CN101665799A - Recombination preparation method and application of Exendin-4 derivative - Google Patents
Recombination preparation method and application of Exendin-4 derivative Download PDFInfo
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Abstract
The invention relates to a recombination preparation method and an application of an Exendin-4 derivative with Exendin-4 activity. The molecular structural formula of the derivative is one of the following six forms of Exendin-4 (1-39)-Xaa40 (Seq ID No.2), Exendin-4 (1-39)-Xaa40-Xaa41 (Seq ID No.3), Exendin-4 (1-39)-Xaa40-Xaa41-Xaa42 (Seq ID No.4), Exendin-4 (1-30)-Xaa31-(Seq ID No.6), Exendin-4(1-30)-Xaa31-Xaa32 (Seq ID No.7) and Exendin-4 (1-30)-Xaa31-Xaa32-Xaa33 (Seq IN No.8), wherein Xaa40, Xaa41, Xaa42, Xaa31, Xaa32 and Xaa33 are respectively any one amino acid of Cys, Ala, Gly, His, Serand Thr. The derivative is prepared by a DNA recombination technology, has a longer half-life period than Exendin-4, is easy to purify with low cost, and is especially suitable to be used as an activecomponent of the medicament in the preparation of a medicament for treating diabetes.
Description
Technical field
The present invention relates to the active Exendin-4 derivative of a kind of Exendin-4 of having and preparation and application, belong to technical field of bioengineering.
Background technology
1992, Eng etc. are separated to a kind of albumen of being made up of 39 amino acid from Gila monster (Heloderma suspectum) saliva that is distributed in State of Arizona, US and Mexico the north, with GLP-1 53% homology is arranged, after with its called after Exendin-4.Studies show that subsequently, Exendin-4 and GLP-1 act on same acceptor, and signal transduction pathway is similar, has identical physiological function in mammalian body.The insulin secretion that Exendin-4 can stimulate glucose to rely on promptly stimulates insulin secretion when blood sugar concentration is high, does not stimulate insulin secretion when lower concentration and normal concentration; Also have the Beta cell proliferation of promotion simultaneously and secrete, slow down effects such as stomach emptying, inhibition food absorption with new life, glucagon suppression.In April, 2005, the GLP-1 analogue Exenatide of chemosynthesis goes on the market through drugs approved by FDA, clinical experiment shows: Exenatide can effectively control N1,N1-Dimethylbiguanide and the unfavorable type ii diabetes of sulfonylurea curative effect, reduce empty stomach and postprandial blood sugar, reduce saccharification hemoglobin content, lose weight, type ii diabetes is had curative effect preferably.
But, because the transformation period of Exenatide (amidated Exendin-4) is lacked (2 ~ 4 hours), need subcutaneous injection every day twice, bring very big misery to the patient.As seen still there is bigger defective in Exenatide in clinical application, therefore presses for exploitation transformation period Exendin-4 derivative longer, that be convenient to clinical use.
Be by its structure being changed, the Exendin-4 derivative that obtains being applied to treating diabetes, to reach the purpose of prolong drug effective drug duration in vivo both at home and abroad to one of research direction of Exendin-4.U.S. Amylin company adopts the epsilon-amino on the polymer-modified Exendin-4 Methionin of one or more polyoxyethylene glycol (PEG), above-mentioned Exendin-4 derivative is compared with natural Exendin-4, can the significant prolongation transformation period, can also reduce immunogenicity, opposing protease hydrolysis in addition, improve bioavailability, reduce toxicity and increase stability, but the preparation technology of this method is complicated, product separation and purification difficulty, and yield is very low, is unfavorable for suitability for industrialized production.
The Exendin-4 derivative can prepare by solid state chemistry is synthetic, (application number is for " 200910045187.6 " to see the inventor's application for a patent for invention, denomination of invention is " a kind of Exendin-4 derivative and solid state chemistry thereof are synthetic "), but this preparation method's cost is higher, therefore, the invention provides a kind of economical and efficient, utilize the DNA recombinant technology to prepare the method for Exendin-4 derivative.
Summary of the invention
The Exendin-4 that the present invention relates to refers to Exendin-4 (1-39), and its sequence (Seq ID No.1) is enumerated as follows:
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。In addition, also relate to the clipped form Exendin-4 (1-30) of Exendin-4, its sequence (Seq ID No.5) is enumerated as follows:
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。
The Exendin-4 derivative m39Exendin-4 that the present invention proposes has following three kinds of structure: Exendin-4 (1-39)-Xaa40 (Seq ID No.2), one of Exendin-4 (1-39)-Xaa40-Xaa41 (Seq ID No.3) and Exendin-4 (1-39)-Xaa40-Xaa41-Xaa42 (Seq ID No.4), Xaa40 wherein, Xaa41 and Xaa42 are respectively Cys, Ala, Gly, His, any one amino acid among Ser and the Thr.
The Exendin-4 derivative m30Exendin-4 that the present invention proposes has following three kinds of structure: Exendin-4 (1-30)-Xaa31 (Seq ID No.6), one of Exendin-4 (1-30)-Xaa31-Xaa32 (Seq ID No.7) and Exendin-4 (1-30)-Xaa31-Xaa32-Xaa33 (Seq ID No.8), Xaa31 wherein, Xaa32 and Xaa33 are respectively Cys, Ala, Gly, His, any one amino acid among Ser and the Thr.
Said derivative has not only kept natural Exendin-4 activity in vivo, and the effective drug duration in the body of having gone back significant prolongation has very good clinical value.In addition, this derivative is by the preparation of DNA recombinant technology, and cost is low, and the output height helps scale operation and use.
Technical scheme of the present invention provides the method for utilizing the DNA recombinant technology to prepare said derivative.
Produce the Exendin-4 derivative by the DNA recombinant technology, cost is low, and the output height is easy to purifying, and loss of activity is less in the purge process.For achieving the above object, technical scheme of the present invention selects for use the DNA recombinant technology to prepare the Exendin-4 derivative.
Now describe technical scheme of the present invention in detail.
A kind of preparation method of Exendin-4 derivative is characterized in that, comprises step:
(1) presses the aminoacid sequence synthetic gene fragment of Exendin-4 derivative;
(2) make up the recombinant expression vector that contains Exendin-4 derivative encoding sequence;
(3) expression vector with the Exendin-4 derivative gene order that contains Exendin-4 derivative or fusion changes procaryotic host cell over to, obtains engineering strain;
(4) with engineering strain through liquid fermenting, centrifugal acquisition contains Exendin-4 derivative fusion rotein or contains the proteic wet thallus of Exendin-4 derivative separately;
(5), separate obtaining to contain Exendin-4 derivative fusion rotein or contain the proteic crude product of Exendin-4 derivative separately with the wet thallus broken wall;
(6) purified, lyophilize makes the Exendin-4 derivative.
Another technical scheme of the present invention provides the Exendin-4 derivative is made active constituents of medicine in the medicine of preparation prevention or treatment diabetes application.
Another technical scheme of the present invention provides the composition that contains significant quantity Exendin-4 derivative and the acceptable composition of pharmacy.
Exendin-4 derivative of the present invention preferably stores with aseptic freeze-dried thing form, before administration, mix with suitable isotonic solution, wherein contain pharmaceutically acceptable composition such as N.F,USP MANNITOL, Sorbitol Powder, sodium-chlor etc., then can be by mode administrations such as intravenous injection, abdominal injection or subcutaneous injections.
Description of drawings
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and following m39Exendin-4 and m30Exendin-4 represent Exendin-4 derivative of the present invention.
Fig. 1 is the structure of plasmid pET32a (+)-m39Exendin-4, and wherein this plasmid contains the Exendin-4 derivative.
Fig. 2 is the structure of plasmid pET32a (+)-m39Exendin-4 (Trx-), and wherein this plasmid contains the Exendin-4 derivative.
Fig. 3 is the structure of plasmid pET32a (+)-m30Exendin-4, and wherein this plasmid contains the Exendin-4 derivative.
Fig. 4 is the structure of plasmid pET32a (+)-m30Exendin-4 (Trx-), and wherein this plasmid contains the Exendin-4 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.
Embodiment 1DNA recombinant technology prepares Exendin-4 derivative of the present invention, and the molecular structural formula of this derivative is Seq ID No.2, i.e. Exendin-4 (1-39)-Xaa40, wherein Xaa40=Cys;
Pressing the aminoacid sequence of Exendin-4 derivative in the step (1), select the intestinal bacteria preference codon for use, is the segment of 164bp by the synthetic following length of PCR method, i.e. Seq ID No.9:
gccctagatc?tggatgacga?tgacaagcat?ggcgaaggca?cctttaccag?cgatctgagc 60
aaacagatgg?aagaagaagc?ggtgcgcctg?tttattgaat?ggctgaaaaa?tggtggtccg?120
agcagcggtg?cgccgccgcc?gagcnnntaa?tgaaagctta?aact 164
Wherein, n145=t, n146=g, n147=c;
Wherein, this segment contains enteropeptidase EK site, Exendin-4 derivative gene, terminator codon TAA and restriction enzyme BglII and HindIII site;
In the step (2), the said gene segment is with restriction enzyme BglII and HindIII double digestion and purifying recovery, reclaim big segment with restriction enzyme BglII and HindIII double digestion escherichia coli plasmid pET32a (+) and purifying, the two mixes the back and adds the T4DNA ligase enzyme, 16 ℃ connect 1-8 hour, make up recombinant plasmid pET32a (+)-m39Exendin-4 that obtains containing Exendin-4 derivative gene order;
In the step (3), recombinant plasmid pET32a (+)-m39Exendin-4 mixes with recipient bacterium e. coli bl21 (DE3), ice bath 15-30 minute, 42 ℃ of water-bath heat shock 60-90 seconds, cultivated 30-45 minute for 37 ℃, the coating of bacterium liquid contains the agarose plate of penbritin, cultivates the bacterium colony that occurs after 12-16 hour for 37 ℃ and is the engineering strain that contains Exendin-4 derivative gene order;
In the step (4), the engineering strain that contains Exendin-4 derivative gene order is cultured to OD600nm=0.5-0.9 in containing the LB liquid nutrient medium of penbritin, add IPTG to final concentration 0.5-1.0mmol/L, induced 3-5 hour centrifugal collection wet thallus for 37 ℃;
In the step (5), thalline is resuspended in the IDA-0 damping fluid, ultrasonication in the ice bath, centrifugal back is collected supernatant and is carried out nickel ion chelating affinity chromatography, collection contains the elution fraction of Exendin-4 derivative fusion rotein, adopt molecular weight cut-off be the Amicon Ultra-15 ultra-filtration centrifuge tube of 5kD with the albumen desalination, be concentrated into more than the 3mg/mL, obtain containing the crude product of Exendin-4 derivative fusion rotein;
In the step (6), in Exendin-4 derivative fusion rotein crude product, add enteropeptidase, 23-25 ℃ cracking 6-12 hour, reaction mixture carries out nickel ion chelating affinity chromatography again, collects to penetrate the peak, lyophilize obtains the Exendin-4 derivative.
Embodiment 2DNA recombinant technology prepares Exendin-4 derivative of the present invention, and the molecular structural formula of this derivative is Seq ID No.3, i.e. Exendin-4 (1-39)-Xaa40-Xaa41, Xaa40=Cys wherein, Xaa41=Gly;
Pressing the aminoacid sequence of Exendin-4 derivative in the step (1), select the intestinal bacteria preference codon for use, is the segment of 167bp by the synthetic following length of PCR method, i.e. Seq ID No.10:
gccctagatc?tggatgacga?tgacaagcat?ggcgaaggca?cctttaccag?cgatctgagc 60
aaacagatgg?aagaagaagc?ggtgcgcctg?tttattgaat?ggctgaaaaa?tggtggtccg?120
agcagcggtg?cgccgccgcc?gagcnnnnnn?taatgaaagc?ttaaact?167
Wherein, n145=t, n146=n148=n149=g, n147=n150=c;
Wherein, this segment contains enteropeptidase EK site, Exendin-4 derivative gene, terminator codon TAA and restriction enzyme BglII and HindIII site;
All the other steps are with embodiment 1.
Embodiment 3DNA recombinant technology prepares Exendin-4 derivative of the present invention, and the molecular structural formula of this derivative is Seq ID No.3, i.e. Exendin-4 (1-39)-Xaa40-Xaa41, Xaa40=Gly wherein, Xaa41=Cys;
In step (1) the synthetic Seq ID No.10 gene segment, n145=n146=n149=g, n147=n150=c, n148=t.
All the other steps are with embodiment 1.
Embodiment 4DNA recombinant technology prepares Exendin-4 derivative of the present invention, and the molecular structural formula of this derivative is Seq ID No.4, i.e. Exendin-4 (1-39)-Xaa40-Xaa41-Xaa42, Xaa40=Cys wherein, Xaa41=Ala, Xaa42=Gly;
Pressing the aminoacid sequence of Exendin-4 derivative in the step (1), select the intestinal bacteria preference codon for use, is the segment of 170bp by the synthetic following length of PCR method, i.e. Seq ID No.11:
gccctagatc?tggatgacga?tgacaagcat?ggcgaaggca?cctttaccag?cgatctgagc 60
aaacagatgg?aagaagaagc?ggtgcgcctg?tttattgaat?ggctgaaaaa?tggtggtccg?120
agcagcggtg?cgccgccgcc?gagcnnnnnn?nnntaatgaa?agcttaaact 170
Wherein, n145=t, n146=n148=n150=n151=n152=g, n147=n149=n153=c;
Wherein, this segment contains enteropeptidase EK site, Exendin-4 derivative gene, terminator codon TAA and restriction enzyme BglII and HindIII site;
All the other steps are with embodiment 1.
Embodiment 5DNA recombinant technology prepares Exendin-4 derivative of the present invention, and the molecular structural formula of this derivative is Seq ID No.4, i.e. Exendin-4 (1-39)-Xaa40-Xaa41-Xaa42, Xaa40=Ala wherein, Xaa41=Cys, Xaa42=Gly;
In step (1) the synthetic Seq ID No.11 gene segment, n145=t, n146=n148=n150=n151=n152=g, n147=n149=n153=c.
All the other steps are with embodiment 1.
Embodiment 6DNA recombinant technology prepares Exendin-4 derivative of the present invention, and the molecular structural formula of this derivative is Seq ID No.4, i.e. Exendin-4 (1-39)-Xaa40-Xaa41-Xaa42, Xaa40=Ala wherein, Xaa41=Gly, Xaa42=Cys;
In step (1) the synthetic Seq ID No.11 gene segment, n145=n147=n148=n149=n152=g, n146=n150=n153=c, n151=t.
All the other steps are with embodiment 1.
Embodiment 7 prepares the Exendin-4 derivative of Seq ID No.2 to knock out the Trx mode;
Plasmid pET32a (+)-m39Exendin-4 that will contain Exendin-4 derivative gene order in the step (2) cuts with restriction enzyme Nde I enzyme and makes up plasmid pET32a (+)-m39Exendin-4 (Trx-) that obtains containing Exendin-4 derivative gene order, and wherein Trx is a Trx.
Embodiment 8DNA recombinant technology prepares Exendin-4 derivative of the present invention, and the molecular structural formula of this derivative is Seq ID No.6, i.e. Exendin-4 (1-30)-Xaa31, wherein Xaa31=Cys;
Pressing the aminoacid sequence of Exendin-4 derivative in the step (1), select the intestinal bacteria preference codon for use, is the segment of 137bp by the synthetic following length of PCR method, i.e. Seq ID No.12:
cctccagatc?tggatgacga?tgacaagcat?ggcgaaggca?cctttaccag?cgatctgagc 60
aaacagatgg?aagaagaagc?ggtgcgcctg?tttattgaat?ggctgaaaaa?tggcggcnnn?120
taatgaaagc?ttcctcc 137
Wherein, n118=t, n119=g, n120=c;
Wherein, this segment contains enteropeptidase EK site, Exendin-4 derivative gene, terminator codon TAA and restriction enzyme BglII and HindIII site;
All the other steps are with embodiment 1.
Embodiment 9DNA recombinant technology prepares Exendin-4 derivative of the present invention, and the molecular structural formula of this derivative is Seq ID No.7, i.e. Exendin-4 (1-30)-Xaa31-Xaa32, Xaa31=Cys wherein, Xaa32=Gly;
Pressing the aminoacid sequence of Exendin-4 derivative in the step (1), select the intestinal bacteria preference codon for use, is the segment of 140bp by the synthetic following length of PCR method, i.e. Seq ID No.13:
cctccagatc?tggatgacga?tgacaagcat?ggcgaaggca?cctttaccag?cgatctgagc 60
aaacagatgg?aagaagaagc?ggtgcgcctg?tttattgaat?ggctgaaaaa?tggcggcnnn?120
nnntaatgaa?agcttcctcc 140
Wherein, n118=t, n119=n121=n122=g, n120=n123=c;
Wherein, this segment contains enteropeptidase EK site, Exendin-4 derivative gene, terminator codon TAA and restriction enzyme BglII and HindIII site;
All the other steps are with embodiment 1.
Embodiment 10DNA recombinant technology prepares Exendin-4 derivative of the present invention, and the molecular structural formula of this derivative is Seq ID No.7, i.e. Exendin-4 (1-30)-Xaa31-Xaa32, Xaa31=Gly wherein, Xaa32=Cys;
In step (1) the synthetic Seq ID No.13 gene segment, n118=n119=n122=g, n120=n123=c, n121=t.
All the other steps are with embodiment 1.
Embodiment 11DNA recombinant technology prepares Exendin-4 derivative of the present invention, and the molecular structural formula of this derivative is Seq ID No.8, i.e. Exendin-4 (1-30)-Xaa31-Xaa32-Xaa33, Xaa31=Cys wherein, Xaa32=Ala, Xaa33=Gly;
Pressing the aminoacid sequence of Exendin-4 derivative in the step (1), select the intestinal bacteria preference codon for use, is the segment of 143bp by the synthetic following length of PCR method, i.e. Seq ID No.14:
cctccagatc?tggatgacga?tgacaagcat?ggcgaaggca?cctttaccag?cgatctgagc 60
aaacagatgg?aagaagaagc?ggtgcgcctg?tttattgaat?ggctgaaaaa?tggcggcnnn?120
nnnnnntaat?gaaagcttcc?tcc 143
Wherein, n118=t, n119=n121=n123=n124=n125=g, n120=n122=n126=c;
Wherein, this segment contains enteropeptidase EK site, Exendin-4 derivative gene, terminator codon TAA and restriction enzyme BglII and HindIII site;
All the other steps are with embodiment 1.
Embodiment 12DNA recombinant technology prepares Exendin-4 derivative of the present invention, and the molecular structural formula of this derivative is Seq ID No.8, i.e. Exendin-4 (1-30)-Xaa31-Xaa32-Xaa33, Xaa31=Ala wherein, Xaa32=Cys, Xaa33=Gly;
In step (1) the synthetic Seq ID No.14 gene segment, n118=n120=n122=n124=n125=g, n119=n123=n126=c, n121=t.
All the other steps are with embodiment 1.
Embodiment 13DNA recombinant technology prepares Exendin-4 derivative of the present invention, and the molecular structural formula of this derivative is SeqID No.8, i.e. Exendin-4 (1-30)-Xaa31-Xaa32-Xaa33, Xaa31=Ala wherein, Xaa32=Gly, Xaa33=Cys;
In step (1) the synthetic Seq ID No.14 gene segment, n118=n120=n121=n122=n125=g, n119=n123=n126=c, n124=t.
All the other steps are with embodiment 1.
Embodiment 14 prepares the Exendin-4 derivative of Seq ID No.6 to knock out the Trx mode;
Plasmid pET32a (+)-m30Exendin-4 that will contain Exendin-4 derivative gene order in the step (2) cuts with restriction enzyme Nde I enzyme and makes up plasmid pET32a (+)-m30Exendin-4 (Trx-) that obtains containing Exendin-4 derivative gene order, and wherein Trx is a Trx.
The hypoglycemic activity of embodiment 15Exendin-4 derivative.
Experiment material and method:
Female Sexual health kunming mice (cleaning level, Fudan University in Shanghai medical college animal center provides);
60% glucose solution;
0.9%NaCl solution;
Exendin-4(1-39)；
Exendin-4(1-30)；
M39Exendin-4 has the structure of embodiment 1 described Exendin-4 derivative;
M30Exendin-4 has the structure of embodiment 8 described Exendin-4 derivatives;
Blood glucose monitoring system (the newly upright medicine equipment company limited in Shanghai produces).
Female Sexual health kunming mice overnight fasting is divided into 5 groups (n=10).1, the physiological saline control group; 2, Exendin-4 (1-39) administration control group; 3, Exendin-4 (1-30) administration control group; 4, m39Exendin-4 administration group, concrete sequential structure is the structure described in the embodiment 1; 5, m30Exendin-4 administration group, concrete sequential structure is the structure described in the embodiment 8.The Exendin-4 (1-39) of Exendin-4 (1-39) administration control group 2 abdominal injection 0.5ug; The Exendin-4 (1-30) of Exendin-4 (1-30) administration control group 3 abdominal injection 0.4ug; M39Exendin-4 administration group 4～9, the m39Exendin-4 of every group of difference abdominal injection 0.5ug; M30Exendin-4 administration group 10～15, the m30Exendin-4 of every group of difference abdominal injection 0.4ug; Note was zero moment at this moment.1～3 group gave 100uL 60% glucose solution in 5.5,9.5,11.5,13.5,23.5,27.5,29.5,35.5 hours, carried out mouse tail vein respectively at 6,10,12,14,24,28,30,36 hours and get blood 10ul, test sugared instrument with blood and measure blood sugar concentration; 4 groups gave 100uL 60% glucose solution in 5.5,11.5,23.5,29.5,35.5 hours, carried out mouse tail vein respectively at 6,12,24,30,36 hours and got blood 10ul, tested sugared instrument with blood and measured blood sugar concentration; 5 groups gave 100uL 60% glucose solution in 5.5,9.5,13.5,23.5,27.5 hours, carried out mouse tail vein respectively at 6,10,14,24,28 hours and got blood 10ul, tested sugared instrument with blood and measured blood sugar concentration.
The result is as shown in the table, shown in numerical value be the average of n=10, shown in the hypoglycemic rate calculate as follows: hypoglycemic rate (%)=(physiological saline control group blood glucose value-administration group blood glucose value)/physiological saline control group blood glucose value.Compare with the physiological saline control group mice, after administration 6 hours, Exendin-4 (1-39) administration control group and Exendin-4 (1-30) administration control group all can not significantly reduce mouse blood sugar, a little higher than Exendin-4 of hypoglycemic activity (1-30) of Exendin-4 (1-39).And m39Exendin-4 administration group can significantly reduce mouse blood sugar in 30 hours, and m30Exendin-4 administration group can significantly reduce mouse blood sugar in 24 hours, and duration of efficacy all prolongs than Exendin-4 (1-39).The result shows that also the m39Exendin-4 and the m30Exendin-4 that prepare by the inventive method all show than synthetic m39Exendin-4 and the more long lasting hypoglycemic activity of m30Exendin-4 for preparing of solid state chemistry.
The hypoglycemic activity of table m39Exendin-4, m30Exendin-4
Embodiment 16 comprises the composition of Exendin-4 derivative
Get the m30Exendin-4 of 1mg, be dissolved in the 0.5mL deionized water by the preparation of DNA recombinant technology; Other gets 50mg N.F,USP MANNITOL, is dissolved in the 0.5mL deionized water; The two mixing is the composition of m30Exendin-4 and N.F,USP MANNITOL, wherein contains the m30Exendin-4 of 1mg/mL and the N.F,USP MANNITOL of 50mg/mL.
Sequence table
＜110〉East China Normal University
＜120〉a kind of recombination and preparation of Exendin-4 derivative and application
<160>8
<210>1
<211>39
<212>PRT
＜213〉Gila monster (Heloderma suspectum)
<400>1
His?Gly?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Lys?Gln?Met?Glu
5 10 15
Glu?Glu?Ala?Val?Arg?Leu?Phe?Ile?Glu?Trp?Leu?Lys?Asn?Gly?Gly
20 25 30
Pro?Ser?Ser?Gly?Ala?Pro?Pro?Pro?Ser
35
<210>2
<211>40
<212>PRT
＜213〉artificial sequence
<220>
＜221〉synthetic construct
<222>(40)
＜223〉Xaa=Cys, Ala, Gly, His, Ser or Thr
<400>2
His?Gly?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Lys?Gln?Met?Glu
5 10 15
Glu?Glu?Ala?Val?Arg?Leu?Phe?Ile?Glu?Trp?Leu?Lys?Asn?Gly?Gly
20 25 30
Pro?Ser?Ser?Gly?Ala?Pro?Pro?Pro?Ser?Xaa
35 40
<210>3
<211>41
<212>PRT
＜213〉artificial sequence
<220>
＜221〉synthetic construct
<222>(40，41)
＜223〉Xaa=Cys, Ala, Gly, His, Ser or Thr
<400>3
His?Gly?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Lys?Gln?Met?Glu
5 10 15
Glu?Glu?Ala?Val?Arg?Leu?Phe?Ile?Glu?Trp?Leu?Lys?Asn?Gly?Gly
20 25 30
Pro?Ser?Ser?Gly?Ala?Pro?Pro?Pro?Ser?Xaa?Xaa
35 40
<210>4
<211>42
<212>PRT
＜213〉artificial sequence
<220>
＜221〉synthetic construct
<222>(40，41，42)
＜223〉Xaa=Cys, Ala, Gly, His, Ser or Thr
<400>4
His?Gly?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Lys?Gln?Met?Glu
5 10 15
Glu?Glu?Ala?Val?Arg?Leu?Phe?Ile?Glu?Trp?Leu?Lys?Asn?Gly?Gly
20 25 30
Pro?Ser?Ser?Gly?Ala?Pro?Pro?Pro?Ser?Xaa?Xaa?Xaa
35 40
<210>5
<211>30
<212>PRT
＜213〉artificial sequence
<400>5
His?Gly?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Lys?Gln?Met?Glu
5 10 15
Glu?Glu?Ala?Val?Arg?Leu?Phe?Ile?Glu?Trp?Leu?Lys?Asn?Gly?Gly
20 25 30
<210>6
<211>31
<212>PRT
＜213〉artificial sequence
<220>
＜221〉synthetic construct
<222>(31)
＜223〉Xaa=Cys, Ala, Gly, His, Ser or Thr
<400>6
His?Gly?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Lys?Gln?Met?Glu
5 10 15
Glu?Glu?Ala?Val?Arg?Leu?Phe?Ile?Glu?Trp?Leu?Lys?Asn?Gly?Gly
20 25 30
Xaa
<210>7
<211>32
<212>PRT
＜213〉artificial sequence
<220>
＜221〉synthetic construct
<222>(31，32)
＜223〉Xaa=Cys, Ala, Gly, His, Ser or Thr
<400>7
His?Gly?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Lys?Gln?Met?Glu
5 10 15
Glu?Glu?Ala?Val?Arg?Leu?Phe?Ile?Glu?Trp?Leu?Lys?Asn?Gly?Gly
20 25 30
Xaa?Xaa
<210>8
<211>33
<212>PRT
＜213〉artificial sequence
<220>
＜221〉synthetic construct
<222>(31，32，33)
＜223〉Xaa=Cys, Ala, Gly, His, Ser or Thr
<400>8
His?Gly?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Leu?Ser?Lys?Gln?Met?Glu
5 10 15
Glu?Glu?Ala?Val?Arg?Leu?Phe?Ile?Glu?Trp?Leu?Lys?Asn?Gly?Gly
20 25 30
Xaa?Xaa?Xaa
<210>9
<211>164
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(145，146，147)
＜223〉n=a or g or c or t
<400>9
gccctagatc?tggatgacga?tgacaagcat?ggcgaaggca?cctttaccag?cgatctgagc 60
aaacagatgg?aagaagaagc?ggtgcgcctg?tttattgaat?ggctgaaaaa?tggtggtccg?120
agcagcggtg?cgccgccgcc?gagcnnntaa?tgaaagctta?aact 164
<210>10
<211>167
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(145，146，147，148，149，150)
＜223〉n=a or g or c or t
<400>10
gccctagatc?tggatgacga?tgacaagcat?ggcgaaggca?cctttaccag?cgatctgagc 60
aaacagatgg?aagaagaagc?ggtgcgcctg?tttattgaat?ggctgaaaaa?tggtggtccg?120
agcagcggtg?cgccgccgcc?gagcnnnnnn?taatgaaagc?ttaaact 167
<210>11
<211>170
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(145，146，147，148，149，150，151，152，153)
＜223〉n=a or g or c or t
<400>11
gccctagatc?tggatgacga?tgacaagcat?ggcgaaggca?cctttaccag?cgatctgagc?60
aaacagatgg?aagaagaagc?ggtgcgcctg?tttattgaat?ggctgaaaaa?tggtggtccg?120
agcagcggtg?cgccgccgcc?gagcnnnnnn?nnntaatgaa?agcttaaact 170
<210>12
<211>137
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(118，119，120)
＜223〉n=a or g or c or t
<400>12
cctccagatc?tggatgacga?tgacaagcat?ggcgaaggca?cctttaccag?cgatctgagc 60
aaacagatgg?aagaagaagc?ggtgcgcctg?tttattgaat?ggctgaaaaa?tggcggcnnn 120
taatgaaagc?ttcctcc 137
<210>13
<211>140
<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>13
cctccagatc?tggatgacga?tgacaagcat?ggcgaaggca?cctttaccag?cgatctgagc 60
aaacagatgg?aagaagaagc?ggtgcgcctg?tttattgaat?ggctgaaaaa?tggcggcnnn 120
nnntaatgaa?agcttcctcc 140
<210>14
<211>143
<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>14
cctccagatc?tggatgacga?tgacaagcat?ggcgaaggca?cctttaccag?cgatctgagc 60
aaacagatgg?aagaagaagc?ggtgcgcctg?tttattgaat?ggctgaaaaa?tggcggcnnn?120
nnnnnntaat gaaagcttcc?tcc?143
Claims (8)
1. the recombination and preparation of an Exendin-4 derivative is characterized in that, comprises step:
(1) presses the aminoacid sequence synthetic gene fragment of Exendin-4 derivative;
(2) make up the recombinant expression vector that contains Exendin-4 derivative encoding sequence;
(3) expression vector with the Exendin-4 derivative gene order that contains Exendin-4 derivative or fusion changes procaryotic host cell over to, obtains engineering strain;
(4) with engineering strain through liquid fermenting, centrifugal acquisition contains Exendin-4 derivative fusion rotein or contains the proteic wet thallus of Exendin-4 derivative separately;
(5), separate obtaining to contain Exendin-4 derivative fusion rotein or contain the proteic crude product of Exendin-4 derivative separately with the wet thallus broken wall;
(6) purified, lyophilize makes the Exendin-4 derivative.
2. according to the preparation method of the described Exendin-4 derivative of claim 1, it is characterized in that the molecular structural formula of this derivative is Seq ID No.2, i.e. Exendin-4 (1-39)-Xaa40, wherein Xaa40=Cys;
In the step (1), press the aminoacid sequence of Exendin-4 derivative, select the intestinal bacteria preference codon for use, synthetic following length is the segment of 164bp, i.e. Seq ID No.9:
gccctagatc?tggatgacga?tgacaagcat?ggcgaaggca?cctttaccag?cgatctgagc?60
aaacagatgg?aagaagaagc?ggtgcgcctg?tttattgaat?ggctgaaaaa?tggtggtccg?120
agcagcggtg?cgccgccgcc?gagcnnntaa?tgaaagctta?aact 164
Wherein, n145=t, n146=g, n147=c;
Wherein, this segment contains enteropeptidase EK site, Exendin-4 derivative gene, terminator codon TAA and restriction enzyme BglII and HindIII site;
In the step (2),, be expression vector, make up recombinant plasmid pET32a (+)-m39Exendin-4 that obtains containing Exendin-4 derivative gene order with escherichia coli plasmid pET32a (+) with restriction enzyme BglII and HindIII double digestion;
In the step (3), the host receptor bacterium is e. coli bl21 (DE3).
3. according to the preparation method of the described Exendin-4 derivative of claim 2, it is characterized in that, prepare the Exendin-4 derivative of Seq ID No.2 to knock out the Trx mode; Wherein in the step (2), plasmid pET32a (+)-m39Exendin-4 that will contain Exendin-4 derivative gene order cuts with restriction enzyme Nde I enzyme, structure obtains containing plasmid pET32a (+)-m39Exendin-4 (Trx-) of Exendin-4 derivative gene order, and wherein Trx is a Trx.
4. according to the preparation method of the described Exendin-4 derivative of claim 1, it is characterized in that the molecular structural formula of this derivative is Seq ID No.6, i.e. Exendin-4 (1-30)-Xaa31, wherein Xaa31=Cys;
In the step (1), press the aminoacid sequence of Exendin-4 derivative, select the intestinal bacteria preference codon for use, synthetic following length is the segment of 137bp, i.e. Seq ID No.12:
cctccagatc?tggatgacga?tgacaagcat?ggcgaaggca?cctttaccag?cgatctgagc?60
aaacagatgg?aagaagaagc?ggtgcgcctg?tttattgaat?ggctgaaaaa?tggcggcnnn?120
taatgaaagc?ttcctcc 137
Wherein, n118=t, n119=g, n120=c;
Wherein, this segment contains enteropeptidase EK site, Exendin-4 derivative gene, terminator codon TAA and restriction enzyme BglII and HindIII site;
In the step (2),, be expression vector, make up recombinant plasmid pET32a (+)-m30Exendin-4 that obtains containing Exendin-4 derivative gene order with escherichia coli plasmid pET32a (+) with restriction enzyme BglII and HindIII double digestion;
In the step (3), recipient bacterium is e. coli bl21 (DE3).
5. according to the preparation method of the described Exendin-4 derivative of claim 4, it is characterized in that prepare the Exendin-4 derivative to knock out the Trx mode, the molecular structural formula of this derivative is Seq IDNo.6; Wherein in the step (2), plasmid pET32a (+)-m30Exendin-4 that will contain Exendin-4 derivative gene order cuts with restriction enzyme Nde I enzyme, structure obtains containing plasmid pET32a (+)-m30Exendin-4 (Trx-) of Exendin-4 derivative gene order, and wherein Trx is a Trx.
6. the preparation method of Exendin-4 derivative according to claim 1, it is characterized in that, recombination engineering bacteria in the step (3) is the bacillus coli DH 5 alpha that carries recombinant plasmid, BL21 (DE3) or BLR (DE3), described recombinant plasmid is four kinds of recombinant plasmid pET32a (+)-m39Exendin-4 that contains the described Exendin-4 derivative of claim 1 gene, pET32a (+)-m39Exendin-4 (Trx-), pET32a (+)-m30Exendin-4 or pET32a (+)-m30Exendin-4's (Trx-) is one of any, and wherein Trx is a Trx.
7. in the medicine of preparation treatment diabetes, do the application of the activeconstituents of this medicine by the Exendin-4 derivative of the described method preparation of claim 1.
8. a composition is characterized in that, described composition contains the Exendin-4 derivative by the described method preparation of claim 1 of significant quantity, and the acceptable composition of pharmacy.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618552A (en) * | 2012-04-01 | 2012-08-01 | 东莞市麦亘生物科技有限公司 | Productive technology of recombined exenatide |
| WO2015127273A1 (en) * | 2014-02-21 | 2015-08-27 | Medimmune, Llc | Anti-pcsk9~glp-1 fusions and methods for use |
| WO2016005903A3 (en) * | 2014-07-08 | 2016-03-10 | Theramyt Novobiologics Private Limited | A process for obtaining exendin-4 |
| CN112608964A (en) * | 2020-12-18 | 2021-04-06 | 北京博康健基因科技有限公司 | Fermentation method for large-scale production of recombinant Exendin-4 polypeptide |
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| CN101298477A (en) * | 2008-05-20 | 2008-11-05 | 大连民族学院 | Novel insulin derivates and preparation thereof |
| CN101463078A (en) * | 2009-01-12 | 2009-06-24 | 华东师范大学 | Exendin-4 derivative and solid phase chemical synthesis thereof |
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| EP1386930A1 (en) * | 2001-05-10 | 2004-02-04 | Shanghai Huayi Bio Lab | Derivatives of exendin |
| CN101298477A (en) * | 2008-05-20 | 2008-11-05 | 大连民族学院 | Novel insulin derivates and preparation thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618552A (en) * | 2012-04-01 | 2012-08-01 | 东莞市麦亘生物科技有限公司 | Productive technology of recombined exenatide |
| CN102618552B (en) * | 2012-04-01 | 2014-08-20 | 东莞市麦亘生物科技有限公司 | Productive technology of recombined exenatide |
| WO2015127273A1 (en) * | 2014-02-21 | 2015-08-27 | Medimmune, Llc | Anti-pcsk9~glp-1 fusions and methods for use |
| US10077319B2 (en) | 2014-02-21 | 2018-09-18 | Medimmune, Llc | Anti-PCSK9-GLP-1 fusions and methods for use |
| WO2016005903A3 (en) * | 2014-07-08 | 2016-03-10 | Theramyt Novobiologics Private Limited | A process for obtaining exendin-4 |
| CN112608964A (en) * | 2020-12-18 | 2021-04-06 | 北京博康健基因科技有限公司 | Fermentation method for large-scale production of recombinant Exendin-4 polypeptide |
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