CN1861635A - Human pancreas hyperglycemiacin relative peptide-2 analogue - Google Patents
Human pancreas hyperglycemiacin relative peptide-2 analogue Download PDFInfo
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- CN1861635A CN1861635A CNA2005100392653A CN200510039265A CN1861635A CN 1861635 A CN1861635 A CN 1861635A CN A2005100392653 A CNA2005100392653 A CN A2005100392653A CN 200510039265 A CN200510039265 A CN 200510039265A CN 1861635 A CN1861635 A CN 1861635A
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Abstract
A human glucagon associated peptide-2 analog Pro-Pro-h[Gly2]GLP-2(1-35) for treating the short bowel syndrome and malabsorption of stomach and intestine is prepared through configuring genetic engineering bacterium for effective expression of Pro- Pro-h[Gly2]GLP-2(1-35) in colibacillus cell, splitting, washing, dissolving urea, depositing in alcohol, separating fusion protein, hydrolyzing it, chromatography by DEAE-52 column, separating by HPLC, and freeze drying.
Description
Technical field
Technology of the present invention is a kind of human pancreas hyperglycemiacin relative peptide-2 analogue Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide.The amalgamation and expression technology of preparing platform of the active polypeptide of the new length 30-70aa that makes up with this research department is structured in efficient amalgamation and expression human pancreas hyperglycemiacin relative peptide-2 analogue Pro-Pro-h[Gly in the Bacillus coli cells
2] genetic engineering bacterium of GLP-2 (1-35), the L-Ala of the N end position 2 of human pancreas hyperglycemiacin relative peptide-2 analogue (
2Ala) residue is replaced by Gly, and the N termination adds 2 proline residues simultaneously.At Pro-Pro-h[Gly
2] the N end design acid-sensitive sense site (Asp-Pro) of GLP-2 (1-35) peptide, with sour cleavage of fusion proteins AnsB-C-Pro-Pro-h[Gly
2] GLP-2 acquisition purpose peptide Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide, Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide generates highly active h[Gly by the cutting of pepx IV and PPCE enzyme in vivo
2] GLP-2.The reorganization bacterium is through cracking → washing → dissolving → ethanol precipitation divides isolated fusion protein → 60mmol/L hydrochloric acid hydrolysis fusion rotein → DEAE-52 column chromatography for separation → HPLC to separate again, records Pro-Pro-h[Gly with electron spray mass spectrometry
2] GLP-2 (1-35) peptide relative molecular mass is 3948Da.Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide need not renaturation, be dissolved in after the freeze-drying among the PBS, to normal male SD rat (160g-200g) subcutaneous administration 14 days, 0.1,0.25,0.5mg/kg/day, small intestine weight is compared with control group, and the biologic activity of short intestines growth in the obvious body is arranged.Might be used to improve short bowel syndrome, the gastrointestinal illness that malabsorption and absorptive function change is put, chemotherapy patients's dietetic alimentation situation, strengthens patient's physique, promotes patients ' recovery, has the good clinical application prospect.
Background technology
Human pancreas hyperglycemiacin relative peptide-2 (or glucagon-like-peptide-2, human Glucagon-like peptide-2, hGLP-2,) be called people's intestines tethelin again, it is the polypeptide that constitutes by 33 amino acid, mainly, has the effect that stimulates the intestines growth by intestinal secretion (exist in a large number at small intestine distal end and proximity junction, rectum, ileum far-end L cell place concentration is the highest).Preclinical study has confirmed that the effect of GLP-2 is receptor-mediated by GLP-2, it is strong that various gastrointestinal illnesss (mainly showing as malabsorption and absorptive function changes) are had the effect specificity, effect is obvious, stability is high, treat wide, might be used to improve put, chemotherapy patients's dietetic alimentation situation, strengthen patient's physique, promote patients ' recovery.Thereby has a good clinical application prospect.
Bibliographical information is abroad arranged: the amino acid that changes GLP-2 is formed, and can change the short intestines growth activity of GLP-2, holds the 2nd amino acid Ala as replacing N-by Gly, 2 Arg is added to the N-end of GLP-2 or with [US05789379] such as the aminations of C-end of GLP-2.
Pepx IV in the mammalian cell (Dipeptidyl peptidase IV, DPP IV) is a kind of Serine exopeptidase, is present in vertebrate many cells and the tissue, can discharge dipeptides from the N-terminal of peptide.The single-minded substrate of its hydrolysis is NH2-X-Pro-(it is Pro that peptide or protein chain N hold the 2nd amino-acid residue), is typical X-prolyl-dipeptide aminopeptidase, and PPCE is the endopeptidase of hydrolysis-Pro-X-, Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide should be classified as the supposition substrate of DPPIV and PPCE enzyme, Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide may be to be generated highly active h[Gly by the cutting of DPPIV and PPCE enzyme in vivo
2] GLP-2 (1-33) peptide and playing a role.The N-terminal X-Ala-that contains among the GLP-1 can be cut away by DPPIV, and the transformation period is less than 2min in its body.And DPPIV circumscribed-speed of Ala-X-key only is 1%～5% of circumscribed-Pro-X-speed, the speed of PPCE inscribe-Ala-X-key only is 0.1%～1% of inscribe-Pro-X-speed.Therefore, Pro-Pro-h[Gly
2] the N-terminal X-Pro-of GLP-2 (1-35) peptide should be in 2min unites by intravital DPPIV and PPCE and cut away.
The external at present main chemical synthesis that adopts prepares small peptide, and cost is still higher, adopts genetically engineered secreting, expressing Pro-Pro-h[Gly
2] GLP-2, still not success.
Summary of the invention
One object of the present invention just provides new human pancreas hyperglycemiacin relative peptide-2 analogue Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide and preparation method thereof, this method can prepare human pancreas hyperglycemiacin relative peptide-2 analogue Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide.
Another object of the present invention provides corresponding coding sequence, carrier and host cell.
Another object of the present invention is Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide has the biologic activity of short intestines growth in the significant body.
In a first aspect of the present invention, provide a kind of human pancreas hyperglycemiacin relative peptide-2 analogue Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide, it includes pepx IV and PPCE restriction enzyme site before human pancreas hyperglycemiacin relative peptide-2 analogue 1-35 peptide, and holds at N
2Ala is replaced by Gly, and the N termination adds 2 proline residues simultaneously.
In a second aspect of the present invention, provide a kind of human pancreas hyperglycemiacin relative peptide-2 analogue Pro-Pro-h[Gly
2] preparation method of GLP-2 (1-35), it is characterized in that this method comprises step:
(a) under conditions suitable for the expression, cultivate claim 6 described plasmid vector and transformed bacterias thereof.
(b) from culture, isolate the human pancreas hyperglycemiacin relative peptide-2 analogue Pro-Pro-h[Gly of aminoacid sequence shown in the claim 2
2] (1-35) peptide of GLP-2 (1-35).
(c) be purified into Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide.
(d) utilize the interior pepx IV enzyme of body to cut Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide, form h[Gly
2] GLP-2 (1-33) peptide.
In a preference, in the method for the invention, described step (b) comprises
By bacteriolyze, washing, methods such as ethanol sedimentation are isolated Pro-Pro-h[Gly
2] fusion rotein formed of GLP-2 (1-35) peptide and Asparaginase C-terminal; From described fusion rotein, downcut the Asparaginase C-terminal with acid hydrolysis process, form Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide;
In another preference, described step (c) comprises by the DEAE-52 ion exchange column isolates Pro-Pro-h[Gly
2] GLP-2 (135) peptide.
In another preference, described step (c) comprises that also HPLC separates and freeze-drying.
In a second aspect of the present invention, Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide is to normal male SD rat (160g-200g) subcutaneous administration 14 days, gets small intestine, cleans with physiological saline, removes dirt and too much liquid, weigh.
Description of drawings
Figure 1A is Pro-Pro-h[Gly
2] the plasmid structure iron of fusion expression plasmid PED-PPGLP-2 of GLP-2 (1-35) peptide.
Figure 1B is for containing Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide fusion gene amalgamation and expression and inducing the back expression product to account for the time dependent situation of percentage composition of whole bacterial protein. swimming lane 1-6 is that PED-PPGLP-2/BL21 is at lactose-induced 0h, 2h, 4h, 6h, 8h, 10h, after the expressing fusion protein situation, swimming lane 7 is the antalzyme protein contrast.
Fig. 1 C is the dna sequencing collection of illustrative plates of PED-PPGLP-2 fusion expression plasmid.
Processing of Fig. 2 fusion rotein and purpose peptide purification collection of illustrative plates.
Fig. 2 A wherein: for containing Pro-Pro-h[Gly
2] the purifying collection of illustrative plates of fusion rotein of GLP-2 (1-35) peptide.Swimming lane 1 is the antalzyme protein contrast; Swimming lane 2 is the contrast of Regular Insulin albumen; Swimming lane 3 is the expressing fusion protein of PED-PPGLP-2/BL21 behind lactose-induced 4h; Swimming lane 4 is the fusion rotein before the acid hydrolysis; Swimming lane 5 is the hydrolyzed solution of fusion rotein behind the 60mmol/L hydrochloric acid hydrolysis.
Fig. 2 B purpose peptide Pro-Pro-h[Gly
2] the purifying collection of illustrative plates of GLP-2 (1-35) peptide.Swimming lane 1 is middle molecular weight protein standard and the contrast of Regular Insulin albumen; Swimming lane 2 and 3 is Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide.
Fig. 3 mass spectroscopy (ESI-MS) determining molecular weight.Pro-Pro-h[Gly
2] the ESI-MS collection of illustrative plates of GLP-2 (1-35) peptide.
Fig. 4 biological activity determination figure (short intestines growth method).The research of urging the intestines growth activity with biological activity determination method in the body.SD rat (160g-200g) is divided into 4 groups at random, and 6/group, 1 group is the PBS control group; 3 groups is Pro-Pro-h[Gly in addition
2] GLP-2 (1-35) administration group, dosage is respectively 0.1,0.25,0.5mg/kg/day, 1 day 2 times (morning 8:00 and afternoon 6:00), abdominal part hypodermic administration 14 days, the fasting in evening was put to death in second day, get small intestine, clean, remove dirt and too much liquid, weigh with physiological saline.Show Pro-Pro-h[Gly
2] the dosage vigor relation of GLP-2 (1-35) peptide.Fig. 5 biological activity determination shows short intestinal villi growth.Wherein Fig. 5 A is the PBS control group, and Fig. 5 B is 0.5mg/kg/dayPro-Pro-h[Gly
2] the administration group of GLP-2 (1-35) peptide, abdominal part hypodermic administration 14 days.
Embodiment
Material:
(1) bacterial strain and plasmid:
Host bacterium E.coli BL21 (DE3) LysS is genetically engineered its bacterial classification of worker commonly used, in the laboratory relevant with genetically engineered research preservation is arranged all generally.
Plasmid pET28a is available from Novagen company.
Plasmid pED is made up and is preserved by this laboratory.PED is this laboratory Liu Jing crystalline substance, Chen Zhenglan makes up is suitable for the recombinant plasmid that small peptide efficiently expresses and processes, this recombinant plasmid system with cancellation make up between the L-Asnase C-terminal 127 peptide genes in unique acid hydrolysis site and NcoI that one group of multiple clone site is introduced plasmid PET28a and the BamHI site and form.
(2) enzyme and reagent:
Molecular cloning toolenzyme and reagent, bacterial genomes, plasmid extraction test kit are Pu Luomaige (Promega) company product;
(3) substratum:
The LB substratum, the document Sambrook J that sees reference that fills a prescription, Fristsh E F, Maniatis T.Molecular Cloning; A Laboratory Manual 2nd ed.NY:Cold Spring Harbor Laboratory Press, 1989.This book is the classical works of genetic engineering technique, in many libraries of the universities collection is arranged all.
The corn steep liquor substratum contains: corn steep liquor 25g/L, beef extract 15g/L, monosodium glutamate 10g/L.
Method
The molecular biology working method.
The recovery of plasmid extraction, polymerase chain reaction, endonuclease digestion, dna segment, connection and transformed into escherichia coli: in the genetically engineered research field, these all are the routine operation methods, referring to Sambrook J, Fristsh E F, Maniatis T.Molecular Cloning; A Laboratory Manual 2nd ed.NY:Cold Spring Harbor Laboratory Press, 1989, pp.16-340.
The mensuration of expression of recombinant proteins amount: referring to Sambrook J, Fristsh E F, Maniatis T.Molecular Cloning; A Laboratory Manual 2nd ed.NY:Cold Spring Harbor Laboratory Press, 1989, method is carried out.
Embodiment 1Pro-Pro-h[Gly
2] design of GLP-2 (1-35) polypeptide gene, synthetic and clone
According to colibacillary codon and design requirements with the L-Ala of N end position 2 (
2Ala) residue is changed into nucleotide sequence by 35 aminoacid sequences that Gly replaces the human pancreas hyperglycemiacin relative peptide-2 of 2 proline(Pro) of while N end extension, and its structural framing is seen Fig. 2.Set up BamH I restriction enzyme site at its 5 ' end, 3 ' end is set up terminator codon TAA and HindIII restriction enzyme site, after computer-aided analysis, determine full length gene 124bp, three oligonucleotide fragments that are divided into 53,54,53 bases, simultaneously, for PCR method primary dcreening operation positive colony, designed downstream primer M4.
Synthetic 4 sequences are as follows:
M1 (5 ' GCGG
G GAT CCG CCG CAC GGT GAC GGT TCT TTC TCT GAC GAA ATG AAC ACC ATC 3 ' contains BamH I restriction enzyme site)
M2(5’GAC?GAA?ATG?AAC?ACC?ATC?CTG?GAC?AAC?CTG?GCT?GCT?CGT?GAC?TTC?ATC?AAC?TGG?3’)
M3 (5 ' GGCC
AAGC TTAATC AGT GAT TTT GGT CTG GAT CAG CCA GTT GAT GAA GTC ACG 3 ' contains the HindIII restriction enzyme site)
M4 (5 ' GGCC
AAGC TTAATC AGT GAT TTT GGT CTG 3 ' contains the HindIII restriction enzyme site)
Synthetic on Beckman oligo-1000DNA synthesizer by Shanghai Bo Ya Bioisystech Co., Ltd.PCR is reflected on the GTC-2 type amplification instrument and carries out.Wherein M1, M2, M3 mix by 10: 1: 10 concentration ratio, in 94 ℃, and 30s; 54 ℃, 60s; 72 ℃, 90s; Totally 30 circulations.1.8% agarose gel electrophoresis is identified the PCR product, the purpose fragment is reclaimed with gel be stored in-20 ℃ after test kit reclaims.
PCR purpose fragment that reclaims through gel and cloned plasmids pUC18 are through BamH I and HindIII double digestion, and agarose gel electrophoresis reclaims the purpose fragment, and 16 ℃ of connections are spent the night in T4DNA ligase enzyme reaction system, Transformed E .coli JM109 competent cell.Cell is applied to the dull and stereotyped last 37 ℃ of overnight incubation of the LB that contains 100 μ g/ml Amp, 40 μ g/ml X-gal and 0.1mmol/L ITPG, picking white colony, extracting and purifying recombinant clone plasmid pUCGLP-2.Respectively get and Pro-Pro-h[Gly
2] gene upstream and downstream nucleotide sequences complementary two special primer M1, M4 respectively of GLP-2, each 100pmol/L, make template respectively with the recombinant plasmid of purifying and in 10 μ l systems, carry out PCR, the primary dcreening operation positive colony checks order positive colony again on the ABI of Dalian Bao Bio-Engineering Company 377 automatic dna sequencers.
As special primer, is template with the pUCGLP-2 of the correct purifying that checks order with M1, M4, the PCR reaction conditions: 94 ℃, and 30s; 54 ℃, 60s; 72 ℃, 90s; Pcr amplification is carried out in totally 30 circulations, and institute's amplification PCR products goes up electrophoresis detection and reclaims the purpose fragment in 1.8%Argarose.Gained PCR purpose fragment is inserted the corresponding restriction enzyme site of pED, and is kept original reading frame after BamH I and the two enzymic digestions of HindIII.Transformed E .coli BL21 (DE3) LysS competent cell.Cell is applied on the LB flat board that contains 50 μ g/mL kantlex, and 37 ℃ of overnight incubation are selected positive recombinant expressed bacterium PED-PPGLP-2/BL21.Behind PCR primary dcreening operation recombinant expression plasmid pED-PPGLP-2 (seeing Figure 1A), check order.Dna sequence analysis also with the expection conform to (gene sequencing the results are shown in Figure 1C).
Embodiment 2Pro-Pro-h[Gly
2] expression of GLP-2 (1-35) polypeptide gene in intestinal bacteria
Recombinant expressed bacterium PED-PPGLP-2/BL21 is 37 ℃ of shaken overnight in liquid LB substratum, transfer in the corn steep liquor fermention medium with 2% inoculum size, cultivate 4h for 37 ℃, add the lactose-induced expression of final concentration 5mmol/l, collect the fermentation thalline behind the 4h.Keep sample and carry out 15%SDS-PAGE analysis and thin layer scanning.At the about 17700Da of molecular weight place tangible protein band (Figure 1B) appears, with fusion rotein AnsB-C-Pro-Pro-h[Gly
2] GLP-2 theoretical calculate value unanimity.Fusion rotein reached stable maximum expression amount in back 8 hours inducing, and Densitometric analysis analyzes and shows that fusion rotein accounts for more than 40% of total bacterial protein, and has formed inclusion body in born of the same parents.
Engineering bacteria behind the abduction delivering is through centrifugal recovery thalline, thalline is suspended in shell-broken liquid (pH8.0 phosphate-buffered salt, 0.02% N,O-Diacetylmuramidase), obtains inclusion body behind the cellular lysate, inclusion body obtains purer fusion rotein AnsB-C-Pro-Pro-h[Gly through washing behind urea dissolving and the ethanol precipitation
2] GLP-2 (Fig. 2 A).
Fusion rotein AnsB-C-Pro-Pro-h[Gly with the ethanol sedimentation acquisition
2] GLP-2 is according to 6% ratio, is configured as solution with 60mmol/LHCl, respectively at 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ of cutting 48h, the Tricin-SDS-PAGE electrophoresis result shows: be lower than 55 ℃ of cleavage of fusion proteins, can obtain purpose peptide band comparatively clearly.Fusion rotein is used 30mmol/L, 60mmol/L respectively according to 6% ratio, 120mmol/L, 180mmol/L, different concns HCl such as 240mmol/L HCl are configured as solution, in 50 ℃ of cutting 48h, the Tricin-SDS-PAGE electrophoresis result shows: with 60mmol/L HCl cleavage of fusion proteins, can obtain comparatively clear and more purpose peptide band.With 60mmol/LHCl fusion rotein is configured as 1%, 2%, 4% respectively, the solution of 6%, 8% (w/v) is in 50 ℃ of cutting 48h, the Tricin-SDS-PAGE electrophoresis result shows: the fusion rotein solution to 6% or 8% carries out the acid cutting, can obtain purpose peptide band comparatively clearly.Fusion rotein according to 6% ratio, is configured as solution with 60mmol/LHCl, cuts 0h respectively in 50 ℃, 24h, 36h, 48h, 72h, 96h, the Tricin-SDS-PAGE electrophoresis result shows: with fusion rotein acid cutting 48h to 72h, can obtain more clear and more purpose peptide band, because of the purpose peptide band of 48h and 72h acquisition is more or less the same, for guaranteeing that the purpose peptide is not destroyed, so select 48h for use.(fusion rotein is configured as solution according to 6% ratio with 60mmol/LHCl, in 50 ℃ of cutting 48h (Fig. 2 A) with the sour cutting technique of optimizing.
Embodiment 5Pro-Pro-h[Gly
2] separation and purification of GLP-2 (1-35) peptide
To contain Pro-Pro-h[Gly
2] the fusion rotein AnsB-C-Pro-Pro-h[Gly of GLP-2 (1-35) peptide
2] acid hydrolysis liquid of GLP-2 is adjusted to the pH value with the level pad basically identical, be diluted to about 10 times of former hydrolyzed solution volume, flow and be splined on the good 0.02mol/L DEAE-52 ion exchange column of 0.02mol/L ammonium acetate balance, the 0.02mol/L ammonium acetate is carried out gradient elution to the 0.25mol/L ammonium acetate, collect the sample peak, freeze-drying; The HPLC separation and purification obtains human pancreas hyperglycemiacin relative peptide-2 analogue Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide (C
18Post, room temperature, sample dissolves with 0.1% trifluoroacetic acid, solvent A:0.1% trifluoroacetic acid aqueous solution, B:99% second eyeball/0.1% trifluoroacetic acid,, second eyeball gradient 30%60% wash-out 45min, flow velocity 0.7ml/min detects wavelength 215nm, the vacuum-drying of HPLC sample) (Fig. 2 B).
Company finishes by the Shanghai gill.
Method is: electrospray ionization mass spectrum.
Record the Pro-Pro-h[Gly of reorganization preparation
2] relative molecular mass of GLP-2 (1-35) is 3948Da (Fig. 3), and is consistent with the theoretical calculate value.
Embodiment 7Pro-Pro-h[Gly
2] biological activity research in the body of GLP-2 (1-35) polypeptide
The research of urging the intestines growth activity with biological activity determination method in the body.Normal male SD rat (160g-200g) is divided into 4 groups at random, and 6/group, 1 group is the PBS control group; 3 groups is Pro-Pro-h[Gly in addition
2] GLP-2 (1-35) administration group, dosage is respectively 0.1,0.25,0.5mg/kg/day, 1 day 2 times (morning 8:00 and afternoon 6:00), abdominal part hypodermic administration 14 days, the fasting in evening was put to death in second day, get small intestine, clean, remove dirt and too much liquid, weigh with physiological saline.The result shows: the small intestine weight of administration group is compared with the PBS contrast, shows the biologic activity of short intestines growth in the obvious body and dosage correlation (Fig. 4) is arranged.Compare with PBS control group (Fig. 5 A) simultaneously, 0.5mg/kg/day dosage group significantly promotes intestinal villi growth (Fig. 5 B).
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition read of the present invention above-mentioned tell about content after, those skilled in the art can make various changes or modification to the present invention, these equivalent form of values fall within the appended claims of the application institute restricted portion equally.
SEQUENCE?LISTING
＜110〉China Medicine University
＜120〉human pancreas hyperglycemiacin relative peptide-2 analogue
<130>ppglp-2
<160>2
<170>PatentIn?version?3.2
<210>1
<211>105
<212>DNA
<213>Artificial
<220>
<223>Artificial?Sequence
<220>
<221>CDS
<222>(1)..(105)
<400>1
ccg?ccg?cac?ggt?gac?ggt?tct?ttc?tct?gac?gaa?atg?aac?acc?atc?ctg 48
Pro?Pro?His?Gly?Asp?Gly?Ser?Phe?Ser?Asp?Glu?Met?Asn?Thr?Ile?Leu
1 5 10 15
gac?aac?ctg?gct?gct?cgt?gac?ttc?atc?aac?tgg?ctg?atc?cag?acc?aaa 96
Asp?Asn?Leu?Ala?Ala?Arg?Asp?Phe?Ile?Asn?Trp?Leu?Ile?Gln?Thr?Lys
20 25 30
atc?act?gat 105
Ile?Thr?Asp
35
<210>2
<211>35
<212>PRT
<213>Artificial
<220>
<223>Synthetic?Construct
<400>2
Pro?Pro?His?Gly?Asp?Gly?Ser?Phe?Ser?Asp?Glu?Met?Asn?Thr?Ile?Leu
1 5 10 15
Asp?Asn?Leu?Ala?Ala?Arg?Asp?Phe?Ile?Asn?Trp?Leu?Ile?Gln?Thr?Lys
20 25 30
Ile?Thr?Asp
35
Claims (9)
1. a human pancreas hyperglycemiacin relative peptide-2 (hGLP-2) analogue Pro-Pro-h[Gly
2] GLP-2 (1-35) is Pro-Pro-h[Gly.sup.2] GLP-2 (1-35).It is characterized in that: this analogue relative molecular mass 3948Da, by L-Ala (Ala) residue of Gly replacement N end position 2, the N termination adds 2 proline(Pro) (Pro) residue simultaneously, and the N end comprises pepx IV and PPCE restriction enzyme site.
2. human pancreas hyperglycemiacin relative peptide-2 analogue Pro-Pro-h[Gly according to claim 1
2] GLP-2 (1-35), it is characterized in that it comprises aminoacid sequence shown in the SEQ ID NO:2.
3. claim 1 and 2 described human pancreas hyperglycemiacin relative peptide-2 analogue Pro-Pro-h[Gly
2] biological activity of GLP-2 (1-35), it is characterized in that: the N end of human pancreas hyperglycemiacin relative peptide-2 (1-33)
2Ala is replaced by Gly, and the human pancreas hyperglycemiacin relative peptide-2 analogue that while N termination adds 2 proline residues formation still can keep notable biological activity, promotes to increase intestines weight, increases the intestinal villus height.
4. one kind is designed the synthetic polynucleotide, and it is characterized in that: it comprises a nucleotide sequence, it is characterized in that, these polynucleotide have nucleotide sequence shown in the SEQ ID NO:1.The human pancreas hyperglycemiacin relative peptide-2 analogue of aminoacid sequence shown in this nucleotide sequence coded claim 2.
5. carrier, it is characterized in that: it contains claim 4 described nucleotide sequence and recombination methods: 127 amino acid whose dna segments of altheine enzyme C-terminal and artificial and PCR method synthetic Asp-Pro-Pro-h[Gly that will be generated by PCR method
2] gene order of h GLP-2 merges, and constitutes new antigen-4 fusion protein gene.This gene is inserted into the downstream of pED carrier T7 promotor.The fusion expression plasmid of this reorganization claims pED-PPGLP-2.PED-PPGLP-2 has comprised a fusion partners and Asp-Pro-Pro-h[Gly
2] gene of GLP-2.
6. genetic engineering bacterium, it is characterized in that: it contains claim 5 described carriers.
7. comprise described this analogue of claim 5 Pro-Pro-h[Gly
2] recombination method of GLP-2 (1-35), it is characterized in that:
This fusion rotein uses 127 amino-acid residues of L-aspartic acid C-terminal as fusion partners, is connected this connection acid labile with asparagus fern-dried meat peptide bond between fusion partners gene and active polypeptide gene.
8. human pancreas hyperglycemiacin relative peptide-2 analogue Pro-Pro-h[Gly
2] preparation method of GLP-2 (1-35), it is characterized in that this method comprises step:
(a) under conditions suitable for the expression, cultivate claim 6 described host cells.
(b) by bacteriolyze, washing, methods such as ethanol sedimentation are isolated Pro-Pro-h[Gly from culture
2] the fusion rotein AnsB-C-Pro-Pro-h[Gly that forms of GLP-2 (1-35) and Asparaginase C-terminal
2] GLP-2; From described fusion rotein, downcut the Asparaginase C-terminal with acid hydrolysis process, form the human pancreas hyperglycemiacin relative peptide-2 analogue Pro-Pro-h[Gly of aminoacid sequence shown in the claim 2
2] GLP-2 (1-35).
(c) isolate Pro-Pro-h[Gly by DEAE-52 ion exchange column and HPLC
2] GLP-2 (1-35).
(d) utilize the interior pepx IV enzyme of body to cut Pro-Pro-h[Gly
2] GLP-2 (1-35) peptide, form h[Gly
2] GLP-2 (1-33).
9. as claim 8 described methods, it is characterized in that: described step (d) is at Pro-Pro-h[Gly
2] after GLP-2 (1-35) enters in the body, cut away Pro-Pro-h[Gly by pepx IV and the rapid enzyme of PPCE enzyme in the body abundant in the body
2] Pro-Pro-of GLP-2 (1-35) N end, form h[Gly
2] GLP-2 (1-33).This long-acting hormone for the anti-pepx IV of development hydrolysis is useful.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102190720A (en) * | 2010-12-28 | 2011-09-21 | 中国药科大学 | Method for preparing human glucagon-like peptide-2 (1-35 peptide) analogue |
| CN102260346A (en) * | 2011-07-20 | 2011-11-30 | 中国药科大学 | Exendin-4 analog |
| CN102796192A (en) * | 2011-05-23 | 2012-11-28 | 中国药科大学 | Human glucagon-like peptide-1 analogue |
| CN113912741A (en) * | 2021-11-08 | 2022-01-11 | 苏州博领干细胞再生医学有限公司 | Fusion protein, recombinant engineering bacterium, culture method and purification method of anti-glucagon related peptide-2 vaccine |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5990077A (en) * | 1995-04-14 | 1999-11-23 | 1149336 Ontario Inc. | Glucagon-like peptide-2 and its therapeutic use |
| DE69716905T2 (en) * | 1996-04-12 | 2003-07-24 | 1149336 Ontario Inc., Toronto | ANALOGS OF THE GLUCAGON-LIKE PEPTIDES -2 |
| EP1060192A2 (en) * | 1998-02-27 | 2000-12-20 | Novo Nordisk A/S | Glp-2 derivatives with helix-content exceeding 25 %, forming partially structured micellar-like aggregates |
| GB9930882D0 (en) * | 1999-12-30 | 2000-02-23 | Nps Allelix Corp | GLP-2 formulations |
| US7411039B2 (en) * | 2002-10-14 | 2008-08-12 | Novo Nordisk A/S | GLP-2 compounds, formulations, and uses thereof |
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2005
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102190720A (en) * | 2010-12-28 | 2011-09-21 | 中国药科大学 | Method for preparing human glucagon-like peptide-2 (1-35 peptide) analogue |
| CN102796192A (en) * | 2011-05-23 | 2012-11-28 | 中国药科大学 | Human glucagon-like peptide-1 analogue |
| CN102260346A (en) * | 2011-07-20 | 2011-11-30 | 中国药科大学 | Exendin-4 analog |
| CN113912741A (en) * | 2021-11-08 | 2022-01-11 | 苏州博领干细胞再生医学有限公司 | Fusion protein, recombinant engineering bacterium, culture method and purification method of anti-glucagon related peptide-2 vaccine |
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| Publication number | Publication date |
|---|---|
| CN100418983C (en) | 2008-09-17 |
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