CN1982336A - Human pancreas glucagon sample peptide-1-derivative, its production and use - Google Patents
Human pancreas glucagon sample peptide-1-derivative, its production and use Download PDFInfo
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Abstract
A human pancreatic hyperglycemic hormone sample peptide-1 derivative, its production and use are disclosed. The molecular formula has 3 structures: GLP-1(7-37)-Xaa38(Seq ID No.2), GLP-1(7-37)-Xaa38-Xaa39(Seq ID No.3) and GLP-1(7-37)-Xaa38-Xaa39-Xaa40(Seq ID No.4); Xaa38, Xaa39 and Xaa40 correspond to any amid-acid of Cys, Ala, Gly, His, Ser and Thr separately. It's simple, cheap and efficient, it adopts DNA recombinant technology and has longer half life period. It can be used to prepare active ingredients of medicine in treatment of diabetes.
Description
Technical field
The present invention relates to a kind of the have active human glucagon-like-peptide-1 derivative of human glucagon-like-peptide-1 (GLP-1) (GLP-1 derivative) and preparation and application, belong to technical field of bioengineering.
Background technology
Diabetes are worldwide diseases, and global onset diabetes rate increases rapidly, have become the chronic disease of the third-largest serious threat human health after tumour, cardiovascular pathological changes.According to international diabetes study institute (IDI) up-to-date address prediction in 2003, global diabetic subject has reached 1.94 hundred million people, and according to present rate of growth, by 2025, number of patients will reach 3.33 hundred million.China has diabetic subject more than 4,000 ten thousand, and in early 1980s, China's onset diabetes rate only is 0.67%, ground sickness rate such as Beijing, Shanghai all surpass 10% now, and this numeral also continue to increase, and diabetes will be serious public health problems of China in following 50 years.According to statistics, the year cost that common diabetic subject is used for the treatment of is 3726 yuan, and the diabetic subject needs lifelong medication, and the Glucovance of therefore developing high performance cheap has tempting prospect.
In recent years, very fast to the progress of insulin secretion accelerating peptide: GLP-1, it is better to be applied to treat the diabetes effect.GLP-1 mainly is that molecular weight is about 3.355kD by the polypeptide hormone of a kind of 31 peptides of the L emiocytosis of far-end ileum, colon and rectum.The major physiological effect of GLP-1 comprises: the release action of (1) glucose dependency pancreotropic hormone.Its mechanism of action be by with the special acceptor interaction on pancreatic beta cell surface, the insulin secretion of glucose induction is significantly increased.(2) stimulate the β proliferation of cells, suppress the apoptosis of β cell, the GLP-1 receptor stimulant has the effect that promotes beta Cell of islet growth and propagation.(3) secretion of release of stimulating growth chalone and glucagon suppression.In blood glucose regulation, the effect and the Regular Insulin of hyperglycemic-glycogenolytic factor are opposite, increase blood sugar concentration by promoting glycogen to decompose.Under the hyperglycemia situation, use the obviously secretion of glucagon suppression of GLP-1.(4) suppress parietal cell secretion hydrochloric acid in gastric juice, the emptying that prolongs stomach.(5) increase satiety, depress appetite reduces the picked-up of energy.The promoting insulin secretion of GLP-1 depends on the concentration of glucose, hypoglycemia can not take place with its treatment diabetes, shows its good prospects for application aspect diabetes especially type ii diabetes treatment.
But GLP-1 is easy to be degraded to and slough His by two acyltransferase polypeptide peptase IV (DipeptidylPeptidase 4, DPP IV) in vivo
7-Ala
8GLP-1 of the non-activity of-residue (9-37) or GLP-1 (9-36) NH
2The quiet notes GLP-1 transformation period in vivo is less than 5min, the about 13mLkg of its metabolic clearance rate
-1Min
-1No matter be quiet notes or subcutaneous injection GLP-1, the GLP-1 of the 10-20% that only has an appointment in the blood plasma is complete, activated form.Therefore still there is very big defective in GLP-1 in clinical application, presses for the long GLP-1 derivative with better clinical use value of exploitation transformation period.
One of research direction of the diabetes of GLP-1 treatment at present is to change by the structure to GLP-1, obtains the GLP-1 derivative, with these derivatives for treatment diabetes, reaches the purpose of prolong drug effective drug duration in vivo.Existing GLP-1 derivative all demonstrates stability to be increased, and the character that biological activity still can be kept in the body.As the GLP-1 derivative of Padil or butyrine is arranged on the 8th.Be that the GLP-1 derivative with lipid acid has and plasma albumin binding ability enhanced characteristic and for example, can prolong its biological activity at the C end.These GLP-1 derivatives are compared with natural GLP-1, and their transformation period is significant prolongation all, and still, still there are defectives such as preparation and purification difficult in they.
Summary of the invention
GLP-1 has two kinds of forms in vivo, and a kind of is GLP-1 (7-36)-NH2, is made up of 30 amino-acid residues, and another kind is GLP-1 (7-37), is made up of 31 amino-acid residues, and the two has identical biologic activity.The GLP-1 that the present invention relates to refers to GLP-1 (7-37), and its sequence (Seq ID No.1) is enumerated as follows: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly.
First purpose of the present invention is to propose a kind of GLP-1 derivative, it is characterized in that, the molecular structural formula of this derivative is following three kinds of structural formula: GLP-1 (7-37)-Xaa38 (Seq ID No.2), one of GLP-1 (7-37)-Xaa38-Xaa39 (Seq ID No.3) and GLP-1 (7-37)-Xaa38-Xaa39-Xaa40 (Seq ID No.4), Xaa38 wherein, Xaa39 and Xaa40 are respectively Cys, Ala, Gly, His, any one amino acid among Ser and the Thr.This derivative has not only kept natural GLP-1 activity in vivo, has also obviously prolonged effective drug duration in the body, and effective drug duration is sustainable at least 3 hours in the body, has very good clinical use value.In addition, this derivative is by the method preparation of recombinant DNA technology, and cost is low, and suitable popularizing used.
Described derivative is further characterized in that, the molecular structural formula of this derivative is following three kinds of structural formula: GLP-1 (7-37)-Xaa38 (Seq ID No.2), one of GLP-1 (7-37)-Xaa38-Xaa39 (Seq ID No.3) and GLP-1 (7-37)-Xaa38-Xaa39-Xaa40 (Seq ID No.4), Xaa38=Cys wherein, Xaa39 and Xaa40 difference=Cys, Ala, Gly, His, any one amino acid among Ser and the Thr.
Described derivative is further characterized in that, the molecular structural formula of this derivative is one of following two kinds of structural formula: GLP-1 (7-37)-Xaa38-Xaa39 (Seq ID No.3) and GLP-1 (7-37)-Xaa38-Xaa39-Xaa40 (Seq ID No.4), Xaa39=Cys wherein, Xaa38 and Xaa40 difference=Cys, Ala, Gly, His, any one amino acid among Ser and the Thr.
Described derivative is further characterized in that the molecular structural formula of this derivative is GLP-1 (7-37)-Xaa38-Xaa39-Xaa40 (Seq ID No.4), wherein Xaa40=Cys, Xaa38 and Xaa39 difference=Cys, Ala, Gly, His, any one amino acid among Ser and the Thr.
Second purpose of the present invention provides the preparation method of said derivative.For achieving the above object, technical scheme of the present invention is to adopt the DNA recombinant technology.
Now describe technical scheme of the present invention in detail.
A kind of preparation method of GLP-1 derivative is characterized in that, the concrete operations step:
The first step, the aminoacid sequence synthetic gene fragment of pressing the GLP-1 derivative;
In second step, encoding sequence is put into expression vector in the mode that is suitable for expressing independent protein or fusion rotein;
In the 3rd step, the suitable procaryotic host cell of expression vector conversion with the GLP-1 derivative gene order that contains GLP-1 derivative or fusion obtains engineering strain;
In the 4th step, engineering strain through liquid fermenting, is contained GLP-1 derivative fusion rotein or contains the proteic wet thallus of GLP-1 derivative separately through centrifugal acquisition;
In the 5th step,, obtain to contain GLP-1 derivative fusion rotein or contain the proteic crude product of GLP-1 derivative separately through separating with the wet thallus broken wall;
In the 6th step, purified, lyophilize makes product G LP-1 derivative.
The 3rd purpose of the present invention is the application that proposes the GLP-1 derivative, and this derivative is made the activeconstituents of this medicine in the medicine of preparation treatment diabetes.
The invention has the advantages that: the GLP-1 derivative that (1) the present invention proposes has the transformation period of being longer than GLP-1; (2) produce the GLP-1 derivative by the method for DNA recombinant technology, be easy to purifying, the yield height, cost is lower.
Thereby prepare the GLP-1 derivative with DNA recombinant technology provided by the invention, and the output height, purifying process is simplified, and production cost is lower.The GLP-1 derivative for preparing, its long half time are suitable for treating the activeconstituents of diabetes medicament in the transformation period of GLP-1.
Description of drawings
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and following mGLP-1 represents GLP-1 derivative of the present invention.
Fig. 1 is the structure of plasmid pET-32a-mGLP-1, and wherein this plasmid contains the GLP-1 derivative.
Embodiment
Below in conjunction with embodiment, be described in further detail the present invention.Used plasmid, thalline etc. in specification sheets and following examples, and the experimental technique of unreceipted actual conditions, condition is carried out routinely, or is undertaken by the condition that goods supplier is advised.
The first step is pressed the aminoacid sequence synthetic gene fragment of GLP-1 derivative:
Press the aminoacid sequence of GLP-1 derivative, select the intestinal bacteria preference codon for use, synthetic following length is the segment of 131bp, i.e. Seq ID No.5:
ccagatctgg acgacgacga caagcatgcc gaaggcacct ttaccagcga tgtgagcagc 60
tatctggaag gccaggccgc caaagaattt attgcctggc tggtgaaagg cagaggcnnn 120
taaccatgga c 121
N118=n120=t wherein, n119=g, this segment contains enteropeptidase EK site, GLP-1 derivative gene, terminator codon TAA and restriction enzyme BglII and NcoI site;
Second step was put into expression vector in the mode that is suitable for expressed fusion protein with encoding sequence:
With gene segment restriction enzyme BglII and the NcoI double digestion that obtains in the first step, purifying reclaims, simultaneously with escherichia coli plasmid pET-32a (+) restriction enzyme BglII and NcoI double digestion, purifying reclaims big segment, above-mentioned enzyme is cut gene segment to be mixed with digested plasmid, add T4DNA ligase enzyme and T4DNA ligase enzyme damping fluid, room temperature connects 3-4 hour, makes up the plasmid pET-32a-mGLP-1 that obtains containing GLP-1 derivative gene order;
The expression vector of the 3rd step with the GLP-1 derivative gene order that contains fusion transforms suitable procaryotic host cell, obtains engineering strain:
Plasmid pET-32a-mGLP-1 is converted in the recipient bacterium bacillus coli DH 5 alpha, ice bath 30 minutes, be warming up to 42 ℃, after keeping 90 seconds, be transferred to the agarose plate that contains penbritin, 37 ℃ are spent the night, and filter out the bacterium colony of ammonia benzyl resistance, obtain containing the engineering strain of GLP-1 derivative gene order;
The 4th step through liquid fermenting, was contained the wet thallus of GLP-1 derivative fusion rotein with engineering strain through centrifugal acquisition:
To build the engineering strain that contains GLP-1 derivative gene order when in the LB liquid nutrient medium, being cultured to OD600nm=0.6-0.8, the adding final concentration is that the IPTG of 0.4-0.8mM carried out abduction delivering 3-5 hour, produce and accumulate the GLP-1 derivative fusion rotein of solubility expression, centrifugal, abandon supernatant, collect wet thallus;
The 5th step is with the wet thallus broken wall, through separating the crude product that obtains to contain GLP-1 derivative fusion rotein:
With NTA0 damping fluid wet thallus of collecting in resuspended the 4th step, after the carrying out ultrasonic bacteria breaking, centrifugal collection supernatant, carry out NTA0 resin affinity chromatography, collect GLP-1 derivative fusion rotein component, with molecular weight cut-off is that the Millipore Amicon Ultra-15 ultrafiltration pipe of 1KD-5KD is concentrated into albumen more than the 2mg/mL, obtains to contain the crude product of GLP-1 derivative fusion rotein;
The 6th step is purified, and lyophilize makes product G LP-1 derivative:
With enteropeptidase enzymolysis GLP-1 derivative fusion rotein crude product, at 4-37 ℃ of cracking 2-16 hour, termination reaction, and then carry out NTA0 resin affinity chromatography, and to collect and penetrate the peak, lyophilize obtains product G LP-1 derivative.
Embodiment 2 DNA recombinant technologys prepare GLP-1 derivative of the present invention, and the molecular structural formula of this derivative is GLP-1 (7-37)-Xaa38-Xaa39 (Seq ID No.3), Xaa38=Cys wherein, and Xaa39=Gly, operation steps:
The first step is pressed the aminoacid sequence synthetic gene fragment of GLP-1 derivative:
Press the aminoacid sequence of GLP-1 derivative, select the intestinal bacteria preference codon for use, synthetic following length is the segment of 134bp, i.e. Seq ID No.6:
ccagatctgg acgacgacga caagcatgcc gaaggcacct ttaccagcga tgtgagcagc 60
tatctggaag gccaggccgc caaagaattt attgcctggc tggtgaaagg cagaggcnnn 120
nnntaaccat ggac 134
N118=n120=t wherein, n119=n121=n122=g, n123=c, this segment contains enteropeptidase EK site, human glucagon-like-peptide GLP-1 derivative gene, terminator codon TAA and restriction enzyme BglII and NcoI site;
Other steps and embodiment's 1 is same.
Embodiment 3 DNA recombinant technologys prepare GLP-1 derivative of the present invention: the molecular structural formula of this derivative is GLP-1 (7-37)-Xaa38-Xaa39 (Seq ID No.3), Xaa38=Gly wherein, Xaa39=Cys, operation steps is with embodiment 2, in the first step synthetic Seq ID No.6 gene segment, n118=n119=n122=g, n120=c, n121=n123=t.
Embodiment 4 DNA recombinant technologys prepare GLP-1 derivative of the present invention: the molecular structural formula of this derivative is GLP-1 (7-37)-Xaa38-Xaa39-Xaa40 (Seq IDNo.4), Xaa38=Cys wherein, and Xaa39=Ala, Xaa40=G1y, operation steps:
The first step is pressed the aminoacid sequence synthetic gene fragment of GLP-1 derivative:
Press the aminoacid sequence of GLP-1 derivative, select the intestinal bacteria preference codon for use, synthetic following length is the segment of 137bp, i.e. Seq ID No.7:
ccagatctgg acgacgacga caagcatgcc gaaggcacct ttaccagcga tgtgagcagc 60
tatctggaag gccaggccgc caaagaattt attgcctggc tggtgaaagg cagaggcnnn 120
Wherein, n118=n120=t, n119=n121=n124=n125=g, n122=n123=n126=c, this segment contains enteropeptidase EK site, GLP-1 derivative gene, terminator codon TAA and restriction enzyme BglII and NcoI site, and the structure of plasmid is seen Fig. 1 in this step;
Other steps and embodiment's 1 is same.
Embodiment 5 DNA recombinant technologys prepare GLP-1 derivative of the present invention: the molecular structural formula of this derivative is GLP-1 (7-37)-Xaa38-Xaa39-Xaa40 (Seq IDNo.4), Xaa38=Ala wherein, Xaa39=Cys, Xaa40=Gly, operation steps is removed in the first step synthetic Seq ID No.7 gene segment, n118=n122=n124=n125=g, n119=n120=n126=c, outside the n121=n123=t, all the other operation stepss and embodiment's 4 is same.
Embodiment 6 DNA recombinant technologys prepare GLP-1 derivative of the present invention: the molecular structural formula of this derivative is GLP-1 (7-37)-Xaa38-Xaa39-Xaa40 (Seq IDNo.4), Xaa38=Ala wherein, Xaa39=Gly, Xaa40=Cys, operation steps is removed in the first step synthetic Seq ID No.7 gene segment, n118=n121=n122=n125=g, n119=n120=n123=c, outside the n124=n126=t, all the other operation stepss and embodiment's 4 is same.
The hypoglycemic activity of embodiment 7 GLP-1 derivatives.
Experiment material and method:
Male and healthy kunming mice (cleaning level, Fudan University in Shanghai medical college animal center provides);
50% glucose solution;
0.9%NaCl solution;
GLP-1；
MGLP-1 has the structure of the described GLP-1 derivative of embodiment 1-6;
Blood glucose monitoring system (the newly upright medicine equipment company limited in Shanghai produces).
Male and healthy kunming mice overnight fasting is divided into 8 groups (n=8).1, the glucose control group; 2, GLP-1 administration control group; 3-8, mGLP-1 administration group, concrete sequential structure is the structure described in the embodiment 1-6.The GLP-1 of GLP-1 administration control group 2 abdominal injections, 100 μ L50% glucose solutions and 0.4 μ g, mGLP-1 administration group 3-8, every group of mGLP-1 of abdominal injection 100 μ L, 50% glucose solution and 0.4 μ g respectively, note is zero constantly this moment.Carried out mouse tail vein respectively at 10,30,60,90,120,150,180 minutes and get blood 10 μ L, measure blood sugar concentration with blood glucose monitoring system.For the long-time mGLP-1 hypoglycemic activity of observing, after getting blood, 60,150 minutes mouse tail veins give 50 μ L 50% glucose solution immediately once more, with check mGLP-1 hypoglycemic activity.The glucose control group is only injected 50% glucose solution, does not give GLP-1 and mGLP-1, by identical time interval determination blood sugar.
The result is as shown in table 1, shown in numerical value be the average of n=8.Hypoglycemic rate shown in the table 1 is calculated as follows: hypoglycemic rate (%)=(glucose control group blood glucose value-administration group blood glucose value)/glucose control group blood glucose value.Compare with the glucose group mouse, within after the administration 30 minutes, GLP-1 administration control group can reduce mouse blood sugar, and mGLP-1 administration group can both reduce mouse blood sugar in 180 minutes, and duration of efficacy prolongs than GLP-1.The result also shows, when blood sugar in the mouse body is low, mGLP-1 demonstrates lower hypoglycemic activity, see Table in 1 60,120,150 minutes result, and when blood sugar was higher in the mouse body, mGLP-1 demonstrated higher hypoglycemic activity, saw Table in 1 30,90 minutes result, prompting mGLP-1 can not cause hypoglycemia, and it is safer to be used for the treatment of diabetes.The result shows that also the mGLP-1 for preparing by the inventive method all shows similar hypoglycemic activity, and has long lasting more hypoglycemic biologic activity than GLP-1.
The hypoglycemic activity of table 1mGLP-1
| Group | Hypoglycemic rate (%) | ||||||
| 10min | 30min | 60min | 90min | 120min | 150min | 180min | |
| 1 2 3 4 5 6 7 8 | 0 5 5 5.1 3.8 3.2 5.3 3.5 3.4 | 0 32 53 48 41 55 45 41 | 0 0 20 19 18 20 17 17 | 0 0 55 50 51 56 49 51 | 0 0 25 24 24 26 23 24 | 0 0 5.4 4.6 4.2 5.4 4.5 4.4 | 0 0 30 28 27 31 27 27 |
Above-mentioned embodiment is intended to set forth preferred forms of the present invention rather than limits the present invention in any form.Those skilled in the art, all drop in the scope of patent application right requirement of the present invention in conjunction with the various changes that the general knowledge of this area is done according to enlightenment of the present invention.
The aminoacid sequence of the GLP-1 derivative that the present invention relates to is in respect of following 7.Now be shown in detail in following sequence table.
Sequence table
＜110〉East China Normal University
＜120〉a kind of human glucagon-like-peptide-1 derivative and preparation and application
<160>7
<210>1
<211>31
<212>PRT
＜213〉people
<400>1
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
5 10 15
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
Gly
<210>2
<211>32
<212>PRT
＜213〉artificial sequence
<220>
＜221〉synthetic construct
<222>(32)
＜223〉Xaa=Cys, Ala, Gly, His, Ser or Thr
<400>2
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
5 10 15
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
Gly Xaa
<210>3
<211>33
<212>PRT
＜213〉artificial sequence
<220>
＜221〉synthetic construct
<222>(32，33)
＜223〉Xaa=Cys or Ala, Gly, His, Ser, Thr
<400>3
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
5 10 15
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
Gly Xaa Xaa
<210>4
<211>34
<212>PRT
＜213〉artificial sequence
<220>
＜221〉synthetic construct
<222>(32，33，34)
＜223〉Xaa=Cys or Ala, Gly, His, Ser, Thr
<400>4
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
5 10 15
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
Gly Xaa Xaa Xaa
<210>5
<211>131
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(118，119，120)
＜223〉n=a or g or c or t
<400>5
ccagatctgg acgacgacga caagcatgcc gaaggcacct ttaccagcga tgtgagcagc 60
tatctggaag gccaggccgc caaagaattt attgcctggc tggtgaaagg cagaggcnnn 120
taaccatgga c 131
<210>6
<211>134
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(118，119，120，121，122，123)
＜223〉n=a or g or c or t
<400>6
ccagatctgg acgacgacga caagcatgcc gaaggcacct ttaccagcga tgtgagcagc 60
tatctggaag gccaggccgc caaagaattt attgcctggc tggtgaaagg cagaggcnnn 120
nnntaaccat ggac 134
<210>7
<211>137
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(118，119，120，121，122，123，124，125，126)
＜223〉n=a or g or c or t
<400>7
ccagatctgg acgacgacga caagcatgcc gaaggcacct ttaccagcga tgtgagcagc 60
tatctggaag gccaggccgc caaagaattt attgcctggc tggtgaaagg cagaggcnnn 120
＜110〉East China Normal University
＜120〉a kind of human glucagon-like-peptide-1 derivative and preparation and application
<160>7
<210>1
<211>31
<212>PRT
＜213〉people
<400>1
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
5 10 15
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
Gly
<210>2
<211>32
<212>PRT
＜213〉artificial sequence
<220>
＜221〉synthetic construct
<222>(32)
＜223〉Xaa=Cys, Ala, Gly, His, Ser or Thr
<400>2
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
5 10 15
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
Gly Xaa
<210>3
<211>33
<212>PRT
＜213〉artificial sequence
<220>
＜221〉synthetic construct
<222>(32，33)
＜223〉Xaa=Cys or Ala, Gly, His, Ser, Thr
<400>3
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
5 10 15
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
Gly Xaa Xaa
<210>4
<211>34
<212>PRT
＜213〉artificial sequence
<220>
＜221〉synthetic construct
<222>(32，33，34)
＜223〉Xaa=Cys or Ala, Gly, His, Ser, Thr
<400>4
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
5 10 15
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
Gly Xaa Xaa Xaa
<210>5
<211>131
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(118，119，120)
＜223〉n=a or g or c or t
<400>5
ccagatctgg acgacgacga caagcatgcc gaaggcacct ttaccagcga tgtgagcagc 60
tatctggaag gccaggccgc caaagaattt attgcctggc tggtgaaagg cagaggcnnn 120
taaccatgga c 131
<210>6
<211>134
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(118，119，120，121，122，123)
＜223〉n=a or g or c or t
<400>6
ccagatctgg acgacgacga caagcatgcc gaaggcacct ttaccagcga tgtgagcagc 60
tatctggaag gccaggccgc caaagaattt attgcctggc tggtgaaagg cagaggcnnn 120
nnntaaccat ggac 134
<210>7
<211>137
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(118，119，120，121，122，123，124，125，126)
＜223〉n=a or g or c or t
<400>7
ccagatctgg acgacgacga caagcatgcc gaaggcacct ttaccagcga tgtgagcagc 60
tatctggaag gccaggccgc caaagaattt attgcctggc tggtgaaagg cagaggcnnn 120
Claims (7)
1, a kind of GLP-1 derivative, it is characterized in that, the molecular structural formula of this derivative is following three kinds of structural formula: GLP-1 (7-37)-Xaa38 (Seq ID No.2), one of GLP-1 (7-37)-Xaa38-Xaa39 (Seq ID No.3) and GLP-1 (7-37)-Xaa38-Xaa39-Xaa40 (Seq ID No.4), Xaa38 wherein, Xaa39 and Xaa40 are respectively Cys, Ala, Gly, His, any one amino acid among Ser and the Thr.
2, GLP-1 derivative according to claim 1, it is characterized in that, the molecular structural formula of this derivative is following three kinds of structural formula: GLP-1 (7-37)-Xaa38 (SeqID No.2), one of GLP-1 (7-37)-Xaa38-Xaa39 (Seq ID No.3) and GLP-1 (7-37)-Xaa38-Xaa39-Xaa40 (Seq ID No.4), Xaa38=Cys wherein, Xaa39 and Xaa40 difference=Cys, Ala, Gly, His, any one amino acid among Ser and the Thr.
3, GLP-1 derivative according to claim 1, it is characterized in that, the molecular structural formula of this derivative is one of following two kinds of structural formula: GLP-1 (7-37)-Xaa38-Xaa39 (Seq ID No.3) and GLP-1 (7-37)-Xaa38-Xaa39-Xaa40 (SeqID No.4), Xaa39=Cys wherein, Xaa38 and Xaa40 difference=Cys, Ala, Gly, His, any one amino acid among Ser and the Thr.
4, GLP-1 derivative according to claim 1, it is characterized in that, the molecular structural formula of this derivative is GLP-1 (7-37)-Xaa38-Xaa39-Xaa40 (Seq IDNo.4), Xaa40=Cys wherein, Xaa38 and Xaa39 difference=Cys, Ala, Gly, His, any one amino acid among Ser and the Thr.
5, the preparation method of the described GLP-1 derivative of claim 1 is characterized in that, the concrete operations step:
The first step, the aminoacid sequence synthetic gene fragment of pressing the GLP-1 derivative;
In second step, encoding sequence is put into expression vector in the mode that is suitable for expressing independent protein or fusion rotein;
In the 3rd step, the suitable procaryotic host cell of expression vector conversion with the GLP-1 derivative gene order that contains GLP-1 derivative or fusion obtains engineering strain;
In the 4th step, engineering strain through liquid fermenting, is contained GLP-1 derivative fusion rotein or contains the proteic wet thallus of GLP-1 derivative separately through centrifugal acquisition;
In the 5th step,, obtain to contain GLP-1 derivative fusion rotein or contain the proteic crude product of GLP-1 derivative separately through separating with the wet thallus broken wall;
In the 6th step, purified, lyophilize makes product G LP-1 derivative.
6, GLP-1 derivative according to claim 5 is characterized in that, the molecular structural formula of this derivative is GLP-1 (7-37)-Xaa38 (Seq ID No.2), Xaa38=Cys wherein, and operation steps:
The first step is pressed the aminoacid sequence synthetic gene fragment of GLP-1 derivative:
Press the aminoacid sequence of GLP-1 derivative, select the intestinal bacteria preference codon for use, synthetic following length is the segment of 131bp, i.e. Seq ID No.5:
ccagatctgg acgacgacga caagcatgcc gaaggcacct ttaccagcga tgtgagcagc 60tatctggaag gccaggccgc caaagaattt attgcctggc tggtgaaagg cagaggcnnn 120taaccatgga c 131
N118=n120=t wherein, n119=g, this segment contains enteropeptidase EK site, GLP-1 derivative gene, terminator codon TAA and restriction enzyme BglII and NcoI site;
Second step was put into expression vector in the mode that is suitable for expressed fusion protein with encoding sequence:
With gene segment restriction enzyme BglII and the NcoI double digestion that obtains in the first step, purifying reclaims, simultaneously with escherichia coli plasmid pET-32a (+) restriction enzyme BglII and NcoI double digestion, purifying reclaims big segment, above-mentioned enzyme is cut gene segment to be mixed with digested plasmid, add T4DNA ligase enzyme and T4DNA ligase enzyme damping fluid, room temperature connects 3-4 hour, makes up the plasmid pET-32a-mGLP-1 that obtains containing GLP-1 derivative gene order;
The expression vector of the 3rd step with the GLP-1 derivative gene order that contains fusion transforms suitable procaryotic host cell, obtains engineering strain:
Plasmid pET-32a-mGLP-1 is converted in the recipient bacterium bacillus coli DH 5 alpha, ice bath 30 minutes, be warming up to 42 ℃, after keeping 90 seconds, be transferred to the agarose plate that contains penbritin, 37 ℃ are spent the night, and filter out the bacterium colony of ammonia benzyl resistance, obtain containing the engineering strain of GLP-1 derivative gene order;
The 4th step through liquid fermenting, was contained the wet thallus of GLP-1 derivative fusion rotein with engineering strain through centrifugal acquisition:
To build the engineering strain that contains GLP-1 derivative gene order when in the LB liquid nutrient medium, being cultured to 0D600nm=0.6-0.8, the adding final concentration is that the IPTG of 0.4-0.8mM carried out abduction delivering 3-5 hour, produce and accumulate the GLP-1 derivative fusion rotein of solubility expression, centrifugal, abandon supernatant, collect wet thallus;
The 5th step is with the wet thallus broken wall, through separating the crude product that obtains to contain GLP-1 derivative fusion rotein:
With NTAO damping fluid wet thallus of collecting in resuspended the 4th step, after the carrying out ultrasonic bacteria breaking, centrifugal collection supernatant, carry out NTAO resin affinity chromatography, collect GLP-1 derivative fusion rotein component, with molecular weight cut-off is that the Millipore Amicon Ultra-15 ultrafiltration pipe of 1KD-5KD is concentrated into albumen more than the 2mg/mL, obtains to contain the crude product of GLP-1 derivative fusion rotein;
The 6th step is purified, and lyophilize makes product G LP-1 derivative:
With enteropeptidase enzymolysis GLP-1 derivative fusion rotein crude product, at 4-37 ℃ of cracking 2-16 hour, termination reaction, and then carry out NTAO resin affinity chromatography, and to collect and penetrate the peak, lyophilize obtains product G LP-1 derivative.
7, the described GLP-1 derivative of claim 1 is done the application of the activeconstituents of this medicine in the medicine of preparation treatment diabetes.
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