CN101003574B - Recombined expression of peptide for lowering blood sugar in long acting, and application in medication for treating diabetes - Google Patents
Recombined expression of peptide for lowering blood sugar in long acting, and application in medication for treating diabetes Download PDFInfo
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Abstract
This invention relates to gene cloning and gene expression, more specially, a method for expressing recombinant hypoglycemic peptide with long-lasting effects. The method comprises: mutating the digestion site of dipeptidyl peptidase in GLP-1 from alanine to glycine, ligating with human albumin gene, and expressing in Pichia expression system to obtain four fusion proteins, which comprise recombinant GLP-1, human albumin and flexible linkers. The fusion proteins have such advantage as retained GLP-1 activity, long half-life period, good hypoglycemic effect, and no hypoglycemia phenomenon.
Description
Technical field:
The present invention relates to gene clone and genetic expression in the biology field, and use in the area of pharmacology.
Background technology:
Diabetes have been listed in the third-largest killer after cardiovascular and cerebrovascular diseases and tumour.Along with the change of life pattern, dietary structure, China's resident's diabetes prevalence is the trend of rapid rising.Expect the year two thousand thirty world's diabetes number of patients and will increase to 3.66 hundred million people, diabetes will become white elephant of developing country.
Most people inevitably will treat with medicine among the type ii diabetes patient, and oral pharmaceutical have three major types: the first kind is yellow ureas.As glyburide, Glurenor, diamicron, glipizide; Yellow ureas medicine can stimulate islet secretion Regular Insulin, but insulin secretion many after, can cause hypoglycemia, hypoglycemia is very important Hazard Factor for patients, and the part medicine has big side effect, as causing liver dysfunction.The second class medicine is a biguanide drug.As phenformin and N1,N1-Dimethylbiguanide; Biguanide drug does not stimulate insulin secretion, the utilization that its stimulates body to improve Regular Insulin on cell levels, but for the diabetic subject of hypoinsulinism, the effect of its performance is just very limited, and untoward reaction such as lactic acidosis in addition.The 3rd class medicine is a glycosidase inhibitor.As Bay g 5421; The effect of this class medicine is to reduce the absorption of human body to sugar in the diet, reduces because absorb, and blood sugar increasing also relatively slowly.But the side effect of this medicine is exactly a gastrointestinal reaction, takes the back as hypoglycemic reaction takes place, and also wants oral or intravenous injection glucose.
Injectable drug mainly is a Regular Insulin at present, and the Regular Insulin of injection plays an important role in treatment of diabetes.But its deficiency is also arranged, be mainly nervous after the medication, expression is unusual, sweat etc., weight person produces insulin shock or hypoglycemia convulsions; The patient of insulin injection, again during medication, more and more big through drug withdrawal once to the expense of Regular Insulin, even once can tolerate 200U, and its reason may be that body has produced insulin antibody, antibody combines with Regular Insulin and weakens its effect.Based on to the understanding in depth of beta Cell of islet physiology and Regular Insulin peripheral action mechanism, the new drug of a collection of in recent years treatment diabetes is succeeded in developing in succession.Glucagon-like-peptide-1 (GLP-1) is the most representative blood sugar reducing peptide wherein, and it stimulates insulin secretion by the special receptors bind with the β cell surface, only needs the dosage of injection Gamma Magnitude can bring into play physiological effect.The discovery of intestines-pancreas islet endocrine axis, the effect that makes GLP-1 is people's attention extremely.The most important physiological action of GLP-1 is the insulin stimulating effect that glucose relies on, the power that is the GLP-1 effect of stimulating insulin secretion depends on glucose concn, GLP-1 could promote insulin secretion under the high concentration glucose, in this hormesis of the next nothing of normal concentration glucose, compare with other hypoglycemic class medicines, its biggest advantage is exactly that side effect is little and hypoglycemic reaction does not take place, and the medicine of much treating diabetes all has untoward reactions such as hypoglycemia, weight increase and oedema.In addition, GLP-1 can also improve surrounding tissue to the release of the susceptibility of Regular Insulin, glucagon suppression, suppress stomach emptying, can increase effect such as β cell quantity.GLP-1 promotes the expression of insulin gene in the mode that glucose relies on and concentration relies on.It is relevant with blood sugar concentration that GLP-1 promotes insulin gene to express, and is the most effectively gi tract secretogogue of generally acknowledging at present.Yet, the transformation period (only 2-6min) that GLP-1 is of short duration in vivo, greatly limited its Clinical Application.GLP-1 short major cause of transformation period is in vivo degraded by dipeptidyl peptidase, and the Another reason of transformation period weak point is exactly because of GLP-1 is a kind of small molecular weight small peptide in addition, will soon be excreted from urine by kidney in vivo.
Summary of the invention:
The objective of the invention is to solve above-mentioned not enough problem, the recombinant expressed of a kind of long-acting blood sugar reducing peptide is provided, not only kept the transformation period in the activity of GLP-1 but also the extension body, stability is high, its application in Remedies for diabetes in addition, the tool hypoglycemic activity is obvious, and no hypoglycemia phenomenon takes place, and effect is lasting.
Long-acting blood sugar reducing peptide (GLPAG--human albumin fusion rotein) recombinant expressed, aminoacid sequence is as follows:
H
2N-HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR-Linker-DAHKSEVAHRFKDLGEENFKALVLIAFAQYL
QQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQ
HKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAAC
LLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDL
LECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAK
DVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE
QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPV
SDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE
QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL-COOH
Above-mentioned ' H
2N-' represents protein amino terminal (N-terminal), and ' COOH ' represents protein carboxyl terminal (C-terminal), and the Linker representative flexibly connects son.Amino acid abbreviations in the sequence: A L-Ala; The R arginine; The N l-asparagine; The D aspartic acid; The C halfcystine; The Q glutamine; E L-glutamic acid; The G glycine; The H Histidine; The I Isoleucine; The L leucine; K Methionin; The M methionine(Met); The F phenylalanine; The P proline(Pro); The S Serine; The T Threonine; The W tryptophane; Y tyrosine; V a word used in person's names propylhomoserin; The aminoacid sequence that flexibly connects sub-Linker is TSGGSGGS or GGSGGS or GGSGGSGGGGS or GGS.
The described GLPAG--human albumin fusion rotein of in pichia spp, expressing:
The acquisition of GLPAG gene:
At first synthetic following primer:
GLP-F1：5’tactcgagaaaagacatggtgaagggacctttaccagtg?3’；
GLP-R1：5’tccaaataagaacttacatcactggtaaaggtccc?3’；
GLP-R2：5’tccttggcagcttggccttccaaataagaacttac?3’；
GLP-R3：5’accagccaagcaatgaactccttggcagcttggcc?3’；
GLP-R4：5’tcggcctttcaccagccaagcaatg?3’；
Adopt the method that merges PCR that they are spliced into complete GLPAG gene, splicing back sequence is as follows:
5’tactcgagaaaagacatggtgaagggacctttaccagtgatgtaagttcttatttggaa
Ggccaagctgccaaggagttcattgcttggctggtgaaaggccga 3 '; It is tactcgagaaaaga that 5 ' end of this sequence has 6 bases of xho I restriction endonuclease sites and alpha factor signal peptide end.
The structure of carrier:
Synthetic following primer:
Alb pf:5 ' ctggtgaaaggccga-Linker dna sequence dna-gatgcacacaagagtgaggttg 3 ';
alb?pr：5’atgcggccgcttaccgcggtaagcctaaggcagcttgac?3’；
5 ' the end of upstream primer alb pf has been introduced 15 bases of GLPAG end and has been flexibly connected son (linker DNA) sequence, and the Linker dna sequence dna is actagtggtggctcaggtggatcc or ggtggctcaggtggatcc or ggtggctcaggtggatccggtggaggcggaagc or ggtggctca.Downstream primer alb pr 5 ' end is introduced Not I restriction endonuclease sites; (albumin gene our company adopts the RT-PCR method to obtain from the human hepatocyte and is cloned in the T carrier with this a pair of primer amplification albumin gene, turn out to be the cDNA of human albumin through order-checking), obtained 5 ' the albumin gene sequence of holding terminal 15 bases of band GLPAG and connexon.
Above-mentioned albumin gene is mixed the back merge PCR with the GLPAG gene with GLP-F1 primer and alb pr primer, amplified production uses Xho I and Not I double digestion rear clone to the pPIC9K carrier, the positive colony that enzyme is cut preliminary affirmation carries out sequencing, confirm that GLPAG--human albumin gene is the downstream that is cloned in pPIC9K carrier a factor signal peptide, the positive colony of confirming extracts plasmid, after Sal I enzyme is cut into linearity, transform the GS115 Pichia yeast, do the G418 resistance screening again, obtain the Yeast engineering bacteria of anti-4mg/ml G418.
Fermentation expression:
Yeast engineering bacteria adopts ordinary method to carry out fermentation expression under methanol induction in fermentor tank, and fermented liquid calculates that through protein electrophoresis scanning expression amount is about 1mg/ml (1000mg/l).
Application in described long-acting the blood sugar reducing peptide application in Remedies for diabetes, particularly type ii diabetes medicine.
The long-acting blood sugar reducing peptide of the present invention (GLPAG--human albumin fusion rotein) is mutated into the critical sites of dipeptidyl peptidase effect among the GLP-1 in other amino acid---second amino acids of GLP-1 is sported glycine (Gly or G) from original L-Ala (Ala or A), dipeptidyl peptidase can't be played a role, can solve the problem of dipeptidyl peptidase enzyme liberating, be referred to as GLPAG after the sudden change, splice at gene level with the human albumin gene again, this splicing (fusion) is not directly GLPAG gene and human albumin gene to be linked together, directly link together and to reduce the activity of GLPAG, but with a kind of connexon (flexible Linker) with flexible structure GLPAG and human albumin are linked together, we have used four kinds of different connexons (TSGGSGGS or GGSGGS or GGSGGSGGGGS or GGS) respectively.In pichia yeast expression system, give expression to four kinds of GLPAG and human albumin at last respectively by flexibly connecting the fusion rotein that son (flexible Linker) is connected, this fusion rotein has not only kept the activity of GLP-1 but also its transformation period is in vivo prolonged greatly, be referred to as " long-acting blood sugar reducing peptide ", i.e. the GLPAG-human albumin.To we recombinant expressed this " GLPAG-human albumin ", the experimentation on animals of carrying out with GK type-II diabetes rat model shows that it is active consistent with GLP-1, hypoglycemic activity is obvious, no hypoglycemia phenomenon takes place, in addition, its outstanding advantage is more lasting than simple GLP-1 effect, and the time length was about about 1 week.
Description of drawings:
Accompanying drawing is that fermented liquid is through the protein electrophoresis scintigram.
Embodiment:
Below in conjunction with embodiment the recombinant expressed of long-acting blood sugar reducing peptide and the application in Remedies for diabetes thereof are further described:
In pichia spp, express GLPAG--human albumin fusion rotein:
The acquisition of GLPAG gene:
At first synthetic following primer (Engineering Co., Ltd is synthetic by the precious biotinylated biomolecule in Dalian):
GLP-F1：5’tactcgagaaaagacatggtgaagggacctttaccagtg?3’；
GLP-R1：5’tccaaataagaacttacatcactggtaaaggtccc?3’；
GLP-R2：5’tccttggcagcttggccttccaaataagaacttac?3’；
GLP-R3：5’accagccaagcaatgaactccttggcagcttggcc?3’；
GLP-R4：5’tcggcctttcaccagccaagcaatg?3’；
Adopt the method that merges PCR that they are spliced into complete GLPAG gene, splicing back sequence is as follows: 5 ' tactcgagaaaagacatggtgaagggacctttaccagtgatgtaagttcttatttg gaaggccaagctgccaaggagttcattgcttggctggtgaaaggccga 3 '; It is tactcgagaaaaga that 5 ' end of this sequence has 6 bases of xho I restriction endonuclease sites and alpha factor signal peptide end.
The detailed process that merges PCR is: the GLP-F1 primer 1 μ l of 25 μ M and 25 μ M GLP-R1 primers, 1 μ l, carry out 10 round-robin sex change (94 ℃ 20 seconds), annealing (55 ℃ 20 seconds) in volume is the PCR reaction solution of 50 μ l, extends (72 ℃ 20 seconds), other component in the reaction solution is identical with conventional PCR.Electrophoresis reclaims the fragment of GLP-F1 primer and GLP-R1 primer amplification, is template with this fragment, carries out secondary pcr amplification with GLP-F1 primer and GLP-R2 primer, and reaction conditions is the same, but is 20 circulations.The PCR product second time that reclaims with electrophoresis is a template, carries out for the third time pcr amplification with GLP-F1 primer and GLP-R3 primer, 20 circulations, and reaction conditions is the same.The product of PCR for the third time that reclaims with electrophoresis is a template, carries out the 4th time pcr amplification with GLP-F1 primer and GLP-R4 primer, 25 circulations, and reaction conditions is the same.The product of the 4th pcr amplification is complete GLPAG gene.
The structure of carrier:
Synthetic following primer (Engineering Co., Ltd is synthetic by the precious biotinylated biomolecule in Dalian):
Alb pf:5 ' ctggtgaaaggccga-Linker dna sequence dna-gatgcacacaagagtgaggttg 3 ';
alb?pr：5’atgcggccgcttaccgcggtaagcctaaggcagcttgac?3’；
5 ' the end of upstream primer alb pf has been introduced 15 bases of GLPAG end and has been flexibly connected son (linker DNA) sequence, and the Linker dna sequence dna is actagtggtggctcaggtggatcc or ggtggctcaggtggatcc or ggtggctcaggtggatccggtggaggcggaagc or ggtggctca.Downstream primer alb pr 5 ' end is introduced Not I restriction endonuclease sites; (albumin gene our company adopts the RT-PCR method to obtain from the human hepatocyte and is cloned in the T carrier with this a pair of primer amplification albumin gene, turn out to be the cDNA of human albumin through order-checking), obtained 5 ' the albumin gene sequence of holding terminal 15 bases of band GLPAG and connexon.
Above-mentioned albumin gene is mixed the back merge PCR with the GLPAG gene with GLP-F1 primer and alb pr primer, reaction volume 50 μ l, 30 circulations, cycling condition: sex change (94 ℃ 50 seconds), annealing (55 ℃ 50 seconds), extend (72 ℃ 2 minutes), archaeal dna polymerase in the reaction solution high-fidelity EX Taq enzyme of TaKaRa company, other component is identical with conventional PCR.Amplified production with Xho I and Not I double digestion rear clone to pPIC9K carrier (will being positioned at another Xho I site mutation at 4475 places in the pPIC9K carrier), the positive colony that enzyme is cut preliminary affirmation carries out sequencing, confirms that GLPAG--human albumin gene is the downstream that is cloned in pPIC9K carrier alpha factor signal peptide.The positive colony of confirming extracts plasmid, after Sal I enzyme is cut into linearity, transforms the GS115 Pichia yeast, carries out the G418 resistance screening again, obtains the Yeast engineering bacteria of anti-4mg/ml G418.The GLPAG--human albumin Yeast engineering bacteria of four kinds of different connexons respectively filters out a strain.
Fermentation expression:
Yeast engineering bacteria adopts ordinary method to carry out fermentation expression under methanol induction.Fermentation is carried out in Germany ' Bei Lang ' 30-L fermentor tank, and fermented liquid calculates that through protein electrophoresis scanning expression amount is about 1mg/ml (1000mg/l), sees accompanying drawing, 1 is standard molecular weight among the figure, is respectively 97.4kd from top to bottom, 66.2kd, 42.7kd, 31kd, 14.4kd; 2 is the human albumin of 2mg/ml; 3 is the human albumin of 1mg/ml; 4 GLPAG-human albumins (linker is TSGGSGGS) for the fermented liquid expression; 5 GLPAG-human albumins (linker is GGSGGS) for the fermented liquid expression; 6 GLPAG-human albumins (linker is GGSGGSGGGGS) for the fermented liquid expression; 7 GLPAG-human albumins (linker is GGS) for the fermented liquid expression.Fermented liquid is used for the treatment experiment of diabetes model animal subsequently as medicine behind centrifugal, ultrafiltration, ion exchange column purifying.
Approaching from the GLPAG-human albumin (4,5,6,7 swimming lane) that accompanying drawing was shown the fermented liquid as can be seen with 1mg/ml human albumin (3 swimming lane) amount, reach the expression amount that contains the 1mgGLPAG-human albumin in every milliliter of fermented supernatant fluid.
The GLPAG-human albumin is used for the treatment experiment of GK type ii diabetes rat model as medicine:
(every body weight is at 200g ± 10g), be divided into six groups, 10 every group for 60 of GK type ii diabetes rats; First group is the GLP-1 positive controls, and second group is physiology saline control group, the 3rd to the 6th group of GLPAG--human albumin experimental group that is respectively Different L inker; All extract blood before 60 GK rat experiments, separation of serum, stored frozen is to be measured.Every rat abdominal cavity inoculation of GLPAG--human albumin experimental group 0.5ml, the GLPAG--human albumin (GLPAG only accounts for the about 1/20 of whole fusion protein molecule amount in GLPAG--human albumin fusion rotein, so also only contain the GLPAG of about 3 μ g in the GLPAG--human albumin of 60 μ g) that contains 60 μ g; Every rat abdominal cavity of GLP-1 positive controls is inoculated 0.5ml, contains the GLP-1 of 3 μ g; The physiological saline of every rat abdominal cavity inoculation of control group 0.5ml; Every day injection once, inject 10 days continuously after, blood drawing immediately, separation of serum, stored frozen is to be measured.After the drug withdrawal 7 days, blood sampling respectively again, separation of serum together with the two batches of stored frozen serum to be measured in front, detects the concentration of blood sugar, the results are shown in following table 1:
Table 1
As can be seen from the above table GLPAG--human albumin experimental group and GLP-1 positive controls successive administration after 10 days blood glucose value all dropped to the level (normal rat blood glucose value 4.9-6.2) of normal value, the activity of GLPAG--human albumin and GLP-1 is identical.But the blood glucose value of drug withdrawal GLP-1 positive controls after 7 days has been returned to outlier again, and the blood glucose value of GLPAG--human albumin experimental group is still at normal value, the activity that the GLPAG--human albumin is described can keep the longer time promptly in vivo specific activity GLP-1 prolong 7 days approximately.
The GLPAG--human albumin stimulates GK rat Langerhans islet β emiocytosis Regular Insulin and stimulates the beta Cell of islet proliferative effect:
The external primary cell culture of carrying out: get GK type ii diabetes rat Langerhans islet β cell, tryptic digestion is counted after becoming individual cells.Every hole adds 10 in 24 orifice plates
5Individual cell, every group 4 hole.' GLPAG--human albumin ' group is (according to 4 kinds of different Linker, divide 4 groups, every group 4 hole) final concentration of every hole adding corresponding ' GLPAG--human albumin ' is 20 μ g/ml, the final concentration that GLP-1 organizes every hole adding GLP-1 is 1 μ g/ml, and the final concentration that the every hole of negative control group adds bovine serum albumin is 20 μ g/ml.Cell is put in 37 ℃ of carbonic acid gas incubators and is cultivated, and nutrient solution is the DMEM/F2 nutrient solution that contains low sugar.Cultivate after 4 hours, every hole continues to cultivate after taking out a small amount of supernatant.
Regular Insulin detected result in 4 hours supernatants is: insulin content 3.4 ± 0.3 μ U in ' GLPAG--human albumin ' group (Linker is TSGGSGGS) supernatant, ' GLPAG--human albumin ' group (Linker is GGSGGS) 3.3 ± 0.3 μ U, the GLPAG--human albumin ' group (Linker is GGSGGSGGGGS) 3.3 ± 0.4 μ U, the GLPAG--human albumin ' group (Linker is GGS) 3.5 ± 0.3 μ U, GLP-1 positive controls 3.3 ± 0.4 μ U, negative control group 0.8 ± 0.1 μ U.This result as can be seen in ' GLPAG--human albumin ' each group and the GLP-1 positive controls supernatant amount of Regular Insulin obviously to be higher than negative control group, illustrate that ' GLPAG--human albumin ' is the same with GLP-1, have the function that stimulates the beta Cell of islet excreting insulin.Cultivate after 7 days the cell dissociation in the hole and counting, GLPAG--human albumin group (Linker is TSGGSGGS) every hole average 9 * 10 as a result
5Individual cell, GLPAG--human albumin group (Linker is GGSGGS) every hole average 8.5 * 10
5Individual cell, GLPAG--human albumin group (Linker is GGSGGSGGGGS) every hole average 9 * 10
5Individual cell, GLPAG--human albumin group (Linker is GGS) every hole average 9.5 * 10
5Individual cell, the every hole of GLP-1 positive controls average 8.5 * 10
5Individual cell, the every hole of negative control group average 1 * 10
5Individual cell.Results suggest GLPAG--human albumin and GLP-1 have the outgrowth effect of stimulation in rats beta Cell of islet.
The hormesis that GLPAG--human albumin glucose relies on:
The hormesis that glucose relies on is exactly that ' GLPAG-human albumin ' fusion rotein does not stimulate insulin secretion, so do not cause hypoglycemia yet when blood sugar in the body during at normal level.(every body weight is at 200g ± 10g), be divided into six groups, 10 every group for 60 of normal SD rats; One group is the GLP-1 control group, and another group is physiology saline control group; All the other four groups is ' GLPAG--human albumin ' group; All extract blood before 60 SD rat experiments, separation of serum is measured blood sugar.' GLPAG--human albumin ' four group every rat abdominal cavity is inoculated 0.5ml, contains ' the GLPAG--human albumin ' of 600 μ g; Every rat abdominal cavity of GLP-1 control group is inoculated 0.5ml, contains the GLP-1 of 30 μ g; The physiological saline of every rat abdominal cavity inoculation of negative control group 0.5ml; In injection blood sampling in back 2 hours, 4 hours, 6 hours, 8 hours, separation of serum is measured blood sugar.Blood sugar detection result such as following table 2 as can be seen from the table, though injected the GLP-1 and ' GLPAG--human albumin ' of 10 times of therapeutic doses to rat, all do not cause hypoglycemic reaction.
Table 2
Claims (5)
1. long-acting blood sugar reducing peptide is recombinant expressed, and it is characterized in that: aminoacid sequence is as follows:
H
2N-HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR-Linker-DAHKSEVAHRFKDLGEENFKALVLIAFAQYL
QQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQ
HKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAAC
LLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDL
LECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAK
DVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE
QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPV
SDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE
QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL-COOH
Above-mentioned ' H
2N-' represents protein amino terminal, and ' COOH ' represents protein carboxyl terminal, and the Linker representative flexibly connects son, the amino acid abbreviations in the sequence: the A L-Ala; The R arginine; The N l-asparagine; The D aspartic acid; The C halfcystine; The Q glutamine; E L-glutamic acid; The G glycine; The H Histidine; The I Isoleucine; The L leucine; K Methionin; The M methionine(Met); The F phenylalanine; The P proline(Pro); The S Serine; The T Threonine; The W tryptophane; Y tyrosine; V a word used in person's names propylhomoserin;
The aminoacid sequence that flexibly connects sub-Linker is TSGGSGGS or GGSGGS or GGSGGSGGGGS or GGS.
2. long-acting blood sugar reducing peptide according to claim 1 is recombinant expressed, it is characterized in that: recombinant expressed carrier is the pPIC9K carrier, and the host bacterium is the GS115 Pichia yeast.
3. long-acting blood sugar reducing peptide according to claim 1 is recombinant expressed, it is characterized in that: the acquisition of GLPAG gene:
At first synthetic following primer:
GLP-F1：5’tactcgagaaaagacatggtgaagggacctttaccagtg?3’；
GLP-R1：5’tccaaataagaacttacatcactggtaaaggtccc?3’；
GLP-R2：5’tccttggcagcttggccttccaaataagaacttac?3’；
GLP-R3：5’accagccaagcaatgaactccttggcagcttggcc?3’；
GLP-R4：5’tcggcctttcaccagccaagcaatg?3’；
Adopt the method that merges PCR that they are spliced into complete GLPAG gene, splicing back sequence is as follows:
5 ' tactcgagaaaagacatggtgaagggacctttaccagtgatgtaagttcttatttg gaaggccaagctgccaaggagttcattgcttggctggtgaaaggccga 3 '; It is tactcgagaaaaga that 5 ' end of this sequence has 6 bases of xhoI restriction endonuclease sites and alpha factor signal peptide end;
The structure of carrier:
Synthetic following primer:
Alb pf:5 ' ctggtgaaaggccga-Linker dna sequence dna-gatgcacacaagagtgaggttg 3 ';
alb?pr：5’atgcggccgcttaccgcggtaagcctaaggcagcttgac?3’；
5 ' the end of upstream primer alb pf has been introduced 15 bases of GLPAG end and has been flexibly connected subsequence, and the Linker dna sequence dna is actagtggtggctcaggtggatcc or ggtggctcaggtggatcc or ggtggctcaggtggatccggtggaggcggaagc or ggtggctca; Downstream primer alb pr 5 ' end is introduced Not I restriction endonuclease sites; With this a pair of primer amplification albumin gene, obtained the albumin gene sequence of 5 ' terminal 15 bases of end band GLPAG and connexon;
Above-mentioned albumin gene is mixed the back merge PCR with the GLPAG gene with GLP-F1 primer and alb pr primer, amplified production uses Xho I and Not I double digestion rear clone to the pPIC9K carrier, the positive colony that enzyme is cut preliminary affirmation carries out sequencing, confirm that GLPAG--human albumin gene is the downstream that is cloned in pPIC9K carrier alpha factor signal peptide, the positive colony of confirming extracts plasmid, after Sal I enzyme is cut into linearity, transform the GS115 Pichia yeast, do the G418 resistance screening again, obtain the Yeast engineering bacteria of anti-4mg/ml G418;
Fermentation expression:
Yeast engineering bacteria adopts ordinary method to carry out fermentation expression under methanol induction in fermentor tank, and fermented liquid calculates that through protein electrophoresis scanning expression amount is about 1mg/ml.
4. the application of long-acting blood sugar reducing peptide according to claim 1 in Remedies for diabetes.
5. the application of long-acting blood sugar reducing peptide according to claim 1 in the type ii diabetes medicine.
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| CN102382191A (en) * | 2011-09-23 | 2012-03-21 | 江南大学 | Preparation method and application of novel braingutpeptide stimulant molecule |
| CN104257696B (en) * | 2014-09-04 | 2017-08-25 | 西安国誉生物科技有限公司 | A kind of steady sugar yeast bacterium powder of hypoglycemic and its preparation method and application |
| CN105153311B (en) * | 2015-07-17 | 2018-09-04 | 山东泉港药业有限公司 | Recombinant human glucagon-like peptide-1 mutant fusion protein and preparation method thereof |
| CN105368806B (en) * | 2015-12-09 | 2016-08-24 | 暨南大学 | A kind of Fixedpoint mutation modified yeast dipeptidyl peptidase II I |
| CN109730270B (en) * | 2019-02-12 | 2022-06-28 | 西安培华学院 | Sugar-reducing yeast fermented pumpkin powder and preparation method and application thereof |
| CN112010941B (en) * | 2019-05-31 | 2022-08-16 | 华南理工大学 | Blood sugar reducing heptapeptide |
| CN115894719B (en) * | 2022-11-24 | 2023-10-20 | 武汉禾元生物科技股份有限公司 | Human serum albumin insulin conjugate and preparation method thereof |
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| CN1405182A (en) * | 2001-08-10 | 2003-03-26 | 中国人民解放军军事医学科学院生物工程研究所 | Serum albumin and granulocyte colony stimulating factor fusion protein |
| CN1405181A (en) * | 2001-08-10 | 2003-03-26 | 中国人民解放军军事医学科学院生物工程研究所 | Serum albumin and interferon fusion protein |
| CN1483041A (en) * | 2000-12-07 | 2004-03-17 | GLP-1 fusion protein |
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| CN1483041A (en) * | 2000-12-07 | 2004-03-17 | GLP-1 fusion protein | |
| CN1405182A (en) * | 2001-08-10 | 2003-03-26 | 中国人民解放军军事医学科学院生物工程研究所 | Serum albumin and granulocyte colony stimulating factor fusion protein |
| CN1405181A (en) * | 2001-08-10 | 2003-03-26 | 中国人民解放军军事医学科学院生物工程研究所 | Serum albumin and interferon fusion protein |
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| 张志珍.重组人胰高血糖素样肽-1突变体的分离纯化及活性分析.中国生物化学与分子生物学学报21 4.2005,21(4),510-515. * |
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