CN117417951A - Prokaryotic expression vector of pharmaceutical grade human fibroblast growth factor-18, and construction method, preparation method and application thereof - Google Patents
Prokaryotic expression vector of pharmaceutical grade human fibroblast growth factor-18, and construction method, preparation method and application thereof Download PDFInfo
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- CN117417951A CN117417951A CN202311337452.4A CN202311337452A CN117417951A CN 117417951 A CN117417951 A CN 117417951A CN 202311337452 A CN202311337452 A CN 202311337452A CN 117417951 A CN117417951 A CN 117417951A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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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
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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- 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
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Animal Behavior & Ethology (AREA)
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Abstract
The invention discloses a prokaryotic expression vector capable of preparing pharmaceutical grade human fibroblast growth factor-18 in a large scale, which comprises a DNA fragment consisting of a nucleotide sequence for encoding fusion protein containing human fibroblast growth factor 18 and a DNA fragment consisting of a nucleotide sequence for encoding small molecule-like ubiquitination modified protease; according to the invention, chemical substances (IPTG) are not required to be used for induction, and meanwhile, the fusion protein can be cut by protease without separation and purification, so that FGF18 protein is obtained, and the steps of separation and purification of recombinant protein in the production process are reduced, thereby reducing the loss of target protein, lowering the production cost and realizing large-scale production. The prokaryotic expression vector can be used for preparing pharmaceutical grade human fibroblast growth factor-18 in a large scale, and can be applied to preparing medicines for treating acute lung injury.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a prokaryotic expression vector capable of preparing pharmaceutical grade human fibroblast growth factor-18 in a large scale, and a construction method, a preparation method and application thereof.
Background
The fibroblast growth factor (Fibroblast growth factors, FGFs) superfamily consists of 22 members and is largely divided into secreted and intracellular types. Secreted fibroblast growth factors include FGF1-10 and FGF16-22, while intracellular fibroblast growth factors include FGF11-14. Secreted fibroblast growth factors must activate downstream signaling pathways by binding to and activating Fibroblast Growth Factor Receptors (FGFRs) of cell surface tyrosine kinase (receptor tyrosine kinases, RTKs) family members, while intracellular fibroblast growth factors interact primarily with other proteins to regulate cell sodium channel switching. FGFs are expressed in almost all tissues in humans and play an important biological role in embryonic development, organogenesis and regeneration, tissue repair, and metabolism of the body. FGFs superfamily can be further divided into FGF1, 4, 7, 8, 9, 11 and 19 subfamilies according to their regulatory functions, sequence similarity and evolutionary relationships. FGF8 subfamily members include FGF8, FGF17, and FGF18, all of which activate receptors FGFR1, FGFR2, FGFR 3, and FGFR4c. FGF18 protein plays an important role in the development of multi-organ mesenchymal components of the body, and is currently known to play a vital role in skeletal development.
The expression of FGF-18 by using a prokaryotic system has been reported, however, the efficient expression of FGF-18 with natural activity is still a difficulty in the current large-scale preparation, including problems of inclusion body formation, complex purification process, low product activity and the like.
Disclosure of Invention
The invention aims at providing a prokaryotic expression vector of human fibroblast growth factor-18, which solves the problem of large-scale expression of human fibroblast growth factor-18 fusion protein and small-molecule like ubiquitination modified protease.
The invention also aims at providing a construction method of the prokaryotic expression vector of the human fibroblast growth factor-18.
The invention also provides a preparation method of the human fibroblast growth factor-18.
The invention aims at providing the application of the prokaryotic expression vector of the human fibroblast growth factor-18 in preparing the pharmaceutical grade human fibroblast growth factor-18 in preparing the acute lung injury drugs.
The technical scheme provided by the invention is as follows:
the prokaryotic expression vector of the pharmaceutical grade human fibroblast growth factor-18 can be prepared in a large scale, and comprises an expression vector and a DNA fragment;
wherein the DNA fragment comprises a DNA fragment consisting of a nucleotide sequence shown as SEQ ID NO.9 and encoding a fusion protein containing a human fibroblast growth factor 18 and a DNA fragment consisting of a nucleotide sequence shown as SEQ ID NO.10 and encoding a small molecule like ubiquitination modified protease;
the amino acid sequence of the fusion protein containing the human fibroblast growth factor 18 is shown as SEQ ID No.5, and the amino acid sequence of the small molecule-like ubiquitination modified protease is shown as SEQ ID No. 4.
Preferably, the fusion protein containing the human fibroblast growth factor 18 comprises a green fluorescent protein consisting of an amino acid sequence shown as SEQ ID NO.1, a small molecule ubiquitin-like modifier mature peptide consisting of an amino acid sequence shown as SEQ ID NO.2 and a human fibroblast growth factor-18 consisting of an amino acid sequence shown as SEQ ID NO.3 from the N end to the C end.
Preferably, the expression vectors are pDawn and pBV220.
The construction method of the prokaryotic expression vector capable of preparing pharmaceutical grade human fibroblast growth factor-18 in a large scale comprises the steps of connecting a DNA fragment encoding small molecule-like ubiquitination modified protease to a first expression vector to obtain a first recombinant expression vector, and connecting a DNA fragment encoding fusion protein containing human fibroblast growth factor 18 to the first recombinant expression vector to obtain the prokaryotic expression vector;
wherein, the DNA fragment encoding the small molecule-like ubiquitination modified protease comprises a DNA fragment consisting of a nucleotide sequence shown as SEQ ID NO.10, and the DNA fragment encoding the fusion protein containing the human fibroblast growth factor 18 comprises a DNA fragment consisting of a nucleotide sequence shown as SEQ ID NO. 9; and
the amino acid sequence of the fusion protein containing the human fibroblast growth factor 18 is shown as SEQ ID No.5, and the amino acid sequence of the small molecule-like ubiquitination modified protease is shown as SEQ ID No. 4.
The pharmaceutical grade human fibroblast growth factor-18 can be prepared in large scale, the prokaryotic expression vector of claim 1 is used for transforming into a host, the recombinant organism is obtained, then the recombinant organism is cultured in a culture solution until the recombinant organism expresses a fusion protein comprising the human fibroblast growth factor 18 and a small molecular ubiquitin-like modified protease, and the pharmaceutical grade human fibroblast growth factor-18 can be prepared in large scale after purification.
The preparation method of pharmaceutical grade human fibroblast growth factor-18 can be realized in a large scale, the prokaryotic expression vector of claim 1 is used for transforming into a host, the recombinant organism is obtained, then the recombinant organism is cultured in a culture solution until a culture containing fusion protein of human fibroblast growth factor 18 and micromolecular ubiquitin-like modified protease is expressed, and the pharmaceutical grade human fibroblast growth factor-18 can be obtained after purification.
Preferably, the host is E.coli.
Preferably, the strain of E.coli is BL21 (DE 3) pLysS.
Preferably, the culture conditions in the culture medium after obtaining the recombinant organism include: the growth temperature is 30-37 ℃, the induction temperature is 42 ℃, and the time is 4-8 hours; the recombinant organisms were then incubated at a temperature of 16 to 24℃for a further 60-120 minutes while blue light radiation at a wavelength of 470 nm was turned on at a light intensity of 50-100 nanowatts.
The application of the pharmaceutical grade human fibroblast growth factor-18 in preparing acute lung injury medicines can be realized in a large scale.
The beneficial effects of the invention are as follows:
the FGF18 protein can be obtained by cutting the fusion protein by protease without using chemical substances (IPTG) for induction and separation and purification, and the steps of separating and purifying recombinant protein in the production process are reduced, so that the loss of target protein is reduced, and the production cost is reduced.
Drawings
FIG. 1 is a schematic diagram of a pL-GS-FGF18-pDawn-SE recombinant plasmid according to the present invention.
FIG. 2 is a graph showing the expression of a fusion protein and the hydrolysis analysis of the fusion protein according to the present invention. ( The figure illustrates: lane M represents a protein molecular weight marker; lane 1 shows the total protein pL-GS-FGF18-pDawn-SE/BL21 prior to induction; lanes 2 and 10 show the total pBV-GFP-SUMO/BL21 protein after heat induction; lane 3 shows the total protein pL-GS-FGF18-pDawn-SE/BL21 for 15 min of photoinduction; lane 4 shows the total pL-GS-FGF18-pDawn-SE/BL21 protein light-induced for 30 min; lane 5 shows the total pL-GS-FGF18-pDawn-SE/BL21 protein photoinduction for 45 min; lane 6 shows the total protein pL-GS-FGF18-pDawn-SE/BL21 for 60 minutes of photoinduction; lane 7 shows the total protein pL-GS-FGF18-pDawn-SE/BL21 photoinduction for 90 min; lane 8 shows the total protein pL-GS-FGF18-pDawn-SE/BL21 for 120 minutes of photoinduction; lane 9 shows pL-GS-FGF18-pDawn-SE/BL21 total protein thermally induced for 4 hours; a fusion protein comprising fibroblast growth factor 18, indicated by the first arrow; hydrolyzing the fusion protein shown by the second arrow to obtain a fusion protein fragment of the green fluorescent protein and the small-molecule ubiquitin-like modifier mature peptide; the third arrow shows the human fibroblast growth factor 18 obtained after hydrolysis of the fusion protein. )
Fig. 3 is a schematic diagram of an animal experiment design according to the present invention.
FIG. 4 is a hematoxylin-eosin staining chart of lung tissue of mice according to the present invention.
FIG. 5a is a graph showing the statistics of expression of the target protein of the present invention in mice.
FIG. 5b is a graph showing the expression statistics of the target protein in mice according to the present invention.
FIG. 6a is a fluorescence image of the expression of the target protein in mice according to the present invention.
FIG. 6b is a graph showing the fluorescence statistics of ICAM-1 protein according to the present invention expressed in mice.
FIG. 6c is a graph showing the fluorescence statistics of VCAM-1 protein of the present invention expressed in mice.
FIG. 7a is a graph showing the analysis of the expression of the target protein in human umbilical vein endothelial cells.
FIG. 7b is a graph showing the statistics of the expression of the target protein in human umbilical vein endothelial cells.
FIG. 8a is a fluorescent image of ICAM-1 protein expressed in endothelial cells of human umbilical vein according to the present invention.
FIG. 8b is a graph showing fluorescence statistics of ICAM-1 protein of the present invention expressed in endothelial cells of human umbilical vein.
FIG. 9a is a fluorescent image of VCAM-1 protein expressed in human umbilical vein endothelial cells according to the present invention.
FIG. 9b is a graph showing the fluorescence statistics of VCAM-1 protein of the present invention expressed in human umbilical vein endothelial cells.
FIG. 10a is a graph showing in vivo expression analysis of the target protein of the present invention in mice.
FIG. 10b is a graph showing the expression statistics of the target protein in mice according to the present invention.
FIG. 10c is a graph showing the expression statistics of the target protein in mice according to the present invention.
FIG. 11a is a graph showing the analysis of the expression of the target protein in endothelial cells of umbilical vein of human.
FIG. 11b is a graph showing the statistics of the expression of the target protein in human umbilical vein endothelial cells.
FIG. 11c is a graph showing the statistics of the expression of the target protein in human umbilical vein endothelial cells.
FIG. 12 is a fluorescent image showing the expression of the p65 protein in the nucleus of endothelial cells of umbilical vein of human.
Detailed Description
The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.
Reagents, enzymes, etc., in embodiments of the invention are commercially available unless otherwise specified.
The E.coli pBV-220 expression vector (cat# MCV 031) was purchased from Beijing Ding Guo prosperous biotechnology limited liability company; coli BL21 (DE 3) pLysS (cat# 69451-M) was purchased from Sigma-Aldrich; the E.coli pDawn expression plasmid (cat# 43796) was purchased from Addgene, inc. of U.S.A.
Example 1
Construction of prokaryotic expression vector of human fibroblast growth factor-18 fusion protein
A DNA fragment (shown as a nucleotide sequence shown as SEQ ID No. 9) consisting of a DNA fragment (shown as a nucleotide sequence shown as SEQ ID No. 6) encoding green fluorescent protein, a DNA fragment (shown as a nucleotide sequence shown as SEQ ID No. 7) encoding small-molecule ubiquitin-like modifier mature peptide and a DNA fragment (shown as a nucleotide sequence shown as SEQ ID No. 8) encoding human fibroblast growth factor-18 is subjected to total gene synthesis by Nanjing Jinsri biotechnology limited company, and the 5 'end of the gene fragment is provided with an EcoRI cleavage site and a protective base consisting of 3 random nucleotides, and the 3' end is provided with a BamHI site and a protective base consisting of 3 random nucleotides (shown as a nucleotide sequence shown as SEQ ID No. 11).
A standard 100 μl cleavage system was established: 1 microgram of DNA fragment encoding fusion protein GFP-SUMO-FGF18 (shown as nucleotide sequence shown in SEQ ID No. 9), 10 microliter of digestion buffer (200 mM Tris-Cl,100M MgCl) 2 10mM dithiothreitol, 1000mM KCl), 3 microliters BamH I (Bao Ri doctor materials technology (Beijing), co., ltd., cat# 1010S), 3 microliters EcoR I (Bao Ri doctor materials technology (Beijing), co., ltd., cat# 1040S) and supplemented with sterile water to 100 microliters. After being placed in a water bath at 30 ℃ for 1 hour, the DNA fragment purification kit (Bao Ri doctor materials technology (Beijing) Co., ltd., product No. 9761) was used to recover the digested fragments for use. The vector pBV220 was expressed by double cleavage (BamH I and EcoR I) using the same method and system, and then the fragment of the large fragment of the cleaved vector was recovered for use using the DNA fragment purification kit.
A standard 20 μl carrier ligation system was established: 100 nanograms of the double-digested large carrier fragment, 300 nanograms of the double-digested DNA fragment encoding the fusion protein GFP-SUMO-FGF18, 10 microliters of Solution I (DNA ligation kit, bao Ri doctor technology (Beijing) Co., ltd., cat# 6022Q), and after mixing, all were transferred to 100 microliters of E.coli DH5 alpha competent cells (Bao Ri doctor technology (Beijing) Co., ltd., cat# 9057), heat-shocked at 42℃for 60 seconds, and then coated on agarose plates containing ampicillin resistance at a final concentration of 100 micrograms/ml for screening. The positive clones were inoculated into fresh LB medium containing ampicillin resistance at a final concentration of 100. Mu.g/ml, and the recombinant plasmids were extracted using a plasmid extraction kit (Bao Ri doctor Material technology (Beijing) Co., ltd., cat. No. 9760) and sent to the Shanghai stock Co., ltd. For sequencing. The correct nucleic acid sequence was detected by nucleic acid sequencing and the recombinant plasmid containing the nucleic acid encoding the fusion protein GFP-SUMO-FGF18 was designated pBV-GFP-SUMO-FGF18. The recombinant plasmid is extracted, an enzyme digestion system is established according to the method, and a Bgl II (Bao Ri doctor technology (Beijing) Co., ltd., product No. 1021A) and BamH I are utilized to double enzyme-cut the pBV-GFP-SUMO-FGF18 recombinant plasmid to obtain a DNA fragment containing pL promoter, nucleic acid encoding green fluorescent protein, small molecule sample ubiquitination modified protein mature peptide and human fibroblast growth factor 18 and rrn B T1 terminator expression cassette. Then, the DNA fragment of the expression cassette was blunt-ended by filling in the ends of the cleavage sites with Klenow polymerase (Takara Shuzo Co., ltd., product No. 2140A). The nucleic acid fragment was used for backup (designated as recombinant fragment A) using the DNA fragment purification kit.
Example 2
Construction of prokaryotic expression vector of small ubiquitin-like modifier protease SUMOase
The DNA fragment (shown as a nucleotide sequence shown as SEQ ID No. 10) for encoding small molecular ubiquitination modified protease SUMOase is subjected to total gene synthesis by Nanjing Jinsri biotechnology limited company, and is provided with an Nde I restriction site and a protective base consisting of 3 random nucleotides at the 5 'end of the gene fragment, and is provided with an Xho I site and a protective base consisting of 3 random nucleotides at the 3' end (shown as a nucleotide sequence shown as SEQ ID No. 12).
Referring to the above-mentioned genetic engineering construction method, a DNA fragment encoding the small ubiquitin-like modifier protease SUMOase and the E.coli expression plasmid vector pDAWn are digested with two enzymes (Nde I, bao Ri doctor materials technology (Beijing) Co., ltd., product No. 1161A; xho I, bao Ri doctor materials technology (Beijing)) Co., product No. 1094A, and the nucleic acid fragment is recovered by using a DNA fragment purification kit. A standard recombinant vector ligation system was established and transformed into E.coli DH5a competence, and recombinants were screened using a final concentration of 50. Mu.g/ml of calicheamicin resistant LB plates. Positive clones were sent to the biological engineering (Shanghai) Co.Ltd for sequencing and the recombinant plasmid with correct sequencing was designated pDAWN-SE.
Example 3
Construction of recombinant expression vector for large-scale production of human fibroblast growth factor-18
A standard 100 μl volume single cleavage system was established: 1. Mu.g of pDAWN-SE recombinant plasmid, 10. Mu.l of digestion buffer (330 mM Tris-Ac,100M MgAc,5mM dithiothreitol, 660mM KAc,0.1% BSA), 5. Mu.l of Acl I (Takara Shuzo Co., ltd., product No. 1108A) were supplemented with sterile water to 100. Mu.l. After being placed in a water bath at 37 ℃ for 1 hour, the DNA fragment purification kit recovers the digested fragments. Subsequently, the ends of the cleavage sites were filled in with Klenow polymerase (Bao Ri doctor Material technology (Beijing), inc., cat. No. 2140A) to form blunt ends. The nucleic acid fragment was used for backup (designated as recombinant fragment B) using the DNA fragment purification kit.
A standard vector ligation system was established as described in example 1, ligating recombinant fragment A (300 nanograms) and recombinant fragment B (100 nanograms) into new recombinant vectors and transforming into E.coli DH5 a. After calicheamicin resistance plate screening and nucleic acid sequencing detection, the recombinant plasmid with correct sequence is named as a prokaryotic expression vector of human fibroblast growth factor-18, namely pL-GS-FGF18-pDawn-SE (shown in figure 1).
Example 4
Obtaining of recombinant strains
The recombinant plasmid pL-GS-FGF18-pDawn-SE is extracted by using a plasmid extraction kit, then transformed into an escherichia coli host BL21 (DE 3) pLysS according to the method of example 1, positive recombinant bacteria are screened by using resistance of calicheamicin, the positive recombinant bacteria are named as pL-GS-FGF18-pDawn-SE/BL21 (comprising an amino acid sequence shown as SEQ ID No.4 and an amino acid sequence shown as SEQ ID No. 5), and green fluorescent protein (shown as an amino acid sequence shown as SEQ ID No. 1), a small molecule ubiquitin-like modifier mature peptide (shown as an amino acid sequence shown as SEQ ID No. 2) and a human fibroblast growth factor-18 (shown as an amino acid sequence shown as SEQ ID No. 3) are sequentially arranged from the N end to the C end.
Example 5
Induction expression of recombinant strains
pL-GS-FGF18-pDawn-SE/BL21 was inoculated at an inoculum size of 0.1% (v/v) into 50 ml of fresh liquid LB medium (peptone 10 g/l, yeast extract 5 g/l, sodium chloride 10 g/l, ampicillin 100 mg/l, pH=7.0), 220 rpm, and cultured with shaking at 37℃for 14 hours. Then, the culture was inoculated into 1000 ml of fresh liquid LB medium at an inoculum size of 5% (v/v), 220 rpm, and shaking-cultured at 37 ℃ for about 2-3 hours until the OD600 = 1.0, and then the culture temperature was adjusted up to 42 ℃,160 rpm for 4 hours. Subsequently, the culture temperature was gradually lowered to 16℃with circulating water while turning on a blue light source (470 nm wavelength, 100 nanowatts) and irradiated for 120 minutes. The recombinant cells were collected by centrifugation at 12000 rpm, and resuspended in phosphate buffer (137 mmol/l sodium chloride, 2.7 mmol/l potassium chloride, 10 mmol/l sodium dihydrogen phosphate, 2 mmol/l potassium dihydrogen phosphate, 0.2% NP-40, ph=7.0) to give a bacterial suspension. The bacterial suspension was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sodium-dodecylsulfatepolyacrylamide gel electrophoresis, SDS-PAGE) for detection. For a specific method of operation of SDS-PAGE reference is made to Sambrook et al, molecular Cloning: a Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, NY,1989.
Comparative example 1
Construction of prokaryotic expression vector of fusion protein GFP-SUMO
A DNA fragment (shown as a nucleotide sequence shown as SEQ ID No. 15) consisting of a DNA fragment (shown as a nucleotide sequence shown as SEQ ID No. 6) encoding green fluorescent protein and a DNA fragment (shown as a nucleotide sequence shown as SEQ ID No. 7) encoding a small-molecule ubiquitin-like modifier mature peptide is subjected to total gene synthesis by Nanjing Jinsrui biotechnology limited company, and the 5 'end of the gene fragment is provided with an EcoR I restriction site and a protective base consisting of 3 random nucleotides, and the 3' end is provided with a BamH I site and a protective base consisting of 3 random nucleotides (shown as a nucleotide sequence shown as SEQ ID No. 14).
The synthesized nucleic acid fragment was then cloned into E.coli expression plasmid vector pBV220, as in example 1, to give a recombinant plasmid, which was transformed into E.coli. The correctness of the nucleic acid sequence is detected by nucleic acid sequencing. The recombinant plasmid containing the nucleic acid encoding GFP-SUMO was designated pBV-GFP-SUMO.
Comparative example 2
Obtaining of recombinant strains
The recombinant plasmid pBV-GFP-SUMO was transformed into E.coli host BL21 (DE 3) pLysS as in examples 3 and 4, and positive recombinant bacteria were selected by ampicillin resistance and designated pBV-GFP-SUMO/BL21 (shown in the amino acid sequence of SEQ ID No. 13), respectively, as a control strain for the expression of the protein pBV-GFP-SUMO-FGF18.
Comparative example 3
Induction expression of recombinant strains
pBV-GFP-SUMO/BL21 was inoculated into 50 ml of fresh liquid LB medium (peptone 10 g/l, yeast extract 5 g/l, sodium chloride 10 g/l, ampicillin 100 mg/l, pH=7.0), 220 rpm, and cultured with shaking at 37℃for 14 hours at an inoculum size of 0.1% (v/v). Then, the culture was inoculated into 1000 ml of fresh liquid LB medium at an inoculum size of 5% (v/v), 220 rpm, and shaking-cultured at 37 ℃ for about 2-3 hours until the OD600 = 1.0, and then the culture temperature was adjusted up to 42 ℃,160 rpm for 4 hours. Subsequently, the culture temperature was gradually lowered to 16℃with circulating water while turning on a blue light source (470 nm wavelength, 100 nanowatts) and irradiated for 120 minutes. The recombinant cells were collected by centrifugation at 12000 rpm, and resuspended in phosphate buffer (137 mmol/l sodium chloride, 2.7 mmol/l potassium chloride, 10 mmol/l sodium dihydrogen phosphate, 2 mmol/l potassium dihydrogen phosphate, 0.2% NP-40, ph=7.0) to give a bacterial suspension. The bacterial suspension was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sodium-dodecylsulfatepolyacrylamide gel electrophoresis, SDS-PAGE) for detection. For a specific method of operation of SDS-PAGE reference is made to Sambrook et al, molecular Cloning: a Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, NY,1989.
Test example 1
As shown in FIG. 2, the results showed that the recombinant strain pL-GS-FGF18-pDAWN-SE/BL21 expressed a distinct band of recombinant protein GFP-SUMO-FGF18 with a molecular weight of about 56 kilodaltons (kDa) (lane 9) after 4 hours of heat shock culture at 42℃as compared with the pre-induction control group (lane 1), consistent with the expectations. The expression of the SUMOase protease was then activated by light-induced methods, and over time, the recombinant protein GFP-SUMO-FGF18 was hydrolyzed to FGF18 and GFP-SUMO proteins (lanes 3 through 8). Cleavage of the fusion protein resulted in a GFP-SUMO protein having a molecular weight of about 38 kilodaltons (kDa) and a size identical to the position of the GFP-SUMO band of the recombinant protein produced by the control strain pBV-GFP-SUMO/BL21 (lanes 2 and 10).
Test example 2
Acute Lung Injury (ALI) and its more severe form, acute Respiratory Distress Syndrome (ARDS), is a life threatening disease of a variety of etiologies including sepsis, trauma and viral or bacterial pneumonia. ALI/ARDS is characterized by an acute episode of hypoxia, injury to the capillary-alveolar interface, initiation of pro-inflammatory cytokine and chemokine storms, and infiltration of inflammatory cells. ALI/ARDS is an acute inflammatory syndrome with mortality rates of up to 40% in human patients. ALI/ARDS is characterized by diffuse alveolar damage that can lead to excessive lung inflammation and alveolar endothelial cell damage. Lipopolysaccharide (LPS) is a key component of the outer membrane of gram-negative bacteria and cyanobacteria, and has been widely used to induce cellular immune responses, including over-expression of pro-inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-1 beta (IL-1 beta). LPS-induced inflammatory responses of the body will lead to severe lung dysfunction.
Recent studies have shown that FGF18 plays a critical role in cellular inflammation. However, its mechanism of action in the progression of acute lung injury is not yet clear. As shown in fig. 3 to 6, to verify whether FGF18 has a protective effect in lung injury, mice in the administration group were intraperitoneally injected with recombinant human FGF18 (rh FGF 18) protein (2 mg/kg/day) two weeks prior to molding (fig. 3). After 2 weeks of stable expression in vivo, mice were further subjected to tracheal instillation of LPS (5 mg/kg) for 12 hours. As expected, hematoxylin-eosin (HE) staining showed that FGF 18-dosed groups significantly improved lung injury, with reduced inflammatory cell infiltration compared to control mice (fig. 4). WB results showed that the protein expression of VCAM-1, icam-1, il-6 and TNF- α was significantly reduced in mice of the operating group injected with rh FGF18 (fig. 5a, 5 b). To further verify the important role of FGF18 in ALI, we performed immunofluorescent staining and as a result found that the expression levels of ICAM-1 and VCAM-1 proteins were significantly reduced in lung tissues of mice injected with rh FGF18 protein (fig. 6 a-6 c).
In vitro results also demonstrate that co-culture of rh FGF18 down-regulates protein expression levels of VCAM-1, ICAM-1, IL-6 and TNF- α following LPS injury (FIGS. 7a, 7 b). As expected, the expression of ICAM-1 and VCAM-1 proteins was significantly reduced in endothelial cells in the rh FGF18 treated group (FIGS. 8a, 8b, 9a, 9 b). Taken together, these data indicate that FGF18 can improve the damage status of lung tissue and inhibit inflammatory damage to endothelial cells.
We then further explored the signaling pathways that FGF18 might be involved in protecting against lung injury. NF-. Kappa.B is a family of inducible transcription factors that regulate a large number of genes involved in various processes of immune and inflammatory responses, as is well known. Typically, NF- κ B p65 protein is activated in the early stages of inflammatory injury and activates downstream inflammatory gene expression, thereby transcribing more inflammatory factors and initiating inflammatory storms. Thus, we performed immunoblot detection on treated mice, and found that phosphorylated p65 and phosphorylated iκbα protein expression was significantly reduced (fig. 10 a-10 c). The in vitro results showed that the protein detection of endothelial cells after 6h stimulation with LPS (100 ng/ml) revealed a decrease in both phosphorylated p65 and phosphorylated IκBα protein expression in the co-cultured rh FGF18 group (FIGS. 11 a-11 c), consistent with the in vivo results. To further demonstrate the effect of rh FGF18 on p65 protein, we analyzed the nuclear status of p65 protein using immunofluorescence techniques and found that the nuclear status of p65 protein was significantly reduced in the dosing group (fig. 12). These results indicate that FGF18 may exert lung tissue protective effects by reducing the activity of activation of p65 and ikbα proteins, improving endothelial cell injury.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (10)
1. The prokaryotic expression vector of the pharmaceutical grade human fibroblast growth factor-18 can be prepared in a large scale and is characterized by comprising an expression vector and a DNA fragment;
wherein the DNA fragment comprises a DNA fragment consisting of a nucleotide sequence shown as SEQ ID NO.9 and encoding a fusion protein containing a human fibroblast growth factor 18 and a DNA fragment consisting of a nucleotide sequence shown as SEQ ID NO.10 and encoding a small molecule like ubiquitination modified protease;
the amino acid sequence of the fusion protein containing the human fibroblast growth factor 18 is shown as SEQ ID NO.5, and the amino acid sequence of the small molecule-like ubiquitination modified protease is shown as SEQ ID NO. 4.
2. The prokaryotic expression vector for preparing pharmaceutical-grade human fibroblast growth factor-18 on a large scale according to claim 1, wherein the fusion protein containing the human fibroblast growth factor 18 comprises a green fluorescent protein consisting of an amino acid sequence shown in SEQ ID NO.1, a small molecule ubiquitin-like modifier mature peptide consisting of an amino acid sequence shown in SEQ ID NO.2 and the human fibroblast growth factor-18 consisting of an amino acid sequence shown in SEQ ID NO.3 from N end to C end.
3. The prokaryotic expression vector for the large-scale production of pharmaceutical grade human fibroblast growth factor-18 according to claim 2, wherein said expression vector is pDawn and pBV220.
4. The construction method of the prokaryotic expression vector capable of preparing pharmaceutical grade human fibroblast growth factor-18 on a large scale is characterized in that a DNA fragment encoding small molecular ubiquitin-like modified protease is connected to a first expression vector to obtain a first recombinant expression vector, and a DNA fragment encoding fusion protein containing human fibroblast growth factor 18 is connected to the first recombinant expression vector to obtain the prokaryotic expression vector;
wherein, the DNA fragment encoding the small molecule-like ubiquitination modified protease comprises a DNA fragment consisting of a nucleotide sequence shown as SEQ ID NO.10, and the DNA fragment encoding the fusion protein containing the human fibroblast growth factor 18 comprises a DNA fragment consisting of a nucleotide sequence shown as SEQ ID NO. 9; and
the amino acid sequence of the fusion protein containing the human fibroblast growth factor 18 is shown as SEQ ID No.5, and the amino acid sequence of the small molecule-like ubiquitination modified protease is shown as SEQ ID No. 4.
5. The pharmaceutical grade human fibroblast growth factor-18 can be prepared in a large scale, and is characterized in that the prokaryotic expression vector of claim 1 is used for being transformed into a host, the recombinant organism is obtained, and then the recombinant organism is cultured in a culture solution until a culture containing fusion protein of human fibroblast growth factor 18 and micromolecular ubiquitin-like modified protease is expressed, and the pharmaceutical grade human fibroblast growth factor-18 can be prepared in a large scale after purification.
6. The preparation method of pharmaceutical grade human fibroblast growth factor-18 is characterized in that the prokaryotic expression vector of claim 1 is used for transforming into a host, the recombinant organism is obtained, then the recombinant organism is cultured in a culture solution until the recombinant organism expresses a culture comprising fusion protein of human fibroblast growth factor 18 and micromolecular ubiquitination modified protease, and the pharmaceutical grade human fibroblast growth factor-18 is obtained after purification.
7. The method for the preparation of pharmaceutical grade human fibroblast growth factor-18 on a large scale according to claim 6, wherein the host is E.coli.
8. The method for preparing pharmaceutical grade human fibroblast growth factor-18 on a large scale according to claim 6, wherein the E.coli strain is BL21 (DE 3) pLysS.
9. The method for the preparation of pharmaceutical grade human fibroblast growth factor-18 on a large scale according to claim 6, wherein the culture conditions in the culture solution after obtaining the recombinant organism include: the growth temperature is 30-37 ℃, the induction temperature is 42 ℃, and the time is 4-8 hours; the recombinant organisms were then incubated at a temperature of 16 to 24℃for a further 60-120 minutes while blue light radiation at a wavelength of 470 nm was turned on at a light intensity of 50-100 nanowatts.
10. Use of human fibroblast growth factor-18 of pharmaceutical grade in the manufacture of a medicament for acute lung injury, which can be prepared on a large scale according to claim 5.
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