selected from:

A) human serum albumin HSA has the aminoacid sequence shown in the SEQ ID NO:3 or with this sequence 90% above homology is arranged;

Or b) human normal immunoglobulin Fc has the aminoacid sequence shown in the SEQ ID NO:4 or with this sequence 90% above homology is arranged.

First peptide zone is by the N-end of its C-end with second peptide zone, and perhaps the C-of second peptide zone N-end terminal and first peptide zone is connected;

This fusion rotein is expressed as: (Exendin-4-GLP-1) n-HSA, (GLP-1-Exendin-4) n-HSA, (Exendin-4-GLP-1) n-Fc, (GLP-1-Exendin-4) n-Fc, the repeat number that on behalf of this tandem sequence, the n in the above-mentioned tandem sequence be arranged in order with same direction, repeat number n is 1-5, is preferably 1.

For example, concrete fusion rotein preparation process that is connected with HSA is can general description as follows:

Preparation process is:

(1) human serum albumin HSA gene clone obtains the HSA gene;

(2) Exendin-4-GLP-1 gene or GLP-1-Exendin-4 gene are synthetic;

(3) Exendin-4-GLP-1 and HSA gene fusion or GLP-1-Exendin-4 and HSA gene fusion;

(4) make up the expression vector of Exendin-4-GLP-1/HSA or the expression vector of GLP-1-Exendin-4/HSA;

(5) contain the structure of Exendin-4-GLP-1/HSA fusion rotein or GLP-1-Exendin-4/HSA expressing fusion protein engineering bacteria;

(6) Exendin-4-GLP-1/HSA or GLP-1-Exendin-4/HSA Expression of Fusion Protein and purifying;

(7) determination of activity of Exendin-4-GLP-1/HSA or GLP-1-Exendin-4/HSA fusion rotein.

Above-mentioned steps (1) to (7) has only been described a kind of preparation method of this fusion rotein specifically, can not be interpreted as to limit the scope of the invention.Having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.

Above-mentioned Exendin-4-GLP-1 tandem sequence amino acid has the aminoacid sequence shown in the SEQ ID NO:1 or with this sequence 90% above homology is arranged.

Above-mentioned GLP-1-Exendin-4 tandem sequence amino acid has the aminoacid sequence shown in the SEQ ID NO:2 or with this sequence 90% above homology is arranged.

The nucleotides sequence of described fusion rotein is classified aminoacid sequence corresponding nucleotide sequences or its complementary nucleotide sequence of fusion rotein as.

The present invention also provides a kind of carrier, and it contains top described nucleotide sequence, and a concrete expression vector is: Exendin-4-GLP-1/HSA-pPIC9K expression vector or GLP-1-Exendin-4/HSA-pPIC9K expression vector.

The present invention also provides a kind of host cell, and it comprises above-mentioned carrier.

Term in the present invention and methods involving

Term " protein ", " peptide " or " polypeptide " are used interchangeably.They refer to the two or more amino acid whose chain that links together by peptide bond or amido linkage no matter whether to pass through posttranslational modification (for example, glycosylation or phosphorylation).

Term " C-of first peptide zone terminal or be connected with the N-of second peptide zone is terminal " is meant that the connection between this two peptide species/protein molecule do not have any connection polypeptide, and this has been avoided the immunogenicity that may cause because of unnecessary connection peptides.Mode of connection can be that the C-end with first peptide zone directly is connected on the N-end of second peptide zone, also can be that the N-end of first peptide zone directly is connected on the C-end of second peptide zone.The C-end that preferred mode of connection is first peptide zone directly is connected the N-end of second peptide zone.

Term " single Exendin-4 or GLP-1 ".Be for " tandem sequence ", it refers to single Exendin-4 or GLP-1 polypeptide.Term " tandem sequence " refers to that each Exendin-4 monomer C-end directly is connected with the monomeric N-of GLP-1 is terminal, or the monomeric C-end of each GLP-1 directly is connected with the monomeric N-of Exendin-4 is terminal, tandem sequence can repeat successively with same direction, repeat number is 1-5, and preferential repeat number is 1.

In a preferred embodiment of the present invention, described fusion rotein of the present invention has and is selected from the aminoacid sequence shown in the aminoacid sequence SEQ ID NO:5;

The present invention provides nucleotide sequence on the other hand, and it has nucleotide sequence or its complementary sequence of the above-mentioned fusion rotein of coding.In addition, also contained in the scope of nucleotide sequence of the present invention and identical or substantially the same nucleotide sequence, if any the homology more than at least 90% even 95% or the nucleotide sequence of homogeny.

Nucleotide sequence of the present invention can obtain with the method for pcr amplification method, recombination method or synthetic usually.And with commercial cDNA storehouse or by the prepared cDNA of ordinary method well known by persons skilled in the art as template, amplification and obtain relevant sequence, and then the fragment that each time amplifies is stitched together by proper order, it is cloned into carrier, change cell again over to, from the host cell after the propagation, separate obtaining relevant sequence then by ordinary method.

Fusion rotein of the present invention prepares with recombination method.Specifically, coding Exendin-4-GLP-1 tandem sequence directly is connected with the nucleotide sequence of HSA polypeptide, form Exendin-4-GLP-1-HSA reorganization fusion gene, coding GLP-1-Exendin-4 tandem sequence directly is connected with the nucleotide sequence of HSA polypeptide, constitutes (GLP-1-Exendin-4) _n-HSA the fusion gene of recombinating; Adopt similar approach, coding Exendin-4-GLP-1 tandem sequence directly is connected with the nucleotide sequence of human normal immunoglobulin Fc polypeptide, constitute (Exendin-4-GLP-1) _n-Fc the fusion gene of recombinating, the GLP-1-Exendin-4 tandem sequence of maybe will encoding directly is connected with the nucleotide sequence of human normal immunoglobulin Fc polypeptide, constitutes (GLP-1-Exendin-4) _n-Fc the fusion gene of recombinating.The repeat number that on behalf of this tandem sequence, the n in the above-mentioned tandem sequence be arranged in order with same direction, repeat number n is 1-5, preferential repeat number is 1.

Then, above-mentioned recombinant nucleic acid sequence is cloned in the expression vector plasmid, the expression plasmid carrier of selecting for use comprises the various plasmids that are adapted at expressed fusion protein in the different cell hosts, for example adopts expression vector plasmid pPIC9K (Invitrogen), can obtain on market.

Then, above-mentioned expression plasmid is transformed in the recombined engineering cell host, thus the recombined engineering cell host of construction expression fusion rotein of the present invention.These recombined engineering cells can be to derive from animal, plant, insect, fungi, yeast, bacterium etc.Plasmid pPIC9K is adapted at expressed fusion protein in the Yeast engineering bacteria, and therefore preferred recombined engineering cell host is a Yeast engineering bacteria, for example adopts Pichia yeast engineering KM71.

Term used herein " conversion " is meant that will contain expression of nucleic acids carrier interested with method well known to those skilled in the art directly imports in the host cell.Method for transformation is different because of the host cell type, generally includes: electricity transforms; Adopt the transfection of calcium chloride, DEAE-dextran or other material; Microparticle bombardment; The fat transfection; Infect and other method (seeing people's such as Sambrook " molecular cloning experiment guide " the 2nd edition, 1989 years).Preferred methods is electric method for transformation.

Subsequently, under the condition that is fit to expressing fusion protein of the present invention, cultivate the host cell that transforms gained.Those skilled in the art just can select according to routine test and conditions such as definite substratum, culture temperature, time.Adopt this area conventional sense means, as SDS-PAGE, Western trace etc. can detect Expression of Fusion Protein of the present invention.At last, the separation and purification of protein technology of available routine is carried out the purifying of fusion rotein, and it comprises centrifugal, and precipitation is filtered means such as chromatography.Particularly, chromatography method comprises affine method again, gel-filtration, and ion-exchange, hydrophobic chromatography, and reverse chromatography etc.The separation purification method of fusion rotein of the present invention provided by the invention also comprises the appropriate combination of above-mentioned the whole bag of tricks.

The present invention also provides a kind of bioactive method of identifying the insulin secretion accelerating peptide fusion rotein.Particularly, adopt sub-HEK-293/GLP-1R cell, GLP-1 and agonist can induce this clone to produce cAMP, and cAMP generation and activity are proportionate.This method is to measure the ability that fusion rotein activates the GLP-1 acceptor, and compares with ability that Exendin-4 activates the GLP-1 acceptor, thereby calculates the fusion rotein biological activity.

Beneficial effect of the present invention: the invention provides a kind of method that produces fusion rotein of the present invention, this method comprises provides a kind of host cell, this host cell comprises expression vector, under the condition that is fit to protein expression, obtain fusion rotein by the host cell generation and through separating, this fusion rotein not only possesses the pharmacological property of insulin secretion accelerating peptide, prolong its transformation period in vivo, has the Exendin-4-HSA of ratio simultaneously, GLP-1-HSA, Exendin-4-Fc, the GLP-1-Fc fusion rotein has higher biological activity, comprises that at pharmaceutical field type ii diabetes and obesity treatment will have good prospects for application.

Description of drawings

Fig. 1 Exendin-4-GLP-1-HSA fusion protein expression plasmid construction strategy figure.

The SDS-PAGE electrophoresis result of Fig. 2 Exendin-4-GLP-1-HSA expressing fusion protein.1, protein molecular weight mark, 2,3,4 is Exendin-4-GLP-1-HSA expressing fusion protein supernatant liquor.

The Western trace result of Fig. 3 Exendin-4-GLP-1-HSA expressing fusion protein.1, protein molecular weight mark, 2, with the Western-blotting of anti-Exendin-4 antibody, 3, with the Western-blotting of anti-GLP-1 antibody, 4, with the Western-blotting of anti-HSA antibody.

Fig. 4 HEK-293/GLP-1R cell stimulates the artifact determination of activity through Exendin-4-GLP-1-HSA, Exendin-4-HSA, Exendin-4, HSA.Show that the Exendin-4-GLP-1-HSA fusion rotein has the biological activity that activates people GLP-1 acceptor, shows that its biological activity is higher than Exendin-4-HSA.

The carbohydrate tolerance test of Fig. 5 mouse.Show that the Exendin-4-GLP-1-HSA biological activity is higher than Exendin-4-HSA.

Embodiment

Be example with Exendin-4-GLP-1/HSA below, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Unless description is arranged in addition, enforcement of the present invention will be adopted molecular biology, microbiology, recombinant DNA and immunologic routine techniques, and these all are known to those skilled in the art.These technology have complete description in following document or other document of having published: for example, and Sambrook " molecular cloning experiment guide " the 2nd edition (1989); " protein purification: principle and put into practice " the 2nd edition (Springer-Verlag, N.Y.), and " experiment immunization is learned handbook I-IV volume (D.C.Weir and C.C.Blackwell edit 1986).Perhaps, can carry out according to the specification sheets that the reagent manufacturer is provided.

Embodiment 1: the human serum albumin HSA gene clone obtains the HSA gene

The human serum albumin HSA gene can obtain by RT-PCR.By being template, with PCR method amplification HSA gene with people's embryo cDNA.According to HSA gene order SEQ ID NO:5 design pcr amplification primer HSA primer 1 and HSA primer 2.Its sequence is as follows:

HSA primer 1:5 '-GATGCACACAAGAGTGAGGTTGCT-3 '

The HSA primer 2: 5 '- GCGGCCGCTTATAAGCCTAAGGCAGCTT-3 '.

The line part is NotI enzyme point of contact.

The PCR reaction system is: people's embryo cDNA: 1 μ L; HSA primer 1:50 μ M, 1 μ L; HSA primer 2: 50 μ M, 1 μ L; 10 * pfu polymerase buffer, 5 μ L; MgCl ₂: 25mM, 5 μ L; DNTP:2mM, 4 μ L; Pfu polysaccharase 1 μ L; DdH ₂O: supplying total reaction system is 50 μ L.

The PCR reaction conditions is: 94 ℃, 2min; Then, 94 ℃, 30sec; 55 ℃, 30sec; 72 ℃, 90sec carry out 30 circulations altogether.Agarose gel with 1% carries out gel electrophoresis to the PCR product, downcuts the target DNA band about 1800bp under ultraviolet lamp, and the PCR product glue with PROMEGA company reclaims test kit recovery HSA gene fragment again.After adopting determined by ultraviolet spectrophotometry to reclaim the concentration of product ,-20 ℃ of preservations.

Embodiment 2:Exendin-4-GLP-1 gene is synthetic

According to the requirement of SEQ ID NO:6,, and make its 3 ' end be with 5 ' terminal sequence of the preceding paragraph HSA gene by the gene order of the synthetic Exendin-4-GLP-1 of the handsome Bioisystech Co., Ltd in Shanghai.

Embodiment 3:Exendin-4-GLP-1 and HSA gene fusion

It is example that present embodiment forms the Exendin-4-GLP-1/HSA fusion gene with Exendin-4-GLP-1 gene and HSA gene fusion, and the cloning process of fusion gene is described.Present method also is fit to the clone that HSA gene and other gene form fusion gene.

According to SEQ ID NO:6, the synthetic primer of design:

EXT?1：5′-TT TACGTACACGGTGA?AGGTACTTTCACT-3′

The line part is SnaB1 enzyme point of contact.

The preparation of antigen-4 fusion protein gene uses the reaction method of Overlap PCR to obtain.Specific as follows.

The reaction system of Overlap PCR is: Exendin-4-GLP-1DNA:2 μ L; HSA DNA:2 μ L; 10 * pfu polymerase buffer, 5 μ L; The MgC1 of 25mM ₂5 μ L; The dNTP 4 μ L of 2mM; Pfu polysaccharase 1 μ L; DdH ₂It is 50 μ L that O supplies total reaction system.

The PCR condition is: beginning, 94 ℃, 2min; Then, 94 ℃, 30sec; 55 ℃, 30sec; 72 ℃, 2min; 5 circulations altogether.Then, add 50 μ M primer EXT1:2 μ L and 50 μ M HSA primer 2s: 2 μ L.Enter next program: 94 ℃, 30sec; 55 ℃, 30sec; 72 ℃, 2min; 30 circulations altogether.

With molecular biology method commonly used, separate obtaining fusion gene cloning then.The sepharose of employing 1% carries out electrophoresis to the PCR product, downcuts the DNA band of molecular weight about 1900bp from running gel, and the PCR product glue with PROMEGA company reclaims test kit recovery Exendin-4-GLP-1/HSA fusion gene fragment again.

The structure of embodiment 4:Exendin-4-GLP-1/HSA fusion protein expression vector Exendin-4-GLP-1/HSA-pPIC9K

Below be example with the Exendin-4-GLP-1/HSA fusion gene, the structure of expression vector is described.

It is expression plasmid that pPIC9K is adopted in this experiment, and its construction process is molecular biology method commonly used.That is, contain the bacterial classification of expression vector, extract and obtain this plasmid by cultivation.The plasmid for preparing-20 ℃ preservation is stand-by.

For the Exendin-4-GLP-1/HSA fusion gene is connected in the pPIC9K expression plasmid, with SnaB1 and Not1 restriction enzyme expression plasmid pPIC9K and Exendin-4-GLP-1/HSA fusion gene are carried out double digestion earlier.Actual conditions is as follows.

It is as follows that 37 ℃ of thermostat water bath internal reactions 3 hours, enzyme are cut system:

The enzyme of Exendin-4-GLP-1/HSA fusion gene is cut system: Exendin-4-GLP-1/HSA DNA:10 μ L; SnaB1:1 μ L; Not1:1 μ L; NEBuffer2:5 μ L; DdH ₂O supplies total reaction volume 50 μ L.

The enzyme of pPIC9K is cut system: expression plasmid pPIC9K DNA:10 μ L; SnaB1:1 μ L; Not1,1 μ L; NEBuffer2:5 μ L; DdH ₂O supplies total reaction volume 50 μ L.

With molecular biology method commonly used, separate obtaining then through the Exendin-4-GLP-1/HSA of double digestion fusion gene and through the pPIC9K of double digestion plasmid DNA.Specifically be with 1% sepharose enzyme to be cut product to carry out electrophoresis, downcut the purpose band, PCR product glue with PROMEGA company reclaims the test kit recovery through the Exendin-4-GLP-1/HSA of double digestion fusion gene fragment again, and pPIC9K plasmid DNA fragment.

Then, enzyme is cut product connect, obtain Exendin-4-GLP-1/HSA integrative gene expression vector Exendin-4-GLP-1/HSA-pPIC9K.Specific as follows: total linked system is 10 μ L, comprise T4DNA ligase enzyme damping fluid 1 μ L, the Exendin-4-GLP-1/HSA gene DNA of double digestion: 2 μ L, double digestion expression plasmid pPIC9K DNA:2 μ L, T4DNA ligase enzyme 1 μ L, sterilized water complements to 10 μ L.16 ℃ of water bath with thermostatic control incubated overnight.

Connect product transformed competence colibacillus DH5 α cell.Get a pipe competence DH5 α cell and add the connection product, place 30min on ice behind the soft mixing, place 2min 42 ℃ of water-baths again.Add 500 μ L LB liquid nutrient mediums and shake 40min at 37 ℃ of 180r/min.Get the LB solid medium that 100 μ L conversion fluids are applied to that resistance of card, be inverted for 37 ℃ and cultivated 12-16 hour.

The evaluation of positive colony is by the single transformant bacterium colony of picking, and the extracting plasmid carries out restriction analysis and sequencing result and determines.

Embodiment 5: the structure that contains Exendin-4-GLP-1/HSA expressing fusion protein engineering bacteria

Present embodiment utilizes electric method for transformation and fusion expression vector (Exendin-4-GLP-1/HSA-pPIC9K) to be example, the engineering bacteria of construction expression Exendin-4-GLP-1/HSA fusion rotein.Concrete grammar is as follows.

At first select the bacterium that contains the Exendin-4-GLP-1/HSA-pPIC9K expression vector and clone, extract expression vector dna, extracting method is molecular biology method commonly used.Then, the method for electricity consumption conversion changes plasmid over to pichia spp KM27.The electricity method for transformation is specific as follows: carry out the preparation of thalline earlier.The single bacterium colony of picking pichia spp KM27 is seeded in the 50mL triangular flask that contains the 5mLYPD substratum 30 ℃, 250-300r/min overnight incubation.The culture of getting 100-500 μ L then is seeded to the 2L triangle that contains the fresh YPD substratum of 500mL and shakes in the bottle, and 28～30 ℃, 250-300r/min overnight incubation are to OD ₆₀₀Reach 11.5.Again with cell culture in 4 ℃, the centrifugal 5min of 1500g, with the sterilized water of the ice precooling of 500mL that bacterial sediment is resuspended.After centrifugal, use the sterilized water of ice precooling of 250mL that bacterial sediment is resuspended again.Centrifugal again, the Sorbitol Solution USP of the 1mol of the ice precooling of usefulness 20mL is resuspended with bacterial sediment.Centrifugal again, the Sorbitol Solution USP of the 1mol of the ice precooling of usefulness 1mL is resuspended with bacterial sediment, and its final volume is about 1.5mL.

Transform with electric-shocking method then.Specific as follows: the DNA of 5～20 μ g expression plasmid Exendin-4-GLP-1/HSA-pPIC9K is dissolved in 5～10 μ L Tris damping fluids, and with the thalline mixing of the above-mentioned steps gained of 80 μ L, the electricity that goes to the precooling of 0.2cm ice transforms in the cup.Electricity is transformed cup ice bath 5min.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 1mL ice went to the thalline mixing in the Eppendorf pipe of 1.5mL, and the thalline suspension is coated on the sorbyl alcohol flat board, per 200～600 μ L coating one flat plate.Flat board is placed 30 ℃ of cultivations, occur until single bacterium colony.

Embodiment 6:Exendin-4-GLP-1/HSA Expression of Fusion Protein and purifying

The Exendin-4-GLP-1/HSA expressing fusion protein engineering bacteria that makes up among the embodiment 5, can be with following method expressed fusion protein: the BMGY substratum (peptone 2% that Exendin-4-GLP-1/HSA expressing fusion protein barms is inoculated in 500mL, yeast extract 1%, glycerine 1%, YNB 1.34%, and D-biotin 4 * 10 ^-5% transfers pH to 6 with the potassium phosphate buffer of 0.1M) in, 30 ℃ of shaking tables, 180r/min cultivated 48 hours.Then, in the fermentor tank of 5 liters of YP (peptone 2%, yeast extract 1%), autoclaving 30min, the cooling back adds the YNB 1.34% after the degerming, and D-biotin 4 * 10 ^-5% and methyl alcohol 1% is made into the BMMY substratum.Pre-incubated bacterium liquid is seeded among the BMMY, and the fermenting process temperature control is at 30 ℃, and greater than 20% saturation ratio, pH is controlled at about 5.5-6 dissolved oxygen all the time, and every 24h adds 1% methyl alcohol, continuously ferments 5 days.Utilize recombinant protein separation method commonly used, obtain above yeast expressed fusion protein primary extract.Particularly, can utilize methods such as centrifugal, ultrafiltration and concentration.Then, can utilize chromatography method, be further purified fusion rotein.Particularly, can adopt Sepharose SP XL chromatography column to be used for catching of fusion rotein, utilize Butyl Sepharose FF chromatography column again, and DEAE Sepharose FF chromatography column, carry out higher purifying.

Expressing fusion protein and purification result are identified by analysis of protein method commonly used, are included, but are not limited to SDS-PAGE, methods such as Western trace.Fig. 2 is the SDS-PAGE electrophoresis of Exendin-4-GLP-1/HSA fusion protein expression products, the result shows the protein band about 71KDa, Western trace method proves that this protein band has Exendin-4, GLP-1, the HSA antigenicity, the proof expressed protein is the Exendin-4-GLP-1/HSA fusion rotein, as shown in Figure 3.

The activity of embodiment 7:Exendin-4-GLP-1/HSA fusion rotein

Adopt HEK-293/GLP-1R cells in vitro measuring method that the HSA/GLP-1 fusion rotein is carried out determination of activity in the present embodiment, GLP-1 and agonist can induce this clone to produce cAMP, and cAMP generation and activity are proportionate.Specifically be to measure the ability that the Exendin-4-GLP-1/HSA fusion rotein activates the GLP-1 acceptor, and compare with ability that Exendin-4-HSA activates the GLP-1 acceptor.Concrete grammar is as follows: by 20,000 to 40, the amount of 000 cells/well (the shiny black base plate in 96 holes), inoculation HEK-293/GLP-1R cell, cultivate and use no blood plasma cultivation adds people's different concns respectively after 15-20 hour Exendin-4-GLP-1/HSA fusion rotein after 1 day instead, Exendin-4 or HSA, measure cAMP content (the cAMP-Screen system of AB company of every porocyte behind the cultivation 2h, ELISA Kit), the result shows that the Exendin-4-GLP-1/HSA fusion rotein has higher biological activity than Exendin-4-HSA fusion rotein, the results are shown in Figure 4.

Embodiment 8: the anti-sugar test of mouse

C57BL/6J mouse overnight fasting (15 hours) back is injected Exendin-4-GLP-1/HSA fusion rotein 0.2mg respectively by per kilogram mouse body weight intraperitoneal, or Exendin-4-HSA fusion rotein 0.2mg.In mouse peritoneal, press per kilogram mouse body weight injectable dextrose monohydrate 2mg/kg mouse body weight behind the 1h again, measure its glucose content at different time period blood samplings, the result shows that the Exendin-4-GLP-1/HSA fusion rotein has the effect of more obvious reduction mouse blood sugar than Exendin-4-HSA fusion rotein, as shown in Figure 5.

Although the invention describes concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.It is for referencial use that this paper is all included in all publications, patent and the patent application that this paper quotes in.

Sequence table

<210>SEQ?ID?NO：1

<211>69

<212>PRT

<213>Exendin-4-GLP-1

 

<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?His Gly?Glu?Gly?Thr?Phe

35 40 45

Thr?Ser?Asp?Val?Ser Ser?Tyr?Leu?Glu?Gly Gln?Ala?Ala?Lys?Glu

50 55 60

Phe?Ile?Ala?Trp?Leu Val?Lys?Gly?Arg

65 69

 

<210>SEQ?ID?NO：2

<211>69

<212>PRT

<213>GLP-1-exendin-4

 

<400>2

His?Gly?Glu?Gly?Thr Phe?Thr?Ser?Asp?Val Ser?Ser?Tyr?Leu?Glu

1 10 15

Gly?Gln?Ala?Ala?Lys Glu?Phe?Ile?Ala?Trp Leu?Val?Lys?Gly?Arg

20 25 30

His?Gly?Glu?Gly?Thr Phe?Thr?Ser?Asp?Leu Ser?Lys?Gln?Met?Glu

35 40 45

Glu?Glu?Ala?Val?Arg Leu?Phe?Ile?Glu?Trp Leu?Lys?Asn?Gly?Gly

50 55 60

Pro?Ser?Ser?Gly?Ala Pro?Pro?Pro?Ser

65 69

 

<210>SEQ?ID?NO：3

<211>585

<212>PRT

<213>HSA

<400>3

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

5 05 560

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：4

<211>223

<212>PRT

<213>IgG?Fc

 

<400>4

Thr?Cys?Pro?Pro?Cys Pro?Ala?Pro?Glu?Leu Leu?Gly?Gly?Pro?Ser

5 10 15

Val?Phe?Leu?Phe?Pro Pro?Lys?Pro?Lys?Asp Thr?Leu?Met?Ile?Ser

20 25 30

Arg?Thr?Pro?Glu?Val Thr?Cys?Val?Val?Val Asp?Val?Ser?His?Glu

35 40 45

Asp?Pro?Glu?Val?Lys Phe?Asn?Trp?Tyr?Val Asp?Gly?Val?Glu?Val

50 55 60

Hi?s?Asn?Ala?Lys?Thr Lys?Pro?Arg?Glu?Glu Gln?Tyr?Asn?Ser?Thr

65 70 75

Tyr?Arg?Val?Val?Ser Val?Leu?Thr?Val?Leu His?Gln?Asp?Trp?Leu

80 85 90

Asn?Gly?Lys?Glu?Tyr Lys?Cys?Lys?Val?Ser Asn?Lys?Ala?Leu?Pro

95 100 105

Ala?Pro?Ile?Glu?Lys Thr?Ile?Ser?Lys?Ala Lys?Gly?Gln?Pro?Arg

110 115 120

Glu?Pro?Gln?Val?Tyr Thr?Leu?Pro?Pro?Ser Arg?Asp?Glu?Leu?Thr

125 130 135

Lys?Asn?Gln?Val?Ser Leu?Thr?Cys?Leu?Val Lys?Gly?Phe?Tyr?Pro

140 145 150

Ser?Asp?Ile?Ala?Val Glu?Trp?Glu?Ser?Asn Gly?Gln?Pro?Glu?Asn

155 160 165

Asn?Tyr?Lys?Thr?Thr Pro?Pro?Val?Leu?Asp Ser?Asp?Gly?Pro?Phe

170 175 180

Phe?Leu?Tyr?Ser?Lys Leu?Thr?Val?Asp?Lys Ser?Arg?Trp?Gln?Gln

185 190 195

Gly?Asn?Val?Phe?Ser Cys?Ser?Val?Met?His Glu?Ala?Leu?His?Asn

200 205 210

His?Tyr?Thr?Gln?Lys Ser?Leu?Ser?Leu?Ser Pro?Gly?Lys

215 220 223

 

<210>SEQ?ID?NO：5

<211>1758

<212>DNA

<213>HSA

 

<400>5

gatgcacaca?agagtgaggt?tgctcatcga?tttaaagatt?tgggagaaga?aaatttcaaa 60

gccttggtgt?tgattgcctt?tgctcagtat?cttcagcagt?gtccatttga?agatcatgta 120

aaattagtga?atgaagtaac?tgaatttgca?aaaacatgtg?ttgctgatga?gtcagctgaa 180

aat1gtgaca?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：6

<211>207

<212>DNA

<213>Exendin-4-GLP-1

 

<400>6

cacggtgaag?gtactttcac?ttctgatttg?tctaagcaaa?tggaagaaga?agctgttaga 60

ttgttcattg?aatggttgaa?gaacggtggt?ccatcttctg?gtgctccacc?accatctcac 120

ggtgaaggta?ctttcacttc?tgatgtttct?tcttacttgg?aaggtcaagc?tgctaaggaa 180

ttcattgctt?ggttggttaa?gggtaga 207

Claims (6)

1. an insulin secretion accelerating peptide fusion rotein is characterized in that adopting Exendin-4 and GLP-1 series connection back to be connected to form fusion rotein with human serum albumin HSA or human normal immunoglobulin Fc, to increase the biological activity of insulin secretion accelerating peptide fusion rotein; This fusion rotein contains 2 peptide zones, wherein the first peptide zone sequence is made up of Exendin-4-GLP-1 or GLP-1-Exendin-4 tandem sequence, series system is C-terminal connection of terminal N-with Exendin-4 of C-end with N-end or the GLP-1 of GLP-1 of Exendin-4, above-mentioned tandem sequence repeats n time successively with same direction, and repeat number n is 1-5;

Second peptide zone is selected from: human serum albumin HSA, or human normal immunoglobulin Fc;

First peptide zone is by the N-end of its C-end with second peptide zone, and perhaps the C-of second peptide zone N-end terminal and first peptide zone is connected;

This fusion rotein is expressed as: (Exendin-4-GLP-1) n-HSA, and (GLP-1-Exendin-4) n-HSA, (Exendin-4-GLP-1) n-Fc, (GLP-1-Exendin-4) n-Fc, n are 1-5.

2. fusion rotein according to claim 1 is characterized in that Exendin-4-GLP-1 tandem sequence amino acid has the aminoacid sequence shown in the SEQ ID NO:1 or with this sequence 90% above homology arranged.

3. fusion rotein according to claim 1 is characterized in that GLP-1-Exendin-4 tandem sequence amino acid has the aminoacid sequence shown in the SEQ ID NO:2 or with this sequence 90% above homology arranged.

4. fusion rotein according to claim 1 is characterized in that second peptide zone is selected from:

A) human serum albumin HSA has the aminoacid sequence shown in the SEQ ID NO:3 or with this sequence 90% above homology is arranged;

Or b) human normal immunoglobulin Fc has the aminoacid sequence shown in the SEQ ID NO:4 or with this sequence 90% above homology is arranged.

5. fusion rotein according to claim 1 is characterized in that the nucleotides sequence of described fusion rotein classifies aminoacid sequence corresponding nucleotide sequences or its complementary nucleotide sequence of fusion rotein as.

6. the preparation method of the described insulin secretion accelerating peptide fusion rotein of claim 1 is characterized in that preparation process is:

(1) human serum albumin HSA gene clone obtains the HSA gene;

(2) Exendin-4-GLP-1 gene or GLP-1-Exendin-4 gene are synthetic;

(3) Exendin-4-GLP-1 and HSA gene fusion or GLP-1-Exendin-4 and HSA gene fusion;

(4) make up the expression vector of Exendin-4-GLP-1/HSA or the expression vector of GLP-1-Exendin-4/HSA;

(5) contain the structure of Exendin-4-GLP-1/HSA fusion rotein or GLP-1-Exendin-4/HSA expressing fusion protein engineering bacteria;

(6) Exendin-4-GLP-1/HSA or GLP-1-Exendin-4/HSA Expression of Fusion Protein and purifying;

(7) determination of activity of Exendin-4-GLP-1/HSA or GLP-1-Exendin-4/HSA fusion rotein.

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