CN1304422C - Non-amidated omega-conotoxin VIIA and its prepn process and application - Google Patents
Non-amidated omega-conotoxin VIIA and its prepn process and application Download PDFInfo
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- CN1304422C CN1304422C CNB2005100607814A CN200510060781A CN1304422C CN 1304422 C CN1304422 C CN 1304422C CN B2005100607814 A CNB2005100607814 A CN B2005100607814A CN 200510060781 A CN200510060781 A CN 200510060781A CN 1304422 C CN1304422 C CN 1304422C
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Abstract
The present invention provides non-amidation omega-conotoxin M VIIA(omega-CTX M VIIA). a DNA segment for encoding the non-amidation omega-CTX M VIIA is combined and designed according to a known omega-CTX M VIIA, the XhoI/EcoRI double-enzyme cutting of an expression carrier pPIC9 is carried out, a recombinant expression plasmid pPIC9-CTX is formed by connection, pichia pastoris is converted by an electric punching method, and bacterial strains with high yield can be obtained by sieving and can be induced and expressed by methanol. The present invention successively expresses the non-amidation omega-conotoxin M VIIA in the pichia pastoris, produces novel non-amidation omega-CTX M VIIA, and has the advantages of simple preparation process and low production cost; the present invention has high analgesic activity from the certification of animal experiment, and the analgesia efficiency is 900 times more than that of morphine; the present invention can be used for the development of analgesia medicine.
Description
Technical field
The invention belongs to genetically engineered, relate to structure omega-conotoxin M VIIA (ω-CTX M VIIA) gene eucaryon expression plasmid, prepare the method for non-amidated omega-CTX M VIIA, and the application in the preparation analgesic.
Background technology
(conotoxin CTX) is the peptide toxoid of the class biologically active that obtains to conotoxin from ocean gasteropod cone shell (conus), so far kind surplus in the of existing 50000.Generally contain 10～30 amino acid, be rich in disulfide linkage mostly,, can be divided into families such as α, ω, μ by its target site and pharmacological activity.(ω-CTX) is by 24～29 amino acid, 3 pairs of little peptides of rigidity that disulfide linkage constitutes to omega-conotoxin, is most important toxin in the conotoxin.Because ω-CTX has high-affinity, highly narrow spectrum characteristics, it is blocking voltage responsive type calcium channel (Voltage sensitive calciumchannel specifically, VSCC), playing a significant role aspect the evaluation of ionic channel hypotype, the sacred disease treatment, become the research focus in fields such as neurobiology, molecular biology, chemiluminescent polypeptide, pharmacopathology.
Maximum ω-CTX of research is M VIIA at present, is made up of 25 amino acid, and wherein the Cys of carboxyl terminal is amidated structure, and its aminoacid sequence is CKGKGAKCSRLMYDCCTGSCRSGKC-NH
2 *(contain 6 cysteine residues, form three pairs of intersection disulfide linkage, promptly the disulfide linkage mode of connection is C1-C16, C8-C20, C15-C25; * the Cys of carboxyl terminal is amidation); Studies have shown that its tool double effect, on the one hand block pain sensation transmission and the tool analgesic effect, suppress to stimulate toxicity (excitotoxic) neurotransmitter to discharge on the other hand, stop stream in the neuronal cell pathologic calcium and neuroprotective is arranged.Because of it directly acts on calcium channel, need not second messenger or G albumen, so habituation not can be used for treating chronic severe pain (Persistent Pain that causes as late malignant tumour and AIDS disease etc.).
At present, the ω of chemosynthesis-CTX M VIIA, go on the market by the FDA approval in the U.S., but because the molecular structure complexity of ω-CTX M VIIA, pairing forms disulfide linkage to have 6 halfcystines (Cys) to need accurately in the molecule, needs 20 polystep reactions, yield is very low, technology is very difficult, the quality instability, and also cost is very high.Therefore, seek a kind of suitable method acquisition omega-conotoxin M VIIA and become needs.
Summary of the invention
The purpose of this invention is to provide the non-amidated omega-conotoxin M VII A of polypeptide organic compound, have the aminoacid sequence of SEQ ID NO:1:
Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys 25
Cys1 and Cys16, Cys8 and Cys20, Cys15 and Cys25 form three pairs of disulfide linkage respectively in this peptide molecule; The C-end is non-amidated Cys.
Another object of the present invention is to utilize gene engineering method to make up omega-conotoxin M VIIA (ω-CTX M VIIA) gene eucaryon expression plasmid, the method for preparing non-amidated omega-CTX M VIIA, directly obtain the little peptide of purpose, the present invention is achieved through the following technical solutions:
(1) design of the dna fragmentation of coding ω-CTX M VIIA is with synthetic: according to known ω-CTXM VIIA aminoacid sequence, press pichia spp preference codon design oligonucleotides segment (CTX1 and CTX2), 5 ' end adds sequence and the phosphorylation of Xho I restriction enzyme site and coding Lys-Arg, and 3 ' end adds TAA terminator codon and EcoR I restriction enzyme site;
(2) structure of expression vector: yeast expression vector pPIC9 is carried out Xho I/EcoR I double digestion, and the gene fragment of ω-CTX M VIIA that the coding of annealing generation is non-amidated is connected on the pPIC9, is built into recombinant expression plasmid pPIC9-CTX.It is carried out PCR and double digestion respectively, identify (referring to Fig. 2,3), clone 2pPIC9-CTX sequencing result is shown correct being inserted among the pPIC9 of dna sequence dna of ω-CTX M VIIA that coding is non-amidated with agarose electrophoresis.
(3) conversion of expression vector in the pichia spp: get the recombinant expression vector that 5～10 μ g have removed RNA, through the linearizing of Sal I single endonuclease digestion, ethanol sedimentation reclaims the back and transforms pichia spp GS115 with electroporation, on MD and MM flat board, screen transformant, used electroporation is Bio-Rad GenePulser, conversion condition is voltage 1500V, electric capacity 25 μ F, resistance 200 Ω;
(4) genetic expression: pichia spp genetic expression host bacterium is selected GS115 for use, picking mono-clonal Mut
+The type bacterium colony, the GS115 (Mut of the operational manual that provides according to Invitrogen company
+) fermentation process introduced carries out shake flask fermentation, sampling regularly is with 18%SDS-PAGE testing goal expression of gene level.
The application of the non-amidated omega that another purpose of the present invention provides-CTX M VIIA in the preparation analgesic.
Characteristics of the present invention are: use genetic engineering technique, successfully expressed non-amidated ω-CTX M VIIA in pichia spp; This method can be produced novel non-amidated ω-CTX M VIIA, preparation technology's simple and stable, and every liter of fermented liquid can obtain the non-amidated ω of about 36mg-CTX M VIIA, and production cost is lower; Experimentation on animals proves: this novel non-amidated ω-CTX M VIIA has very high analgesic activities, and 900 times (by mole analgesia strength ratio) of its analgesia renders a service that the chances are morphine can be used for the exploitation of analgesic.
Description of drawings
The structure of Fig. 1, carrier for expression of eukaryon pPIC9-CTX.
The agarose electrophoretic analysis figure of Fig. 2, PCR product.
The agarose electrophoretic analysis figure of the double digestion product of Fig. 3, recon.
Fig. 4, clone's 2 sequencing results.
The agarose electrophoresis figure of Fig. 5, PCR product.
Fig. 6, with the electrophorogram of SDS-PAGE screening high expression level bacterial strain.
Fig. 7, non-amidated ω-CTX M VIIA ion-exchange purification figure.
The SDS-PAGE figure of Fig. 8, non-amidated ω-CTX M VIIA purified components.
Fig. 9, ELISA identify target protein: non-amidated ω-CTX M VIIA.
Non-amidated ω-CTX M VIIA is identified in Figure 10, MALDI-TOF-MS mass spectroscopy.
Figure 11, mouse hot plate analgesic test: the percentage broken line graph is improved in administration time-pain territory.
Figure 12, the hot tail test of rat: the percentage broken line graph is improved in administration time-pain territory.
Embodiment
The present invention reaches accompanying drawing in conjunction with the embodiments and is described further.
1. materials and methods
1.1 material and reagent
1.1.1 bacterial strain and plasmid
Pichia spp GS115, carrier for expression of eukaryon pPIC9 are available from Invitrogen company.
1.1.2 toolenzyme and chemical reagent: various restriction enzymes and other toolenzymes are given birth to worker company available from Shanghai, and other all ingredients are homemade analytical pure.
1.1.3 animal: female NIH mice, male Sprague-Dawley rat are all available from the Zhejiang Academy of Medical Sciences animal center.
1.2 method
1.2.1 the design of the dna fragmentation of coding ω-CTX M VIIA is with synthetic: according to known ω-CTXMVIIA aminoacid sequence, press pichia spp preference codon design oligonucleotides segment (CTX1 and CTX2), 5 ' end adds the sequence of Xho I restriction enzyme site and phosphorylation and coding Lys-Arg, 3 ' end adds TAA terminator codon and EcoR I restriction enzyme site, gives birth to worker bio-engineering corporation synthetic (seeing Table 1) by Shanghai.
| Fragment(bp) DNA sequences(5’→3’) |
| CTX1(90bp) TCGAGAAAAGATGTAA AGGTAAAGGTGCTAAATGTTCTCG TCTTATGTATGATTGTTGTACTGGTTCTTGTCGTTCTGGTAA ATGTtaa G CTX2(90bp) AATTCttaACATTTACCTGAACGACAAGAACCAGTACAACAA TCATACATAAGACGAGAACATTTAGCACCTTTACCTTTACA TCTTTT C |
It is Xho I and EcoR I restriction enzyme site that table 1, italic have the sequence of underscore, and black matrix is the sequence of coding Lys-Arg, and taa is a terminator codon.
Moles such as CTX1 and CTX2 are mixed, be heated to 90 ℃, naturally cool to room temperature, promptly form the dna fragmentation of coding ω-CTX M VIIA.
1.2.2 the structure of expression vector: yeast expression vector pPIC9 is carried out Xho I/EcoR I double digestion, the dna fragmentation of the coding ω-CTX M VIIA that generates annealing is connected on the pPIC9, be built into recombinant expression plasmid pPIC9-CTX (referring to Fig. 1), it carried out PCR and double digestion respectively identify.
1.2.3DNA order-checking: sample presentation Shanghai is given birth to worker bio-engineering corporation and is measured.
1.2.4 the conversion of expression vector in the pichia spp: get the recombinant expression vector that 5～10 μ g have removed RNA, through the linearizing of SalI single endonuclease digestion, ethanol sedimentation reclaims the back and transforms pichia spp GS115 with electroporation, on MD and MM flat board, screen transformant, used electroporation is Bio-Rad GenePulser, conversion condition is voltage 1500V, electric capacity 25 μ F, resistance 200 Ω.
1.2.5 the PCR method of recombinant conversion is identified: the genome with recombination yeast is a template, is primer with AOX1-up and AOX1-down, and the operational manual that provides according to Invitrogen company carries out PCR.
1.2.6 the screening of genetic expression and superior strain: pichia spp genetic expression host bacterium is selected GS115 for use, picking mono-clonal Mut
+The type bacterium colony, the GS115 (Mut of the operational manual that provides according to Invitrogen company
+) fermentation process introduced carries out shake flask fermentation, sampling regularly is with 18%SDS-PAGE testing goal expression of gene level.
1.2.7 the mensuration of expression product concentration: measure protein content according to the Bradford method.
1.2.8 the purifying of expression product: fermented liquid centrifuging and taking supernatant, relative molecular mass are the sodium acetate dialysis to the 0.02M of pH 6.0 of 1000 dialysis tubing, and dialyzate adopts weak cation exchange column chromatography, collects detached peaks and carries out cataphoretic determination purity.
1.2.9ELISA evaluation target protein: because target protein is little peptide, molecular weight is less, thus can not wrap by to enzyme plate with the method for coating of routine, still adopt the crosslinked method bag of PLL by the polarity small molecular protein
[4]Establish four groups of blank group, sample sets (high, medium and low three concentration) altogether, every group contains 5 multiple holes.How anti-the one anti-anti-ω of specificity-CTX M VIIA for this laboratory oneself preparation is, and two to resist then be the goat-anti rabbit anti-serum of horseradish peroxidase-labeled.TMB Color Appearance System colour developing 10min, 0.5mol/LH
2SO
4Termination reaction, (450nm 630nm) measures the OD value to dual wavelength.
1.2.10 the mass spectrum of non-amidated ω-CTX M VIIA identifies that purifying is collected the liquid freeze-drying delivers to Chinese Academy of Sciences Shanghai biochemical cell institute, does molecular weight identification by MALDI-TOF-MS.
1.2.11 bioactive detection:
1. classical hot plate analgesia method detects its biologic activity.Healthy female NIH mouse, body weight 18～22g, regulate 55 ± 0.5 ℃ of waters bath with thermostatic control, mouse is put on the hot plate, select have in 5～20s lick metapedes action or lift metapedes and later the animal of (pain sensation reaction) be qualified animal, this reaction times is normal thresholding bitterly before the medicine, selects 60 of qualified animals, be divided into 6 groups at random, adopt the tricorn administration.The blank group gives equivalent physiological saline, positive controls gives the morphine (25 μ g/kg) of low dosage and the morphine (125 μ g/kg) of high dosage respectively, and experimental group 1,2,3 gives non-amidated ω-CTX M VIIA albumen of 1.5 μ g/kg, 0.75 μ g/kg, 0.25 μ g/kg respectively.0.5h, 1h, 2h, 3h repeat said determination behind the medicine, the record pain sensation reaction times.By formula calculate the percentage of pain territory raising: average thresholding T1*100% bitterly before pain territory raising percentage=(the preceding average thresholding T1 bitterly of average pain thresholding T2-medication after the medication)/medication.
2. the hot tail method of rat (Hot-tail flick) detection of biological is active.Healthy male Sprague-Dawley rat, body weight 250 ± 20g, the tricorn pipe laying is injected penicillin every day, carries out pharmacological testing after raising a week.Regulate 55 ± 0.5 ℃ of waters bath with thermostatic control, administration survey half an hour pain territory.Normal mice pain territory is generally 2s, and pain territory 5s is analgesia fully.Select 30 of qualified animals, be divided into 6 groups at random, by the sleeve pipe administration.The blank group gives equivalent physiological saline, positive controls gives the morphine (25 μ g/kg) of low dosage and the morphine (125 μ g/kg) of high dosage respectively, and experimental group 1,2,3 gives non-amidated ω-CTX M VIIA albumen of 2.0 μ g/kg, 1.2 μ g/kg, 0.4 μ g/kg respectively.0.5h, 1h, 2h, 3h repeat said determination after the administration, the record pain sensation reaction times, by formula calculate the percentage that improve in the pain territory, and method of calculation are the same.
2 results
2.1 the structure of expression vector: yeast expression vector pPIC9 is carried out Xho I/EcoR I double digestion, and the gene fragment of ω-CTX M VIIA that the coding of annealing generation is non-amidated is connected on the pPIC9, is built into recombinant expression plasmid pPIC9-CTX.It is carried out PCR and double digestion respectively, identify (referring to Fig. 2,3) with agarose electrophoresis, among Fig. 2,1 representative clone, 1 PCR product, 2 representative clones, 2 PCR products, 3 representative clone 3PCR products, 4 represent the pPIC9PCR product, and 5 represent pPIC9, M, small molecule DNA molecular weight standard.1 is clone's 1 double digestion product among Fig. 3; 2 are clone's 2 double digestion products; 3 are clone's 3 double digestion products; 4 is pPIC9 double digestion product; M is the small molecule DNA molecular weight standard.Clone's 2pPIC9-CTX sequencing result is shown correct being inserted among the pPIC9 of dna sequence dna of ω-CTX M VIIA that coding is non-amidated, and referring to Fig. 4, Fig. 4 is clone's 2 sequencing results.Dna sequence dna 317 to 391, TGT AAA GGT AAA GGT GCT AAA TGT TCT CGT CTT ATGTAT GAT TGT TGT ACT GGT TCT TGT CGT TCT GGT AAA TGT is the structure gene of coding non-amidated omega-CTX M VIIA.
2.2 the conversion of expression vector and evaluation: the method by electroporation obtains 107 clones altogether, successively is applied to MM, MD plate screening Mut
+Type, having 102, to be cloned on two kinds of flat boards growing way better, is used for screening the high expression level bacterial strain.By PCR result as can be known recombinant conversion 1,2 all have a band consistent with plasmid pPIC9-CTX, and also have a band of about 2200bp, locating, prove absolutely goal gene CTX homologous recombination be incorporated into and be Mut in the yeast GS115 genome
+Type, but recombinant conversion 3 does not have band at the 2200bp place, illustrates in conversion process, and the existence of AOX1 has destroyed wild-type AOX1 gene in the carrier, is Mut
sType, referring to Fig. 5, Fig. 5 is the agarose electrophoresis figure of PCR product, rise on a left side: 1 is plasmid pPIC9-CTX; 2 are reorganization transformant 1; 3 are reorganization transformant 2; 4 are reorganization transformant 3; M is a dna molecular amount standard.
2.3 the screening of genetic expression and high expression level bacterial strain: each Mut
+The supernatant of transformant after shake flask fermentation is cultivated 96h detects through 18%SDS-PAGE, find that No. 6 clonal expression product band color is darker relatively, tentatively be decided to be the high expression level bacterial strain, referring to Fig. 6, Fig. 6 is that rise on a left side with the electrophorogram of SDS-PAGE screening high expression level bacterial strain: 1 is that reorganization transformant 1 is expressed supernatant; 2 are reorganization transformant 2 expression supernatants; 3 are reorganization transformant 3 expression supernatants; 4 are reorganization transformant 4 expression supernatants; 5 are reorganization transformant 5 expression supernatants; 6 are reorganization transformant 6 expression supernatants.Adopt the Bradford method to measure the non-amidated ω-CTXMVIIA protein yield of expressing, be about every liter of fermented liquid 36mg.
2.4 the purifying of expression product: electrophoretogram is the machine gray scale scanning as calculated, and non-amidated ω-CTXMVIIA expression amount accounts for about 60% of pichia spp secretion total protein concentration.Express supernatant and behind the dialysis desalination, cross the weak cation exchange column, collect elution peak, referring to Fig. 7, wherein---represent the detection curve of 280nm,-----be the detection curve of 215nm ... represent the electric conductivity value detection curve.18%SDS-PAGE purification Identification effect is referring to Fig. 8, and rise on a left side: 1 is the non-amidated ω-CTX M VIIA of purifying; 2 is that clone's 6 expression supernatants penetrate liquid; 3 are clone's 6 expression supernatants; M is the small molecular protein molecular weight standard.
Wherein contain target protein 2.5ELISA identify the target protein purified product through elisa assay: non-amidated ω-CTX M VIIA (referring to Fig. 9).(be provided with: basic, normal, high three kinds of dosage), the OD value is in rising trend by the increasing of non-amidated ω-CTX M VIIA concentration along with bag.
2.6MALDI-TOF-MS analytical results MALDI-TOF-MS result shows, non-amidated ω-CTX M VIIA molecular weight by the yeast expression purifying is 2638.89Da (referring to Figure 10), 2639.22Da meets with theoretical molecular, point out 6 sulfydryls of non-amidated ω-CTX M VIIA all to be oxidized to disulfide linkage, and not by glycosylation modified.
2.7 bioactive detection: the hot tail method of mouse hot plate analgesic experiment and rat confirms that all among Figure 11: Saline is the physiological saline group by the non-amidated ω of yeast expression-CTX M VIIA tool analgesic effect (Figure 11,12); Morphine (L) is low dosage morphine group (25 μ g/kg); Morphine (H) is high dosage morphine group (125 μ g/kg); CTX (L) is that the non-amidated ω of low dosage-CTX M VIIA organizes (0.25 μ g/kg); CTX (M) is that the non-amidated ω of middle dosage-CTX M VIIA organizes (0.75 μ g/kg); CTX (H) is that the non-amidated ω of high dosage-CTX M VIIA organizes (1.5 μ g/kg).Among Figure 12: Saline is the physiological saline group; Morphine (L) is low dosage morphine group (25 μ g/kg); Morphine (H) is high dosage morphine group (125 μ g/kg); CTX (L) is that the non-amidated ω of low dosage-CTX M VIIA organizes (0.4 μ g/kg); CTX (M) is that the non-amidated ω of middle dosage-CTX M VIIA organizes (1.2 μ g/kg); CTX (H) is that the non-amidated ω of high dosage-CTX M VIIA organizes (2.0 μ g/kg), and has dose-dependently.Estimate that from above-mentioned zooperal dose curve by mole analgesia strength ratio, it is 900 times (the morphine molecular weight is 285, and the molecular weight of non-amidated omega-CTX M VIIA is 2639) of morphine that the analgesia of this non-amidated ω-CTX M VIIA is renderd a service.
ω-CTX M VIIA acts on nervous tissue N-type VSCC, and tool " lower molecular weight, high reactivity; high specificity " characteristics are the highly effective probes of neuroscience, influences many cell processes by regulating calcium concn, as neurotransmission, hormone secretion, ionic channel and enzymic activity etc.The double effect of its no addiction analgesia and neuroprotective makes it in anodyne of new generation and neuroprotective drug field tool broad prospect of application.
The little peptide that ω-CTX M VIIA is made up of 25 amino acid contains three pairs of disulfide linkage, synthetic cost height, makes three pairs of correct method of matching of disulfide linkage loaded down with trivial details; If adopt the direct eukaryotic expression of gene engineering method then can reduce cost greatly,, in secretion process, can form correct disulfide linkage pairing because there is the modification ability after translating in pichia spp.This experiment adopt the Pichia anomala expression system utilized its with respect to prokaryotic expression system incomparable advantage: 1. use the pichia spp system, can obtain the little peptide of direct secretion; Because the albumen that pichia spp self is secreted into substratum seldom, high excretory foreign protein is easy to separation and purification; 2. have the rhetorical function after the translation, can directly obtain active result; 3. have complete fermentation process, can the high-density cultured continuously, expressing protein output height.In addition, also has a bold trial in our experiment: the GAGGCTGAAGCTTACGTA behind the carrier pPIC9 signal peptide cutting site is cut away and the direct upward goal gene that connects, the target protein of expressing like this will can not facts have proved that this trial is successful with any unnecessary amino acid.
ω-the expression of CTX M VIIA in pichia spp do not appear in the newspapers so far as yet, because the carboxyl terminal of natural ω-CTXM VIIA is amidated, and present protokaryon that gene engineering method adopted or eukaryotic expression system lack amidated function, thereby nobody carries out the trial of this respect.Our invention shows: the non-amidated ω-CTX M VIIA with the pichia spp direct secretion is expressed, have the intensive analgesic activities equally, and it is 900 times (by mole analgesia strength ratio) of morphine that its analgesia is renderd a service; And express the output height, and this research adopts shake flask fermentation to obtain higher expression, if adopt the fermentor tank high density fermentation, its expression amount will reach higher level.
In sum, we use genetic engineering technique, have successfully expressed non-amidated ω-CTX M VIIA in pichia spp; This method can be produced novel non-amidated ω-CTX M VIIA, and preparation technology is simple, and every liter of fermented liquid can obtain the non-amidated ω of about 36mg-CTX M VIIA, and production cost is lower; Experimentation on animals proves: this novel non-amidated ω-CTX M VIIA has very high analgesic activities, can be used for the exploitation of analgesic.
Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.Therefore, the embodiment of front is interpreted as only illustrating, but not limits the scope of the invention by any way.
Claims (3)
1. non-amidated omega-conotoxin M VII A, it is characterized in that: aminoacid sequence with SEQ ID NO:1, molecular weight is 2639, and Cys1 and Cys16, Cys8 and Cys20, Cys15 and Cys25 form three pairs of disulfide linkage respectively in this peptide molecule, and the C-end is non-amidated Cys.
2. the preparation method of non-amidated omega-conotoxin M VII A according to claim 1 is characterized in that: utilize gene engineering method to make up omega-conotoxin M VII A gene eucaryon expression plasmid and fermentation expression, be achieved through the following technical solutions:
(1) design of the dna fragmentation of coding ω-CTX M VIIA is with synthetic: according to known ω-CTXM VIIA aminoacid sequence, press pichia spp preference codon design oligonucleotides segment CTX1 and CTX2,5 ' end adds sequence and the phosphorylation of Xho I restriction enzyme site and coding Lys-Arg, and 3 ' end adds TAA terminator codon and EcoR I restriction enzyme site;
(2) structure of expression vector: yeast expression vector pPIC9 is carried out Xho I/EcoR I double digestion, the gene fragment of ω-CTX M VIIA that the coding of annealing generation is non-amidated is connected on the pPIC9, be built into recombinant expression plasmid pPIC9-CTX, it is carried out PCR and double digestion respectively, identify with agarose electrophoresis;
(3) conversion of expression vector in the pichia spp: get the recombinant expression vector that 5～10 μ g have removed RNA, through the linearizing of Sal I single endonuclease digestion, ethanol sedimentation reclaims the back and transforms pichia spp GS115 with electroporation, on MD and MM flat board, screen transformant, used electroporation is Bio-Rad GenePulser, conversion condition is voltage 1500V, electric capacity 25 μ F, resistance 200 Ω;
(4) genetic expression: pichia spp genetic expression host bacterium is selected GS115 for use, picking mono-clonal Mut type bacterium colony, the fermentation process that the operational manual that provides according to Invitrogen company is introduced carries out shake flask fermentation, and sampling regularly is with 18%SDS-PAGE testing goal expression of gene level.
3. the non-amidated application of omega-conotoxin M VIIA in the preparation analgesic according to claim 1.
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| CN102964451B (en) * | 2012-12-12 | 2014-06-11 | 黑龙江大学 | msCT-CTx fusion protein for treating osteoporosis and reliving pain and nucleic acid encoding fusion protein |
| CN103013901B (en) * | 2012-12-31 | 2014-05-28 | 黑龙江大学 | OGP-CTx fusion protein transgenic engineering strain |
| CN110358770B (en) * | 2019-07-27 | 2021-08-03 | 福建农林大学 | Method for biologically synthesizing conotoxin by using yeast |
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| WO1991007980A1 (en) * | 1989-11-22 | 1991-06-13 | Neurex Corporation | Compositions for treating ischemia-related neuronal damage |
| WO2005000285A2 (en) * | 2003-06-13 | 2005-01-06 | Dynogen Pharmaceuticals, Inc. | METHODS OF TREATING NON-INFLAMMATORY GASTROINTESTINAL TRACT DISORDERS USING Cav2.2 SUBUNIT CALCIUM CHANNEL MODULATORS |
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