CN104293834B

CN104293834B - GLP 1 or its analog and antibody Fc fragment fusion protein preparation method

Info

Publication number: CN104293834B
Application number: CN201410531801.0A
Authority: CN
Inventors: 许必雄; 郭颀然; 赵红阳
Original assignee: SYNDEGEN (SHANGHAI) BIOTECHNOLOGY CO Ltd
Current assignee: SYNDEGEN (SHANGHAI) BIOTECHNOLOGY CO Ltd
Priority date: 2014-10-11
Filing date: 2014-10-11
Publication date: 2018-03-23
Anticipated expiration: 2034-10-11
Also published as: CN104293834A

Abstract

The present invention relates to biological technical field, more particularly to a kind of GLP 1 or its analog and antibody Fc fragment fusion protein preparation method.The present invention provides the preparation method of a kind of GLP 1 or its analog and antibody Fc fragment fusion protein, comprises the following steps：GLP 1 or its analog are cloned into expression vector with antibody Fc fragment fusion protein coded sequence；Expression vector is transfected into CHO DXB11 cells, cultivates and filters out positive cell strain；Gained cell is expressed, after purification, produces the fusion protein.Preparation method provided by the present invention expresses the Fc molecules of GLP 1 in Chinese hamster ovary celI system DXB11, will not produce degradation problem, and follow-up purification yield greatly improves, and biological activity does not weaken, and is especially suitable for technique industry production requirement.

Description

GLP-1 or its analog and antibody Fc fragment fusion protein preparation method

Technical field

The present invention relates to biological technical field, is merged more particularly to a kind of GLP-1 or its analog with antibody Fc fragment The preparation method of albumen.

Background technology

Glucagon-like-peptide-1 (GLP-1) can promote insulin releasing, reduce the Plasma Glucagon Level in blood plasma, drop The speed of low gastric emptying, promote satietion and stimulate the biosynthesis of pancreas islet and the propagation of β cells, available for diabetes B Treatment.In China, diabetes B morbidity is extensive, if treated not in time, is readily converted into type 1 diabetes, to patient and Country causes very big burden.Therefore, the demand for developing this kind of medicine is extremely urgent.The Exenatide of Lilly Co., Eli. at present The Liraglutide (Nuo Heli) of (hundred secrete reach) and Novo Nordisk drugmaker of Denmark obtains import registration, two kinds of medicines in China Thing all has the function that hypoglycemic and lost weight, but needs daily 1-2 subcutaneous administrations.

For increased Half-life in vivo, make GLP-1 long-actingization so as to reduce unit interval administration number of times and have become state The focus of inside and outside research.The method of the GLP-1 studied at present and the like long-actingization mainly has following three kinds of modes：(1) PEGylation It is long-acting, be represented as Nove Nordisk companies (Novo Nordisk Co., Ltd of Denmark) research and development weekly hypodermic extended GLP-1- 1 analog Suo Malu peptides；(2) GLP-1 and albumin fusion protein, it is represented as patent CN10665538A；(3) GLP-1 and antibody Fc fragment fusion proteins, be represented as patent CN1802386B.The GLP-1-Fc (LY2189265) of Li Lai companies is currently beautiful State's three phases clinical stage, according to the data report of clinical trial, LY2189265 can be effectively increased GLP-1 half-life period, realize Weekly administration once, improves the compliance of patient's treatment.

Recombinant human glucagon-like peptide-1 (GLP-1) analog and the antibody Fc with the effect of prolonged human Half-life in vivo The fusion protein is applied to human clinical trial's energy by the fusion protein (abbreviation GLP-1-Fc) of fragment composition, external drugmaker Lost weight while enough effective treatment diabetes Bs.

With the development of antibody technique, it in CHO-S or CHO-K1 cells is original that the antibody class medicines of many listings, which is all, Engineering cell is built on the basis of cell, uses the method for concentration cultivation to realize that antibody target protein exists in industrial production High expression in final cell cultivation liquid.Because antibody molecule has the characteristics of molecular structure stabilized, concentration cultivation process In the protease that is discharged due to part engineering cell apoptosis very little is influenceed on antibody target protein.Because GLP-1-Fc is merged There is part of the antibody Fc fragment as molecule in albumen, its molecule has the characteristic of some antibody, many people think that CHO-S Or it is most convenient and rational selection that CHO-K1 cells should also be as structure engineering cell on the basis of initial cell.But Some information of this technical field obtain, and different degrees of degraded occurs in cells express stage for the fusion protein, is degraded GLP-1-Fc later separation purifying prepare in be very difficult to remove, not only having influence on the biological activity of product can also have a strong impact on Product prepare yield, its reason the GLP-1 parts of N-terminal most critical in GLP-1-Fc fusion proteins 31 amino acid very It is unstable, the hydrolysis of the protease discharged after easily being cracked by Apoptosis and degrade.Qiao-Zhen Lu et al. are in text Offer Characterization of the Proteases Involved in the N-Terminal Clipping of Mentioned in Glucagon-Like-Peptide-1-Antibody Fusion Proteins, GLP-1-Fc is in CHO-S cell lines The imperfect form more than 50%, that is, band of degrading at most occurs in middle expression.Due to the caused N- in cell fermentation Hold the GLP-1-Fc fusion proteins for occurring degrading effectively to be removed in the manufacturing process of final medicine, cause qualified finished product Yield between 10%-50%.

Mentioned in same piece article and protease inhibitors (benzamidine hcl) control degraded added in cell cultivation process, But risk is also the increase in the patent medicine of product, in follow-up technique protease inhibitors is inactivated and eradicated and also can So that the preparation technology of whole product is more complicated and uneconomical, and addition protease inhibitors can only reduce GLP-1-Fc points The degree of sub- N- ends degraded, it is impossible to effectively suppress its generation, it is impossible to really solve degradation problem.Another kind removes GLP-1-Fc points The method of sub- N- ends catabolite is that the purifying of multi-step is carried out using a variety of column chromatographies, although can also obtain very pure GLP-1-Fc fusion proteins, but the yield of overall process is greatly reduced to less than 20%, and it is big to be significantly less than therapeutic antibodies medicine Yield in 70% is horizontal.

The content of the invention

In view of the above the shortcomings that prior art, it is an object of the invention to provide a kind of GLP-1 or its analog with The preparation method of antibody Fc fragment fusion protein, for solving the problems of the prior art.Inventor is by GLP-1-Fc Expression plasmid DNA is imported in different mammal cell line, is had surprisingly found that expressed in Chinese hamster ovary celI system DXB11 GLP-1-Fc fusion proteins occur without degraded, so that the yield of qualified finished product is more than 70%.

In order to achieve the above objects and other related objects, first aspect present invention provide a kind of GLP-1 or its analog with The preparation method of antibody Fc fragment fusion protein, comprises the following steps：

1) GLP-1 or its analog are cloned into expression with antibody Fc fragment fusion protein (GLP-1-Fc) coded sequence to carry In body；

2) expression vector is transfected into CHO-DXB11 cells, cultivates and filter out positive cell strain；

3) expressed, after purification using step 2 gained cell, produce the fusion protein.

Preferably, the GLP-1 or its analog and antibody Fc fragment fusion protein by restructuring human glucagon-like-peptide- 1 (GLP-1) or its analog to be formed by connecting peptide with having the antibody Fc fragment that prolonged human Half-life in vivo acts on to be connected.

It is furthermore preferred that the amino acid sequence of the GLP-1 or its analog and SEQ ID No.14 (GLP-1 prototype sequences) With more than 80% homology, it is furthermore preferred that with more than 90% homology, more preferably with more than 97% Homology.

It is furthermore preferred that the particular sequence of the GLP-1 or its analog such as SEQ ID No.14, SEQ ID No：1 or SEQ ID No：Shown in 2.

It is furthermore preferred that the amino acid quantity of the connection peptide is >=2, the connection peptide is by glycine (Gly) and silk ammonia Sour (Ser) is combined.

It is furthermore preferred that the amino acid quantity of the connection peptide is 6-36.

It is furthermore preferred that the amino acid quantity of the connection peptide is 14-36.

It is furthermore preferred that the amino acid sequence such as SEQ ID No of the connection peptide：Shown in 3-8.

It is further preferred that the amino acid sequence such as SEQ ID No of the connection peptide：Shown in 7.

It is furthermore preferred that the antibody Fc fragment is the non-cracking performance Fc for not having ADCC effector functions of this area.

It is further preferred that the amino acid sequence of the antibody Fc fragment such as SEQ ID No：Shown in 9.

In a preferred embodiment of the invention, the amino of the GLP-1 or its analog and antibody Fc fragment fusion protein Acid sequence such as SEQ ID No：10 and SEQ ID No：Shown in 11；The GLP-1 or its analog merge egg with antibody Fc fragment White coded sequence such as SEQ ID No：12 and SEQ ID No：Shown in 13.

Preferably, the expression vector is selected from the expression vector of DHFR systems and/or the expression vector of GS systems.

It is further preferred that the expression vector of the DHFR systems is selected from pIRES-DHFR, the expression of the GS systems carries Body is selected from pee12.4.

Preferably, it is described by GLP-1 or its analog and antibody Fc fragment fusion protein (GLP-1-Fc) code sequence lek The grand method in expression vector is specially：In GLP-1 or its analog and the two of antibody Fc fragment fusion protein coded sequence End design and the restriction enzyme site corresponding to the multiple cloning sites of carrier, then by restriction enzyme site corresponding on coded sequence and carrier Connection, realizes the connection of target protein coding DNA fragment and expression vector.

It is further preferred that described encode GLP-1 or its analog with antibody Fc fragment fusion protein (GLP-1-Fc) The method that sequence is cloned into expression vector is specially：By the coded sequence both ends of GLP-1-Fc fusion proteins devise NheI and Two restriction enzyme sites of MluI, are connected with NheI the and MluI restriction enzyme sites on expression vector, realize target protein coding DNA respectively The connection of fragment and expression vector.

Preferably, it is described that expression vector is transfected into CHO-DXB11 cells, cultivate and filter out the method tool of positive cell strain Body is：Using DNA transfection reagents by plasmid DNA transfection CHO-DXB11 cells, by determining the table of fusion protein after Secondary Culture Up to amount, cell line is formed so as to filter out positive cell.

It is further preferred that the specific method of the expression quantity of the measure fusion protein is：For GLP1 point trace, pin Western blotting Western blots to GLP1 and one kind or more in being detected for the Elisa methods of Fc fragments The combination of kind, so as to determine the expression quantity of fusion protein.

Preferably, the method for the cell expression is the cell expression suitable for CHO-DXB11 cells.

It is furthermore preferred that the method for the cell expression is specially：The positive cell strain filtered out is subjected to serum-free domestication, Then shaking table culture in shaking flask is seeded to, takes supernatant to carry out protein purification gained medium centrifugal.

More there is the step of choosing, the shaking table culture to be preferably：36.5-37.5 DEG C of temperature, 4.8-5.2% CO₂Environment Middle shaking table culture to density reaches 3.5-4.5*10⁶, temperature is reduced to 31.5-32.5 DEG C, nutriment is supplemented daily, after 5-6 days Cell viability is reduced to 80-85% and stops culture.

More there is choosing, described the step of taking supernatant to carry out protein purification gained medium centrifugal is preferably, by gained After cell is taken out in nutrient solution 1000-3000RCF centrifugations, then 4000-6000RCF centrifuging and takings supernatant carries out protein purification.

Preferably, the purifying is purified specifically by membrane filtration, affinity column and gel permeation chromatography.

Second aspect of the present invention provides purposes of the CHO-DXB11 cell lines in GLP-1 albumen preparation fields.

Preferably, the purposes is specially the purposes in GLP-1-Fc fusion protein preparation fields.

Third aspect present invention provides a kind of fusion protein, is merged by the GLP-1 or its analog with antibody Fc fragment The preparation method of albumen prepares.

Fourth aspect present invention provides a kind of pharmaceutical composition, including the GLP-1 of effective dose or its analog with Antibody Fc fragment fusion protein.

Preferably, the composition may also include the carrier of this area, diluent, excipient, stabilizer, thickener etc., And such as tablet, capsule, powder, syrup, solution or the various medicines well-known to those skilled in the art of suspension can be prepared to Agent type.

Fifth aspect present invention provides a kind of CHO-DXB11 host cells of restructuring, the host cell contain GLP-1 or The expression vector of its analog and antibody Fc fragment fusion protein, the expression vector are specially to include GLP-1 or its analog With the expression vector of antibody Fc fragment fusion protein coded sequence.

Inventor is directed to current GLP-1/mTf, the GLP-1-Fc particularly blended with antibody Fc fragment Expression of the molecule in zooblast generally occurs that (degradation fragment goes out the trend of the times for situation about being degraded by albumen enzyme effect Very big influence must be brought to isolating and purifying for downstream, the yield for reducing the quality of product and preparing, by adding protease Inhibitor can partly suppress, but can not be fully solved the problem of GLP-1-Fc molecules are degraded in cell cultivation process； The protease inhibitors of addition needs effectively to remove in subsequent purification process, adds the difficulty of purifying), there is provided a kind of GLP- 1 or the preparation method of its analog and antibody Fc fragment fusion protein.Preparation method provided by the present invention is in Chinese hamster ovary celI system GLP-1-Fc molecules are expressed in DXB11, degradation problem will not be produced, follow-up purification yield greatly improves, and biological activity is simultaneously Do not weaken, be especially suitable for technique industry production requirement.

Brief description of the drawings

Fig. 1 is shown as present invention process flow chart.

Embodiment

Inventor imports GLP-1-Fc expression plasmids DNA in different mammal cell lines, surprised hair Expressed GLP-1-Fc fusion proteins occur without degraded in present Chinese hamster ovary celI system DXB11, so that the yield of qualified finished product More than 70%, the present invention is completed on this basis.

The present invention provides the preparation method of a kind of GLP-1 or its analog and antibody Fc fragment fusion protein, including as follows Step：

GLP-1 provided by the present invention or its analog with the preparation method of antibody Fc fragment fusion protein, GLP-1 or Its analog is passed through with antibody Fc fragment fusion protein by recombinant human glucagon-like peptide-1 (GLP-1) or its analog to be connected Peptide is connected to be formed with the antibody Fc fragment with the effect of prolonged human Half-life in vivo.

In GLP-1 or its analog and antibody Fc fragment fusion protein, GLP-1 analog can be restructuring GLP-1, The amino acid sequence of GLP-1 or its analog has more than 80% homology with SEQ ID No.14 (GLP-1 prototype sequences), It is furthermore preferred that with more than 90% homology, more preferably with more than 97% homology.It is similar for GLP-1 Thing (be such as intended to improve GLP-1 molecule performances so as to GLP-1 molecules increase its biological activity, reduce its immunogenicity, Deng the GLP-1 analogs that are obtained of any modification) with for antibody Fc fragment fusion protein, when GLP-1 or its analog Amino acid sequence and prototype GLP-1 homology >=80% (being preferably >=90%, more preferably >=97%), can be The problem of stable expression is obtained in DXB-11 initial cells, and is degraded without the hydrolysis of worry protease, so that Very high yield is also ensured while increasing biology performance.Specifically, because GLP-1 protein products are degraded, it is main former Because 31 amino acid of the GLP-1 parts for coming from N-terminal most critical are very unstable, the egg discharged after easily being cracked by Apoptosis The hydrolysis of white enzyme and degrade, so when modify prototype GLP-1 molecules after, can cause GLP-1 molecules in theory The probability being degraded reduces, and the probability that its reason is to form new degradation site after modifying is low-down.The specific such as U.S. The GLP-1-Fc products Dulaglutide of Li Lai companies has put forward the stability of GLP-1-Fc products just by molecular modification GLP-1 biological activity is not influenceed while liter also.So should be understood that to those skilled in the art, When GLP-1-Fc fusion proteins provided by the present invention preparation method can stablize expression with GLP-1 prototype sequences and with Prototype sequence has more than 97% homology sequence (SEQ ID No：1), there is more than 90% homology sequence (SEQ ID No：2) during GLP-1-Fc, the preparation method can stablize other GLP-1 of expression with suitable or lower slightly homology and modify The fusion protein of fragment is expected.

In a preferred embodiment, the amino acid sequence of the GLP-1 or its analog such as SEQ ID No.14, Shown in SEQ ID No.1 or SEQ ID No.2.

In GLP-1 or its analog with antibody Fc fragment fusion protein, connecting the various connections that peptide can be this area Peptide, as long as not producing limitation to the goal of the invention of the present invention.Preferably, the connection peptide is by glycine (Gly) and silk ammonia Sour (Ser) is combined, and the connection peptide is not Gly and Ser any combination, but generally uses with G4S (i.e. amino acid sequences Arrange GGGGS) to be realized for more units connection of unit, the design principle of such connection peptide has been the known skill of those skilled in the art Art.It for details, reference can be made to the prior arts such as CN 102875683B, CN 102875683B, application number 201110193210.3.Connection The amino acid quantity of peptide is >=2, more preferably 6-36, more preferably 14-36.

In a preferred embodiment, the amino acid sequence such as SEQ ID No of peptide are connected：Shown in 3-8.

In an optimum embodiment of the invention, the amino acid sequence such as SEQ ID No of peptide are connected：Shown in 7.

In GLP-1 or its analog and antibody Fc fragment fusion protein, antibody Fc fragment does not have ADCC for this area The non-cracking performance Fc of effector function, as long as not producing limitation to the goal of the invention of the present invention.Due to GLP-1 or its change The Fc fragments that structure body is connected by flexible peptide linker derive from the constant region of Immunoglobulin IgG, and it exempts from eliminating pathogen Played an important role in epidemic disease defence.In four kinds of human IgG hypotypes, IgG1 and IgG2, IgG3, IgG4 can be with GLP-1 or its changes Structure body is combined by flexible peptide linker.Fc is merged primarily to increase inside it circulating half-life and reduce production cost, And " Antibody -dependent cell cytotoxicity effect " (antibody-dependent cell-mediated of Fc mediations Cytotoxicity, ADCC) function is unnecessary, in some instances it may even be possible to produce harmful side effect.In order to not had ADCC effects The non-cracking performance Fc of subfunction, inventor are carried out to the IgG4 of people three sites of Fc fragments (P01861_IGHG4) Modification, first modification is S228P of the Fc amino acid sequences according to EU numbering systems, and this modification can consolidate interchain disulfide bond Structure is so as to having the function that stable dimeric structure；Second modification is the L235E of EU numbering systems, and this modification thoroughly eliminates Fc fragment Fc R combination, so as to which ADCC be preferably minimized.3rd modification is the L445P of EU numbering systems, and this modification makes IgG4 Fc c terminal amino acid sequence is identical with IgG1 and IgG2 Fc c terminal amino acid sequence, so as to increase C-terminal amino acid The homogeneity of sequence.Fc sequence designations after we this modification are " IgG4 (S228P, L235E, L445P) ", after wherein IgG4 Amino acid in the bracket of face is numbered according to EU.

In a preferred embodiment, the amino acid sequence of antibody Fc fragment such as SEQ ID No：Shown in 9.

In a preferred embodiment, the amino of the GLP-1 or its analog and antibody Fc fragment fusion protein Acid sequence such as SEQ ID No：10 and SEQ ID No：Shown in 11；The GLP-1 or its analog merge egg with antibody Fc fragment White coded sequence such as SEQ ID No：12 and SEQ ID No：Shown in 13.

GLP-1 provided by the present invention or its analog are with the preparation method of antibody Fc fragment fusion protein, expressing and carrying Body may be selected from the carrier system that this area is adapted to the expression of CHO-DXB11 cells, as long as not producing limit to the goal of the invention of the present invention System.Preferably, the expression vector is selected from the expression vector of DHFR systems and/or the expression vector of GS systems.

In a preferred embodiment, the expression of the expression vector of DHFR systems such as pIRES-DHFR, GS system carries Body such as pee12.4.

In the preparation method of GLP-1 provided by the present invention or its analog and antibody Fc fragment fusion protein, by GLP-1 Or its analog and antibody Fc fragment fusion protein (GLP-1-Fc) are as long as coded sequence is cloned into method in expression vector not Limitation is produced to the goal of the invention of the present invention.Specifically, can be merged in GLP-1 or its analog with antibody Fc fragment Restriction enzyme site of the both ends design of albumen coded sequence corresponding to the multiple cloning sites of carrier, then by coded sequence and carrier Corresponding restriction enzyme site connection, realizes the connection of target protein coding DNA fragment and expression vector.

In a preferred embodiment, by GLP-1 or its analog and antibody Fc fragment fusion protein (GLP-1- Fc) method that coded sequence is cloned into expression vector is specially：The coded sequence both ends of GLP-1-Fc fusion proteins are designed NheI and MluI two restriction enzyme sites, are connected with NheI the and MluI restriction enzyme sites on expression vector, realize target egg respectively The connection of white coding DNA fragment and expression vector.

GLP-1 provided by the present invention or its analog are with the preparation method of antibody Fc fragment fusion protein, will express Carrier transfects CHO-DXB11 cells, as long as the method cultivated and filter out positive cell strain is not produced to the goal of the invention of the present invention Raw limitation.

In a preferred embodiment, expression vector is transfected into CHO-DXB11 cells, cultivates and filter out positive thin The method of born of the same parents' strain is specially：Using DNA transfection reagents by plasmid DNA transfection CHO-DXB11 cells, Secondary Culture is after about 3 weeks, By determining the expression quantity of fusion protein, cell line is formed so as to filter out positive cell.

The expression quantity specific method by determining fusion protein is：Pass through point trace, the western for GLP1 Blotting Western blots and Elisa methods for Fc fragments are detected, so as to determine the expression quantity of fusion protein.This Outside, by the western blotting Western blots for GLP1, the correct expression of GLP1 albumen is also confirmed.

In the preparation method of GLP-1 provided by the present invention or its analog and antibody Fc fragment fusion protein, cell table As long as the method reached does not produce limitation to the goal of the invention of the present invention, specifically, this area can be selected and be applied to CHO- The cell expression of DXB11 cells.Preferably, the method for the cell expression is specially：The positive cell strain that will be filtered out Serum-free domestication is carried out, is then seeded to shaking table culture in shaking flask, takes supernatant to carry out protein purification gained medium centrifugal.

The step of shaking table culture is preferably：36.5-37.5 DEG C of temperature, 4.8-5.2% CO₂Shaking table culture in environment Reach 3.5-4.5*10 to density⁶, temperature is reduced to 31.5-32.5 DEG C, supplements nutriment daily, Cell viability drops after 5-6 days As little as 80-85% stops culture.

Described the step of taking supernatant to carry out protein purification gained medium centrifugal, is preferably, by gained nutrient solution After cell is taken out in 1000-3000RCF centrifugations, then 4000-6000RCF centrifuging and takings supernatant carries out protein purification.

In a preferred embodiment, the method for cell expression is specially：The positive cell strain filtered out is carried out Serum-free is tamed, with 2*10⁵Density is passaged in 1L shaking flasks, 37 DEG C of temperature, 5% CO₂In environment, 120RPM shaking table cultures are extremely Density reaches 4*10⁶, temperature is reduced to 32 DEG C, supplements the nutriment such as glucose, amino acid daily, Cell viability drops after 5-6 days As little as 80-85% stops culture；Cell liquid 2000RCF is centrifuged after taking out cell, then 5000RCF centrifuging and takings supernatant carries out albumen Purifying.

In the preparation method of GLP-1 provided by the present invention or its analog and antibody Fc fragment fusion protein, for melting The purification process of hop protein does not have concrete restriction, as long as not producing limitation to the goal of the invention of the present invention.Fusion protein Purification process be the state of the art, the purifying of fusion protein is mainly using the method purifying of chromatography, such as make With anion-exchange chromatography or cation-exchange chromatography or using gel permeation chromatography, or using hydrophobic chromatography, reversed phase chromatography, go back It can be chromatographed using hydroxylapatite adsorption, metal chelate chromatography etc., those skilled in the art can be to above-mentioned all purification steps Appropriate selection and combination are carried out, purity of protein is reached substantially uniform.In addition, utilize the spy containing the fusion protein The affinity column of heterogenetic antibody, acceptor or the part such as rProteinA, the nProteinA natural rProteinG that recombinate, The MabSelectSure of nProteinG or improved alkali resistants is purified to the fusion protein of expression can also be used to purify The fusion protein.

In a preferred embodiment, purify specifically by membrane filtration, affinity column and gel permeation chromatography Purified.

The present invention also provides purposes of the CHO-DXB11 cell lines in GLP-1 albumen preparation fields, and the purposes is preferred For in the purposes of GLP-1-Fc fusion protein preparation fields, GLP-1- is expressed specifically by CHO-DXB11 cell lines cell line Fc fusion proteins.

The GLP-1-Fc fusion proteins are GLP-1 or its analog and antibody Fc fragment fusion protein.

The amino acid sequence of the GLP-1 or its analog have 80% with SEQ ID No.14 (GLP-1 prototype sequences) Homology above, it is furthermore preferred that with more than 90% homology, more preferably with more than 97% homology.

The present invention also provides a kind of fusion protein, by the GLP-1 or its analog and antibody Fc fragment fusion protein Preparation method prepares.

The present invention also provides a kind of pharmaceutical composition, includes the GLP-1 or its analog and antibody Fc of effective dose Fragment fusion protein.The composition may also include the carrier that those skilled in the art commonly use, diluent, excipient, stabilizer, Thickener etc., and such as tablet, capsule, powder, syrup, solution or the medicament class of the various this areas of suspension can be prepared to Type.

The present invention further provides a kind of CHO-DXB11 host cells of restructuring, the host cell contain GLP-1 or its The expression vector of analog and antibody Fc fragment fusion protein, the expression vector be specially comprising GLP-1 or its analog with The expression vector of antibody Fc fragment fusion protein coded sequence.

Illustrate embodiments of the present invention below by way of specific instantiation, those skilled in the art can be by this specification Disclosed content understands other advantages and effect of the present invention easily.The present invention can also pass through specific realities different in addition The mode of applying is embodied or practiced, the various details in this specification can also be based on different viewpoints with application, without departing from Various modifications or alterations are carried out under the spirit of the present invention.

Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless in text Explicitly point out in addition, singulative "one", " one " and " this " include plural form.

When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range Any one numerical value can be selected between point and two end points.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except used in embodiment specific method, equipment, Outside material, according to grasp of the those skilled in the art to prior art and the record of the present invention, it can also use and this Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.

Unless otherwise indicated, disclosed in this invention experimental method, detection method, preparation method using this technology lead Domain conventional molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..

Embodiment 1

The coded sequence of recombined human GLP-1-Fc fusion proteins

1.GLP-1 Amino acid profile

Natural GLP-1 and its any GLP-1 molecules for changing structure can be by different flexible peptide linkers and different Fc Fragment, such as IgG1, IgG2, IgG3, IgG4 and change structure etc. connection form fusion protein, these fusion proteins have biology work Property and long-term effect.

Natural GLP-1 amino acid sequence is as follows：

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG(SEQ ID No：14).

The fusion protein molecule of the present invention is the variant that modification was carried out on original shape GLP-1 molecular basises, is specially The variant of two kinds of different modifyings, its title and molecular structure have been carried out on the basis of 31 amino acid of original shape GLP-1 molecules Respectively：

Molecular name：G1 (have in the GLP-1 molecules of 31 amino acid 1 amino acid modified)

Modification：Gly8-GLP-1 (7-37), original shape GLP-1 first amino acid number be 7 (the 7th), then the 8th Amino acid (actually corresponding to second amino acid) is changed to Gly.Particular sequence such as SEQ ID No：Shown in 1：

HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG(SEQ ID No：1)

Molecular name：G3 (have in the GLP-1 molecules of 31 amino acid 3 amino acid modified)

Modification：Gly8-Glu22-Gly36-GLP-1 (7-37), original shape GLP-1 first amino acid number are 7, then the The amino acid of 8,22,36 is changed to Gly, Glu, Gly respectively.Particular sequence such as SEQ ID No：Shown in 2：

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG(SEQ ID No：2)

2. flexible peptide linker sequence

Peptide ammino acid number is connected herein from 2 to 31, predominantly glycine (Gly) and mutual group of serine (Ser) Close.

Flexible peptide linker sequence containing 6 amino acid is GSGGGS (SEQ ID No：3), it is named as " L1 "；

Flexible peptide linker sequence containing 11 amino acid is GSGGGSGGGGS (SEQ ID No：4), it is named as " L2 "；

Flexible peptide linker sequence containing 16 amino acid is GSGGGSGGGGSGGGGS (SEQ ID No：5), it is named as “L3”；

Flexible peptide linker sequence containing 21 amino acid is GSGGGSGGGGSGGGGSGGGGS (SEQ ID No：6), order Entitled " L4 "；

Flexible peptide linker sequence containing 26 amino acid is GSGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID No： 7), it is named as " L5 "；

Flexible peptide linker sequence containing 31 amino acid is GSGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID No：8), it is named as " L6 ".

Flexible peptide linker L5 is preferred scheme.Experiment shows that the GLP-1-Fc expressed with this flexible peptide linker detects biology Active highest.

3.IgG4 Fc fragments

The IgG4 of people three sites of Fc fragments (P01861_IGHG4) are modified, first modification is Fc amino Acid sequence is according to the S228P of EU numbering systems, and second modification is the L235E of EU numbering systems, and the 3rd modification is EU numberings The L445P of system.Fc sequence designations after our this modification are " IgG4 (S228P, L235E, L445P) ", and particular sequence is such as SEQ ID No：Shown in 9：

SKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID No：9)

4. the amino acid sequence of two kinds of fusion proteins

Two kinds of Argine Monohydrochlorides that two kinds of different GLP-1 Isoforms sequences are formed with flexible peptide linker and IgG4-Fc Sequence is respectively：

Name：G1-L5-Fc；

Structure：G1-L5-IgG4 (S228P, L235E, L445P), particular sequence such as SEQ ID No：Shown in 10：

HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGGGSGGGGSGGGGSGGGGSGGGGSSKYGPPCPPCPAPEFEGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No：10)

Name：G3-L5-Fc；

Structure：G3-L5-IgG4 (S228P, L235E, L445P), particular sequence such as SEQ ID No：Shown in 11：

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGSGGGSGGGGSGGGGSGGGGSGGGGSSKYGPPCPPCPAPEFEGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No：11)

Embodiment 2

GLP-1-Fc fusion proteins are expressed in CHO-DXB11 and CHO-DG44 cells：

Two kinds of albumen (G1- that two kinds of different GLP-1 Isoforms sequences and flexible peptide linker and IgG4-Fc are formed L5-Fc and G3-L5-Fc) coded sequence SEQ ID No：12 and SEQ ID No：13, which are cloned into the expression containing IRES-DHFR, carries In body sequence, expression vector expresses GLP-1-Fc under the driving of CMV promoter.

G1-L5-Fc coded sequence such as SEQ ID No：Shown in 12：(signal peptide containing 19aa)

atggagtggtcctgggtgttcctgttctttctgtccgtgaccacaggagtccacagccatggtgaagggacctttac cagtgatgtaagttcttatttggaaggacaagctgccaaggagttcattgcttggctggtgaaaggccgtggaggat ccggtggcggttccggcggtggaggatcaggaggcggtggctctggaggtggtggaagtggtggcggcggttcgtct aagtacgggcccccttgccctccttgcccagctcctgaatttgagggcggacccagcgtgttcctgttccccccaaa gcccaaggacaccctgatgatcagcagaacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccg aggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaac agcacctacagagtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggt gtccaacaagggcctgcccagcagcatcgaaaagaccatcagcaaggccaagggccagcctcgcgagccccaggtgt acacactgcctccaagccaggaagagatgaccaagaaccaggtgtccctgacctgtctcgtgaagggcttctacccc tccgacatcgccgtggaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacag cgacggctcattcttcctgtacagcagactgaccgtggacaagagcagatggcaggaaggcaacgtgttcagctgca gcgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgagccccggcaaa(SEQ ID No： 12)

G3-L5-Fc coded sequence such as SEQ ID No：Shown in 13：

atggagtggtcctgggtgttcctgttctttctgtccgtgaccacaggagtccacagccatggtgaagggacctttac cagtgatgtaagttcttatttggaagagcaagctgccaaggagttcattgcttggctggtgaaaggcggtggaggat ccggtggcggttccggcggtggaggatcaggaggcggtggctctggaggtggtggaagtggtggcggcggttcgtct aagtacgggcccccttgccctccttgcccagctcctgaatttgagggcggacccagcgtgttcctgttccccccaaa gcccaaggacaccctgatgatcagcagaacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccg aggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaac agcacctacagagtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggt gtccaacaagggcctgcccagcagcatcgaaaagaccatcagcaaggccaagggccagcctcgcgagccccaggtgt acacactgcctccaagccaggaagagatgaccaagaaccaggtgtccctgacctgtctcgtgaagggcttctacccc tccgacatcgccgtggaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacag cgacggctcattcttcctgtacagcagactgaccgtggacaagagcagatggcaggaaggcaacgtgttcagctgca gcgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgagccccggcaaa(SEQ ID No： 13)

By coded sequence (the SEQ ID No of GLP-1-Fc fusion proteins：12 and SEQ ID No：13) both ends devise Two restriction enzyme sites of NheI and MluI, are connected with NheI the and MluI restriction enzyme sites on expression vector, realize target protein respectively The connection of coding DNA fragment and expression vector, obtain DNA.

Transfect CHO-DXB11 and CHO-DG44 cells to carry out in 6 orifice plates, CHO-DXB11 blanc cells are with 1：10 are passaged to 6 In orifice plate, with containing 10% hyclone (FBS) and 1XHT (HT Supplement, HT culture medium additives, the production of gibco companies Product) DMEM culture mediums cultivated.Transfection assay is carried out when cell confluency reaches 90%.Cell culture is removed first Liquid, to each fresh 10%FBS+1X HT of 2 milliliters of Kong Lijia DMEM nutrient solutions.14 g plasmid DNA are diluted to 150 microlitres DMEM culture mediums, 12 microlitres of Lipofectamine reagent dilutions to 150 microlitres of DMEM culture mediums, by DNA and It is stored at room temperature 20 minutes, is then uniformly added into the cell culture fluid of 6 orifice plates, Ran Houfang after the mixing of Lipofectamine reagents Enter 37 degree of cell culture incubators to be cultivated, be 300 microlitres per hole addition.Remove nutrient solution after 6 hours, change into 2 milliliters it is fresh FBS+HT culture mediums.After 24 hours, suspended with after trypsin digestion cell with fresh FBS+HT culture mediums, with 1：5 are diluted to two Continue to cultivate in T25 square vases.Culture continues to cultivate after 24 hours with the DMEM culture mediums of the 10%FBS without HT, is carried out per 2-3 days Liquid is once changed, positive cell forms cell line after about 3 weeks, now carries out detecting for the ELISA of Fc fragments.ELISA is detected Specific method is：It is 1~10 μ g/ that anti-human IgG antibody is diluted into antibody concentration with 0.05M PH9 carbonate coating buffer solution Ml (antibody buying Sigma companies, article No. 18885)；Add 0.1ml in the reacting hole of each 96 orifice plate, 4 DEG C overnight；Next day, discard Solution in hole, washed 3 times with lavation buffer solution PBST, 3 minutes every time (referred to as washing, similarly hereinafter)；Add the measuring samples necessarily diluted 0.1ml puts 37 DEG C and is incubated 1 hour, be washed out in the above-mentioned reacting hole being coated with；Washing is faced after addition in each reacting hole When tmb substrate solution (it is ShiJi Co., Ltd to purchase in health) 0.1ml for preparing, 37 DEG C 10~30 minutes, most after in each reacting hole Add 2M sulfuric acid 0.05ml terminating reactions, ELIASA reading OD 450nm value (taking OD value >=0.15 to continue to pass on).Use simultaneously Dilution method is by passage into 96 orifice plates, and after 2-3 weeks, monoclonal can be picked out from 96 plates, pass in T25 and pressurize Culture.Pressurizeed and cultivated with the 10%FBS of the MTX containing 100nM DMEM nutrient solutions first, about 2 pericytes adapt to 100nM's After MTX, cell cryopreservation, then proceed to continue to cultivate with 250nM, 500nM, 1000nM MTX pressure, at the same with point a trace, WB (western blotting) and Elisa methods for Fc fragments are screened.The point marking and WB primary antibody select anti-GLP1 Monoclonal antibody (Sigma buyings), secondary antibody select with horseradish peroxidase mark goat anti-mouse monoclonal antibody (green skies company adopts Purchase), same as above for the Elisa methods of Fc fragments, specific experiment step and method also refer to Molecular Cloning: A Laboratory and referred to South, finally pick out 3-6 strains cell and carry out serum-free domestication.

The cell line for expressing different GLP-1-Fc fusion proteins can adapt in serum free medium after suspension culture, with 2*10⁵Density is passaged in 1L shaking flasks, temperature 37C, 5% CO₂In environment, 120RPM shaking table cultures to density reach 4*10⁶, Temperature is reduced to 32C, supplements the nutriment such as glucose, amino acid daily, Cell viability is reduced to 80-85% stoppings after 5-6 days Culture.Cell liquid 2000RCF is centrifuged after taking out cell, then 5000RCF centrifuging and takings supernatant carries out protein purification.

Embodiment 3

GLP-1-Fc fusion proteins are expressed in CHO-S and CHO-K1 cells：

Two kinds of albumen (G1- that two kinds of different GLP-1 Isoforms sequences and flexible peptide linker and IgG4-Fc are formed L5-Fc and G3-L5-Fc) coded sequence SEQ ID No：12 and SEQ ID No：13 are cloned into containing glutamine synthelase In the pee12.4 expression vectors of GS expression systems, expression vector expresses GLP-1-Fc under the driving of CMV promoter.

Cloning process is same as Example 2, i.e. the coded sequence both ends of GLP-1-Fc fusion proteins devise NheI and Two restriction enzyme sites of MluI, are connected with NheI the and MluI restriction enzyme sites on expression vector, realize target protein coding DNA respectively The connection of fragment and expression vector.

CHO-S and DMEMs of the CHO-K1 containing 10% hyclone medium culture, cell was carried out in 1 day before transfection Rear overhang overstatement number is digested, cell is diluted to 1*10⁵Individual/ml, 6 orifice plates are transferred to, cultivate to the 2nd day degree of converging and reach 60%, Transfected.A liquid：DMEM250 microlitres of serum-free is used respectively, dilutes 1,2,4 g plasmid DNA；B liquid：Use serum-free DMEM250 microlitres, 10 microlitres of Lipofectamine2000 reagents are added, room temperature is placed 5 minutes.A liquid is mixed with B liquid, room temperature After placing 20 minutes, add in 6 orifice plates, be put into incubator and cultivated.Nutrient solution is discarded after 6 hours, 2 milliliters of addition is fresh The DMEM nutrient solutions of 10% hyclone.After continuing culture 48 hours, digestion suspension hole inner cell, using (800 is micro- containing G418 Gram every milliliter) DMEM complete mediums suspend after move in culture dish and cultivate, change liquid once within every three days.It is positive after about 3 weeks Cell forms cell line, now carries out the Elisa detections (specific method is with embodiment 2) for Fc fragments, while use dilution method By passage into 96 orifice plates, after 2-3 weeks, monoclonal can be picked out from 96 plates, pass on and continue to train into 12 orifice plates Support, detect the content (specific method is with embodiment 2) of Fc in cell culture fluid, so as to pick out the cell line of high expression, finally Pick out 3-6 strains cell and carry out serum-free domestication.

Embodiment 4

GLP-1-Fc fusion proteins isolate and purify：

(GLP-1-Fc expression quantity is about for cell culture fluid containing GLP-1-Fc fusion proteins (G1-L5-Fc and G3-L5-Fc) 1mg/ml) 2L, 0.22 micron of nitrocellulose filter in-depth filtration, 10kD molecular cut off pvdf membrane flat board cross-flows be concentrated by ultrafiltration to The rProteinA FF affinity columns purifying of 200ml, GE company, 50mM citrate buffer solutions pH3.3 elutions, often collect 10ml and wash De- liquid addition 1ml 1M Tris pH8.5 are neutralized.Collect eluent to purify by GE companies Sephacryl S-200HR, obtain Final sample, product quality are respectively：G1-L1-IgG4200mg、G1-L2-IgG4200mg、G1-L3-IgG4200mg、G1- L4-IgG4200mg、G1-L5-IgG4200mg、G1-L6-IgG4200mg、G3-LY-IgG4200mg.Carry out N-terminal sequencing and Analysis detection, N-terminal sequencing result show G1-L5-Fc and G3-L5-Fc in CHO-K1, CHO-S, CHO-DG44, CHO-DXB11 The equal frame of the fusion protein of expression is errorless.

Embodiment 5

GLP-1-Fc degraded bands and multimer analysis：

1.SDS-Page electrophoresis：

The SDS-Page running gels of 15v/v% acrylamide concentrations are prepared, dilute GLP- with the sample buffer of 5 times of concentrations (5 times of concentration SDS sample buffers compositions are 60mM Tris-HCl pH6.8 to 1-Fc samples, 25% glycerine, 2%SDS, 14.4mM 2 mercapto ethanol, 0.1% bromophenol blue, the aqueous solution；Electrophoresis Sample uses 40 microlitres of 5 times of concentrating sample buffer solutions and 10 microlitres of samples Product are sufficiently mixed), sample is boiled 5 minutes in boiling water, is centrifuged after cooling.Take supernatant loading (10-30 microlitres of loading) after centrifuging, control Constant pressure 150V processed terminates electrophoresis in about 80 minutes, and coomassie brilliant blue staining is observed after terminating electrophoresis.

2. molecular exclusion method (SEC-HPLC)

Molecular exclusion uses TSK3000-SW-XL (5 microns, 7.8*300 millimeters) high pressure liquid phase analysis of eastern Cao Da companies Post, analysis mobile phase are PBS, pH7.4,10% acetonitrile buffer, and (sample uses the preparation method of embodiment 4 to sample, and sample is dense Degree control is in 0.5mg/ml -1mg/ml, and sample is dissolved in 20mM citric acids, 150mM sodium chloride, in the pH6-7 aqueous solution) enter Sample flow velocity 0.5ml/ minutes, Detection wavelength 214nm.

3. rp-hplc method (RP-HPLC) (sample uses the preparation method of embodiment 4)

Reversed-phase HPLC is analyzed using Zorbax 300SB-C8 (4.6*50 millimeters) analytical column, and A phases mobile phase is 0.1% (v/ V) TFA buffer solutions (aqueous solution), B phases mobile phase are 100% acetonitrile, 0.085% (v/v) TFA buffer solutions, run and are existed with Gradient Detected under 214nm Detection wavelengths.The graded of mobile phase is as shown in table 1.

Table 1

Time	%B phases
		0	30
5	30
		35	45
40	90
		45	90
50	25
		65	25

Four kinds of different cell line CHO-S, CHO-K1, CHO-DG44, the GLP-1-Fc samples after CHO-DXB11 expression and purifications Purity has carried out analysis detection (having used three kinds of different determination methods), and concrete outcome is shown in Table 2：

Table 2

Being detected by different detection methods proves, GLP-1-Fc obtains having on product quality after expressing in different cell lines Very big difference, with identical purification process after purification, CHO-DXB11 cell lines expression GLP-1-Fc purity reaches after purification More than 96%, other cell lines all can be with the presence of substantial amounts of degraded band, can be big if be removed using other means of purification The yield of whole production is reduced greatly, can be also affected even if the specific activity of product so after purification.

Embodiment 6

Extracorporeal receptor GLP-1 activation experiments：

External activity detection (specific detection method reference is carried out using the mankind GLP-1 acceptor HEK293 cells of overexpression Wolfgang Glaesner；Engineering and characterization of the long-acting glucagon-like peptide-1analogue LY2189265,an Fc fusion protein；Diabetes Metab Res Rev 2010；26:287–296.；In method), using after GLP-1-Fc, the activation of these cells GLP-1 acceptors is drawn The activation of adenyl cyclase is played, the activation of the enzyme induces the expression of the reporter gene by the driving of ring Amp response elements again.We It is to cause the concentration value of 50% ceiling effect to characterize GLP-1-Fc activity by EC50 values.We to CHO-S, CHO-K1, GLP-1-Fc samples after CHO-DG44, CHO-DXB11 expression and purification have carried out Activity determination, and discovery contains a large amount of catabolites GLP-1-Fc activity be minimum, and possess very high biological activity in the GLP-1-Fc of CHO-DXB11 expression, specifically It the results are shown in Table 3：

Table 3

From table 3, fusion protein GLP-1-Fc prepared by the present invention has biological activity, this biological activity Referring to that GLP-1-Fc is combined and activated GLP-1 acceptors and cause the ability of reaction in vivo, this reacts the secretion for including insulin, Suppression of hyperglycemic factor etc..

Two kinds of not homotactic DNA (the GLP-1-Fc molecules of two kinds of gene configurations) have imported into several by inventor In the different Chinese hamster ovary celI system of kind and its hypotype, such as CHO-S, CHO-K1, CHO-DG44, CHO-DXB11, and made egg respectively White expression and purification.Further the albumen that two kinds of different genes sequence tables reach in different cell lines is being purified and analyzed Afterwards, comparative evaluation is carried out to the content of target protein in cell culture fluid and its catabolite, cytology activity.By evaluation result It can be seen that GLP-1-Fc fusion proteins are substantially better than other cell lines in Chinese hamster ovary celI system DXB11, unexpected skill is achieved Art effect.

Embodiment 7

G1-L1-Fc amino acid such as SEQ ID No：Shown in 15：

MEWSWVFLFFLSVTTGVHSHGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGGGSSKYGPPCPPCPAPEFEGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No：15)

Coded sequence such as SEQ ID No：Shown in 21：

atggagtggtcctgggtgttcctgttctttctgtccgtgaccacaggagtccacagccatggtgaagggacctttac cagtgatgtaagttcttatttggaaggacaagctgccaaggagttcattgcttggctggtgaaaggccgtggaggat ccggtggcggttcctctaagtacgggcccccttgccctccttgcccagctcctgaatttgagggcggacccagcgtg ttcctgttccccccaaagcccaaggacaccctgatgatcagcagaacccccgaagtgacctgcgtggtggtggacgt gtcccaggaagatcccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagccca gagaggaacagttcaacagcacctacagagtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaa gagtacaagtgcaaggtgtccaacaagggcctgcccagcagcatcgaaaagaccatcagcaaggccaagggccagcc tcgcgagccccaggtgtacacactgcctccaagccaggaagagatgaccaagaaccaggtgtccctgacctgtctcg tgaagggcttctacccctccgacatcgccgtggaatgggagagcaacggccagcccgagaacaactacaagaccacc ccccctgtgctggacagcgacggctcattcttcctgtacagcagactgaccgtggacaagagcagatggcaggaagg caacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgagccccg gcaaa(SEQ ID No：21)

G1-L2-Fc amino acid sequence such as SEQ ID No：Shown in 16：

MEWSWVFLFFLSVTTGVHSHGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGGGSGGGGSSKYGPPCPPCPAPEFE GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No：16)

Coded sequence such as SEQ ID No：Shown in 22：

atggagtggtcctgggtgttcctgttctttctgtccgtgaccacaggagtccacagccatggtgaagggacctttac cagtgatgtaagttcttatttggaaggacaagctgccaaggagttcattgcttggctggtgaaaggccgtggaggat ccggtggcggttccggtggaggcggaagctctaagtacgggcccccttgccctccttgcccagctcctgaatttgag ggcggacccagcgtgttcctgttccccccaaagcccaaggacaccctgatgatcagcagaacccccgaagtgacctg cgtggtggtggacgtgtcccaggaagatcccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacg ccaagaccaagcccagagaggaacagttcaacagcacctacagagtggtgtccgtgctgaccgtgctgcaccaggat tggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcccagcagcatcgaaaagaccatcagcaa ggccaagggccagcctcgcgagccccaggtgtacacactgcctccaagccaggaagagatgaccaagaaccaggtgt ccctgacctgtctcgtgaagggcttctacccctccgacatcgccgtggaatgggagagcaacggccagcccgagaac aactacaagaccaccccccctgtgctggacagcgacggctcattcttcctgtacagcagactgaccgtggacaagag cagatggcaggaaggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagtccc tgagcctgagccccggcaaa(SEQ ID No：22)

G1-L3-Fc amino acid sequence such as SEQ ID No：Shown in 17：

MEWSWVFLFFLSVTTGVHSHGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGGGSGGGGSGGGGSSKYGPPCPPCP APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No：17)

Coded sequence such as SEQ ID No：Shown in 23：

atggagtggtcctgggtgttcctgttctttctgtccgtgaccacaggagtccacagccatggtgaagggacctttac cagtgatgtaagttcttatttggaaggacaagctgccaaggagttcattgcttggctggtgaaaggccgtggaggat ccggtggcggttccggtggaggcggaagcggcggtggaggatcatctaagtacgggcccccttgccctccttgccca gctcctgaatttgagggcggacccagcgtgttcctgttccccccaaagcccaaggacaccctgatgatcagcagaac ccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtgcagttcaattggtacgtggacggcg tggaagtgcacaacgccaagaccaagcccagagaggaacagttcaacagcacctacagagtggtgtccgtgctgacc gtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcccagcagcatcga aaagaccatcagcaaggccaagggccagcctcgcgagccccaggtgtacacactgcctccaagccaggaagagatga ccaagaaccaggtgtccctgacctgtctcgtgaagggcttctacccctccgacatcgccgtggaatgggagagcaac ggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctgtacagcagact gaccgtggacaagagcagatggcaggaaggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccact acacccagaagtccctgagcctgagccccggcaaa(SEQ ID No：23)

G1-L4-Fc amino acid sequence such as SEQ ID No：Shown in 18：

MEWSWVFLFFLSVTTGVHSHGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGGGSGGGGSGGGGSGGGGSSKYGPP CPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No：18)

Coded sequence such as SEQ ID No：Shown in 24：

atggagtggtcctgggtgttcctgttctttctgtccgtgaccacaggagtccacagccatggtgaagggacctttac cagtgatgtaagttcttatttggaaggacaagctgccaaggagttcattgcttggctggtgaaaggccgtggaggat ccggtggcggttccggtggaggcggaagcggcggtggaggatcaggaggcggtggctcttctaagtacgggccccct tgccctccttgcccagctcctgaatttgagggcggacccagcgtgttcctgttccccccaaagcccaaggacaccct gatgatcagcagaacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtgcagttcaatt ggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaacagcacctacagagtg gtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcct gcccagcagcatcgaaaagaccatcagcaaggccaagggccagcctcgcgagccccaggtgtacacactgcctccaa gccaggaagagatgaccaagaaccaggtgtccctgacctgtctcgtgaagggcttctacccctccgacatcgccgtg gaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattctt cctgtacagcagactgaccgtggacaagagcagatggcaggaaggcaacgtgttcagctgcagcgtgatgcacgagg ccctgcacaaccactacacccagaagtccctgagcctgagccccggcaaa(SEQ ID No：24)

G1-L5-Fc amino acid sequence such as SEQ ID No：Shown in 10, coded sequence such as SEQ ID No：Shown in 12：

atggagtggtcctgggtgttcctgttctttctgtccgtgaccacaggagtccacagccatggtgaagggacctttac cagtgatgtaagttcttatttggaaggacaagctgccaaggagttcattgcttggctggtgaaaggccgtggaggat ccggtggcggttccggtggaggcggaagcggcggtggaggatcaggaggcggtggctctggaggtggtggaagttct aagtacgggcccccttgccctccttgcccagctcctgaatttgagggcggacccagcgtgttcctgttccccccaaa gcccaaggacaccctgatgatcagcagaacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccg aggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaac agcacctacagagtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggt gtccaacaagggcctgcccagcagcatcgaaaagaccatcagcaaggccaagggccagcctcgcgagccccaggtgt acacactgcctccaagccaggaagagatgaccaagaaccaggtgtccctgacctgtctcgtgaagggcttctacccc tccgacatcgccgtggaatgggagagcaacggccagcccgagaacaactacaagaccaccccccctgtgctggacag cgacggctcattcttcctgtacagcagactgaccgtggacaagagcagatggcaggaaggcaacgtgttcagctgca gcgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgagccccggcaaa(SEQ ID No： 12)

G1-L6-Fc amino acid sequence such as SEQ ID No：Shown in 19：

MEWSWVFLFFLSVTTGVHSHGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGGGSGGGGSGGGGSGGGGSGGGGSG GGGSSKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG K(SEQ ID No：19)

Coded sequence such as SEQ ID No：Shown in 25：

atggagtggtcctgggtgttcctgttctttctgtccgtgaccacaggagtccacagccatggtgaagggacctttac cagtgatgtaagttcttatttggaaggacaagctgccaaggagttcattgcttggctggtgaaaggccgtggaggat ccggtggcggttccggtggaggcggaagcggcggtggaggatcaggaggcggtggctctggaggtggtggaagtggt ggcggcggttcgtctaagtacgggcccccttgccctccttgcccagctcctgaatttgagggcggacccagcgtgtt cctgttccccccaaagcccaaggacaccctgatgatcagcagaacccccgaagtgacctgcgtggtggtggacgtgt cccaggaagatcccgaggtgcagttcaattggtacgtggacggcgtggaagtgcacaacgccaagaccaagcccaga gaggaacagttcaacagcacctacagagtggtgtccgtgctgaccgtgctgcaccaggattggctgaacggcaaaga gtacaagtgcaaggtgtccaacaagggcctgcccagcagcatcgaaaagaccatcagcaaggccaagggccagcctc gcgagccccaggtgtacacactgcctccaagccaggaagagatgaccaagaaccaggtgtccctgacctgtctcgtg aagggcttctacccctccgacatcgccgtggaatgggagagcaacggccagcccgagaacaactacaagaccacccc ccctgtgctggacagcgacggctcattcttcctgtacagcagactgaccgtggacaagagcagatggcaggaaggca acgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagtccctgagcctgagccccggc aaa(SEQ ID No：25)

LY2189265 amino acid sequence such as SEQ ID No：Shown in 20：

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID No：20)

Coded sequence such as SEQ ID No：Shown in 26：

atggagtggtcctgggtgttcctgttctttctgtccgtgaccacaggagtccacagccatggtgaagggacctttac cagtgatgtaagttcttatttggaagagcaagctgccaaggagttcattgcttggctggtgaaaggcggtggaggag gcggtggctctggaggtggtggaagtggtggcggcggttcggctgaatctaagtacgggcccccttgccctccttgc ccagctcctgaagctgcaggcggacccagcgtgttcctgttccccccaaagcccaaggacaccctgatgatcagcag aacccccgaagtgacctgcgtggtggtggacgtgtcccaggaagatcccgaggtgcagttcaattggtacgtggacg gcgtggaagtgcacaacgccaagaccaagcccagagaggaacagttcaacagcacctacagagtggtgtccgtgctg accgtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcccagcagcat cgaaaagaccatcagcaaggccaagggccagcctcgcgagccccaggtgtacacactgcctccaagccaggaagaga tgaccaagaaccaggtgtccctgacctgtctcgtgaagggcttctacccctccgacatcgccgtggaatgggagagc aacggccagcccgagaacaactacaagaccaccccccctgtgctggacagcgacggctcattcttcctgtacagcag actgaccgtggacaagagcagatggcaggaaggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaacc actacacccagaagtccctgagcctgagcctgggctaatag(SEQ ID No：26)

Each fusion protein in CHO-DXB11 expression (such as coded sequence carrier connection, transfection, cell expression, Screening technique) it is same as Example 2, purification process is same as Example 4.Extracorporeal receptor is carried out to purifying gained fusion protein

GLP-1 activation experiments (specific method is same as Example 6), as a result as shown in table 4：

Table 4

Fusion protein (G1-L-Fc)

EC50 (activity) μM

G1-L1-Fc	0.382 ± 0.02 (n=3)
		G1-L2-Fc	0.165 ± 0.02 (n=3)
G1-L3-Fc	0.047 ± 0.005 (n=3)
		G1-L4-Fc	0.021 ± 0.005 (n=3)
G1-L5-Fc	0.0018 ± 0.001 (n=3)
		G1-L6-Fc	0.0038 ± 0.001 (n=3)
LY2189265	0.0076 ± 0.001 (n=3)

From table 4, flexible peptide linker L5 is preferred scheme.Experiment shows the GLP-1-Fc expressed with this flexible peptide linker Detect biological activity highest.

In summary, the present invention effectively overcomes various shortcoming of the prior art and has high industrial utilization.

The above-described embodiments merely illustrate the principles and effects of the present invention, not for the limitation present invention.It is any ripe Know the personage of this technology all can carry out modifications and changes under the spirit and scope without prejudice to the present invention to above-described embodiment.Cause This, those of ordinary skill in the art is complete without departing from disclosed spirit and institute under technological thought such as Into all equivalent modifications or change, should by the present invention claim be covered.

Claims

1. a kind of GLP-1 or its analog and antibody Fc fragment fusion protein preparation method, comprise the following steps：

1) GLP-1 or its analog are cloned into expression vector with antibody Fc fragment fusion protein coded sequence；

3) expressed, after purification using step 2 gained cell, produce the fusion protein；

The GLP-1 or its analog and antibody Fc fragment fusion protein are by recombinant human glucagon-like peptide-1 or its analog To be formed by connecting peptide with having the antibody Fc fragment that prolonged human Half-life in vivo acts on to be connected；

The amino acid sequence of GLP-1 or its analog such as SEQ ID No：Shown in 1；

Connect the amino acid sequence such as SEQ ID No of peptide：Shown in 7；

Antibody Fc fragment is the non-cracking performance Fc, the amino acid sequence such as SEQ ID of antibody Fc fragment for not having ADCC effector functions No：Shown in 9.

2. a kind of GLP-1 as claimed in claim 1 or its analog and antibody Fc fragment fusion protein preparation method, it is special Sign is, the amino acid sequence such as SEQ ID No of the GLP-1 or its analog and antibody Fc fragment fusion protein：Shown in 10； The GLP-1 or its analog and antibody Fc fragment fusion protein coded sequence such as SEQ ID No：Shown in 12.

3. a kind of GLP-1 as claimed in claim 1 or its analog and antibody Fc fragment fusion protein preparation method, it is special Sign is that the expression vector is selected from the expression vector of DHFR systems or the expression vector of GS systems.

4. a kind of GLP-1 as claimed in claim 1 or its analog and antibody Fc fragment fusion protein preparation method, it is special Sign is, side GLP-1 or its analog being cloned into antibody Fc fragment fusion protein coded sequence in expression vector Method is specially：More grams with carrier are designed at GLP-1 or its analog and antibody Fc fragment fusion protein coded sequence both ends Restriction enzyme site corresponding to grand site, then coded sequence is connected with restriction enzyme site corresponding on carrier, realize target protein The connection of coding DNA fragment and expression vector.

5. a kind of GLP-1 as claimed in claim 1 or its analog and antibody Fc fragment fusion protein preparation method, it is special Sign is, described that expression vector is transfected into CHO-DXB11 cells, and the method cultivated and filter out positive cell strain is specially：Make With DNA transfection reagents by plasmid DNA transfection CHO-DXB11 cells, by determining the expression quantity of fusion protein after Secondary Culture, from And filter out positive cell and form cell line.

6. a kind of GLP-1 as claimed in claim 1 or its analog and antibody Fc fragment fusion protein preparation method, it is special Sign is that the method for the cell expression is specially：The positive cell strain filtered out is subjected to serum-free domestication, is then seeded to Shaking table culture in shaking flask, supernatant is taken to carry out protein purification gained medium centrifugal.

7. a kind of GLP-1 as claimed in claim 1 or its analog and antibody Fc fragment fusion protein preparation method, it is special Sign is that the purifying is purified specifically by membrane filtration, affinity column and gel permeation chromatography.

A kind of 8. fusion protein, as the GLP-1 as described in claim 1-7 any claims or its analog and antibody Fc piece The preparation method of section fusion protein prepares.

9. a kind of pharmaceutical composition, include the fusion protein as claimed in claim 8 of effective dose, in addition to carrier, dilution One or more combinations in agent, excipient, stabilizer, thickener.