CN1935846A - Fusion protein for treating diabetes, and its preparing method and use - Google Patents
Fusion protein for treating diabetes, and its preparing method and use Download PDFInfo
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Abstract
The invention relates to fusion protein used to prevent and cure I type and II type diabetes and its preparation method and application. It supplies the fusion protein which is formed by glucagon analogy peptide GLP-1, GLP-1 mutant and IgG-Fc, its analogy factor lizard salivary gland polypeptide extendin-4 and IgG-Fc, and the application of the above fusion protein and its DNA used in diabetes prevention and therapy. The fusion protein can increase not only GLP-1 effect, but also the affinity and immunological tolerance of ligand, which is excreted with the form of homogeneity dimmer to improve polypeptide drug effect, overcomes the defect of the GLP-1 for short half-life, and simplifies purification process.
Description
Technical field
The present invention relates to biological technical field.Particularly, the present invention relates to be used to prevent and treat the fusion rotein of I type and type ii diabetes, and its production and application.
Background technology
Diabetes are to cause human dead main diseases therefore, seriously jeopardize human health.Type i diabetes and type ii diabetes are two kinds of principal modes of diabetes.Apoptosis is that programmed death is I type and type ii diabetes islet cells main causes of death.I type and type ii diabetes people have showed progressive pancreas islet B-cell quality minimizing and the B-cell function reduces.
Insulin treatment is the main means of treatment type 1 diabetes.Pancreatic islets transplantation also is a kind of effective methods of treatment, but is restricted owing to donor is limited, moreover acceptor (patient) also will replenish immunosuppressor throughout one's life.The ordinary method of treatment type ii diabetes, comprise and going on a diet, physical exercise, and use the sulfo group urea, metformin and Regular Insulin are treated, but most of patients all can't reverse the lasting decline of glycemic control ability, the minimizing of B cell quality and the reduction of function usually.Finally also can't keep the glycemic control level at the stepped methods of treatment that type ii diabetes adopted clinically, repeat inevitably from reduce feed, strengthen taking exercise, to the process of using the single medicine treatment, this course of disease of insulinize being increased the weight of gradually to combination drug treatment and Zhongdao.These treatments can not be alleviated the process of type ii diabetes effectively, can not prevent complication effectively again, and tracing it to its cause may be that traditional treatment lays particular emphasis on and alleviates as cardinal symptoms such as hyperglycemia, rather than at the real pathogenic factor of type ii diabetes.
Therefore, adopt a kind of effective therapeutic strategy, be target with the molecule mechanism that discloses disease formation, and be not the PD that only prevents and reverse type ii diabetes, just seem very urgent at symptom.Although whether disorderly and insulin resistant effect is exactly that the cause of disease still has considerable arguement around the B cell function, the two is all generally acknowledged in the onset diabetes process and has been played vital role at present.
The similar peptide of hyperglycemic-glycogenolytic factor (GLP-1, the aminocompound from 7 to 36 amino acids) is a kind of main physiological insulin-like hormone.Because the plain sample effect of the pancreotropic hormone sample effect of natural GLP-1 uniqueness and hyperglycemia, and, therefore representing great pharmaceutical application prospect aspect the treatment diabetes to the trophism of pancreas B cell.The therapeutic goal that this point and clinical treatment diabetes are reformulated is perfectly in harmony.The significance of its pharmacy also is, compares with a line medicine of treatment diabetes such as the sulfo group urea of current use, Regular Insulin, and it can't produce side effect such as put on weight.In fact,, can reduce the picked-up of food naturally, therefore limit the increase of weight in patients, even might cause the reduction of body weight because GLP-1 can promote the full sensation that rises.
The main difficulty for the treatment of is that some enzymes (as dipeptides acyl pepx (DPPIV)) can make the GLP-1 rapid deactivation, adds the discharge effect of kidney to GLP-1 again, make that the transformation period of GLP-1 itself is very short (t1/2＜2min).So the subcutaneous perfusion of taking persistence can be kept the effect of GLP-1 in the body.Although the DPPIV inhibitor can prolong the transformation period of GLP-1 and just test in clinical experiment, but this method still lacks specificity, this is because DPPIV can also make other several polypeptide hormones and chemokine inactivation simultaneously, and it is suppressed to produce various adverse effects.
Exendin-4 (Ex-4) is a kind of lizard sialisterium polypeptide of 39 residues, and pharmaceutical properties is similar to GLP-1.Opposite with GLP-1, Ex4 can form the monomer spiral in the aqueous solution.This spiral shows rare thermostability because the head and the tail of hydrophobicity C end interact.Because GLP-1 lacks the interaction of C end head and the tail, himself snappiness and 16 the sour residue of sweet base (being meant that glycine the still is amino acid whose residue) factor that is considered to reduce monomer spiral status stability.And the normotopia effect of the stability of Ex4 spiral inside can reduce bind receptor the time, this is in conjunction with needing C-terminal to keep spiral status.
In addition, albumin link coupled GLP-1 (Albugon) has and the long transformation period and the function for the treatment of diabetes too.
Showing very big pharmaceutical use prospect although have the GLP-1 receptor stimulant of DPP-IV-resistance as Ex4 aspect the treatment diabetes, these polypeptide need to inject 1-2 time every day or cooperate oral pharmaceutical to use.Because the Albugon molecule is bigger, and is less by the kidney output, thereby also have the long transformation period.Yet the experiment in vitro result shows that the effect of fusion rotein is not as Ex-4.And, the more important thing is that polypeptide drug can produce dangerous sensitivity response.This makes people drop into more energy and goes to develop that effect is more lasting in vivo, the effector molecule that stability is higher.
Summary of the invention
One of purpose of the present invention provides a kind of nucleotide sequence that can prevent and treat the fusion rotein of I type and type ii diabetes and this fusion rotein of encoding, such fusion rotein has the DPPIV resistance, can promote B cell propagation, regeneration and reduce apoptosis, thereby increase the islet cells quality.Such fusion rotein can promote pancreas B emiocytosis Regular Insulin and to the tolerance of glucose, activate cAMP and its coupling second messenger's approach that joins and the expression that promotes protein kinase (Aktl and MAPK) in the B cell and increase its content, reduce the activity of caspase-3.
Described fusion rotein can be the fusion rotein of hyperglycemic-glycogenolytic factor similar peptide GLP-1-1 with IgG-Fc, and IgG-Fc wherein is IgG1, IgG2 or IgG3; Also can be for having the same merit factor lizard sialisterium polypeptide extendin-4 of similar sequence and biological property and the fusion rotein of IgG-Fc with GLP-1, IgG-Fc wherein is IgG1, IgG2, IgG3 or IgG4; Described fusion rotein can also be mutant GPL-1-A8G-IgG-Fc, wherein the 8th of the aminoacid sequence of GPL-1 the L-Ala is replaced by glycine or by other aminoacid replacement, perhaps other amino acid modified mutant GPL-1 that makes of GLP-1 sequence is tolerated its degrading enzyme (as dipeptides acyl pepx, DPPIV).
A further object of the present invention provides the expression vector that comprises above-mentioned fusion and as the application of nucleic acid vaccine.
A further object of the present invention provides the mammalian expression system and the bacterial expression system of above-mentioned fusion rotein.
A further object of the present invention provides a kind of method for preparing the above-mentioned fusion rotein of biologically active.
A further object of the present invention provides the application that above-mentioned fusion rotein and dna sequence dna and analogue thereof are used to prepare prevention and treat the medicine of diabetes.
The present inventor proposes and finishes the present invention to achieve these goals.
According to technical scheme of the present invention, the invention provides a kind of fusion rotein, described fusion rotein is hyperglycemic-glycogenolytic factor similar peptide GLP-1-1 or has the same merit factor of 50% above homology and the fusion rotein of IgG-Fc with it.Described IgG-Fc is mouse IgG-Fc or human IgG-Fc, and IgG wherein is IgG1, IgG2 or IgG3.
According to fusion rotein of the present invention, the same merit factor of described GLP-1 can be lizard sialisterium polypeptide extendin-4, resulting fusion rotein is Extendin4-IgG-Fc, and wherein IgG-Fc is mouse IgG-Fc or human IgG-Fc, and IgG wherein is IgG1, IgG2, IgG3 or IgG4.
According to technical scheme of the present invention, by using the crossover round pcr with people GLP-1 (7-37, its sequence and mouse GLP-1 are in full accord) thereby and mouse IgG-Fc nucleotide sequence reorganization expressed fusion protein, therefore the invention provides the fusion rotein of a kind of hyperglycemic-glycogenolytic factor similar peptide GLP-1-1 and mouse IgG-Fc, be GLP-1-IgG1-Fc, it has the aminoacid sequence shown in SEQ ID No.1.The IgG-Fc district of fusion rotein comprises the constant heavy chain district (portion C H1, hinge, CH2 and CH3) of IgG1.The present invention also uses with the recombinated fusion rotein of GLP-1 and human IgG-Fc of quadrat method, i.e. GLP-1 and human IgG2-Fc nucleotide sequence reorganization produces people GLP-1-IgG2-Fc fusion rotein, and it has the aminoacid sequence shown in SEQ ID No.2.The IgG-Fc district of fusion rotein comprises the constant heavy chain district (hinge, CH2 and CH3) of IgG2.Therefore the invention provides the fusion rotein of a kind of hyperglycemic-glycogenolytic factor similar peptide GLP-1-1 and mouse or human IgG-Fc,
SEQ?ID?No.1：
GLP-1
His?Ala?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gln?Ala?Ala
Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg-NH
2
Mouse IgG-Fc
Pro?Ser?Glu?Thr?Val?Thr?Cys?Asn?Val?Ala?His?Pro?Ala?Ser?Ser
Thr?Lys?Val?Asp?Lys?Lys?Ile?Val?Pro?Arg?Asp?Cys?Gly?Cys?Lys?Pro
Cys?Ile?Cys?Thr?Val?Pro?Glu?Val?Ser?Ser?Val?Phe?Ile?Phe?Pro?Pro
Lys?Pro?Lys?Asp?Val?Leu?Thr?Ile?Thr?Leu
Thr?Pro?Lys?Val?Thr?Cys?Val?Val?Val?Asp?Ile?Ser?Lys?Asp?Asp
Pro?Glu?Val?Gln?Phe?Ser?Trp?Phe?Val?Asp?Asp?Val?Glu?Val?His
Thr?Ala?Gln?Thr?Gln?Pro?Arg?Glu?Glu?Gln?Phe?ASn?Ser?Thr?Phe
Arg?Ser?Val?Ser?Glu?Leu?Pro?Ile?Met?His?Gln?Asp?Trp?Leu?Asn
Gly?Lys?Glu?Phe?Lys?Cys?Arg?Val?Asn?Ser?Ala?Ala?Phe?Pro?Ala
Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Thr?Lys?Gly?Arg?Pro?Lys?Ala
Pro?Gln?Val?Tyr?Thr?Ile?Pro?Pro?Pro?Lys?Glu?Gln?Met?Ala?Lys
Asp?Lys?Val?Ser?Leu?Thr?Cys?Met?Ile?Thr?Asp?Phe?Phe?Pro?Glu
Asp?Ile?Thr?Val?Glu?Trp?Gln?Trp?Asn?Gly?Gln?Pro?Ala?Glu?Asn
Tyr?Lys?Asn?Thr?Gln?Pro?Ile?Met?Asn?Thr?Asn?Gly?Ser?Tyr?Phe
Val?Tyr?Ser?Lys?Leu?Asn?Val?Gln?Lys?Ser?Asn?Trp?Glu?Ala?Gly
Asn?Thr?Phe?Thr?Cys?Ser?Val?Leu?His?Glu?Gly?Leu?His?Asn?His
His?Thr?Glu?Lys?Ser?Leu?Ser?His?Ser?Pro?Gly?Lys
IgK
Met?Glu?Thr?Asp?Thr?Leu?Leu?Leu?Trp?Val?Leu?Leu?Leu?Trp?Val?Pro?Gly?Ser
Thr?Ser?Asp
SEQ?ID?No.2：
GLP-1
His?Ala?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu?Glu?Gln?Ala?Ala
Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg-NH
2
Human IgG2-Fc
Glu?Arg?Lys?Cys?Cys?Val?Glu?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Pro?Val?Ala?Gly
Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg
Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val
Gln?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro
Arg?Glu?Glu?Gln?Phe?Asn?Ser?Thr?Phe?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Val
His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Gly
Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Thr?Lys?Gly?Gln?Pro?Arg?Glu
Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Glu?Glu?Met?Thr?Lys?Asn?Gln?Val
Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Gln?Trp
Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Met?Leu?Asp
Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp
Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His
Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys
GHRH
Val?Leu?Trp?Val?Phe?Phe?Phe?Val?Ile?Leu?Thr?Leu?Ser?Asn?Ser?Ser?His?Cys
Ser
The present invention's fusion rotein designed and that make up is the new molecule that the GLP-1 molecule combines with the IgG-Fc molecule and forms, and it has double dominant, has not only strengthened the effect of GLP-1 but also the avidity and the immunotolerance of the part that increased owing to the IgG-Fc molecule.
The present invention's fusion rotein designed and that make up also is to have the GLP-1 molecule derivant of DPPIV resistance such as a kind of new molecule of GLP-1A8G and the formation of IgG-Fc molecule, and therefore constructed fusion rotein has enhanced GLP-1 effect and above-mentioned IgG-Fc molecule advantage.
Similarly, according to another technical scheme of the present invention, the present invention is designed to be to have similar aminoacid sequence of GLP-1 and the bioactive a kind of recruit who forms with merit factor lizard sialisterium polypeptide extendin-4 and people's IgG2-Fc molecule with construction of fusion protein, be Ex4-IgG-Fc, it has the aminoacid sequence shown in SEQ ID No.3.This fusion rotein has the effect of strong similar GLP-1 and keeps the advantage of IgG-Fc molecule, and the sample of wherethrough reason is consistent with the effect of the sample that GLP-1/IgG-Fc handles.
SEQ?ID?No.3：
Ex4
His?Gly?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Lys?Gln?Met?Glu?Glu?Glu?Ala
Val?Arg?Leu?Phe?Ile?Glu?Trp?Leu?Lys?Asn?Gly?Gly?Pro?Ser?Ser?Gly?Ala?Pro
Pro?Pro?Ser-NH
2
Human IgG2-Fc
Glu?Arg?Lys?Cys?Cys?Val?Glu?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Pro?Val?Ala?Gly
Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg
Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val
Gln?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro
Arg?Glu?Glu?Gln?Phe?Asn?Ser?Thr?Phe?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Val
His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Gly
Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Thr?Lys?Gly?Gln?Pro?Arg?Glu
Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Glu?Glu?Met?Thr?Lys?Asn?Gln?Val
Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp
Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Met?Leu?Asp
Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp
Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His
Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys
GHRH
Val?Leu?Trp?Val?Phe?Phe?Phe?Val?Ile?Leu?Thr?Leu?Ser?Asn?Ser?Ser?His?Cys
Ser
The present invention also provides and has expressed above-mentioned Expression of Fusion Protein carrier, can express above-mentioned fusion rotein in the preferred COS-7 clone of mammalian expression system and conventional other clone used and engineering bacterium expression system.
Therefore, the present invention also provides a kind of method for preparing the above-mentioned fusion rotein of biologically active, this method comprises that the advantage of making the IgG-Fc fusion rotein is can obtain product GLP-1-IgG-Fc fusion rotein by a simple step program in the laboratory with GLP-1 or Ex4 fusion in the secretion leader peptide sequences of IgK or GHRH and the above-mentioned fusion rotein.
According to the experimental result of specific embodiments of the present invention, the DNA of fusion rotein of the present invention or encoding fusion protein can be used to prepare the medicine that is used for the treatment of I type and type ii diabetes.
Preparation according to the present invention has the method for bioactive fusion rotein, can directly the synthetic polypeptide be secreted in the substratum by secretion leader peptide sequences and the GLP-1 sequence that merges IgK or GHRH, fusion rotein in secretion process after proteolytic cleavage removes its leading peptide, the active histidine residues of fusion rotein N-terminal will be merged mutually with GLP-1 (or Ex4), utilize G albumen dextrane gel can finish the purification of fusion rotein a step then.The advantage of this fusion method is can obtain product GLP-1-IgG-Fc fusion rotein by a simple step program in the laboratory, and this method also can be used for industrial production.Simultaneously, this method can reach: 1) GLP-1-IgG-Fc (Ex-4-IgG-Fc) fusion rotein is with the dimeric form secretion of homogeneity, overcome GLP-1 short shortcoming of existing transformation period, and owing to its higher part affinity makes fusion rotein have higher effect; 2) a large amount of GPCR exist with dimeric precursor forms at cell surface, thereby have strengthened the drug effect of polypeptide; 3) simplified purge process.Utilize G albumen dextrane gel a step to finish purification, be beneficial to the separation and purification of fusion rotein.
According to technical scheme of the present invention, integrated the non-variable region gene of GLP-1 active gene and immunoglobulin G (IgG) heavy chain, give expression to fusion rotein GLP-1-IgG-Fc, both prolonged its physiology transformation period, strengthened its GLP-1 effect again and strengthened immunotolerance.
One of concrete application of the present invention be fusion rotein DNA as a kind of therapeutic preparation, be applied to simple and safe non-virus carrier gene therapy.Virus vector is adopted in most so far gene therapy, though this method transfer efficiency is very high, limitation is to be easy to generate the pathogenic virus anaphylaxis.It is more convenient not only to organize by other great majority by the exposed DNA plasmid of muscle tissue injection, and genetically modified expression is more permanent usually.According to specific embodiments of the present invention, adopt the continuous mode that applies low strength of electric field (100-200V/cm) for 6-8 time, relatively grows (20-50mS) square wave electric pulse to transmit the plasmid vector that comprises fusion rotein of the present invention by intramuscular routes.Though this low voltage electricimpulse might make muscle damage to a certain extent, it is general gentle and instantaneous, and result of study shows electrotransfer after 2 weeks, and it is normal that muscle recovers basically.
According to of the present invention in intramuscular routes transmits plasmid vector and combination the non-viral gene methods of treatment of the treatment diabetes of electrotransfer be proved to be effective, safe and easy.This method is versatile and flexible, and is applicable to and uses cytokine, polypeptide hormone, and soluble receptors and many membranins and cytoplasm protein carry out gene therapy.This method general provide effective especially aspect protein regulatory factor such as the GLP-1-IgG-Fc, and overcome and use virus vector to carry out sensitization and the pathogenic aspect critical limitations that gene therapy brought up to now.The application of gene therapy described in the present invention shows GLP-1-IgG-Fc, GLP-1A8G-IgG-FC and the Ex4-IgG-Fc carrier is single or both are in conjunction with carrying out intramuscular injection 1-2 time, can obtain with in two weeks every day peritoneal injection long-acting/the identical result of treatment of GLP-1 analogue Ex-4 of brute force.
Therefore, application of the present invention provides a kind of gene therapy of uniqueness to prevention and treatment type i diabetes and type ii diabetes.This gene therapy, has overcome and has used virus vector to carry out sensitization and the pathogenic aspect critical limitations that gene therapy brought up to now as the carrier of transgenosis being gone into body without the virus of any kind.
The present invention is fit to obtain identical result of treatment with above-mentioned fusion rotein direct injection equally.
In addition, the GLP-1-IgG-Fc fusion rotein of prepared according to the methods of the invention divalence has following characteristics: 1) increased transformation period t1/2; 2) high-affinity and vigor; 3) minimized immunogenicity.
Description of drawings
Fig. 1: make up GLP-1-IgG-Fc plasmid synoptic diagram, wherein scheme A and represent mouse GLP-1-IgG1-Fc with figure B; Figure C and figure D represent people GLP-1-IgG2-Fc.
Fig. 2: expression and the detection of IgG-Fc fusion rotein in the COS-7 cell.
Fig. 3: the GLP-1 that detects transfection COS-7 emiocytosis
Fig. 4: (A): COS-7 cell great expression IgG-Fc fusion rotein; (B): the expression of GLP-1 in mammalian cell and intestinal bacteria.
Fig. 5: GLP-1 is in the COS-7 of stably express fusion rotein clone.
Fig. 6: the GLP-1-IgG-Fc fusion rotein influences INS-1 cell Regular Insulin excretory.
Fig. 7: GLP-1-IgG-FC produces cAMP to the INS-1 cell has similar effect to Ex-4.
The effect that Fig. 8: GLP-1-IgG-Fc is tried to express in the mouse at diabetes B model db/db.
Fig. 9: in the effect of the type i diabetes model mouse expression in vivo GLP-1-IgG-Fc of the defect of insulin secretion of inducing formation through STZ, (A) CD1 is tried each 50 μ g of plasmid of mouse intramuscular injection coding GLP-1-IgG-Fc, Ex4-IgG-Fc or IgG-Fc gene, and original position applies electricimpulse to promote the transfection effect simultaneously; (B) tried mouse pancreas tissue slice.
Figure 10: GLP1-IgG-Fc expresses the influence to blood sugar in the large mammal pig
Embodiment
1, the structure of plasmid
For making up GLP-1 and mouse IgG-Fc cDNAs sequence, with GLP-1/PCR2.1 and IgG plasmid is template, is that primer carries out first round crossover PCR (overlapPCR) with following specific sequence 5 '-ccgGATATCgccaccATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTG GGTTCCAGGTTCCACTGGTGACca-3 ' and 5 '-TgctgaagggACctttaccagtg-3 '.With the PCR product is that template is carried out second and taken turns crossover PCR and produce the GLP-1-IgG-FccDNA sequence.With the product subclone of amplification to Bam HI and the Eco RV site of plasmid Vrnew.For making up GLP-1 and human IgG-Fc cDNAs sequence, be template with GLP-1/PCR2.1 and IgG plasmid, make up human IgG with following specific sequence
2(hinge-ch2-ch3), the primer is 5 '-AAGGATATCGATCGCAAATGTTGTGTCGAGTGCCCA-3 ' and 5 '-CGTAAGCTTCATTTACCCGGAGACAGGGAGAG-3 ' to FC.Used carrier is the same.For making up the IgG-Fc control plasmid, with following specific sequence 5 '-ccgGATATCgccaccATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTG GGTTCCAGGTTCCACTGGTGACcccagcgagaccgtcacc-3 ' and 5 '-cgcggatccctaTCATTTACCAGGAGAGTGGGAGAGG-3 ' is that primer carries out PCR, and extension amplification outcome is to Bam HI and the Eco RV site of plasmid Vrnew.
Carrier Vrnew contains cmv enhancer and promotor, single eukaryote transcriptional units, minimum beta sphaeroprotein poly VITAMIN B4 tail of rabbit and transcription termination sequence are the derivative vectors by VR1255 carrier deletion luciferase reporter gene, and have added restriction endonuclease sites.For making the secretion of GLP-1 or Exendin-4 sequence, ' hold and introduce mouse Ig κ chain signal peptide sequence or people GHRH signal peptide sequences in 5 by PCR.For in bacterium, expressing the GLP-1-IgG-Fc fusion rotein, by the fusion cDNA sequence of pcr amplification Vrnew carrier, and subclone to the pET-28a plasmid (Novagen, EMD Bioscience, San Diego, CA).
2, mammal cell line is expressed the GLP-1-IgG-Fc fusion rotein
1) Expression of Fusion Protein
Expression in mammalian cell is that (CA) transfection is to COS-7 clone for Invitrogen, Carlsbad by liposome Lipofectamine2000 with the cDNA of GLP-1-IgG-Fc or IgG-Fc in contrast.Its process is to be every hole 2.5 * 10 with density earlier
5Individual COS-7 cell is grown in 6 porocyte culture plates.Adding the serum-free contain 4 μ gGLP-1-IgG-Fc cDNAs and 10 μ L transfection liposomes and do not have the DMEM nutrient solution (Invitrogen) of antibiotic cultivates at 37 ℃ CO2 incubator.Behind the 6h, be converted to the full substratum of DMEM.48h collects nutrient solution and cell respectively after the transfection.In order to express the GLP-1-IgG-Fc fusion rotein on a large scale, with the COS-7 cell cultures at the 150mm culture dish, use 150mM NaCl with positively charged ion transfection reagent Poly (ethyleneimine) (PEI respectively, 25kDa) with the dilution of 80 μ g Related cDNAs, mix the back and place 20min. and the DNA/PEI mixture is added to fills Tissue Culture Dish that serum-free do not have antibiotic DMEM nutrient solution after 37 ℃ CO2 incubator is placed 6h then, replace with the DMEM nutrient solution that is added with 10%FBS and 1%P/S.The transfection efficiency of this method can be up to 85%.
For setting up the COS-7 clone of stably express GLP-1-IgG-Fc fusion rotein, cell is with every hole 2.5 * 10
5Density cultivate at 6 porocyte culture plates, and according to linearizing GLP-1-IgG-Fc plasmid of above-mentioned steps transfection 4 μ g or IgG-Fc plasmid in contrast.24h after the transfection, cell is cultivated at the DMEM nutrient solution that contains G418 (500 μ g/mL), to screen the cell of the external source of stable integration reorganization cDNA.In the culturing process, every 3d changes nutrient solution and once forms until the cell clone of stable expression of exogenous gene.Separate monoclonal cell and cultivate into stable expression cell line.Cultivation detects fusion rotein with mouse GLP-1RIA test kit respectively at the cell and the cell culture fluid of 24 well culture plates.Select the cell that to secrete fusion rotein and be used as later specificity analysis.
2) analysis of expressing fusion protein characteristic
In order to evaluate plasmid in the ability of expressing with secretion GLP-1-Fc fusion rotein, the fusion rotein plasmid transfection of structure transient expression to the COS-7 cell.48h after the transfection extracts total RNA and assesses expression effect with RT-PCR in transfectional cell.The applying gene Auele Specific Primer, detect the GLP-1-Fc antigen-4 fusion protein gene on transcriptional level expression and the expression of IgG-Fc crt gene (Fig. 2 a).In the untransfected control sample, do not detect the expression of transcriptional level.
The COS-7 cell lysate of transfection and cell culture medium have been detected to determine the expression of fusion rotein on translation skill with Western immunity marking method.The results are shown in Figure 2b.Simultaneously in nutrient solution and cell lysate, detect fusion rotein.The electrophoretic migration position of fusion rotein is the monomeric molecular weight of fusion rotein under the SDS-PAGE condition greatly about 37kDa.In nutrient solution and cell lysate, detect fusion rotein simultaneously and show fusion rotein synthetic justacrine in mammalian cell.The GLP-1 fusion rotein also further uses radioimmunology (RIA) to be identified that this method can detect the GLP-1 that exists with form of ownership.COS-7 clone also respectively transfection the GLP-1-Fc/Vmew plasmid of different concns or transfection Fconly/Vrnew plasmid in contrast.48h collecting cell nutrient solution is used for that GLP-1RIAs analyzes so that total GLP-1 secretory volume of determining to take advantage of a situation and expressing after the transfection.And in the COS-7 cell of transfection Fc-only/Vrnew vector plasmid in contrast, do not detect the existence of GLP-1.The content and the DNA transfection amount that also detect GLP-1 from the cell of transfection GLP-1-Fc/Vrnew vector plasmid are proportionate.The results are shown in Figure 3.The DNA that has used 0.8 μ g is to 1.25 * 10
5Individual cell/mL carries out transfection.Then from the COS-7 emiocytosis substratum of 50mL, detect the GLP-1 that total amount is 100 μ mol.
3, purifying GLP-1-Fc fusion rotein from mammalian cell cultures
For a large amount of fusion roteins that obtain, use the protein purification that G albumen sephadex column can carry out middle scale.The nutrient solution of the COS-7 cell of the transfection fusion rotein plasmid that the column volume of 50ml is grown in can purifying 15cm culture dish.Behind the albumen wash-out with purifying, cumulative volume is 1mL after the neutralization, desalination, the results are shown in (Fig. 4 A), show under natural condition, to have dimeric GLP-1-Fc fusion rotein.
And two step elution processs can be with most of fusion rotein by eluting on the sephadex column.The component of sample dyes with appraisal productive rate and purifying rate through the SDS-PAGE separation and through Coomassie brilliant blue.The result shows every ml nutrient solution inoculation 1.25 * 10
5Behind the cell cultures 2d, can obtain the fusion rotein of about every milliliter 6 μ g.
Particularly, the specific practice of extracting purifying on a small scale is that the 2.5mL substratum is got in every hole from 6 well culture plates of cultivating the Mammals transfectional cell, be transferred to what the damping fluid balance that contains 100mM Tris pH8.0 and 150mM NaCl was crossed and the full volume of 70 μ L be housed in advance in conjunction with the proteic dextrane gel 4 quick wash-out resin (Amersham-Pharmacia of G, Piscataway, NJ).After spending the night through 4 ℃,, contain the sample buffer of SDS with the fusion rotein wash-out with 30 μ L then with the flushing of Tris damping fluid.
It is to be used for obtaining relatively large fusion rotein that middle scale is extracted purifying.Its concrete steps are the mammalian cells of 48h after the transfection, or from the cell of stably express, collect the DMEM nutrient solution of 50mL.The full volume of the 1mL that packs into is in advance in conjunction with the proteic sephadex column of G (Amersham-Pharmacia). and under 1%Triton X-100 4 ℃ spend the night.Then repeatedly with the PBS flushing that contains 0.1%Triton X-100, final step is washed with 150mM NaCl.With 1mL 0.1M glycine (pH2.7) wash-out fusion rotein.Elutriant is also used the PBS wash-out with Tris damping fluid (pH9.0) neutralization, fusion rotein with PD-10 post (Amersham-Pharmacia) desalination.The final volume of albumen behind above wash-out, neutralization, desalination through the gel column purifying is 1mL.
4, bacterial expression GLP-1-IgG-Fc fusion rotein
In bacterial cell system, express the GLP-1-IgG-Fc fusion rotein; be with GLP-1-IgG-Fc/pET28a plasmid or general IgG-Fc/pET28a plasmid transformation escherichia coli Rosettagami 2 clones (Novagen in contrast; EMD biosciences, San Diego, CA).Method can be traditional Calcium Chloride Method or use electroporation apparatus and carry out the electricity conversion.Its concrete parameter can be found from any molecular biology experiment handbook.Bacterium after the conversion is coated the culture dish incubated overnight, picks and screen the bacterium bacterial plaque of optimum expression fusion rotein.Pick 2X YT nutrient solution that single bacterial plaque is added with kantlex at 50mL in 37 ℃ with the 225rpm incubated overnight.Then nutrient solution was diluted to new nutrient solution with 1: 50, after cell density reaches O.D600 reading value 0.6, in nutrient solution, adds the IPTG (EMD) of 1mM again to induce expressing fusion protein.Add the back centrifugal collection bacterial cell of 3hr and spend preservation-80.
Fusion rotein by purifying in mammalian cell and the bacterium further passes through total GLP-1RIA, determines the expression of GLP-1.Resulting fusion rotein quantity all becomes positive correlation with the dna vector quantity that transforms in two kinds of express cells, but the amount of the GLP-1 fusion rotein of expressing in bacterium is lower than the expression amount that obtains in mammalian cell, the results are shown in Figure 4 (B).
5, purifying GLP-1-IgG-Fc fusion rotein from bacterial cultures
Use with the above-mentioned method that purified fusion protein is identical from mammalian cell cultures also can be from bacterium purified fusion protein.The cold PBS damping fluid that conversion is had the bacterium incubated overnight of antigen-4 fusion protein gene plasmid, the precipitation after next day is centrifugal to be dissolved in again to contain the multiple protein enzyme inhibitors and by the ultrasonic treatment cell.It is centrifugal behind the adding 1%Triton X-100 maintenance 30min in the damping fluid that (12,000g 10min), obtains containing the supernatant liquor of fusion rotein.Its through the process of the process of gel column purifying and wash-out, neutralization, desalination to above-mentioned similar.
6, the COS-7 emiocytosis GLP-1-IgG-Fc fusion rotein of stably express
Secrete GLP-1 through the G148 screening with RIA checking clone, set up the COS-7 clone of stably express GLP-1-Fc fusion rotein.With the baseline of total GLP-1 level in the growth stable cell lines nutrient solution, the results are shown in Figure 5 as assessment emiocytosis GLP-1-Fc fusion rotein level.All are only expressed the proteic control group excretory of Fc GLP-1 level and all are lower than nutrient solution GLP-1 reference line.Separate secretion GLP-1-Fc fusion rotein amount and be higher than a few strain stably express cells of baseline and set up stable expression cell line, the results are shown in Figure 5.However, secretion level only is higher than 2 times in baseline.
7, the external feature experiment of GLP-1-IgG-Fc fusion rotein
Natural GLP-1 can rely on glucose level stimulates Mammals B emiocytosis Regular Insulin.For whether assessment has biological function by the GLP-1-Fc fusion rotein of mammal cell line secretion purifying, the influence of the secreting function of the INS-1 clone of the tool insulin secretion function that the clone is cultivated is assessed.Apply GLP-1-Fc fusion rotein behind the purifying of various dose through the INS-1 cell of serum-free culture and glucose hunger, add 0.5 or the glucose of 20mM simultaneously.Shown in Figure 6, in the absence that glucose exists, the GLP-1-Fc fusion rotein can not promote the insulin secretion of beta cell, yet, under the situation of 5mM or the existence of 20mM glucose, the GLP-1-Fc fusion rotein promotes B cell secretion of insulin according to the height of dosage.This result shows that the GLP-1-Fc fusion rotein has biologic activity, according to the height promotion INS-1 emiocytosis Regular Insulin of dosage.
8, the GLP-1-Fc fusion rotein is induced cAMP
The INS-1 cell is cultivated at 24 porocyte culture plates with the density of 62,500 cells in every hole.Before the experiment of next day, cell is cultivated 5h in the serum-free SF-RPMI substratum of the 450 μ mL that are added with 100 μ M IBMX.Then cell adds the freezing ethanol of 1mL and finishes reaction after the SF-RPMI nutrient solution of Fc fusion polypeptide (120nM) that is added with purification or Exendin-4 (100nM) is cultivated 10min.Extract remains under-20 ℃ of conditions 3h or spends the night.Then with per 200 μ L packing and freeze-drying.Freeze dried sample be dissolved in again 50 μ L sodium-acetate buffers and with cAMP RIAs test kit detect (Biomedical Technologies, Stoughton, MA).
Fig. 7 shows, when glucose content is zero, measures cAMP with cAMP level in the INS-1 cell of 120nM GLP-1-Fc fusion rotein processing as baseline values.Under the condition of 5mM glucose, the INS-1 cell cAMP level of handling through the GLP-1-Fc fusion rotein significantly increases, with the cell cAMP level of handling through exendin-4 much at one.This result shows, the generation that the GLP-1-Fc fusion rotein stimulates cAMP in the INS-1 cell in the mode according to the glucose concn height.
9, the generation of GLP-1-IgG-Fc fusion rotein prevention db/db mouse diabetes
Carried out functional experiment in the body of GLP-1/IgG fusion rotein with diabetes animal model db/db mouse.The db/db mouse in 4 ages in week respectively intramuscular injection GLP-1-IgG-Fc fusion rotein plasmid and IgG-Fc plasmid in contrast.And the electricimpulse of using the generation of electrotransfer instrument is strengthened the transfection effect.In 2 weeks of back of injection for the first time, carried out the 2nd injection again.Body weight and the glucose level of being tried mouse are monitored weekly.In 2 weeks before injection and after the injection for the first time, 12 weeks were collected fasting Regular Insulin and glucagon content in the saphena blood measuring body.The intravital expression amount of GLP-1-IgG-FcGLP-1-IgG-Fc fusion rotein is to determine with the active GLP-1 content of the Linco GLP-1 Elisa of company kit detection cell matter.To GLP-1 in the gene therapy mouse body, Regular Insulin and the demonstration of glucagon measuring result, in 2 weeks of back of injection for the first time, the mouse of being tried of injection GLP-1-IgG-FcGLP-1-IgG-Fc plasmid significantly improves than injection contrast IgG-Fc plasmid serum GLP-1 level.And after 16 weeks, being had its value of falling after rise still to be higher than baseline values though try mouse serum GLP-1 level, the result is shown in Fig. 8 A.
In experimentation, the significant variation do not take place in two groups of body weight of being tried mouse.In first month after the injection, two groups are tried mouse fasting serum glucose level and are not also all had to take place significant the variation.Yet after 12 weeks of injection, the mouse fasting serum glucose concentration of being tried of injecting the GLP-1-IgG-FcGLP-1-IgG-Fc plasmid significantly is lower than control group (P＜0.001), and result such as Fig. 8 B show.And the mouse fasting insulin level that tried of GLP-1-IgG-Fc carrier is significantly higher than the control group of injecting the IgG-Fc carrier, shows as Fig. 8 C.Fasting glucagon level is tried mouse at GLP-1 and is lower than contrast mouse (P＜0.05) (Fig. 8 D).This result shows that expression in vivo GLP-1-IgG-Fc albumen is tried the effect that mouse truly has lowering blood glucose to diabetes, and its reason is likely owing to promoted secretion of insulin and reduced due to the secretion of basic glucagon.
10, the GLP-1-IgG-Fc fusion rotein can prevent the generation (type i diabetes model) by STZ inductive insulin deficit diabetes
Nearest result of study shows that secretin has effect to the adjusting and the treatment of type i diabetes blood sugar.In order to determine the provide protection of GLP-1-IgG-Fc gene therapy to the B cell damage, we study the CD1 model mouse with streptozotocin (STZ).With GLP-1-Fc, Ex4-Fc or Fc plasmid (50 μ g/ individuality) intramuscular injection is tried mouse to CD1 respectively, strengthens the transfection effect with the electricimpulse that the electrotransfer instrument produces.After the DNA injection 7 days, and continuous 5 days injection STZ (55mg/kg, i.p.).The result shows; Fc control group blood-sugar content significantly increases; only the standard that just reached diabetes in several days (＞17mM), but the mouse of being tried of injecting GLP-1-IgG-Fc (or Ex4-IgG-Fc) is protected after STZ handles, its glucose level remains on the lower limit (Fig. 9) of diabetes.
Pancreas Histochemical studies result shows the infringement that is tried all to have showed with control group B cell in various degree, and 2 groups are tried the profit of invading that monocyte (lymphocyte and scavenger cell) all taken place the mouse pancreas islet.But at GLP-1-IgG--Fc (or Ex4-IgG-Fc) treatment group degree of damage light (Fig. 9).Although what is interesting is that degraded has resistant function to EX4-Fc to DPPIV, result and GLP-1-Fc treatment effect that Ex4-Fc handles are about the same.Although these discoveries show pancreas islet and are inflamed (insulitis) that the expression of GLP-1-IgG-Fc (or Ex4-IgG-Fc) has been protected by STZ inductive islet cells and damaged.
11, expression in vivo GLP1-IgG-Fc and to the influence of large mammal pig blood sugar
GLP-1-IgG-Fc or the intramuscular injection of contrast IgG-Fc plasmid are arrived the Yorkshire boar, and every 4mg/23kg carries out electrotransfer with ADViSYS electrotransfer instrument subsequently.Inject after 3 days, can cause the Alloxan hydrate (80mg/kg of hyperglycemia, Sigma) be dissolved in 25ml salt solution, PH transfers neutral, intravenous injection under anesthesia. because of this neutral solution can not effectively cause hyperglycemia, inject to cause moderate hyperglycemia at the control group of only injecting blank IgG-Fc plasmid again subsequently.Measure fasting plasma glucose weekly 2 times (Figure 10 A) and detect Fc albumen (Figure 10 B) with ELISA
In sum, we have researched and developed a kind of special GLP-1 analogue with people GLP-1 (people is consistent with mouse GLP-1 amino acid coding) and mouse IgG1-Fc or human IgG2-Fc derivative fusion.This analogue can increase the transformation period, improves activity in vivo and reduces immunogenicity (immunogenicity).Under state of nature, the GLP-1-IgG-Fc fusion rotein exists with the form of divalence.Fusion rotein shows that the high low degree that can lean on glucose concn promotes the generation of Ins-1B-cell secretion of insulin and cAMP.Show with experimental result in the animal model, we can deliver this albumen long-term expression in vivo with a kind of gene therapy method of non-virus, and confirm streptozotocin (STZ) inductive diabetes (the type i diabetes model of beta cell damage) and effective to diabetes and obesity (type ii diabetes model).
＜110〉Wang Qinghua, Huang Xiaohang
＜120〉a kind of fusion rotein that is used for the treatment of diabetes and its production and application
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Claims (10)
1, a kind of fusion rotein is characterized in that, described fusion rotein is hyperglycemic-glycogenolytic factor similar peptide GLP-1-1 or has the same merit factor of at least 50% homology and the fusion rotein of IgG-Fc with it.
2, the nucleotide sequence of the described fusion rotein of coding claim 1.
3, fusion rotein as claimed in claim 1 is characterized in that described IgG-Fc is mouse IgG-Fc or human IgG-Fc.
4, fusion rotein as claimed in claim 1 is characterized in that, described fusion rotein is the fusion rotein of hyperglycemic-glycogenolytic factor similar peptide GLP-1-1 and IgG-Fc, and IgG-Fc wherein is IgG1, IgG2 or IgG3; Or the same merit factor lizard sialisterium polypeptide extendin-4 of GLP-1 and the fusion rotein of IgG-Fc, IgG-Fc wherein is IgG1, IgG2, IgG3 or IgG4.
5, the mutant GPL-1-A8G-IgG-Fc of fusion rotein as claimed in claim 1 is characterized in that, the 8th L-Ala of the aminoacid sequence of GPL-1 is replaced by glycine, and IgG-Fc wherein is IgG1, IgG2 or IgG3.
6, comprise the described Expression of Fusion Protein carrier of claim 1.
7, the expression vector of claim 6 is used to prepare the application of the medicine for the treatment of diabetes.
8, mammal cell line expression system or insect expression system or the bacterial expression system or the yeast expression system of the COS-7 clone of the described fusion rotein of claim 1, Chinese hamster ovary celI system or other present laboratory use.
9, a kind of method for preparing the fusion rotein as claimed in claim 1 of biologically active is characterized in that may further comprise the steps:
1) leader peptide sequences and the fusion rotein as claimed in claim 1 with IgK or GHRH merges;
2) transform mammal cell line or the engineering strain that use in the conventional COS-7 clone of using in this area, Chinese hamster ovary celI system or other present laboratory with the above-mentioned fusion rotein that obtains;
3) by carrying out purifying in conjunction with the proteic sephadex column of G.
10, fusion rotein as claimed in claim 1 and be used to prepare the application of the medicine of prevention and treatment I type and type ii diabetes individually or with other composition with its like derivatives with at least 50% homology with mixing.
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| CN102311501A (en) * | 2010-07-08 | 2012-01-11 | 天津药物研究院 | Fusion protein comprising GLP-1 (Glucagon-Like Peptide) or analog thereof and preparation method as well as application thereof |
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| WO2011153965A1 (en) * | 2010-06-11 | 2011-12-15 | 北京精益泰翔技术发展有限公司 | Fusion protein of exendin-4 and its analog, preparation method and use thereof |
| JP2013531488A (en) * | 2010-06-11 | 2013-08-08 | 北京東方百泰生物科技有限公司 | Exendin-4 fusion proteins and analogs thereof, methods for their preparation and uses |
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| EA023037B1 (en) * | 2010-06-11 | 2016-04-29 | Бейджинг Дунфан Байотек Ко., Лтд. | Fusion protein of exendin-4 and its analog, preparation method and use thereof |
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