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CN107880133A - Corticotropin(ACTH) and type-1 insulin like growth factor fusion protein and preparation method thereof - Google Patents

Corticotropin(ACTH) and type-1 insulin like growth factor fusion protein and preparation method thereof Download PDF

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Publication number
CN107880133A
CN107880133A CN201711074686.9A CN201711074686A CN107880133A CN 107880133 A CN107880133 A CN 107880133A CN 201711074686 A CN201711074686 A CN 201711074686A CN 107880133 A CN107880133 A CN 107880133A
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acth
igf
ekst
histag
linker
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王大勇
黄永林
裴业春
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Hainan University
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Hainan University
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Priority to CN202110624088.4A priority patent/CN113214410A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C07K14/695—Corticotropin [ACTH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

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Abstract

The present invention relates to a kind of gene recombinant human corticotropin(ACTH) (ACTH) and type-1 insulin like growth factor (IGF 1) fusion protein and preparation method thereof.The present invention using the optimization codon for being adapted to express in engineering bacteria, constructs the expression plasmid for encoding ACTH and the fusion proteins of IGF 1 first：The pET30a plasmids of HisTag linker EKst ACTH IGF 1, histidine-tagged (HisTag) sequence, catenation sequence (linker), enterokinase substrate (EKst) sequence and ACTH and the sequences of IGF 1 are included in coded sequence (CDS).In E. coli expression strains BL21 DE3 after induced expression, the polypeptide chains of HisTag linker EKst ACTH IGF 1 are isolated and purified out from the inclusion body of broken dissolving with nickel (Ni) post, afterwards HisTag linker EKst fragments are cut with histidine-tagged enterokinase, then enterokinase and HisTag linker EKst fragments are removed with Ni posts again, obtains the fusion proteins of ACTH IGF 1.

Description

Corticotropin(ACTH) and type-1 insulin like growth factor fusion protein and preparation method thereof
Technical field
The invention belongs to field of biological pharmacy.More particularly to DNA and prlmary structure of protein, expression vector structure and Expression and the separation and purifying of protein.
Background technology
Corticotropin(ACTH) (Corticotropin), also referred to as corticotropin (ACTH, adrenocorticotropic Hormone), it is 39 amino of one kind by tissue secretions such as the frontal lobe of hypophysis, hypothalamus, adrenal medella, enteron aisle and placentas The peptide hormone of acid, there is the biosynthesis and metabolism of regulation sugar, fat and protein, regulation cardio and vascular function, carry High resistance, the synthesis and secretion of regulation and control glucocorticoid (GC), neurotrosis recovers and regeneration, anti-inflammatory, immunosupress, resists Toxin, Hemorrhagic shock etc. act on.ACTH is unique specific drug of eclampsia infantum disease below clinical treatment two years old, the sick incidence of disease 0.5%~1%, 6~33% eclampsia infantum disease infant is dead before 3 years old, and 70~90 % infant intelligence developments are slow, and 30% Develop into self-closing disease.ACTH is also used for lupus erythematosus, multiple sclerosis acute exacerbation, nephrotic syndrome, systemic dermatomyocytis With the treatment of sarcoidosis；It is additionally operable to psoriatic arthritis, rheumatic arthritis, ankylosing spondylitis and severe ocular allergy With the auxiliary treatment of inflammation etc..ACTH is unstable, and Half-life in vivo only has 15 minutes；The ACTH of clinical practice at present is driven Extracted in thing tissue, production cost is high and has the potential risk for propagating animal virus and mycoplasma.The present invention is from engineering bacteria Middle expression prepares genetic recombination ACTH fusion proteins, and production cost is low, will not propagate animal virus and mycoplasma substantially.
Type-1 insulin like growth factor (IGF-1, insulin-like growth factor 1), also referred to as growth promotion The factor or somatomdein (Somatomedin), containing 70 amino acid, relative molecular weight is 7649 peptide hormone, Its long half time is up to 20 hours, and it is acted on insulin type seemingly, and IGF-1, which is used alone, to improve 20 by ACTH effect Times.Main functions of the IGF-1 in human body is regulation blood glucose, growth promotion, promotees cell differentiation, wound repair etc..IGF-1 preparations Clinically have been used for treating a variety of persistent ailments such as diabetes, insulin resistance syndrome, nanism and the nervous system disease, take Obtained good effect.The present invention is by ACTH and IGF-1 amalgamation and expressions and purifies.Obtained fusion protein is not only able to strengthen ACTH effect, the also double pharmacological action with ACTH and IGF-1.
Initial table up to framework be HisTag-linker-EKst-ACTH-IGF-1.Wherein HisTag to be histidine-tagged, Linker is random catenation sequence, and EKst is enterokinase (Enterokinase) substrate sequence (amino acid sequence DDDDK). Enterokinase specific can cut its substrate sequence amino acid sequence end, obtain the ACTH-IGF- without additional amino acid 1 fusion protein.
The content of the invention
1. the purpose of invention
Expressed in utilizing works bacterium and be purified into a kind of gene recombinant fusion protein (ACTH-IGF-1), and its prepare and Purification process.
2. the technical scheme of invention
First, ACTH is connected with IGF-1 using overlapping pcr, structure both ends with restriction enzyme site (BamH1 and Sal1 cDNA frameworks)：BamH1-HisTag-linker-EKst-ACTH-IGF-1-Sal1, inserted before ACTH in this framework The base sequence of coding enterokinase substrate (EKst), EKst are previously inserted into the base sequence of encoding histidine label (HisTag) Row, are connected between ACTH and IGF-1 with flexible linker sequence GGGS.
2nd, double digestion Bamh1-HisTag-linker-EKst-ACTH-IGF-1-Sal1 and carrier pET30a, produce viscous Property end.
3rd, the BamH1-HisTag-linker-EKst-ACTH-IGF- 1- of cohesive end will be cut out with T4 ligases structure Sal1 and plasmid pET30a connections, construct expression HisTag-linker-EKst-ACTH-IGF-1 pET30a plasmids (Fig. 1).
4th, the plasmid built is transferred to E. coli expression strains BL21-DE3, screens high expression bacterial strain, induce table Reach.
5th, thalline is crushed, inclusion body is collected and washs, then broken and dissolving inclusion body, obtain being rich in HisTag- Linker-EKst-ACTH-IGF-1 solution.
6th, it is purified into HisTag-linker-EKst-ACTH-IGF-1 using nickel (Ni) post.
7th, HisTag-EKst-ACTH-IGF-1 is cut with the enterokinase with His labels, obtains ACTH-IGF-1 and melt Hop protein.
8th, the enterokinase with His labels and the HisTag-linker-EKst scaled off are removed with nickel post, obtained The ACTH-IGF-1 of purifying.
3. the beneficial effect of invention
The present invention can obtain the fusion protein IGF-1-ACTH with ACTH and IGF-1 double effects, can strengthen ACTH effect.
Brief description of the drawings
Fig. 1 .HisTag-linker-EKst-ACTH-IGF-1pET30a plasmid maps.
The structure result verification of Fig. 2 .HisTag-EKst-linker-ACTH-IGF-1-pET30a expression plasmids.A. piece Section BamH1-HisTag-linker-EKst-ACTH PCR amplifications.M：marker；Swimming lane 1：Empty swimming lane, swimming lane 2：BamH1- HisTag-linker-EKst-ACTH pcr amplification product.B. fragment IGF-1-Sal1 PCR amplifications.M：marker；Swimming lane 1：Empty swimming lane, swimming lane 2：IGF-1-Sal1 pcr amplification product.C. fragment BamH1-HisTag-linker-EKst-ACTH- IGF-1-Sal1 PCR amplifications.M：marker；Swimming lane 1：Empty swimming lane, swimming lane 2：Bamh1-HisTaglinker--EKst- Site-ACTH-IGF-1-Sal1 pcr amplification product.D. carrier pET30a digestion.M：marker；Swimming lane 1：PET30a is carried Body, swimming lane 2：Bamh1, Sal1 double digestion carrier pET30a.E. corticotropin(ACTH)-type-1 insulin like growth factor expressing fusion protein Carrier HisTag-linker-EKst-ACTH-IGF-1-pET30a digestion identification.M：marker；Swimming lane 1：PET30a is empty Carrier, swimming lane 2,3：Bamh1, Sal1 double digestion have inserted HisTag-EKst-linker-ACTH-IGF-1-pET30a sequences Plasmid.
Fig. 3 .HisTag-linker-EKst-ACTH-IGF-1 sequencer maps.
Ni-sepharose purification result figure is used after Fig. 4 recombination fusion proteins HisTag-linker-EKst-ACTH-IGF-1 expression. After HisTag-linker-EKst-ACTH-IGF-1-pET30a plasmids are expressed in BL21-DE3 engineering bacterias, through ultrasonication, After supernatant ni-sepharose purification, after SDS-PAGE (15%Tris-Glycine polyacrylamide gels) separation, coomassie is bright Blue R-250 native stainings.M：Molecular weight marker.Swimming lane 1-14：The HisTag-linker-EKst-ACTH- of Ni posts after purification The separation component of IGF-1 fusion proteins, obtained fusion protein HisTag-linker- is purified for Ni posts in component 4-13 EKst-ACTH-IGF-1。
The ACTH-IGF-1 of Fig. 5 after purification Western blot detection figures.ACTH-IGF-1 SDS- PAGE (10% Tris-tricine glue) separation after, be transferred to low Poison background pvdf membrane detection, primary antibody is the anti-human ACTH (Santa of mouse Cruz) specific antibody, secondary antibody is Alexa488 fluorescence labelings sheep anti-mouse antibody (Abcam), with Typhoon FLA 9500 (GE Healhcare) detects fluorescent bands.
Embodiment
Embodiment one：Present embodiment is expressed and isolates and purifies genetic recombination ACTH-IGF-1 fusion proteins, presses Following steps are carried out：
First, the codon preference optimization design ACTH CDs sequences in prokaryotic expression system, are suitable in large intestine High efficient expression in bacillus expression bacterial strain BL21-DE3.Upstream and downstream using BamH1 and Sal1 as amalgamation and expression PROTEIN C Ds sequences The restriction enzyme site at both ends, fusion protein prokaryotic expression sequence HisTag- of the structure with His labels and enterokinase cleavage site linker-EKst-ACTH-IGF-1-Sal1.Comprise the following steps that：
Design of primers：Design amplification BamH1-HisTag-linker-EKst-ACTH sense primer BamH1- HisTag-linker-EKst-ACTH-F, anti-sense primer ACTH-R；Design amplification IGF-1 sense primer IGF-1-F, downstream Primer I GF-1-Sal1-R.Primer sequence is shown in nucleotides sequence list.
With over-lap PCR method primer BamH1-HisTag-linker-EKst-ACTH-F and ACTH-R, with artificial synthesized ACTH sequences be template, amplify BamH1-HisTag-linker-EKst-ACTH (see Fig. 2 a)；With the gene containing IGF-1 Plasmid is template, then amplifies IGF-1-Sal1 with primer I GF-1-F and primer I GF-1-Sal1-R (see Fig. 2 b)；Finally use The two fragments (BamH1-HisTag-EKst-ACTH and IGF-1-Sal1) of amplification are template, with primer BamH1- HisTag-EKst-ACTH-F and IGF-1-Sal1-R amplify purpose fragment： BamH1-HisTag-EKst-ACTH-IGF-1- Sal1 (see Fig. 2 c), the nucleotide sequence encoding amino acid flexible connection fragment GGGS between ACTH and IGF-1.
2nd, endonuclease bamhi BamH1-HisTag-EKst-ACTH-IGF-1-Sal1 and blank are distinguished with BamH1 and Sal1 Carrier pET30a (see Fig. 2 d).
3rd, with T4DNA ligases junction fragment and carrier, structure plasmid HisTag-Linker-EKst-ACTH-IGF- 1-pET30a.Digestion is identified (see Fig. 2 e), draws fusion protein expression plasmid collection of illustrative plates (see Fig. 1), sequencing result is shown in Fig. 3.
4th, 40 DEG C, plasmid HisTag-linker-EKst-ACTH-IGF-1-pET30a was transferred to Escherichia coli in 30 seconds Express in bacterial strain BL21-DE3,37 DEG C are recovered thalline 1 hour, take 50uL to apply the LB solid culture plates containing kanamycins, 37 DEG C It is inverted culture 16h.Select the single bacterium colony on plate, be transferred in the conical flask added with 50mL LB liquid mediums, in culture medium added with Kanamycins, 37 DEG C shake bacterium to OD 600 be 0.6 when, it is 0.05mM IPTG in 37 DEG C of 180rpm expression 5h to add concentration.
5th, above-mentioned engineering bacteria is collected in centrifuge tube, with 12,000rpm, under the conditions of 4 DEG C, centrifuges 15min, discard Supernatant.Non denatured lysate (50mM Tris-HCl, 300mM NaCl, 10mM imidazoles, pH 8.0) soft washing thalline is added, 12,000rpm, 4 DEG C of centrifugation 10min collect precipitation, wash 3 times, each 15min.Every gram of weight in wet base adds the non denatured cracking of 8mL Liquid, disperse thalline with rotary mixer.Bacterium solution is placed on ice, crushes thalline with sonicator, broken power is 400W, broken 3s stop 5s and crushed 200 times altogether.12,000rpm, 4 DEG C of centrifugation 20min.Under condition of ice bath, centrifugation is hanged Float on inclusion body cleaning solution I (50mM Tris-HCl, 100mM NaCl, 2mM EDTA, 1mM DTT, the 0.5% (v/ of 9 times of volumes V) Triton X-100, pH 8.0), ultrasound 3 × 10 seconds, room temperature places 10min, and at 4 DEG C, 20min is centrifuged with 12,000rpm, Supernatant and precipitation are collected respectively.Inclusion body cleaning solution II (50mM Tris-HCl, the 100mM for being suspended in 9 times of volumes will be precipitated NaCl, 2mM EDTA, 1mM DTT, pH 8.0), room temperature places 10min after mixing, after ibid condition centrifuges 20min again, receives Collection merges supernatant and is used for protein purification.
6th, after the NI posts regenerated are washed with 2 times of ethanol of volume 20%, gone over 2V deionized water.Change flowing It is mutually level pad (50mM Tris-cl, 300mM Nacl, 10mM imidazoles, pH 8.0), balances 5 column volumes.Loading, Replacing mobile phase is rinsing liquid (50mM Tris-cl, 300mM Nacl, 100mM imidazoles, pH 8.0), rinses 40 column volumes. Elution, replacing mobile phase is eluent (50mM Tris-cl, 300mM Nacl, 250mM imidazoles, pH 8.0), collects eluent Component, after 15%SDS-PAGE electrophoresis, R-250 is dyed (see Fig. 4).
7th, the enterokinase with His labels, final concentration of 1mM, in 37 DEG C of temperature are separately added into component in eluent 1 hour is educated with the HisTag-linker-EKst-ACTH-IGF-1 polypeptide chains in cutting sample, obtains free ACTH-IGF- 1 and His-Tag-linker-EKst.
8th, Ni posts are balanced using the method for step 6, the eluant component after enterokinase is cut adds to Ni posts, 4 DEG C of standings 30-60min is connected with the enterokinase and HisTag-linker-EKst polypeptides of His labels to remove in digestion eluent. Western blot identify that ACTH-IGF-1 results are as shown in Figure 5 in each eluant component.
Embodiment two：Present embodiment does not use pET30a plasmids unlike embodiment one, and It is to be used as expression vector by the use of pET21a.
Embodiment three：Present embodiment do not used unlike embodiment one or two pET30a or PET21a plasmids, but expression vector is used as by the use of pET28a.
Sequence table
<110>University Of Hainan
<120>Corticotropin(ACTH) and type-1 insulin like growth factor fusion protein and preparation method thereof
<130> 2016.6.1
<160> 8
<170> SIPOSequenceListing 1.0
<210> 9
<211> 534
<212> DNA
<213> Artificial Sequence
<220>
<221> terminator
<222> (526)..(534)
<400> 9
atgcaccatc atcatcatca ttcttctggt ctggtgccac gcggttctgg tatgaaagaa 60
accgctgctg ctaaattcga acgccagcac atggacagcc cagatctggg taccgacgac 120
gacgacaagg ccatggctga tatcggatcc atgcatcacc atcaccatca cgacgacgac 180
gacaaatctt actctatgga acacttccgt tggggtaaac cggttggtaa aaaacgtcgt 240
ccggttaaag tttacccgaa cggtgctgaa gacgaatctg ctgaagcttt cccgctggaa 300
ttcggtggtg gttctggacc ggagacgctc tgcggggctg agctggtgga tgctcttcag 360
ttcgtgtgtg gagacagggg cttttatttc aacaagccca cagggtatgg ctccagcagt 420
cggagggcgc ctcagacagg catcgtggat gagtgctgct tccggagctg tgatctaagg 480
aggctggaga tgtattgcgc acccctcaag cctgccaagt cagcttaata ataa 534
<210> 9
<211> 175
<212> PRT
<213> Artificial Sequence
<220>
<221> CHAIN
<222> (63)..(175)
<400> 9
Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser
1 5 10 15
Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp
20 25 30
Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile
35 40 45
Gly Ser Met His His His His His His Asp Asp Asp Asp Lys Ser Tyr
50 55 60
Ser Met Glu His Phe Arg Trp Gly Lys Pro Val Gly Lys Lys Arg Arg
65 70 75 80
Pro Val Lys Val Tyr Pro Asn Gly Ala Glu Asp Glu Ser Ala Glu Ala
85 90 95
Phe Pro Leu Glu Phe Gly Gly Gly Ser Gly Pro Glu Thr Leu Cys Gly
100 105 110
Ala Glu Leu Val Asp Ala Leu Gln Phe Val Cys Gly Asp Arg Gly Phe
115 120 125
Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro
130 135 140
Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg
145 150 155 160
Arg Leu Glu Met Tyr Cys Ala Pro Leu Lys Pro Ala Lys Ser Ala
165 170 175
<210> 9
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 9
cgcggatcca tgcatcacca tcaccatcac gacgacgacg acaaa 45
<210> 9
<211> 32
<212> DNA
<213> Artificial Sequence
<400> 9
tagcgtcgac ttattattaa gctgacttgg ca 32
<210> 9
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 9
ctggaattcg gtggtggttc tggaccggag a 31
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 9
gcgtcgactt attattagaa ttccagcggg 30
<210> 9
<211> 348
<212> DNA
<213> Artificial Sequence
<220>
<221> terminator
<222> (340)..(348)
<400> 9
tcttactcta tggaacactt ccgttggggt aaaccggttg gtaaaaaacg tcgtccggtt 60
aaagtttacc cgaacggtgc tgaagacgaa tctgctgaag ctttcccgct ggaattcggt 120
ggtggttctg gaccggagac gctctgcggg gctgagctgg tggatgctct tcagttcgtg 180
tgtggagaca ggggctttta tttcaacaag cccacagggt atggctccag cagtcggagg 240
gcgcctcaga caggcatcgt ggatgagtgc tgcttccgga gctgtgatct aaggaggctg 300
gagatgtatt gcgcacccct caagcctgcc aagtcagctt aataataa 348
<210> 9
<211> 113
<212> PRT
<213> Artificial Sequence
<400> 9
Ser Tyr Ser Met Glu His Phe Arg Trp Gly Lys Pro Val Gly Lys Lys
1 5 10 15
Arg Arg Pro Val Lys Val Tyr Pro Asn Gly Ala Glu Asp Glu Ser Ala
20 25 30
Glu Ala Phe Pro Leu Glu Phe Gly Gly Gly Ser Gly Pro Glu Thr Leu
35 40 45
Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe Val Cys Gly Asp Arg
50 55 60
Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg
65 70 75 80
Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp
85 90 95
Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu Lys Pro Ala Lys Ser
100 105 110
Ala

Claims (10)

1. genetic recombination corticotropin(ACTH) (ACTH, corticotropin, adrenocorticotropic hormone) and pancreas islet Plain like growth factor 1 (IGF-1, insulin-like growth factor) fusion protein.A characterized in that, polypeptide chain It is upper to include ACTH amino acid sequences and IGF-1 amino acid sequences simultaneously.
2.ACTH is connected with IGF-1.
3.ACTH is connected with IGF-1 by the short peptide chain of some Amino acid profiles.
4. the amino acid sequence of the ACTH-IGF-1 fusion proteins described in claim 1.
5. the nucleotide sequence of the ACTH-IGF-1 fusion proteins described in claim 1.
6. 5 ' the ends for encoding the nucleotide sequence of ACTH-IGF-1 fusion proteins are connected with coding enterokinase (Enterokinase) bottom The nucleotide sequence of thing (EKst).
7.EKst amino acid sequences are connected in the N-terminal of ACTH-IGF-1 fusion proteins.
8. genetic recombination corticotropin(ACTH) (ACTH) and type-1 insulin like growth factor (IGF-1) fusion protein described in claim 1 Vivoexpression method, it is characterised in that using inducible expression plasmid expression in escherichia coli ACTH-IGF-1 merge egg In vain.
9. genetic recombination corticotropin(ACTH) (ACTH) and type-1 insulin like growth factor (IGF-1) fusion protein described in claim 1 Isolation and purification method, it is characterised in that with Ni posts purify with histone label (HisTag) ACTH-IGF-1 merge egg In vain.
10. according to claim 9, during ACTH-IGF-1 fusion proteins are isolated and purified, cut off and connected with enterokinase Additional amino acid sequence on ACTH-IGF-1 fusion protein polypeptide chains.
CN201711074686.9A 2017-11-04 2017-11-04 Corticotropin(ACTH) and type-1 insulin like growth factor fusion protein and preparation method thereof Pending CN107880133A (en)

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CN202110624088.4A CN113214410A (en) 2017-11-04 2017-11-04 Recombinant plasmid for expressing corticotropin and insulin-like growth factor 1 fusion protein and construction method of recombinant bacterium

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