CN1181095C - Gene relative peptide derivant having calcitonin and preparation process thereof - Google Patents
Gene relative peptide derivant having calcitonin and preparation process thereof Download PDFInfo
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- CN1181095C CN1181095C CNB001250485A CN00125048A CN1181095C CN 1181095 C CN1181095 C CN 1181095C CN B001250485 A CNB001250485 A CN B001250485A CN 00125048 A CN00125048 A CN 00125048A CN 1181095 C CN1181095 C CN 1181095C
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Abstract
The present invention forms derivate genes of calcitonin gene related peptide (CGRP) through the gene engineering technology, and produces three derivates of the CGRP through colibacillus fermentation. The CGRP has significant physiological functions of reducing blood pressure, etc., but is easy to be degraded in animal bodies and has short acting time. The present invention designs a DNA sequence to be connected with a colibacillus plasmid vector, and the genes containing the CGRP are formed. Fusion protein produced by the colibacillus fermentation is processed through different separation and extraction processes to be prepared into CGRP derivates I and derivates II. A plurality of CGRP genes connected in series are fermented and separated to obtain CGRP derivates III.
Description
The invention belongs to field of genetic engineering.
CGRP is distributed widely in the human central nervous system, peripheral nervous system, and cardiovascular systems is in the histoorgans such as urinary system.It has many-sided physiological function in animal body, is strong vasodilator, can vasodilation, bring high blood pressure down, strengthen cardiac contractility ability, and improve the blood work output.Have treatment hypertension, regulate arrhythmia, treatment heart failure, stenocardia, the huge clinical value of cardiovascular disordeies such as coronary heart disease and myocardial necrosis.But CGRP itself is little peptide, easily is degraded in animal body, and action time is short, production cost is high, price is expensive, thereby has limited the using value of CGRP in medical practice.
The objective of the invention is to develop three kinds and have calcitonin-gene-related peptide derivative and preparation method thereof.
The objective of the invention is to develop the above-mentioned medicine that has the treatment cardiovascular disorder of calcitonin-gene-related peptide derivative.
The objective of the invention is to develop the above-mentioned route of administration that has the treatment cardiovascular disorder of calcitonin-gene-related peptide derivative.
The present invention uses recombination, changes it over to Bacillus coli cells, after fermentation, is separated to three kinds of CGRP derivatives from Bacillus coli cells.These three kinds of CGRP derivatives all are peptide or protein.
The CGRP derivative I is by 38 bioactive peptides that amino acid is formed of coding, and the N end is the same with natural CGRP, only increases a methionine(Met) at its C end,
Its aminoacid sequence is:
AlaCysAspThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSerGly
GlyValValLysAsnAsnPheValProThrAsnValGlySerLysAlaPheMet
Also can be: (β)
AlaCysAsnThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSerGly
GlyMetValLysSerAsnPheValProThrAsnValGlySerLysAlaPheMet
The molecular weight of this derivative is 3.9Ku.
CGRP derivative I I is made up of 53 amino acid, is made up of A and B two portions peptide section, and the A peptide has 38
Individual amino acid is the CGRP derivative I, and the B peptide is made up of 5-15 amino acid,
15 aminoacid sequences of B peptide are:
GlySerProGlyIleArgAlaArgTyrProGlyIleArgSerMet
Its aminoacid sequence is:
GlySerProGlyIleArgAlaArgTyrProGlyIleArgSerMetAlaCysAspThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSerGlyGlyValValLysAsnAsnPheValProThrAsnValGlySerLysAlaPheMet
Also can be: (β)
GlySerProGlyIleArgAlaArgTyrProGlyIleArgSerMetAlaCysAsnThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSerGlyGlyMetValLysSerAsnPheValProThrAsnValGlySerLysAlaPheMet
The molecular weight of this derivative is 4.5-5.8Ku.
The CGRP derivative III is made up of 137 amino acid, and two respectively contain four amino acid whose joints with the A peptide of three molecules C peptide that is connected in series, and the C peptide has 122 amino acid, and the B peptide is made up of 5-15 amino acid, its amino
Acid sequence is:
GlySerProGlyIleArgAlaArgTyrProGlyIleArgSerMetAlaCysAspTbrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSerGlyGlyValValLysAsnAsnPheValProThrAsnValGlySerLysAlaPheMetSerValGlyMetAlaCysAspThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSerGlyGlyValValLysAsnAsnPheValProThrAsnValGlySerLysAlaPheMetSerValGlyMetAlaCysAspThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSerGlyGlyValValLysAsnAsnPheValProThrAsnValGlySerLysAlaPheMet
Also can be: (β)
GlySerProGlyIleArgAlaArgTyrProGlyIleArgSerMetAlaCysAsnThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSerGlyGlyMetValLysSetAsnPheValProThrAsnValGlySerLysAlaPheMetSerValGlyMetAlaCysAsnThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSerGlyGlyMetValLysSerAsnPheValProThrAsnValGlySerLysAlaPheMetSerValGlyMetAlaCysAsnThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSerGlyGlyMetValLysSerAsnPheValProThrAsnValGlySerLysAlaPheMet
The molecular weight of this derivative is 13.9-14.5Ku.
A kind of preparation method that calcitonin-gene-related peptide is the CGRP derivative that has of the present invention mainly contains preparation, separation, extraction of CGRP gene etc., and its concrete steps are:
(1) human CGRP's gene of chemosynthesis or 3-5 CGRP gene series connection back links to each other with coli expression carrier and changes coli strain over to;
(2) bacterial strain is placed bacteria culture medium, culture temperature incubation time 8-25 hour, is collected somatic cells then between 28-42 ℃;
(3) smudge cells passes through anion-exchange column or gel chromatography column or affinity column with the target protein in the cell;
(4) target protein obtains the CGRP derivative through zymoplasm or Xa factor enzymolysis and CNBr cracking behind the mistake post
I; Target protein obtains CGRP derivative I I through zymoplasm or Xa factor enzymolysis after crossing post; After the CGRP tandem gene was crossed post, target protein obtained the CGRP derivative III through zymoplasm or Xa factor enzymolysis.
The CGRP derivative I for preparing among the present invention, derivative I I and derivative III all contain the CGRP gene.
By 37 amino acid whose polypeptide of this genes encoding, people's CGRP has two kinds of α and β.Wherein the 3rd of α-CGRP the, 22,25 is respectively Asp, Val, Asn; The 3rd, 22,25 is respectively Asn, Met, Ser and all the other sequences are identical among β-CGRP.With α-CGRP gene or β-CGRP gene chemosynthesis, then the CGRP gene is inserted e. coli plasmid vector pUC18, this recombinant plasmid called after pUC18-CGRP during gene clone.Through subclone, change this recombinant plasmid over to the intestinal bacteria recipient bacterium then.It can be C600, TG1, MC1061, JM101 etc. that this recipient bacterium is made competent cell.With double digestion or pcr amplification and nucleotide sequence analysis, the sequence of conclusive evidence transformant is consistent with designed sequence.Transformant can be cultivated on the LB substratum, cultivates 8-15 hour in 28 ℃～42 ℃ temperature, collects bacterial cell then, and broken thalline is collected fusion rotein from supernatant liquor.Then with fusion rotein through heating 45 ℃～90 ℃ and 30%～50% (NH
4)
2SO
4Precipitation.Fusion rotein with preliminary purification passes through Q-Sepharose post and Glutathione Sepherose4B chromatography column again.Behind the chromatography fusion rotein cut with zymoplasm and just obtain CGRP derivative I I.Cut with CNBr again after cutting with zymoplasm and just obtain the CGRP derivative I.
Preparation CGRP derivative III is to downcut the joint that the CGRP gene adds chemosynthesis from the pUC18-CGRP plasmid.Link to each other with three CGRP gene series connection and with the protokaryon plasmid vector, change the intestinal bacteria recipient bacterium over to.This recipient bacterium is a competent cell, and they can be bacterial strains such as C600, TG1, MC1061, JM101.With double digestion and pcr amplification and nucleotide sequence analysis, the sequence of conclusive evidence transformant is consistent with designed sequence.Transformant is cultivated on the LB substratum, cultivates 8-15 hour under 28 ℃～42 ℃ temperature, collects bacterial cell then, and broken thalline is collected fusion rotein.Then fusion rotein is suspended in urea, crosses Q-Sepharose post and Glatathionesepharose 4B chromatography column then, cut through zymoplasm again and just obtain the CGRP derivative III.The CGRP derivative I has 38 amino acid, and molecular weight is 3.9ku.CGRP derivative I I contains 43-53 amino acid altogether, and the N end of comparing it with natural CGRP increases 5-15 amino acid, and the C end increases a methionine(Met), and its molecular weight is from 4.5-5.8ku.Derivative III is compared it with natural CGRP be to be in series by three CGRP, and its N end has increased 5-15 amino acid, and the C end increases a methionine(Met).Contain 127-137 amino acid altogether, its molecular weight is from 13.9-14.5ku.
The gene structure of the present invention's design fully takes into account the CGRP vasodilation, brings high blood pressure down, strengthens myocardial contraction, improves the effect of blood work output, has improved short deficiency CGRP action time again.As the medicine of treatment cardiovascular disorder, CGRP derivative of the present invention extremely helps efficiently, treats hypertension easily, arrhythmia, heart failure, stenocardia cardiovascular system diseases.
The present invention utilizes genetic engineering technique, and the design gene structure by fermentation using bacteria and different separation and purification processes, obtains three kinds of CGRP derivatives.These CGRP derivatives can drip spray by injection, oral cavity, nasal cavity drips approach such as spray, mucous membrane and supplies medicine for patient.Separately or be mixed into after patient supplies with the CGRP derivative, not only produce the function the same in vivo, and action time is more lasting with natural CGRP.
Above-mentioned three kinds of CGRP derivatives have the physiological function same with natural CGRP.With artificial renal hypertension dog ARHB is animal model, and every dog dosage is 2 micrograms, and the blood pressure of hypertension dog just descends rapidly.The CGRP derivative I causes that the blood pressure drops amplitude reaches 50%, and blood pressure drops was held time 22 minutes.CGRP derivative I I causes that the blood pressure drops amplitude is 25.4%, and it is 30 minutes that mean blood pressure decline is held time.The CGRP derivative III causes that the blood pressure drops amplitude is 17.2%, it is 80 minutes that mean blood pressure decline is held time, especially the natural CGRP of biological activity ratio of CGRP derivative I I and CGRP derivative III is more superior, and the blood pressure drops that causes is in animal body held time, significant prolongation.
Embodiment
To then this gene be inserted the pUC18 plasmid with chemical method synthetic CGRP gene 38 amino acid of encoding altogether by claim 1, the clone obtains pUC18-CGRP.Use the BamHI+SalI restriction enzyme site then, above-mentioned CGRP is inserted among the plasmid pGEX-4T obtain recombinant plasmid pGEX-CGRP.Again pGEX-CGRP is converted into intestinal bacteria recipient bacterium JM101.Transformant was cultivated 8 hours under 40 ℃ temperature, collecting cell then, and broken thalline is collected supernatant.Then with 55 ℃ of supernatant liquor heating and add 50% (NH
4)
2SO
4Precipitation, just obtain the fusion rotein of preliminary purification, collect the 2nd peak by Q-Sepharose with buffer solution elution again, pass through Glutathione Sepharose 4B post after concentrating again, fully behind the wash-out, cut the CGRP derivative I I that just obtains purifying with zymoplasm, it contains 53 amino acid, the purity of this kind CGRP derivative I I Tris-Tricine small molecular weight protein electrophoresis.Purity is checked in reverse post analysis of HPLC C-14 and mass spectroscopy, and analysis revealed CGRP derivative I I only has a band in protein electrophoresis, and HPLC detects has only a peak purity to reach 95%, and mass spectrometry results proof molecular weight is that 5.804ku is consistent with theoretical value.
15 amino acid of N-terminal are removed in the CGRP of the purifying thing II CNBr cracking of spreading out, and just obtain the CGRP derivative I.It contains 38 amino acid is that N-terminal is the same with natural CGRP, but C-terminal is Duoed a Met than natural CGRP.The purity of this kind CGRP derivative I is very high, has only a band in Tris-Tricine small molecular weight electrophoresis, and molecular weight 3.94ku is determined in mass spectroscopy.
Preparation CGRP derivative III, separation quality grain pUC18-CGRP gene from Bacillus coli cells at first, the joint that adds two chemosynthesis, connect into the polyphone gene that has 3 CGRP, be connected with plasmid pGEX-4T again with the EcoRI+SalI point of contact, change the transformant that obtains over to intestinal bacteria JM101 bacterial strain, transformant was cultivated 8 hours at 30 ℃, collected thalline.Smudge cells obtains fusion rotein, suspension fusion rotein and cross Q-Sepharose layer folding post in urea and phosphoric acid buffer then, collect composition in the 3rd peak, go up Glutathione Sepharose 4B layer folding post again, collect the 2nd peak and cut, just obtain the CGRP derivative III of purifying with zymoplasm.The purity of this kind derivative III has only a band with Tris-Tricine small molecular weight protein electrophoresis detection.Single main peak appears with the analysis of HPLC C-18 post.Mass spectrometry results proof molecular weight is that 14.5ku has 137 amino acid.Using this separation and purification route, can to obtain purity be pure goods more than 95%.
These three kinds of CGRP derivatives all have biological activity, are animal model with kidney blood pressure dog (ARHB), drip by injection or oral cavity spray all to cause blood pressure drops, its step-down feature such as following table:
The step-down feature of three kinds of CGRP derivatives
| Kind | Administration concentration | Duration of efficacy |
| The CGRP derivative I | 2μg | 22 minutes |
| CGRP derivative I I | 2μg | 30 minutes |
| The CGRP derivative III | 2μg | 80 minutes |
Claims (5)
1. one kind has the structure that calcitonin-gene-related peptide is the CGRP derivative I, it is characterized in that the N end is the same with natural CGRP by 38 bioactive peptides that amino acid is formed of coding, only increases a methionine(Met) at its C end,
The aminoacid sequence of this CGRP derivative is:
AlaCysAspThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSerGlyGlyValValLysAsnAsnPheValProThrAsnValGlySerLysAlaPheMet
The molecular weight of this derivative is 3.9Ku.
2. one kind has the structure that calcitonin-gene-related peptide is CGRP derivative I I, it is characterized in that by two ones of A and B
Divide the peptide section to form, it is the CGRP derivative I that the A peptide has 38 amino acid, and the B peptide is made up of 5-15 amino acid,
15 aminoacid sequences of B peptide are:
GlySerProGlyIleArgAlaArgTyrProGlyIleArgSerMet
The aminoacid sequence of this derivative I I is:
GlySerProGlyIleArgAlaArgTyrProGlyIleArgSerMetAlaCysAspThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSerGlyGlyValValLysAsnAsnPheValProThrAsnValGlySerLysAlaPheMet
The molecular weight of this derivative is 4.5-5.8Ku.
3. one kind has the structure that calcitonin-gene-related peptide is the CGRP derivative III, it is characterized in that respectively containing four amino acid whose joints with the A peptide of three molecules C peptide that is connected in series by two, and the C peptide has 122 amino acid, and the B peptide is by 5-15
Individual amino acid is formed,
The aminoacid sequence of this derivative III is:
GlySerProGlyIleArgAlaArgTyrProGlyIleArgSerMetAlaCysAspThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSerGlyGlyValValLysAsnAsnPheValProThrAsnValGlySerLysAlaPheMetSerValGlyMetAlaCysAspThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSerGlyGlyValValLysAsnAsnPheValProThrAsnValGlySerLysAlaPheMetSerValGlyMetAlaCysAspThrAlaThrCysValThrHisArgLeuAlaGlyLeuLeuSerArgSerGlyGlyValValLysAsnAsnPheValProThrAsnValGlySerLysAlaPheMet
The molecular weight of this derivative is 13.9-14.5Ku.
4. one kind has the preparation method that calcitonin-gene-related peptide is the CGRP derivative, mainly contains preparation, separation, extraction of CGRP gene etc., it is characterized in that:
(1) human CGRP's gene of chemosynthesis or 3-5 CGRP gene series connection back links to each other with coli expression carrier and changes coli strain over to;
(2) bacterial strain is placed bacteria culture medium, culture temperature incubation time 8-25 hour, is collected somatic cells then between 28-42 ℃;
(3) smudge cells passes through anion-exchange column or gel chromatography column or affinity column with the target protein in the cell;
(4) target protein obtains the CGRP derivative I through zymoplasm or Xa factor enzymolysis and CNBr cracking behind the mistake post; Target protein obtains CGRP derivative I I through zymoplasm or Xa factor enzymolysis after crossing post; After the CGRP tandem gene was crossed post, target protein obtained the CGRP derivative III through zymoplasm or Xa factor enzymolysis.
5. according to claim 1 or 2 or 3 described CGRP derivatives, it is characterized in that this derivative is used for the medicine as treatment hypertension and cardiovascular systemic disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001250485A CN1181095C (en) | 2000-09-07 | 2000-09-07 | Gene relative peptide derivant having calcitonin and preparation process thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001250485A CN1181095C (en) | 2000-09-07 | 2000-09-07 | Gene relative peptide derivant having calcitonin and preparation process thereof |
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| Publication Number | Publication Date |
|---|---|
| CN1285358A CN1285358A (en) | 2001-02-28 |
| CN1181095C true CN1181095C (en) | 2004-12-22 |
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|---|---|---|---|
| CNB001250485A Expired - Fee Related CN1181095C (en) | 2000-09-07 | 2000-09-07 | Gene relative peptide derivant having calcitonin and preparation process thereof |
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