CN106366201A - Gene sequence, carrier, recombination strain and recombination protein of fusion protein DAMP4-IGF-1 and preparing method thereof - Google Patents
Gene sequence, carrier, recombination strain and recombination protein of fusion protein DAMP4-IGF-1 and preparing method thereof Download PDFInfo
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- CN106366201A CN106366201A CN201610835702.0A CN201610835702A CN106366201A CN 106366201 A CN106366201 A CN 106366201A CN 201610835702 A CN201610835702 A CN 201610835702A CN 106366201 A CN106366201 A CN 106366201A
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- 238000000034 method Methods 0.000 title abstract description 11
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- 238000000746 purification Methods 0.000 claims description 16
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Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Diabetes (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a gene sequence of fusion protein DAMP4-IGF-1. The invention also discloses fusion protein DAMP4-IGF-1 and a preparing method thereof. The invention also discloses a recombinant expression carrier or a transgenic cell system or a transgenic recombinant bacterium of the gene sequence of the fusion protein DAMP4-IGF-1. The invention discloses the transgenic recombinant bacterium. The invention discloses a host cell. The invention discloses applications of the gene sequence and the recombinant expression carrier or the transgenic cell system or the transgenic recombinant bacterium to producing the fusion protein DAMP4-IGF-1. Novel fusion label DAMP4 and IGF-1 are subjected to fusion expression in bacillus subtilis, and the fusion protein is heated and purified through the particular characteristics of the DAMP4.
Description
Technical field
The present invention relates to technical field of agriculture science, and in particular to the gene order of fusion protein DAMP4-IGF-1, load
Body, recombinant bacterial strain, recombiant protein and preparation method thereof.
Background technology
Early weaning affects the economic interests that pig produces industry to the ablactation stress that piglet brings.Many research finds, female
Some somatomedin in pig breast can pass through the intestinal for promoting piglet such as newborn source somatomedin para-insulin like growth factor IGF-1
Road development is so as to reduce the harm brought by ablactation stress, while can repair impaired intestinal tissue again.Therefore in order to deeply grind
Study carefully the relation between pig source IGF-1 and intestinal growth, people need to obtain a large amount of pig source IGF-1 with biologic activity.Phase
Direct slave internal body portion is compared to from heterologous recombination being carried out using the higher technique for gene engineering of cost performance and be expressed as people
Obtain the first-selection of pig source IGF-1.But in practical operation, for the purification of recombiant protein is the bottle of restriction industrialized production all the time
Neck problem.Some affinity tag albumen can be selected more than traditional purification, for example：6 × His, GST etc..After these label proteins
Phase purification generally requires the instrument and loaded down with trivial details purification step for relying on costliness.These factors improve cost and constrain in herding
Application in industry.
Content of the invention
Goal of the invention：The technical problem to be solved of the present invention there is provided a kind of fusion protein DAMP4-IGF-1's
Gene order.
Present invention technical problem also to be solved there is provided a kind of fusion protein DAMP4-IGF-1.
Present invention technical problem also to be solved there is provided the weight of the gene order of above-mentioned fusion protein DAMP4-IGF-1
Group expression vector, transgenic cell system or transgenic recombinant bacterium.
Present invention technical problem also to be solved there is provided a kind of transgenic recombinant bacterium.
Present invention technical problem also to be solved there is provided a kind of host cell.
Present invention technical problem also to be solved there is provided above-mentioned gene order, above-mentioned recombinant expression carrier, turn
The application of gene cell system or transgenic recombinant bacterium in production fusion protein DAMP4-IGF-1.
Present invention technical problem also to be solved there is provided a kind of preparation method of fusion protein DAMP4-IGF-1.
Technical scheme：In order to solve above-mentioned technical problem, the technical scheme is that there is provided a kind of fusion protein
The gene order of DAMP4-IGF-1, its base sequence such as SEQ ID NO：Shown in 1.
The gene order of fusion protein DAMP4-IGF-1 is as follows：
GGATCCATGGACCCTAGTATGAAGCAGCTGGCAGATAGCCTTCATCAGCTGGCTAGGCAAGTATCAAGG
TTGGAGCATGCAGATCCAAGTATGAAACAGCTCGCTGATTCACTGCACCAACTGGCAAGACAAGTGTCTCGTCTTGA
GCATGCTGATCCATCAATGAAACAGCTGGCCGATAGCCTCCATCAGTTGGCACGACAAGTTTCTCGTCTCGAACATG
CAGACCCTTCTATGAAACAACTTGCCGATTCTTTACACCAGTTAGCGAGACAAGTCTCGAGATTAGAACACGCCGAC
GATGACGACAAATACTTCAACAAACCGACGGGTTATGGATCGAGCAGCCGGCGCGCTCCTCAAACCGGCATTGTTGA
TGAATGCTGCTTTCGTTCCTGTGATTTGCGGCGGCTTGAAATGTATTGTGCGCCGTTAAAGCCGGCGAAATCCGCGA
GATCAGTCCGCGCCCAACGCCATACTGATATGCCGAAAGCGCAGAAAGAAGTGCATCTTAAGAATACATAATAATCT
AGA
Present invention also includes a kind of fusion protein DAMP4-IGF-1, its aminoacid sequence such as SEQ ID NO：2 institutes
Show.
The recombinant expression carrier of the gene order of above-mentioned fusion protein DAMP4-IGF-1, transgenic cell system turn
Gene recombination bacterium.
Above-mentioned gene order is inserted in bacillus subtilises expression vector and is obtained containing fusion protein DAMP4-
The recombinant expression carrier of the gene order of IGF-1.
Above-mentioned bacillus subtilises expression vector is shuttle expression carrier pHT43.
Present invention also includes a kind of transgenic recombinant bacterium, and the recombinant bacterium is to import described recombinant expression carrier
In escherichia coli, screening obtains transgenic recombinant bacterium.
Present invention also includes a kind of host cell, and the host cell is will be withered for described recombinant expression carrier importing
Obtain in careless bacillus cereuss.
Wherein, above-mentioned bacillus subtilises are bacillus subtilises WB800n.
Wherein, above-mentioned gene order, above-mentioned recombinant expression carrier, transgenic cell system or transgenic recombinant bacterium exist
Application in production fusion protein DAMP4-IGF-1.
Present invention also includes a kind of preparation method of fusion protein DAMP4-IGF-1, comprises the following steps：
1), the gene order of fusion protein DAMP4-IGF-1 is designed according to the aminoacid sequence of DAMP4 and IGF-1, described
The gene order of fusion protein DAMP4-IGF-1 such as SEQ ID NO：Shown in 1；
2) recombinant expression carrier of the gene order, containing fusion protein DAMP4-IGF-1 builds；
3), recombinant expression carrier is transformed in bacillus subtilises carries out abduction delivering acquisition fusion protein DAMP4-IGF-
1；
4), fusion protein DAMP4-IGF-1 purification is obtained final product.
The specific preparation method of the fusion protein DAMP4-IGF-1 of the present invention is as follows：
1) gene order of fusion protein DAMP4-IGF-1, whole piece are designed according to the aminoacid sequence of DAMP4 and IGF-1
Nucleotide sequence carries out full genome synthesis in Shanghai life work；
2) by full genome synthesize fusion gene and shuttle expression carrier pHT43 with two identical restriction enzymes (BamHI with
XabI) double digestion process is carried out, and the fragment after process is carried out back to target gene fragment using agarose gel QIAquick Gel Extraction Kit
Receive, be finally attached using T4 ligases；
3) proceeding to linked system carries out the structure of recombinant vector in E. coli competent DH5a, picking positive colony enters
Performing PCR, double digestion and DAN sequencing analysis；
4) by correct for sequencing result recombinant plasmid transformed in expressive host bacterium bacillus subtilises WB800n, picking sun
Property is cloned in incubated overnight in 5mL LB culture medium.Next day, it is inoculated in the fresh 2YT culture medium of 100mL by 1% inoculum concentration
Row amplification culture, adds IPTG derivants (final concentration 1mM) to carry out the abduction delivering of recombiant protein after 4 hours；
5) bacterium solution of 12 hours after induction carried out centrifugation carry out thalline separating with supernatant.Obtain a certain amount of bacterium solution supernatant
After sample, Na is added in bacterium solution supernatant2SO4(final concentration 1M).Na will be added2SO4Bacterium solution supernatant carry out high-temperature heating treatment
Sample recentrifuge, collection precipitation and Supernatant samples were carried out electrophoresis detection after 30 minutes by (90 DEG C of water-baths).
Beneficial effect：The present invention relative to prior art, with advantages below：The present invention utilizes New Fusion label
DAMP4 carries out amalgamation and expression with IGF-1 first in bacillus subtilises, fusion protein is entered using the special nature of DAMP4
Row is heating purified.The present invention test result indicate that, 1, fusion protein DAMP4-IGF-1 successful first in bacillus subtilises
Secreting, expressing is obtained, Western blotting testing results show the immunogen activity sheet in fusion protein with IGF-1
The maximum innovative point of test is that the use of New Fusion label DAMP4, this test are existed to IGF-1 using the fusion tag first
Realize in bacillus subtilises recombinant expressed.In addition in purge process, due to make use of the fusion tag save complexity
Purification instrument purification is carried out to fusion protein, reduce obtain IGF-1 cost, also a certain degree of extend its apply neck
Domain.2nd, supernatant is heated using high temperature and high salt.Most albuminous degenerations are precipitated but fusion protein DAMP4- under this condition
IGF-1 still keeps the solvable purpose so as to reach purification.
Description of the drawings
Fig. 1, the recombinant expression carrier of the gene order containing fusion protein DAMP4-IGF-1 build figure；
Fig. 2, the recombinant expression carrier double digestion qualification figure of the gene order containing fusion protein DAMP4-IGF-1；Swimming lane
1：The genetic fragment of the DAMP4-IGF-1 after for double digestion, 534bp, swimming lane 2：DNAmaker for 15000bp；
Fig. 3, the DNA sequencing comparison result of the recombinant expression carrier of the gene order containing fusion protein DAMP4-IGF-1
Figure；
Fig. 4, the recombinant expression carrier of the gene order of the correct fusion protein DAMP4-IGF-1 of checking are successfully transferred to withered
The monoclonal bacterium colony obtained in careless spore WB800n；
Fig. 5, SDS-PAGE and Western blot analyzes 1mM 12 hours abduction delivering result figures of IPTG；M：200KDa
Albumen Marker；1：Bacillus subtilises WB800n supernatants；2：On bacillus subtilises WB800n containing empty carrier pHT43
Clearly；3：Bacillus subtilises WB800n supernatants (induction) containing recombinant vector pHT43-IGF-1；4、5：Containing recombinant vector
The bacillus subtilises WB800n (induction) of pHT43-IGF-1；
Fig. 6, SDS-PAGE analyze variable concentrations Na2SO4Heating purified effect to fusion protein in 12 hours induction supernatants
Figure；M：200KDa albumen Marker；1：(1mM IPTG are lured bacillus subtilises WB800n supernatants containing empty carrier pHT43
Lead)；2：Bacillus subtilises WB800n containing recombinant vector pHT43-IGF-1 (1mMIPTG inductions)；3：1M Na2SO4Heating
Purification postprecipitation；4：0.5M Na2SO4Heating purified rear supernatant；5：0.8M Na2SO4Heating purified rear supernatant；6：1M Na2SO4
Heating purified rear supernatant；7：Thalline is through 1M Na2SO4Heating purified rear supernatant；
Fig. 7, SDS-PAGE analyze the fusion protein and western blot of enteral zymogenesis and analyze after purification
IGF-1；a：Fusion protein is cut using enterokinase；b：IGF-1 and western blot analyses after purification.
Specific embodiment
Below in conjunction with the accompanying drawings the present invention is further described.
T4DNA ligases in the embodiment of the present invention, DNA marker, restricted enzyme XbaI and BamHI, 4 ×
Tricine-SDS sample buffers, Taq archaeal dna polymerases and albumen marker are purchased from limited purchased from precious biological engineering (Dalian)
Company；DNA agarose gel QIAquick Gel Extraction Kit and the little extraction reagent kit of plasmid are purchased from TIANGEN Biotech (Beijing) Co., Ltd.；
Low melting-point agarose, Ammonium persulfate. (Ammonium Persuifate) etc. are purchased from Sigma companies；Tryptone is purchased from Oxoid
Company；Yeast extract is purchased from Difco companies；Other reagents are domestic pure analysis pure.Total gene synthesis, primer synthesis and survey
Sequence is completed by Sangon Biotech (Shanghai) Co., Ltd..
The 10mL GMI culture medium adopted in the embodiment of the present invention：10 × minimum saline solution of 1mL, 10% yeast of 0.1mL
Powder, 20% glucoses of 0.25mL, 1% caseinhydrolysates of 0.2mL supplement sterile purified water to cumulative volume 10mL.
II culture medium of 10mL GM：10 × minimum saline solution of 1mL, 10% yeast powders of 0.05mL, 20% Fructus Vitis viniferaes of 0.25mL
Sugar, 1% caseinhydrolysates of 0.04mL, 0.05mL 0.1mo l/L CaCl2, 1mL 25mmo l/L MgCl2, supplement sterilizing and steam
Distilled water is to cumulative volume 10mL.
10 × minimum saline solution (Spizizen salt)：K2HPO470g, KH2PO430g, (NH4)2SO410g,
Na3C6H5O7·2H2O 5g, MgSO4·7H2O 1g, dissolve in distilled water successively, add water to 500ml.Filtration sterilization or 113
DEG C sterilizing 30min after be stored in brown bottle, wrapped up in black paper bag.
The clone of gene of the embodiment 1 containing fusion protein DAMP4-IGF-1 and its structure of recombinant expression carrier
1) gene order of fusion protein DAMP4-IGF-1, whole piece are designed according to the aminoacid sequence of DAMP4 and IGF-1
Nucleotide sequence carries out full genome synthesis in Sangon Biotech (Shanghai) Co., Ltd.；
2) by full genome synthesize fusion gene and shuttle expression carrier pHT43 with two identical restriction enzymes (BamHI with
XbaI) double digestion process is carried out, and the fragment after process is carried out back to target gene fragment using agarose gel QIAquick Gel Extraction Kit
Receive, be finally attached using T4 ligases；
Genes of interest and expression vector plasmid are carried out by following enzyme action system：
37 DEG C of water-bath 30min.
Genes of interest is as follows with the linked system of expression vector：
4 DEG C overnight connect.
3) proceeding to linked system carries out the structure of recombinant vector in E. coli competent DH5a, overnight choose positive gram
Grand carry out plasmid extraction and to its carry out double digestion identification and DNA sequencing analysis；
(3a) 10 μ L connection products are added in 200 μ L competent cell E.coli (DH5 α), place 30min on ice；
(3b) 42 DEG C of heat shock 90s；
(3c) rapid taking-up 1～2min of ice bath；
(3d) 800 μ L LB culture medium, 37 DEG C of 200rpm 1～1.5h of shaken cultivation are added in centrifuge tube；
(3e) 7000rpm centrifugations 1min, absorbs 700 μ L of supernatant；
(3f) will be uniform for the piping and druming of remaining converted product with micropipette rifle；
(3g) converted product is coated with the LB flat boards containing 25 μ g/mL chloromycetin, room temperature places 3～5min, treats that liquid is inhaled
After receipts, 37 DEG C are inverted culture to visible single bacterium colony.
Double digestion checking system is as follows：
The fusion gene of the present embodiment synthesis is successfully embedded in expression vector pHT43, and DNA sequencing result shows matter of recombinating
Grain pHT43/DAMP4-IGF-1 is successfully constructed.Concrete outcome as shown in Figure 1, Figure 2, Fig. 3.
Embodiment 3：The structure of the recombinant bacterial strain of expressed fusion protein DAMP4-IGF-1
The correct recombinant vector of sequencing result in embodiment 2 is transformed into expressive host bacterium bacillus subtilises WB800n senses
By in state cell, picking positive colony incubated overnight in 5mL LB culture medium obtains recombinant bacterial strain.Comprise the following steps that：
The recombinant expression carrier successfully constructed in escherichia coli is transferred to expressive host bacterium hay bud using chemical method
In spore bacillus WB800n (Fig. 4), after picking positive colony incubated overnight, abduction delivering is carried out.
The preparation of bacillus subtilises WB800N competent cells and the chemical conversion of recombiant plasmid
(1) the bacillus subtilises WB800N single bacterium colonies of one fresh cultured of picking are inoculated in I culture medium of 25mL GM, and 30
DEG C 130～150r/min shaken cultivation is overnight；
(2) step (1) overnight culture is inoculated in fresh I culture medium of GM of 25mL by 10% inoculum concentration, 37 DEG C 200
～230r/min shaken cultivation 3.5h；
(3) culture that step (2) culture is obtained is carried out second pass generation again, the fresh GM of 50mL are inoculated in 10%
In II culture medium, 37 DEG C of 200～230r/min shaken cultivation 90min；
(4) culture that 1mL steps (3) culture is obtained is taken, and 5000r/min room temperatures are centrifuged 5min, with 1/10 volume supernatant
Again suspended bacterial is precipitated liquid, as bacillus subtilis bacterium competence cell.
(5) 5 μ L are sequenced correct recombinant vector to be added in 100 μ L bacillus subtilises WB800N competent cells；
30～60min is stood in (6) 37 DEG C of water-baths；
(7) 37 DEG C of 200r/min 2～4h of concussion and cultivate；
(8) competence is coated on the LB solid mediums containing chloromycetin (10 μ g/mL), every 100 μ L transformation systems are applied
Flat board is arrived, overnight incubation is inverted for 37 DEG C and is obtained recombinant bacterial strain.
Embodiment 4：IPTG abduction delivering fusion protein DAMP4-IGF-1 and its purification
The recombinant bacterial strain that embodiment 3 is obtained is inoculated in the fresh 2YT culture medium of 100mL by 1% inoculum concentration and is expanded
Big culture, adds IPTG derivants (final concentration 1mM) to carry out the abduction delivering of recombiant protein after 4 hours.12 hours after induction
Bacterium solution carries out centrifugation and carries out thalline separating with supernatant.After obtaining a certain amount of bacterium solution Supernatant samples, add in bacterium solution supernatant
Na2SO4(final concentration 1M).Na will be added2SO4Bacterium solution supernatant carry out high-temperature heating treatment (90 DEG C of water-baths) after 30 minutes by sample
Product recentrifuge, collecting precipitation and Supernatant samples carries out electrophoresis detection.
The present embodiment is using 1mM IPTG to the bacillus subtilises containing recombiant plasmid pHT43/DAMP4-IGF-1
WB800n carries out abduction delivering.As a result through SDS-PAGE analysis shows (Fig. 5), through 12 hour induction rear fusion proteins
DAMP4-IGF-1 (about 19Kda) is secreted in culture medium.Western blot detections (Fig. 5) is carried out to specific band, is tied
Fruit shows that fusion protein DAMP-IGF-1 has IGF-1 immunogen activities.
The present embodiment utilizes difference Na2SO4(0.5M, 0.8M, 1M) albumen of secreting, expressing is carried out heating purified (90 DEG C,
30 minutes), concrete outcome such as Fig. 6, in supernatant the band of 19Kda high temperature and high salt process after keep in the solution solvable (Fig. 6,
4th, 5,6 swimming lanes), and other albuminous degenerations precipitation (Fig. 6, the 3rd swimming lane) in supernatant.As a result show 1MNa2SO4Concentration is pure
Change effect preferably (Fig. 6, the 6th swimming lane).Fusion protein DAMP4-IGF-1 concentration after purification is per liter of 47mg/.
7th swimming lane is the sample that the thalline mixed sodium sulfate after centrifugation heats sum, and being primarily used to observe inside thalline is
The no residual for having fusion protein.
The present embodiment is concentrated using super filter tube and is changed using the fusion protein of hot purification and entered using enterokinase after buffer
Row cutting (20mM Tris-HCl, 200mM NaCl, 2mM CaCl2, pH=7.2,4 DEG C).Concrete outcome such as Fig. 7 a, merge egg
Analyzed using SDS-PAGE after Enterokinase cleavage in vain, occur the bar of DAMP4 and IGF-1 between 14.4Kda and 6.5Kda
Band.
(1) sample by high temperature and high salt after purification carries out four degree of dialysed overnights, and purpose cuts buffer for changing
(20mMTris-HCl, 200mM NaCl, 2mM CaCl2, pH=7.2)；
(2) by dialysis after sample concentrated for the super filter tube of 3Kda using combined closure system, 12000rpm, 10 minutes；
(3) enterokinase is added in the concentrating sample of above-mentioned 100uL, cut the ratio of 1mg to dialysis solution according to every 8 unit
Middle addition enterokinase.Overnight enzyme action is carried out under the conditions of 4 DEG C；
(4) by enzyme action after sample detected using SDS-page.
The present embodiment utilize above-mentioned cutting method, by cutting after sample 10mL dialysed in less salt buffer
(0.125mM NaSO4).Using the insoluble principles in low salt solutions of DAMP4 by the label protein DAMP4 in cutting system
Centrifugation is removed.Supernatant after by centrifugation is detected using SDS-page, is analyzed by SDS-page it is observed that being cutting
DAMP4 after the fusion protein DAMP4-IGF-1 for opening and cutting cracking is removed, can be seen between 6.5Kda and 14.4Kda
Band IGF-1 theoretical moleculars to IGF-1 are 7.6Kda.Concrete outcome such as Fig. 7 b.The concentration of the IGF-1 albumen after cutting is
Per liter of 3mg/.
The above is only the preferred embodiment of the present invention, it should be pointed out that：Ordinary skill people for the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of gene order of fusion protein DAMP4-IGF-1, it is characterised in that its base sequence such as SEQ ID NO：1 institute
Show.
2. a kind of fusion protein DAMP4-IGF-1, it is characterised in that its aminoacid sequence such as SEQ ID NO：Shown in 2.
3. the recombinant expression carrier of the gene order containing the fusion protein DAMP4-IGF-1 described in claim 1, transgenic are thin
Born of the same parents' system or transgenic recombinant bacterium.
4. recombinant expression carrier according to claim 3, it is characterised in that the gene order described in claim 1 is inserted
Enter to obtain the recombinant expressed load of the gene order containing fusion protein DAMP4-IGF-1 in bacillus subtilises expression vector
Body.
5. recombinant expression carrier according to claim 4, it is characterised in that the bacillus subtilises expression vector is for wearing
Shuttle expression vector pHT43.
6. a kind of transgenic recombinant bacterium, it is characterised in that the recombinant bacterium is by the recombinant expressed load described in claim 4 or 5
Body is imported in escherichia coli, and screening obtains transgenic recombinant bacterium.
7. a kind of host cell, it is characterised in that the host cell is by the recombinant expression carrier described in claim 4 or 5
Import and obtain in bacillus subtilises.
8. host cell according to claim 7, it is characterised in that the bacillus subtilises are bacillus subtilises
WB800n.
9. the gene order described in claim 1, the recombinant expression carrier described in claim 3, transgenic cell system or turn
Application of the gene recombination bacterium in production fusion protein DAMP4-IGF-1.
10. a kind of preparation method of fusion protein DAMP4-IGF-1, it is characterised in that comprise the following steps：
1), the gene order of fusion protein DAMP4-IGF-1, the fusion is designed according to the aminoacid sequence of DAMP4 and IGF-1
The gene order of protein D AMP4-IGF-1 SEQ ID NO as claimed in claim 1：Shown in 1；
2) recombinant expression carrier of the gene order, containing fusion protein DAMP4-IGF-1 builds；
3), recombinant expression carrier is transformed in bacillus subtilises carries out abduction delivering acquisition fusion protein DAMP4-IGF-1；
4), fusion protein DAMP4-IGF-1 purification is obtained final product.
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| CN117164722A (en) * | 2023-08-09 | 2023-12-05 | 东北农业大学 | Spiral bundle DAMP4-PR-FO fusion protein D2L and preparation method and application thereof |
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| CN117164722A (en) * | 2023-08-09 | 2023-12-05 | 东北农业大学 | Spiral bundle DAMP4-PR-FO fusion protein D2L and preparation method and application thereof |
| CN117164722B (en) * | 2023-08-09 | 2024-04-09 | 东北农业大学 | Spiral bundle DAMP4-PR-FO fusion protein D2L and preparation method and application thereof |
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