CN107474141A - Glicetin 1 acceptor excitomotor fusion protein and its application - Google Patents

Abstract

The invention discloses a kind of fusion protein, and it includes protein for treatment agent and receptor-binding agents, wherein the protein for treatment agent is the peptides of GLP 1 or its analog, the receptor-binding agents are b subunit of cholera toxin or its analog.The fusion protein energy oral administration of the present invention is to treat diabetes etc..

Description

Glucagon-like peptide-1 receptor stimulant fusion protein and its application

Technical field

The present invention relates to a kind of fusion protein, more particularly it relates to a kind of fusion protein, it includes albumen and controlled Agent and receptor-binding agents are treated, wherein the protein for treatment agent is GLP-1 peptides or its analog, the receptor-binding agents are cholera poison Plain B subunits or its analog.The fusion protein energy oral administration of the present invention is to treat diabetes etc..

Background technology

Diabetes (Diabetes Mellitus) are a kind of global diseases, and number of patients sharply increases in recent years, is people One of chief threat of class health.Diabetes are divided into type 1 diabetes and diabetes B.Supplementation with insulin is the master of type 1 diabetes Want treatment means.Diabetes B (Diabetes Mellitus, DM) is the disease for seriously endangering human health, and number of patients is just With the improvement of living standards, aging population, and improving for diagnostic techniques and increase sharply.In recent years research shows pancreas islet Element resistance and hypoinsulinism are the pathophysiological changes of diabetes B characteristic, two-way interaction, ultimately result in 2 The generation of patients with type Ⅰ DM.Moreover, insulin resistance also take part in chronic complicating diseases of diabetes such as cardiovascular complication, glycosuria Generation, the development of sick nephrosis etc., it is contact diabetes, hypertension, hyperlipidemia, an important pivot of coronary heart disease.Therefore pin To the pathogenesis of diabetes B, insulin resistance, increase insulin secretion are mitigated for 2 type glycosurias using effective medicine The treatment of disease and its complication has great importance.Existing diabetes B medicine mainly includes biguanides, promotees pancreas Island element secretion medicine and insulin sensitizer, they can be used alone or coordinate insulin combination to be treated.Since 1921 Since year insulin is found and is applied to clinic, the progress of insulin preparation quickly, high-purity and it is short-acting, middle imitate and The development and application of long-acting preparation, but insulin is administered to be subcutaneously injected, in a manner of intramuscular injection or intravenous injection, there is no Oral administration medicine lists.And the oral hypoglycemic drug used at present can not solve the problems, such as islet beta-cell apoptosis, and Serious side reaction be present in treatment, as sulfonamides can cause hypoglycemia.Nineteen eighty-three is in analysis hyperglycemic factor precursor-pancreas Glucagon-like-peptide-1 (glucagon-like is found that during the gene order of proglucagon (proglucagon) Peptide-1, referred to as GLP-1), it is most strong more of promoting insulin secretion in the incretin being currently known to find it Peptide.GLP-1 has different physiological roles, promotes propagation and the differentiation of beta Cell of islet, suppresses β Apoptosis；Increase glucose according to Rely property insulin secretion and release and its sensitiveness；Increase glucose uptake and utilization, suppress the secretion of postprandial hyperglycemic factor With release；Delay gastric emptying, appetite-suppressing, be advantageous to mitigate weight, reduce blood glucose.GLP-1 is mainly by enteron aisle L cells point Secrete, by translation process after the precursor of L cells synthesis GLP-1 former glucagon-like peptide, discharge into blood circulation.GLP- 1 is played a role by GLP-1 acceptors (GLP-1 receptor, the GLP-1R) combination with beta Cell of islet.GLP-1 acceptors are one Individual g protein coupled receptor, belong to beta receptor family, the GLP-1 acceptors in islet tissue are mainly expressed in β cells, also deposit extensively It is kidney, lung, intestines and stomach, heart and central nervous system.Complete people GLP-1 polypeptides are by 37 Amino acid profiles, in vivo It is GLP-1 (7-37) and GLP-1 (7-36) acid amides that GLP-1, which has the form of bioactivity, and complete GLP-1 (1-37) is in vivo After generation, by two step digestions, remove 6 amino acid of N-terminal and form the amidatioon of C-terminal, ultimately generate with high biological GLP-1 (7-36) acid amides of activity.However, GLP-1 half-life period is extremely short, once it is released from intestines L cells, will be by blood Enzyme degraded, be 2-5 minutes in plasma half-life.The enzyme for playing principal degradation effect is referred to as DPP IV (dipeptidyl peptidase IV,DPP IV).GLP-1 and the like (being referred to as GLP-1 receptor stimulating agents) and GLP-1 Acceptor combine after, cyclic adenosine monophosphate (cAMP) and blocking effect of mitogen activated protein kinases (MAPK) path in active cell film and play physiology Effect.At present, have Exenatide Byetta as the approved GLP-1 analogs of medicine), Liraglutide (Victoza), degree Shandong peptide (Trulicity), albiglutide (Tanzeum) etc. are drawn, the pancreotropic hormone effect of GLP-1 receptor stimulating agent medicines depends on Blood sugar concentration, GLP-1 will not cause hypoglycemia, can increase the secretion of insulin, reduce empty stomach and postprandial blood sugar, reduce saccharification blood Lactoferrin, on treatment diabetes B there is good clinical value, Liraglutide etc. to have been demonstrated that painstaking effort manage-style can be reduced Danger.Exenatide listed in 2005, for the first GLP-1 analogs in the whole world.GLP-1 of the Exenatide from lizard, because not depositing In people GLP-1 DPP-IV restriction enzyme sites, half-life period is obviously prolonged, but still needs to daily subcutaneous administration twice.Novo Nordisk is developed Unique fatty acid chain modification technique, Liraglutide are based on technology platform exploitation.Liraglutide reaches with people's GLP-1 homologys To 97%, administration frequency for it is subcutaneous once a day, dominated the market rapidly after listing.2015 annual sales amounts reach 2,700,000,000 dollars. LEADER studies have shown that Liraglutides cause mortality risk to reduce by 22%.Merge the long-acting of albumin or antibody Fc GLP-1 receptor stimulating agents have also listed, and albiglutide (GlaxoSmithKline PLC company product), Du Lalu peptides (gift carrys out Products) are It is administered once within one week, administering mode is to be subcutaneously injected.Suo Malu peptides (Semaglutide, the medicine code name of Novo Nordisk development OG217SC) in clinical studies, there are injecting pathway and oral route about medicine, clinical data shows Suo Malu peptides peroral dosage form such as Fruit to play preferable blood sugar reducing function, it is necessary to active component dosage be 300 times of subcutaneous injection agent, that is, Suo Malu is subcutaneously injected Peptide (Semaglutide) is weekly to once each to be administered 1 milligram.If be administered orally, Suo Malu peptides are taken daily (Semaglutide) 40 milligrams.Even if therefore peroral dosage form can be listed successfully, spend inevitable also high.In view of diabetic population Huge (only there are 96,000,000 diabetes patients in China at present) and as chronic disease administration time length, and ejection preparation administration Pain, inconvenience, patient dependence difference etc., the oral protein medicine right and wrong of good, the salable treatment diabetes of development efficacy It is often necessary and urgent need.

Cholera toxin is the enterotoxin as caused by comma bacillus, is formed by 1 A unit (CTA) and 5 B units (CTB) Polymer is formed by connecting.CTA is toxicity subunit, and CTB is nontoxic acceptor combination subunit, can be with all karyocyte films On gangliosides (GM1) acceptor combine.CTB as immunologic adjuvant, have been used for recombinating B subunits/thalline cholera vaccine (on Sea joint Sai Er bioengineering Co., Ltd), dosage is every dose of 1mg, it was demonstrated that is safe.CTB can also be used as effective Protein drug is in delivery carrier, Arati et al. (Arati Limaye, Vijay Koya, Mohtashem Samsam, and Henry Daniell.Receptor-mediatedoral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system.FASEB J.2006, May；20(7):959-961.) by CTB and green fluorescent protein GFP in tobacco chloroplast Amalgamation and expression, with this feeding Balb/c mouse that grow tobacco, author has found that fusion protein has been attached to the GM1 acceptors of cell membrane surface On, GFP enters Lymphatic Circulation, travels further into blood circulation system.

The content of the invention

It is an aspect of the present invention to provide a kind of fusion protein, and it includes protein for treatment agent and receptor-binding agents, wherein institute It is GLP-1 peptides or its analog to state protein for treatment agent, and the receptor-binding agents are b subunit of cholera toxin or its analog.

In certain embodiments, fusion protein of the invention also includes being used to connect protein for treatment agent and receptor-binding agents Connexon, the connexon cracked by the Furin enzymes being widely present into the cell.

Another aspect of the present invention is to provide the nucleotide sequence of encoding fusion protein, and the fusion protein includes protein for treatment Agent and receptor-binding agents, wherein the protein for treatment agent is GLP-1 peptides or its analog, the receptor-binding agents are cholera toxins B subunits or its analog.In certain embodiments, fusion protein of the invention also include be used for connect protein for treatment agent and by The connexon of body bonding agent, the connexon are cracked by intracellular enzyme or environmental factor.

In some embodiments, the nucleotide sequence of encoding fusion protein of the invention includes the core of encoding proteins therapeutic agent The nucleotide sequence of acid sequence and coding receptor-binding agents.In some embodiments, the nucleic acid of encoding fusion protein of the invention Also include the nucleotide sequence of coding connexon in sequence.

On the other hand, the invention provides the expression vector of the nucleotide sequence comprising the present invention.

On the other hand, the invention provides the host cell of the expression vector comprising the present invention.

On the other hand, the invention provides the composition of the fusion protein comprising the present invention.In some embodiments, The composition is pharmaceutical composition.

On the other hand, the invention provides the method that protein for treatment agent is conveyed to study subject, this method includes will be by The epithelial cell for trying object is in contact with the fusion protein of the present invention, and the fusion protein includes protein for treatment agent and acceptor combines Agent, wherein the protein for treatment agent is GLP-1 peptides or its analog, the receptor-binding agents are b subunit of cholera toxin or its class Like thing.

On the other hand, fusion protein of the invention or fusion protein compositions can be used for preparing medicine.

Brief description of the drawings

Fig. 1 is the collection of illustrative plates of OPG expression plasmids

Fig. 2 is fusion protein purification electrophoretogram

Fig. 3 is hypoglycemic activity curve maps of the CTB with GLP-1 analog fusions to diabetic KKAY mice

Embodiment

Definition

Term " protein for treatment agent " refers to protein, polypeptide, peptide or fragment or its variant, has one or more therapeutic And/or biological activity.Therapeutic protein included in the present invention includes but is not limited to protein, polypeptide, peptide and antibody And biological agent.Term peptide, proteins and peptides can be used interchangeably herein.

Term " receptor-binding agents " is the compound for specifically binding acceptor, and exemplary receptor-binding agents include but unlimited In peptide, fragments of peptides, antibody, cell factor, matrix and/or signaling molecule etc.." acceptor " herein refers to energy in study subject body The target molecule specifically bound by receptor-binding agents, exemplary acceptor include the acceptor on cell surface.

Term " connexon " refers to connecting by covalent bond or ion or Van der Waals force or hydrogen bond and two other molecules The molecule connect, for example, 5 ' end with a complementary sequence hybridization and 3 ' end with another complementary sequence hybridization, so as to connect two The nucleic acid molecules of non-complementary sequence." connexon of cleavable " refers to that the connexon can be degraded, or otherwise and Make two components separation that the connexon of the cleavable is connected.The connexon of cleavable is generally general by enzymatic lysis, the enzyme For peptase, protease, nuclease, lipase etc..The connexon of cleavable by environmental factor also to be cracked, such as temperature, pH, salt Change of concentration, etc..This environmental factor change occurs to transport it in the born of the same parents that wear of structure for conveying through polarized epithelial cells film Afterwards.

Term " pharmaceutical composition " refers to the Pharmaceutical composition for being adapted to animal to use.It is effective that pharmaceutical composition contains pharmacy The active agent and pharmaceutically acceptable carrier of amount." pharmacy effective dose " refers to that the amount of reagent can produce the pharmacy of needs Effect." pharmaceutically acceptable carrier " refers to any standard pharmaceutical carriers, medium, buffer solution and excipient, such as phosphoric acid Salt buffer, 5% D/W and emulsifying agent such as oil/water or water/fat liquor and different types of wetting agent And/or auxiliary agent.MackPublishing Co., Easton publish the 21st edition Remington ' s published in 2005 Suitable pharmaceutical carrier and formulation are disclosed in PharmaceuticalSciences." pharmaceutically acceptable salt " refers to can be with It is configured to the salt of medicinal compound, including metal salt (sodium, potassium, magnesium, calcium etc.) and ammonia salt or organic amine salt.

" study subject " of diagnosis, treatment or administration medicine refers to people or non-human animal, including mammal or spirit length Class animal, preferably it is people.

Term " treatment " refers to prophylactic treatment or therapeutic treatment." prophylactic treatment " is directed to not show disease The symptom of disease or the study subject administration for only showing early symptom, for the purpose of the occurrence risk for reducing disease.It is " therapeutic to control Treat " it is directed to show the individual administration of pathological state, for the purpose of less or eliminate these symptoms.

When term " homogeneity " refers to being compared with alignment programs, the amino acid sequence between two or more peptides The consistency level of nucleotide sequence between the consistency level of row or two or more nucleic acid.For example, in the present invention, by 80% homogeneity of the algorithm measure of restriction, implies that given sequence and another sequence at least 80% is identical.Sequence The exemplary horizontal of homogeneity include but is not limited to given sequence have 60%, 70%, 80%, 85%, 90%, 95%, The 98% or higher phase same sex.Homogeneity between sequence can use conventional method well-known to those skilled in the art to carry out Measurement, such as, but not limited to blast program, as the public can from BLASTN, BLASTX that NCBI websites obtain, TBLASTX, BLASTP and TBLASTN programs.

Term " expression vector " refers to plasmid, phasmid or the virus of any encoding exogenous nucleic acid.The term is also explained It is to include non-plasmid, non-phasmid and non-viral compound, nucleic acid is conveniently transferred in virion or cell by it.Carrier can To be suitable as nucleic acid or its mutant being delivered to the viral vector of the delivery medium of cell, or the carrier can be suitable Non-virus carrier for identical purpose.The known virus that can be used to for DNA to be delivered to cell and tissue of those skilled in the art With the example of non-virus carrier.The example of viral vector includes but is not limited to, recombinant poxvirus carrier, recombined adhenovirus, again Group retrovirus, recombinant adeno-associated virus, restructuring avipoxvirus etc..The example of non-virus carrier includes, but are not limited to, fat The polyamine derivative etc. of plastid, DNA.

Term " nucleic acid ", " nucleotide sequence ", " nucleotide sequence " or " polynucleotides " refers to what is be made up of nucleotide units The polymer of single-stranded or double-stranded form.Within the scope of the present invention, " nucleic acid ", " nucleotide sequence ", " nucleotide sequence " or " more nucleosides Acid " can be used interchangeably.Polynucleotides include the nucleotide sequence naturally occurred, such as DNA (" DNA ") and ribose Nucleic acid (" RNA "), and nucleic acid analog.Nucleic acid analog includes those bases for containing non-natural formation and other nucleosides With the nucleosides of the key connection outside the phosphodiester bond that naturally occurs, or include the alkali by the key connection outside phosphodiester bond The nucleic acid analog of base.Therefore, nucleic acid analog includes, such as, but not limited to thiophosphate, phosphorodithioate, phosphorus three Ester, phosphoramidate, boron substituted phosphate, methyl phosphorodithioate, chirality methyl phosphate, 2-O- methyl ribonucleic acids, peptide-nucleic acid (PNA) etc..These polynucleotides can be synthesis, such as be synthesized with automatic dna synthesizer.It is appreciated that work as nucleotide sequence When being represented with DNA sequence dna (i.e. A, T, G, C), it also includes RNA sequence (A, U, G, C), wherein " U " replacement " T ".

Polynucleotide sequence is described using conventional labels herein：The left-hand end of single stranded polynucleotide sequence is 5 ' ends, double The left hand direction of chain polynucleotide sequence is referred to as 5 ' directions.

Term " complementary " match together by the topological compatibility or its contact surface for referring to two polynucleotides.Therefore, should Two molecules are complementary, moreover, being complimentary to one another the characteristics of the contact surface.If the nucleotide sequences of the first polynucleotides and The nucleotide sequences of the binding partners of the polynucleotide sequence of two polynucleotides are essentially identical, or the first polynucleotides are strict Hybridization conditions under can hybridize the second polynucleotides, then the first polynucleotides and the second polynucleotides are complementary.Therefore, sequence - the TATAC-3 ' of row 5 ' polynucleotides and the-GTATA-3 ' of sequence 5 ' polynucleotides are complementary.

" coding " is the specific nucleotide sequences in polynucleotides, such as gene, cDNA or mRNA intrinsic property, for use as life The template of other polymer and macromolecular synthesis during thing, other polymer or macromolecular have the nucleotide sequences limited (i.e. rRNA, tRNA and mRNA) or limit amino acid sequence, and by caused biological property.Thus, if in cell Or the transcription and translation of mRNA caused by gene produces protein, then gene code protein in other biosystems.This Kind of gene or cDNA coding strand and noncoding strand are also regarded as encoding proteins matter or encode the other of the gene or cDNA The nucleotide sequences of product, wherein coding strand are identical with mRNA sequence, provided generally in the form of sequence table, and noncoding strand is used as The template of transcription.Unless specifically stated otherwise, " nucleotide sequence of encoding amino acid sequence " includes degenerate sequences all to each other All nucleotide sequences with coding with amino acid sequence.Encoding proteins matter and RNA nucleotide sequence include introne.

" primer " refers to specifically hybridizing and can provide synthesis complementary multinuclear with specified polynucleotide template The polynucleotides of the initiation site of thuja acid.When polynucleotide primers are in the condition of induction synthesis, i.e., in nucleosides, complementary multinuclear As existing for archaeal dna polymerase during situation, this synthesis will occur for thuja acid template and polymerization agent.Primer be typically it is single-stranded, But it can also be double-strand.Primer is typically DNA, but a large amount of different synthetic primers and natural primer can For multiple use.Primer and the template designed to be hybrid with it are the complementary initiation sites for use as synthesis, but not It must be the template exact sequence.In this case, primer and the specific hybrid of template depend on the stringency of hybridization conditions. Primer can use as chromogenic part, radioactive moiety or fluorescing fractions mark, and be used as detectable part.

" polypeptide " refers to the polymer being made up of amino acid residue, the structural variant of related natural generation and passes through peptide The non-natural hair of the analog that the non-natural of its synthesis of key connection occurs, the structural variant of related natural generation and its synthesis The polymer of raw analog composition.The polypeptide of synthesis can be with such as with the Peptide synthesizer synthesis of automation.Herein with conventional symbol Number peptide sequence described, the starting point of peptide sequence is aminoterminal, and the end of polypeptide is c-terminus.

Term " protein " is commonly referred to as big polypeptide, for example, containing the polypeptide for having more than 50 amino acid.Term " egg White matter " also refers to the dimer containing more than one polypeptide, tripolymer, polymer.

Term " wild type " refers to naturally occurring polynucleotides or peptide sequence.

Term " conservative replacement " is referred to the amino acid in functionally similar 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor polypeptide.Following six groups respective Contain the amino acid that can mutually substitute：

Alanine (A), serine (S) and threonine (T)；

Aspartic acid (D) and glutamic acid (E)；

Asparagine (N) and glutamine (Q)；

Arginine (R) and lysine (K)；

Isoleucine (I), leucine (L), methionine (M) and valine (V)

Phenylalanine (F), tyrosine (Y) and tryptophan (W)

Term " pharmaceutically acceptable " refers to the material and composition being physiologically resistant to, and generally gives people being administered Shi Buhui produces allergy or similar uncomfortable reaction such as gastric disorder causing nausea, feeling dizzy.

Term " therapeutically effective amount " refers to the content of the fusion protein comprising therapeutic molecules, needs this when being administered to When individual, it is sufficient to reach therapeutic purposes.Form the fusion protein of " therapeutically effective amount " content will be with it is used therapeutic Protein, illness or disease seriousness, be treated individual age and body weight and change, this can pass through ordinary skill Personnel routinely determine according to oneself knowledge and the disclosure.

Unless otherwise specified, the scope that term " about " exponential quantity used in the present invention fluctuates is no more than 10%. For example, " about 10 μ g/kg " are meant that between 9 μ g/kg to 11 μ g/kg term.

GLP-1 relevant diseases

In some embodiments, protein for treatment agent is GLP-1 activators.Such compound can be used for any treatment Using wherein activity of the GLP-1 activators in brain or in particular organization is desired.GLP-1 agonist activities are adjoint There are the stimulation (that is, as GLP-1) and glucagon suppression secretion of insulin secretion, so as to help to limit Postprandial glucose excursion processed.GLP-1 activators can also suppress gastrointestinal peristalsis and secretion, so as to as enterogastrone and " ileum A part for braking " mechanism.GLP-1 also appear to be appetite and food intake physiological regulation agent.Due to these effects, GLP-1 It can be used for treating metabolic disease with GLP-1 receptor stimulating agents.Above-mentioned disease include obesity, hyperglycemia, dyslipidemia, Hypertriglyceridemia, X syndrome, insulin resistant, IGT, diabetic dyslipidemia, hyperlipidemia, angiocarpy Disease and hypertension.GLP-1 also has an effects on neural system, including calm or angst resistance effect, such as in U.S. Patent number 5,846, Described in 937.Therefore, GLP-1 activators can be used for treat anxiety, attack, mental disease, epileptic attack, panic attack, Hysteria or sleep-disorder.GLP-1 activators can also be used to treat Alzheimer disease, because have shown can for GLP-1 activators The apoptosis of amyloid-β peptides and glutamate induction is avoided to protect neuron.

Other therapeutical uses of GLP-1 activators include improving study, enhancing neuroprotection and mitigate central nervous system The disease of system or the symptom of obstacle, such as by adjusting nerve to occur, and such as Parkinson's, Alzheimer disease, the prosperous court of a feudal ruler Pause disease, ALS, apoplexy, ADD and neuropsychiatric symptoms.

GLP-1/ fusion proteins comprising at least one GLP-1 or its analog and its fragment can be used for 1 type for the treatment of and 2 types Diabetes and obesity.

Term " GLP-1 molecules " refers to GLP-1, GLP-1 analog or GLP-1 derivatives.

Term " GLP-1 analogs " is defined as, compared with GLP-1, have one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, deletion, The molecule be inverted or added.Many GLP-1 analogs are known in the art, and including, such as GLP-1 (7-34), GLP-1 (7-35), GLP-1 (7-36), Val8-GLP-1 (7-37), Gly8-GLP-1 (7-37), Ser8-GLP-1 (7-37), Gln9-GLP1 (7-37), D-Gln9-GLP-1 (7-37), Thr16-Lys18-GLP-1 (7-37) and Lys18-GLP-1 (7- 37).Other analogs include the GLP-1 of dipeptidase resistance form, and the wherein N-terminal of the peptide is protected.This analog bag Include, but be not limited to the GLP-1 with other amino acid, such as be added to N-terminal or substitute and enter N-terminal amino acid The histidine of (amino acid 7 or 8 in GLP-1 (7-36) or GLP-1 (7-37)).In these analogs, N-terminal can include Residue His-His-Ala, Gly-His-Ala, His-Gly-Glu, His-Ser-Glu, His-Ala-Glu, His-Gly-Glu, His-Ser-Glu、His-His-Ala-Glu、His-His-Gly-Glu、His-His-Ser-Glu、Gly-His-Ala-Glu、 Gly-His-Gly-GIu, Gly-His-Ser-Glu, His-X-Ala-Glu, His-X-Gly-Glu, His-X-Ser-Glu, its Middle X is any amino acid.

Term " GLP-1 derivatives " is defined as the amino acid sequence of GLP-1 or GLP-1 analogs, but one There are other chemistry to repair for individual or multiple amino acid side groups, alpha -carbon atom, terminal amino group or the upper of terminal carboxylic acid group The molecule of decorations.Chemical modification includes, but are not limited to, and addition chemical part, produces new keys and removes chemical part.Nineteen eighty-three exists GLP-1. is found that during the gene order of analysis hyperglycemic factor precursor-Proglucagon (proglucagon) (G.I.Bell,R.Sanchez-Pescador,P.J.Laybourn,R.C.Najarian,Exon duplicationand divergence in the human preproglucagon gene,Nature 304(1983)368-371)。Exendin- 4 be isolated from a kind of huge lizard of Mexico (Heloderma horridum) saliva to have 39 amino acid Polypeptide, its sequence have 53% homology with GLP-1) J.Eng, W.A.Kleinman, L.Singh, G.Singh, J.P.Raufman,Isolation andcharacterization of exendin-4,an exendin-analogue, from Heloderma suspectumvenom:further evidence for an exendin receptor on dispersed acini from guinea pigpancreas,J Biol Chem 267(1992)7402-7405)。

Particularly preferred receptor-binding agents include b subunit of cholera toxin in the present invention.Such as GenBank:U25679.1.Make It can include amino acid sequence as described above or by nucleotides sequence as described above for currently preferred b subunit of cholera toxin Arrange coding amino acid sequence, or with above-mentioned amino acid sequence have at least 80%, at least 85%, at least 90%, extremely The analog of few 95% or at least more than 98% homogeneity.

Such b subunit of cholera toxin can not only include native sequences, can also include mutation.It can use and for example add Add, lack, substitute, and/or insert 1 or a small number of amino acid (such as it is several, within 3, within 5, within 10, Amino acid within 15 or within 20) obtained from amino acid sequence b subunit of cholera toxin, as long as with using natural The situation of the B subunits of type is compared, and will not significantly decrease the expression quantity of fusion protein.In addition it is also possible to use N-terminal and/or C End have 1~several residues (such as 2,3,4,5,6,10,15 or 20 residues) amino acid deletions or addition and obtain Polypeptide and 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor with 1~several residues (such as 2,3,4,5,6,10,15 or 20 residues) and obtain Polypeptide etc..Fragment, analog, derivative and the native protein of the variant that can be used including such as native protein with The fused protein of other polypeptides (such as polypeptide containing extra heterologous signal peptide or antibody fragment).Specifically, it is included in Obtained from substituting in the amino acid sequence of wild-type cholera toxin B subunits, lacking and/or add one or more amino acid Sequence, and with the active polypeptide of the increase fusion expression of recombinant proteins suitable with wild-type cholera toxin B subunits, Ke Yizuo Used for the b subunit of cholera toxin of the present invention.The preferred holding of such modification type cholera toxin Toxin B Subunit forms pentamer Activity.In the case of using the fragment of wild-type protein, it generally comprises the wild type peptide (situation of secretory protein Down be adult form form) more than 70%, preferably more than 80%, more preferably more than 90% (or more than 95%) continuum Domain.

The number of amino acid for being modified is not particularly limited, e.g. whole ammonia of the adult form of natural polypeptides Within the 30% of base acid, within preferably 25%, within more preferably 20%, within more preferably 15%, within more preferably 10%, 5% Within, within 3% or within, such as within 15 amino acid, within preferably 10 amino acid, within more preferably 8 amino acid, Within more preferably 5 amino acid, within more preferably 3 amino acid.When substituting to amino acid amino acid, by being replaced to The similar amino acid of side chain properties can be expected to keep the activity of protein.

The signal peptide (typically 21 initial amino acid) of cholera toxin Toxin B Subunit inherently can be intactly Merged with recombinant protein.Or it can also be removed.

Connexon

The protein for treatment agent of the fusion protein of the present invention and receptor-binding agents can be merged directly or using various length Connexon merge, connexon is rich in glycine (G) and serine (S), can also contain glutamic acid (E), alanine (A), dried meat ammonia Sour (P) and threonine (T).Such as GGGGSGGGGS GGGGSRARR or GGGGSRARR, GGGS are flexible polypeptide, can ensure Protein for treatment agent and the space structure of receptor-binding agents, PARR are enzyme Furin restriction enzyme sites.)

Expression vector

Expression vector for the present invention generally comprises following element, is operably connected with 5 ' to 3 ' directions：Transcription Promoter, coding GLP-1 activators nucleotide sequence, encode connexon nucleotide sequence, encode cholera toxin B nucleic acid sequence Row, and transcription terminator..Suitable promoter includes, such as T7, araBAD, phoA, trc, OL, OR, PL and PR start Son.Preferably,

The promoter is T7 viral promotors.Coli expression carrier, such as pET, pGEX may be selected in expression vector, PBAD etc., bacillus expression vector pHT etc.；Also Yeast expression carrier pPIC9, pYEp etc., lactation breast animal expression may be selected Carrier pcDNA3.1 etc., and expression vector pFastBac of cell line (Sf9 and Sf21) of the ovary tissue of noctuid etc., plant Expression vector pBI101, LBA4404 etc..

Host cell

Present invention additionally comprises being converted to express the host cell of the fusion protein of the present invention, including large intestine bar is not limited to Cell line (the Sf9 of bacterium, bacillus, saccharomyces cerevisiae, Pichia pastoris, mammalian cell CHO, and the ovary tissue of noctuid And Sf21), plant, filamentous fungi, adenovirus, slow disease etc., preferably Escherichia coli.

Present invention also offers the pharmaceutical composition comprising fusion protein, also include auxiliary material in described pharmaceutical composition.

Auxiliary material available for the present invention includes as follows：

Surfactant promotes polypeptide to be absorbed by mucous membrane or internal layer.Useful surfactant include aliphatic acid and its Salt, bile salt, phosphatide or alkyl sugar.The example of aliphatic acid and its salt include sad (C8), capric acid (C10), laurate (C12) and Sodium salt, sylvite and the lysine salt of myristic acid (C14).The example of bile salt includes cholic acid, chenodesoxycholic acid, glycocholic acid, ox Sulphur cholic acid, glycochenodeoxycholate, Irish moss (chondrux), deoxycholic acid, glycodesoxycholic acid, tauroursodeoxycholic acid, stone courage Acid and ursodesoxycholic acid.The example of phosphatide includes single-stranded phosphatide, such as lysophosphatidyl choline, lysophosphatidyl glycerol, haemolysis Phosphatidyl-ethanolamine, hemolytic phosphatidyl inose and hemolytic phosphatidylserine；Or dichain phospholipids, such as two phosphatidyl cholines, Two acyl phosphatidyl glycerols, two acyl phosphatidyl-ethanolamines, two acyl phosphatidyl inoses and two acyl phosphatidylserines.The example of alkyl sugar Including alkyl-glucoside or alkyl mannoside, such as decyl glucoside and lauryl glucosyl.

Can be used as the drug excipient of carrier includes stabilizer, such as human serum albumins (HSA)；Filler such as carbohydrate, Amino acid and polypeptide；PH adjusting agent or buffer；Salt such as sodium chloride, etc..These carriers can be crystallization or amorphous Form or two kinds of mixture.

The example of carbohydrate as filler includes monose such as galactolipin, D-MANNOSE, sorbose etc.；Disaccharides, such as breast Sugar, trehalose etc.；Cyclodextrin, such as 2- hydroxypropyl-β .- cyclodextrins；And polysaccharide, such as gossypose, malt magma Essence, glucan etc.；Aldehyde alcohol, such as mannitol, xylitol etc..The example of polypeptide as filler includes aspartoyl phenylpropyl alcohol ammonia Sour methyl esters.Amino acid includes alanine and glycine, and glycine is preferable.

Additive is the minority component in composition, carrys out the Stable conformation in spray-drying process comprising them and improves powder The dispersiveness at end.These additives include hydrophobic amino acid, such as tryptophan, tyrosine, leucine, phenylalanine etc..

Suitable pH adjusting agent or buffer include the organic salt being prepared from organic bronsted lowry acids and bases bronsted lowry, such as citric acid Sodium, sodium ascorbate etc., sodium citrate are preferable.

The pharmaceutical composition of the present invention can be dosage unit form, for example, being tablet or capsule.In this form, Composition is subdivided into the unit dose containing proper content active component；Unit dosage form can be packaged combination Thing, for example, packaged powder, liquid medicine bottle, injection, the syringe of prepackage or the sack containing liquid.Unit dosage form can For example, capsule or tablet are in itself, or can be any this composition of appropriate number of packaged form to be.It is used for Dosage in treatment must be determined by the doctor in charge is subjective.

Fusion protein oral formulations generally use capsule formulation, it can also use tablet, granule, inhale mist agent and injection Deng formulation, be most preferred formulation with capsule.

Present invention additionally comprises the method for the pharmaceutical composition for orally administering the present invention.This method can include still unlimited In by patient or the step of orally administering of nursing staff's implementation.This step of applying, which can include interval, to be applied, such as daily Once or twice, depending on fusion protein, disease or patient condition or few patients.This method also includes applying various doses The various fusion proteins of amount.For example, first dosage of pharmaceutical composition can be high level, desired effects, such as blood are produced Sugar level declines.Once obtain desired effects, it is possible to reduce subsequent dosage.To these of application program, be altered or modified can To be realized by the doctor in charge or health care personnel.In some cases, application program can be changed by each patient.

Solid Dosage Forms for oral administration include capsule, tablet, pill, powder and particle.In this solid formulation In amount form, reactive compound mixes together with least one pharmaceutically acceptable inert carrier, such as sucrose, lactose or starch Close.For generally practice, this dosage form can also include the other materials outside inert diluent, such as lubricant such as tristearin Sour magnesium.In the case of for capsule, tablet and pill, the dosage form can also include buffer.Tablet and pill can be another Other places is prepared with casing.

Liquid dosage forms for orally administering include pharmaceutically acceptable emulsifying agent, solution, suspension, syrup, With the inert diluent for being generally used for containing in the pharmacopeia of this area, such as water.In addition to these inert diluents, composition In can also include adjuvant, such as wetting agent, emulsifying agent and suspending agent, and sweetener, flavor enhancement and aromatic.

The dosage of active component in the composition of the present invention is variable；But the dosage of active component must cause Suitable dosage form can be obtained.Dosage choice, which depends on desired therapeutic effect, administration route and desired treatment, to be continued Phase.Generally, the dosage level between 0.001-10mg/kg body weight is administered to mammal.

The present invention provides the kit containing fusion protein, and the kit can be used for, for example, therapeutic or non-treatment Property application.Kit includes the container with label.Suitable container includes, for example, bottle, bottle and test tube.Container can be with Prepared with various materials, such as glass or plastics.Equipped with the composition for including fusion protein in container, as described above, fusion egg It is in vain effective for therapeutic or non-therapeutic application.Active agent in composition is therapeutic protein.On container Label indicate that said composition is used for specific treatment or non-therapeutic application, may also indicate for internal or external Purposes, such as those purposes as described above.

The kit of the present invention generally also includes above-mentioned container, and one or more include commercially it is expected with user Material other containers, including buffer solution, diluent, filter, syringe needle, syringe and the packaging with description of use are inserted Thing.

Be not described any further, by using description above and following illustrative, it is believed that this area it is general Logical technical staff can utilize the present invention and implement claimed method.For example, when the therapeutic moieties with not being fused When comparable activity is compared, technical staff can be readily determined the in vitro and in vivo of the fusion protein construct of the present invention Bioactivity.Similarly, those skilled in the art can be readily determined according to the present invention construct serum half-life and Serum stability.Therefore, following working Examples specifically indicate the preferred embodiments of the invention, are not construed as to appoint Where formula limitation disclosed in residual right.

Embodiment

Material and instrument

Material

The experimental plasmid of use：Plasmid pUC57, plasmid pET42a

The enzyme of use：Nde I restriction endonucleases, Not I restriction endonucleases

Other materials：DH5a competence bacterias, DNA glue reclaims kit, the small extraction reagent kit of plasmid, LB culture mediums, BL21 (DE3) competence bacterium, Tris salt, sodium chloride, hydrochloric acid, high pressure homogenizer, GST affinity chromatographies Glutathione Sepharose HP, Q Sepharose HP, butyl hydrophobic chromatography Butyl Sepharose FF, Superdex75 gels, KKAy mouse, hundred secrete reach, mouse anti human GLP-1 antibody, goat anti-mouse igg-HRP, DAB developers

Key instrument

PCR instrument, thermostat, superclean bench, shaking table, centrifuge, refrigerator, protein purification system, glucometer

Embodiment 1：The structure of expression vector

By shown in connector gene shown in the gene shown in SEQ ID NO.2 and SEQ ID NO.9 and SEQ ID NO.4 GLP-1 genes connected with fully synthetic method and (be named as OPG-A i.e. CTB-L1-G) as shown in SEQ ID NO.13, be placed in load In body pUC57, pUC57-OPG-A is named as.Distinguished with SEQ ID NO.16primer F and SEQ ID NO.17primer R For upstream and downstream primer, using pUC57-OPG-A plasmids as masterplate, PCR amplifications, PCR primer reclaims through Nde I and Not I double digestions Afterwards with Nde I and Not I double digestions recovery pET42a be connected, connection product transformed competence colibacillus DH5a, card containing 50ug/ml that Monoclonal is chosen on the LB flat boards of mycin, it is pET-OPG-A expression vectors that correct rear extraction plasmid, which is sequenced,.

By shown in connector gene shown in the gene shown in SEQ ID NO.2 and SEQ ID NO.9 and SEQ ID NO.6 GLP-1-a genes connected with fully synthetic method and (be named as OPG-B i.e. CTB-L1-G a) as shown in SEQ ID NO.14, put In carrier pUC57, pUC57-OPG-B is named as.With SEQ ID NO.16primer F and SEQ ID NO.17primer R Respectively upstream and downstream primer, using pUC57-OPG-B plasmids as masterplate, PCR amplifications, PCR primer is through Nde I and Not I double digestions PET42a after recovery with the recovery of Nde I and Not I double digestions is connected, connection product transformed competence colibacillus DH5a, containing 50ug/ml Monoclonal is chosen on the LB flat boards of kanamycins, it is pET-OPG-B expression vectors that correct rear extraction plasmid, which is sequenced,.

By connector gene shown in the gene shown in SEQ ID NO.2 and SEQ ID NO.11 and SEQ ID NO.7 institutes The GLP-1 analog E4 genes shown are connected with fully synthetic method and (are named as OPG-C i.e. CTB- as shown in SEQ ID NO.15 L2-E4), it is placed in carrier pUC57, is named as pUC57-OPG-C.With SEQ ID NO.16primer F and SEQ ID NO.18primer R are respectively upstream and downstream primer, and using pUC57-OPG-C plasmids as masterplate, PCR amplifications, PCR primer is through Nde I PET42a after being reclaimed with Not I double digestions with the recovery of Nde I and Not I double digestions is connected, connection product transformed competence colibacillus DH5a, monoclonal is chosen on the LB flat boards of the kanamycins containing 50ug/ml, it is pET-OPG-C tables that correct rear extraction plasmid, which is sequenced, Up to carrier.

It is respectively upstream and downstream primer with SEQ ID NO.19primer F and SEQ ID NO.18primer R, with pUC57- OPG-C plasmids are masterplate, PCR amplifications, PCR primer after the recovery of BamH I and Not I double digestions with the double enzymes of BamH I and Not I The pET42a connections cut back to close, connection product transformed competence colibacillus DH5a, choose on the LB flat boards of the mycin of kanamycins containing 50ug/ml Monoclonal, it is pET-OPG-D expression vectors that correct rear extraction plasmid, which is sequenced,.

Embodiment 2：The expression of fusion protein

Correct expression vector pET-OPG-A, pET-OPG-B, pET-OPG-C will be built, pET-OPG-D is converted respectively In BL21 (DE3) competence bacterium, monoclonal is chosen on the LB flat boards of the kanamycins containing 50ug/ml respectively.Monoclonal is inoculated in In the LB fluid nutrient mediums of the kanamycins containing 50ug/ml, 37 DEG C of 200rpm shaken cultivations, work as OD₆₀₀When reaching 1.0, add dense eventually Spend the IPTG induced expressions for 0.5mM.30 DEG C are continued to cultivate 4-6 hours, and thalline is collected by centrifugation, puts -20 DEG C and freezes.

Embodiment 3：The purification of fusion protein

By OPG-A, OPG-B, OPG-C thalline are by weight 1:15 are dissolved in pH8.0 20mMTris-HCl buffer solutions In, wherein NaCl concentration is 0.1M, crushes secondary under 600 bar pressures, and the fragment upper QFF after 0.4um is filtered again is removed in 8000g centrifugations Anion chromatography post, after balance, eluted with the 20mMTris-HCl of the pH8.0 containing 0.5MNaCl.Eluent is adjusted with NaCl solids Salinity is 1.8M, then upper butyl hydrophobic chromatography, is eluted after balance with pH7.5 20mMTris-HCl.Protein eluate with Superdex S100 gel chromatographies on 10mMpH7.2PB buffer solutions, collect main protein peak, as target protein refined solution.

By OPG-D thalline by weight 1:15 are dissolved in pH8.0 20mMTris-HCl buffer solutions, and wherein NaCl is dense Spend for 0.1M, crush secondary under 600 bar pressures, the fragment upper GST affinity chromatographic columns after 0.4um is filtered again are removed in 8000g centrifugations, put down After weighing apparatus, eluted with the pH8.0 of reduced glutathione containing 10mM 20mMTris-HCl buffer solutions.With desalination chromatographic column by buffer solution In the 20mMTris-HCl for changing the pH8.0 containing 0.05MNaCl into, add appropriate EK digestions and fall GST label proteins, solution flows through GST affinity chromatographic columns remove GST label proteins.QFF anion chromatographies post on liquid is flowed through, after balance, with containing 0.5MNaCl's PH8.0 20mMTris-HCl elutions.It is 1.8M that eluent adjusts salinity with NaCl solids, then upper butyl hydrophobic chromatography, balance Eluted afterwards with pH7.5 20mMTris-HCl.Protein eluate is with Superdex S100 gels on 10mMpH7.2PB buffer solutions Chromatography, collects main protein peak, as target protein OPG-D refined solutions.

Embodiment 4：The Bioactivity of fusion protein

The Chinese hamster ovary celI for taking GLP-1R genes to transfect, is separately added into testing sample, using PBS solution as negative control, in selection It is positive control that city's medicine hundred, which is secreted up to (Exenatide, GLP-1 analogs), purifying protein OPG-A, OPG-B, OPG-C and OPG-D It is added to after appropriate dilution in the Chinese hamster ovary celI in 96 orifice plates, with the intracellular cAMP of ELISA method detection content.As a result show, melt Hop protein OPG-A, OPG-B, OPG-C and OPG-D are secreted up to similar to hundred, the rise of the detectable content for causing cAMP.

Western-blot methods detect immunocompetence.By fusion protein OPG-A, OPG-B, OPG-C and OPG-D and control egg White CTB is separated by electrophoresis and is transferred on pvdf membrane, adds mouse anti human GLP-1 antibody, then with enzyme target sheep anti-Mouse secondary antibody Reaction, DAB colour developings.As a result, fusion protein sample is positive, and CTB albumen does not develop the color, show fusion protein have with Immune response activity similar people GLP-1.

Embodiment 5：Hypoglycemic activities of the CTB with GLP-1 analog fusions to diabetic KKAY mice

KKAy diabetes pattern mouse are taken, random packet, every group 4, totally 6 groups.PBS solution is negative control group, positive Control group is secreted from marketed drug hundred and reaches (Exenatide, GLP-1 analogs), subcutaneous administrations, dosage 25nmol/ Kg body weight/only, negative control group administered volume is identical with positive controls；Test group OPG-A, OPG-B, OPG-C and OPG-D are filled Stomach is administered orally, and dosage is a 50nmol/kg body weight/.20min pneumoretroperitoneums injectable dextrose monohydrate (i=1.5g/kg body weight). Blood is taken to measure blood glucose value from the tail vein of mouse in t=0min, 20min, 40min, 60min, 120min respectively.As a result as schemed Shown in 3.Compared with PBS control group, experimental group has certain blood sugar reducing function.

Although being illustrated and described particular implementation of the present invention, to those skilled in the art show and What is be clear to is that can carry out various other changes and change without departing from the spirit and scope of the present invention.Therefore, it is intended that covering institute All such changes and change in attached claims, it is fallen within the scope of the present invention.

SEQUENCE LISTING

<110>Remittance bio tech ltd of the China of Beijing hundred hundred

<120>Glucagon-like peptide-1 receptor stimulant fusion protein and its application

<130> 2017

<160> 19

<170> PatentIn version 3.3

<210> 1

<211> 103

<212> PRT

<213>Comma bacillus

<400> 1

Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Tyr His Asn Thr Gln

1 5 10 15

Ile His Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala

20 25 30

Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Thr Phe

35 40 45

Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala

50 55 60

Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala

65 70 75 80

Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile

85 90 95

Ala Ala Ile Ser Met Ala Asn

100

<210> 2

<211> 309

<212> DNA

<213>Comma bacillus

<400> 2

acacctcaaa atattactga tttgtgtgca gaataccaca acacacaaat acatacacta 60

aatgataaga tattttcgta tacagaatct ctagctggaa aaagagagat ggctatcatt 120

acttttaaga atggtgcaac ttttcaagta gaagtaccag gtagtcaaca tatagattca 180

caaaaaaaag cgattgaaag gatgaaggat accctgagga ttgcatatct tactgaagct 240

aaagtcgaaa agttatgtgt atggaataat aaaacacctc atgcgattgc cgcaattagt 300

atggcaaat 309

<210> 3

<211> 31

<212> PRT

<213>Artificial sequence

<400> 3

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

1 5 10 15

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

20 25 30

<210> 4

<211> 93

<212> DNA

<213>Artificial sequence

<400> 4

catgccgagg gcaccttcac cagcgacgta agcagctatc tggagggaca ggccgctaag 60

gagttcatcg cttggcttgt aaaaggaaga gga 93

<210> 5

<211> 31

<212> PRT

<213>Artificial sequence

<400> 5

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

1 5 10 15

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

20 25 30

<210> 6

<211> 93

<212> DNA

<213>Artificial sequence

<400> 6

catggtgagg gcaccttcac cagcgacgta agcagctatc tggagggaca ggccgctaag 60

gagttcatcg cttggcttgt aaaaggaaga gga 93

<210> 7

<211> 117

<212> DNA

<213>Artificial sequence

<400> 7

catggtgaag gaacatttac cagtgacttg tcaaaacaga tggaagagga ggcagtgcgg 60

ttatttattg agtggcttaa gaacggagga ccaagtagcg gggcacctcc gccatcg 117

<210> 8

<211> 39

<212> PRT

<213>Artificial sequence

<400> 8

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

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 9

<211> 57

<212> DNA

<213>Artificial sequence

<400> 9

ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcgcgcgc taggcgg 57

<210> 10

<211> 19

<212> PRT

<213>Artificial sequence

<400> 10

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg

1 5 10 15

Ala Arg Arg

<210> 11

<211> 27

<212> DNA

<213>Artificial sequence

<400> 11

ggcggtggcg gatcgcgcgc taggcgg 27

<210> 12

<211> 9

<212> PRT

<213>Artificial sequence

<400> 12

Gly Gly Gly Gly Ser Arg Ala Arg Arg

1 5

<210> 13

<211> 459

<212> DNA

<213>Artificial sequence

<400> 13

acacctcaaa atattactga tttgtgtgca gaataccaca acacacaaat acatacacta 60

aatgataaga tattttcgta tacagaatct ctagctggaa aaagagagat ggctatcatt 120

acttttaaga atggtgcaac ttttcaagta gaagtaccag gtagtcaaca tatagattca 180

caaaaaaaag cgattgaaag gatgaaggat accctgagga ttgcatatct tactgaagct 240

aaagtcgaaa agttatgtgt atggaataat aaaacacctc atgcgattgc cgcaattagt 300

atggcaaatg gtggaggcgg ttcaggcgga ggtggctctg gcggtggcgg atcgcgcgct 360

aggcggcatg ccgagggcac cttcaccagc gacgtaagca gctatctgga gggacaggcc 420

gctaaggagt tcatcgcttg gcttgtaaaa ggaagagga 459

<210> 14

<211> 459

<212> DNA

<213>Artificial sequence

<400> 14

acacctcaaa atattactga tttgtgtgca gaataccaca acacacaaat acatacacta 60

aatgataaga tattttcgta tacagaatct ctagctggaa aaagagagat ggctatcatt 120

acttttaaga atggtgcaac ttttcaagta gaagtaccag gtagtcaaca tatagattca 180

caaaaaaaag cgattgaaag gatgaaggat accctgagga ttgcatatct tactgaagct 240

aaagtcgaaa agttatgtgt atggaataat aaaacacctc atgcgattgc cgcaattagt 300

atggcaaatg gtggaggcgg ttcaggcgga ggtggctctg gcggtggcgg atcgcgcgct 360

aggcggcatg gtgagggcac cttcaccagc gacgtaagca gctatctgga gggacaggcc 420

gctaaggagt tcatcgcttg gcttgtaaaa ggaagagga 459

<210> 15

<211> 453

<212> DNA

<213>Artificial sequence

<400> 15

acacctcaaa atattactga tttgtgtgca gaataccaca acacacaaat acatacacta 60

aatgataaga tattttcgta tacagaatct ctagctggaa aaagagagat ggctatcatt 120

acttttaaga atggtgcaac ttttcaagta gaagtaccag gtagtcaaca tatagattca 180

caaaaaaaag cgattgaaag gatgaaggat accctgagga ttgcatatct tactgaagct 240

aaagtcgaaa agttatgtgt atggaataat aaaacacctc atgcgattgc cgcaattagt 300

atggcaaatg gcggtggcgg atcgcgcgct aggcggcatg gtgaaggaac atttaccagt 360

gacttgtcaa aacagatgga agaggaggca gtgcggttat ttattgagtg gcttaagaac 420

ggaggaccaa gtagcggggc acctccgcca tcg 453

<210> 16

<211> 31

<212> DNA

<213>Artificial sequence

<400> 16

gttacatatg acacctcaaa atattactga t 31

<210> 17

<211> 33

<212> DNA

<213>Artificial sequence

<400> 17

cttagcggcc gctaatcctc ttccttttac aag 33

<210> 18

<211> 34

<212> DNA

<213>Artificial sequence

<400> 18

cttagcggcc gctaacgatg gcggaggtgc cccg 34

<210> 19

<211> 42

<212> DNA

<213>Artificial sequence

<400> 19

gttaggatcc gatgacgatg ataaaacacc tcaaaatatt ac 42

Claims (11)

1. a kind of fusion protein, it includes protein for treatment agent and receptor-binding agents, wherein the protein for treatment agent be GLP-1 peptides or Its analog, and the receptor-binding agents are b subunit of cholera toxin or its analog.

2. fusion protein as claimed in claim 1, wherein the protein for treatment agent is with the sequence as described in SEQ ID NO.3 The GLP-1 peptides of row, or with the sequence have 80% homogeneity GLP-1 peptide analogues.

3. fusion protein as claimed in claim 1, wherein the receptor-binding agents are with the sequence as described in SEQ ID NO.1 The b subunit of cholera toxin of row, or with the sequence have 80% homogeneity cholera mycin B subunit analogs.

4. fusion protein as claimed in claim 1, in addition to for connecting the connexon of protein for treatment agent and receptor-binding agents, The connexon is cracked by intracellular enzyme or environmental factor.

5. the nucleotide sequence of coding fusion protein as claimed in claim 1.

6. the expression vector of expression fusion protein as claimed in claim 1, it includes nucleic acid sequence as claimed in claim 5 Row.

7. fusion protein as claimed in claim 2, wherein described GLP-1 peptide analogues, it includes SEQ ID NO.3 The mutant of GLP-1 peptides.

8. fusion protein as claimed in claim 7, wherein the mutant of described GLP-1 peptides, is to have such as SEQ ID NO.5 The GLP-1 peptides of described sequence.

9. fusion protein as claimed in claim 2, wherein the GLP-1 peptide analogues, it includes SEQ ID NO.8 GLP-1 The analog Ai Saina peptides (E4) of peptide.

10. a kind of fusion protein compositions, it includes fusion protein and auxiliary material as claimed in claim 1, and the auxiliary material includes： Capsulae enterosolubilis, balance salt, sugar and amino acid.

11. the fusion protein or fusion protein compositions as claimed in claim 10 as described in claim any one of 1-9 exist Prepare for treat following disease medicine in application, the disease includes：Type i diabetes, II diabetes, alzheimer ' Silent disease, senile dementia, parkinsonism etc..