WO2017101221A1 - 重组人成纤维细胞生长因子-17的生产方法 - Google Patents
重组人成纤维细胞生长因子-17的生产方法 Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
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- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Definitions
- the invention relates to the field of genetic engineering, and in particular to a method for producing recombinant human fibroblast growth factor-17.
- Fibroblast Growth Factor is a kind of polypeptide cell growth factor widely distributed in various tissues. Currently, 23 members have been found with molecular weights ranging from 16kD to 34kD, and their central regions contain high homology ( Approximately 120 amino acid sequences of 30% to 70%) have broad biological activity on various types of cells derived from mesoderm and neuroectoderm. FGFs are very small but widely distributed in the body, and each factor regulates a series of developmental processes, including brain development, limb differentiation, and trunk formation. Most members of FGFs are closely related to embryonic development, tissue damage repair, tumor occurrence, development and metastasis, and have considerable application value.
- FGF-17 belongs to the FGF8 subfamily (FGF8, FGF17, FGF18) and belongs to the paracrine factor as well as other FGF members. In prostate cancer and liver cancer, FGF17 is also involved in autocrine. It plays an important role in the formation of tissues and organs during embryonic development.
- Japanese scientist Masamitsu Hoshikawa et al. isolated a cDNA encoding 216 amino acids of FGF17 from rat embryos and efficiently obtained rat FGF17 using High Five insect cells transfected with cDNA-containing baculovirus. protein.
- FGF17mRAN is preferentially expressed in the ischemic brain tissue of the rat embryonic brain tissue and the septum.
- FGF17 is essential for the development of embryonic brain tissue.
- Human FGF17 has two subtypes, FGF17a and b, b are the main forms, and human FGF17 to be expressed in this experiment is also FGF17b.
- the human FGF17 gene is located on chromosome 8p21.3, with three exons and six mutation sites, consisting of 216 amino acids, with 30% to 70% homology with other found family members.
- FGF-17 shares 93% high homology with the amino acid sequence of the mouse.
- the molecular weight of human FGF17 is about 25kD. In this experiment, the signal peptide was excised and only the mature protein partial sequence was obtained.
- the final protein had a molecular weight of 22.6kD and an isoelectric point of 10.43, which was a basic protein.
- FGF17 is similar to FGF8 and FGF18 and belongs to the same family member. Its homology is 60% and 50%, respectively. Both have signal peptide sequences, which need to be excised when expressed in E. coli.
- FGF17 is of great significance for the normal development of the brain tissue nervous system. FGF17 is a key factor in the normal development of brain tissue structure, and its expression partially overlaps with FGF8 in the same family. Both play important roles in the MHB region (mid-hindbrain, mid-brain) and cortex. Cholfin et al. found that FGF17 is an important part of the mouse frontal cortex. Knocking out FGF17 resulted in a reduction in the dorsal frontal cortex and a reduction in the degree of projection of the frontal cortex to the subcortical target site; Jingsong Xu et al. used FGF17 knockout mice as a model to find either young or adult mice two days after birth.
- FGF17 has potential value in the study of neuropsychiatric diseases.
- the location of FGF17 on the chromosome is involved.
- a 2009 report reviewed chromosome 8p as a neuropsychiatric susceptibility locus, with neuropsychiatric disorders (schizophrenia, autism), neurodegenerative diseases (Alzheimer's disease, Parkinson's disease), Carl Mann syndrome is closely related; 8p21-23 is a key site for cerebral dysplasia, which is closely related to cognitive deficits, convulsions, autism, and schizophrenia.
- the FGF17 gene is located on chromosome 8p21.3 and is of great significance for the study of the above conditions. Zanni et al.
- FGF17 The expression level of FGF17 was significantly decreased, which was the first report on human cerebellar malformation and FGF17 gene transcription down-regulation; Scearce-Levie and other studies found that FGF17 -/- mice had abnormal social behavior, which showed that the vocalization of young rats was reduced, and social interaction defects in adult rats And found that in the human body, interference with the FGF17 signaling pathway may lead to a series of neuropsychiatric diseases such as schizophrenia and autism, and these abnormal behaviors may be due to the reduction of Fos gene activation in the frontal cortex structure of FGF17 deletion. However, the related mechanisms of the neural circulation and signaling pathways are still poorly understood and need further investigation.
- FGF17 has important value in bone research. May be related to the action of FGF17 on the receptor FGFR3.
- FGFR3 plays an important role in skeletal development and is the main receptor expressed in chondrocytes. Its activating mutations lead to a series of skeletal development-deficient diseases such as achondroplasia (ACH), rib dysplasia (HCH) and lethality. Bone dysplasia (TD), persistent activation of FGFR3 in bone cells leads to dwarfism. Krejci P et al. use immunohistochemical method The expression of FGF17 protein was detected in normal human growth plate cartilage, and the level was consistent with FGF19.
- FGF17 and FGF1 were found to inhibit the proliferation of chondrocytes of RCS (rat chondroblast chondrocytes).
- RCS rat chondroblast chondrocytes
- FGF17 significantly inhibited the proliferation of RCS by combination with heparin, while the inhibitory effect of FGF17 alone was not obvious.
- FGF17 could induce phosphorylation of ERK signaling pathway. Therefore, FGF17 is involved in the activation of FGFR3 signaling pathway in chondrocytes, which induces phosphorylation of ERK and inhibits the growth of chondroma cells.
- FGF17 is also a potential carcinogen.
- FGF17 specifically binds to the tyrosine kinase receptors FGFR3c, FGFR4, FGFR2c and FGFR1c on the cell surface (FGFR3c>FGFR4>FGFR2c>FGFR1c), initiates DNA synthesis and promotes cell proliferation through the MAPK pathway, and promotes DNA synthesis.
- FGF17 induced mitosis twice as much as FGF8; Jingsong Xu et al.
- FGF17a and FGF17b (the main expression form of human FGF17) gene into NIH3T3 cells, and after one week, cells containing FGF17b were 40%. Tumorization occurred. After two weeks, all cells formed tumors, and cells containing FGF17a remained normal.
- Polnaszek and Heer detected that FGF17 was overexpressed in prostate cancer cells, especially in DU145 cell line. Gao, Heer et al. also found that FGF17 mRNA is up-regulated at a level four times that of BPH (benign prostatic hyperplasia) in highly differentiated prostate cancer cells. Polnaszek et al.
- FGF17 is generally high in tumor tissue concentration.
- the expression level is lower than that of FGF7 (FGF7 is 28 ng/g, FGF17 is 0.4-0.5 ng/g); for breast cancer, there is still not enough evidence to prove that FGF17 is expressed in breast cancer cells, but Meijer et al.
- the use of tamoxifen in the treatment of breast cancer found that the receptor FGFR4 and its ligand FGF17 signaling caused tamoxifen resistance. The discovery of this pathway provides guidance for targeted therapy in patients with endocrine therapy for breast cancer. Gauglhofer et al.
- FGF8 family including FGF17 is up-regulated, and this phenomenon may be related to the down-regulation of wnt pathway.
- the expression of the FGF8 family was significantly increased.
- the apoptosis of the hepatocellular carcinoma cells was reduced.
- FGF17 is currently known to be closely related to tumors, so it has broad prospects as a target molecule for tumor diagnosis and treatment.
- FGF-17 has important significance for brain tissue, nervous system, bones and tumors, its research and clinical application prospects are broad, and it is receiving more and more attention from the scientific research community. As a new growth factor, FGF-17 has relatively few reports at home and abroad.
- the patent of the present application obtains a high-efficiency expression strain of Escherichia coli by gene optimization, expression vector and host strain screening; optimize culture conditions and bacterial liquid breaking method to increase the ratio of soluble protein; optimize inclusion body washing, denaturation and complex method, to obtain a higher concentration and purity of the inclusion body protein preferably; cation exchange chromatography (HiTrap TM SP HP), a heparin affinity chromatography column (heparin Sepharose CL-6B), to obtain the final protein purity of more than 95%
- the recombinant human FGF-17 protein with high biological activity lays a foundation for further research on the mechanism of action of FGF-17 and research and development of new drugs.
- FGF17 protein expression is low, and it is difficult to prepare a protein with high activity. These have constituted bottlenecks in basic research and clinical applications of FGF18.
- a cell lysis buffer having a pH of 7.5 is usually used, and the temperature at the time of culture is not strictly controlled, thereby resulting in a final culture purity and concentration which are not sufficiently high.
- the present invention constructs a FGF-17 cell expression system using E. coli expression plasmid as a vector, and mainly improves the culture method and purification method thereof, and optimizes the culture temperature.
- one of the objects of the present invention is to provide a method for producing recombinant human fibroblast growth factor-17 protein, which solves the problem of low concentration and purity of FGF-17 protein production;
- Another object of the present invention is to provide a method for culturing a protein of both the soluble FGF-17 protein and the inclusion body FGF-17 protein.
- a method for producing recombinant human fibroblast growth factor-17 protein comprises at least the following steps: Step 1: Recombining and amplifying a native sequence of human fibroblast growth factor-17 protein FGF-17 according to E. coli preference, The recombinant human fibroblast growth factor-17 protein rhFGF-17 gene is obtained, and the base sequence thereof is shown in SEQ ID NO: 1.
- Step 2 ligating the recombinant rhFGF-17 gene into an E. coli expression plasmid to form a recombinant expression vector, and then transforming the recombinant expression vector into an E. coli host strain to obtain an FGF-17 protein expression engineering strain;
- Step 3 cultivating the expression engineering bacteria, followed by purification
- the cell lysis buffer of the following concentration components is used: 45-55 mM Tris, 280-320 mM sodium chloride NaCl, 1.5-2.5 mM ethylenediaminetetraacetic acid DTA 0.1 to 0.3 M sucrose, and 0.8 to 1.2% by volume of polyethylene glycol octylphenyl ether Triton X-100, 0.1 to 0.3% sodium deoxycholate, 5% to 10% glycerol, It is 0.8 to 1.2% of phenylmethylsulfonyl fluoride PMSF; and the pH of the cell lysis buffer is adjusted to be between 7.3 and 7.49.
- the culturing the expression engineering bacteria comprises: culturing the soluble FGF-17 protein at 16 ° C, and at 37 ° C The inclusion body FGF-17 protein was cultured under the conditions.
- the E. coli expression plasmid is pET3a
- the E. coli host strain is BL21(DE3)plysS.
- the cell lysis buffer comprises: 50 mM Tris, 300 mM sodium chloride, 2 mM. Ethylenediaminetetraacetic acid DTA, 0.2 M sucrose, and 1% by volume of polyethylene glycol octylphenyl ether Triton X-100, 0.2% sodium deoxycholate, 5%-10% glycerol Is 1% phenylmethylsulfonyl fluoride PMSF; and the pH of the cell lysis buffer is adjusted to 7.4.
- the culturing the expression engineering bacteria comprises the following steps:
- the FGF-17 protein expression engineering bacteria were inoculated to the first generation LB medium at a ratio of 1:50-100 to obtain the first generation bacteria; the first generation bacteria were cultured for 8h to 12h, and the bacteria concentration A 600 reached 1.2. When it is about 2.0, it is inoculated into a second-generation LB medium containing phosphate buffer at a ratio of 1:100 to 200;
- the temperature is 16 ° C, and the time 16 to 30 h; and if the inclusion body FGF-17 protein is obtained, in the step 2), the temperature is 37 ° C, and the time is 4 to 6 h.
- the obtained FGF-17 protein is frozen at -20 °C.
- the first generation medium comprises: tryptone 10 g/L, and yeast powder 5 g/L. And sodium chloride 10g / L;
- the second generation medium includes: tryptone 10g/L, yeast powder 10g/L, dipotassium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 1g/L, sodium chloride 4g/ L, and glucose 5g / L;
- the second generation medium includes: tryptone 10g/L, yeast powder 4g/L, dipotassium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 1g/L, and sodium chloride. 10g/L.
- the inclusion body FGF-17 protein when the inclusion body FGF-17 protein is cultured, only the heparin column is used for purification without using an ion exchange column.
- a soluble FGF-17 protein is obtained, a 25-fold g/V cell lysis buffer is added, wherein the glycerol is 5% full. Suspension, then low-temperature ultrasonic disruption, wherein the power is 300w, the vibration frequency output is 40%; then the ultrasound is stopped for 5s for 5s, total ultrasound for 10mins;
- the invention constructs a method for producing FGF-17 by using FGF-17 E. coli host cell with Escherichia coli expression plasmid as a carrier, mainly optimizes the concentration component of the cell lysis buffer, and optimizes the culture temperature. Ultimately, the purity and concentration of the product are greatly improved.
- the invention also optimizes and stipulates a plurality of parameters of the culture process and the purification process, and finally obtains high-concentration and high-purity soluble proteins and inclusion body proteins.
- FIG. 1 is a structural diagram of an E. coli expression plasmid PET-3a according to an embodiment of the present invention, wherein an underline is an enzyme cleavage site;
- FIG. 2 is an electrophoresis analysis diagram showing the results of inducing expression of FGF-17 protein by SDS-PAGE, wherein reference numeral 1 is a protein standard marker and reference numeral 2-5 is at 37 ° C according to an embodiment of the present invention. Results after induction of expression under the conditions, reference numerals 6-9 are results after induction of expression at 16 ° C, and reference numeral 10 is the result before induction;
- Figure 3 is a view showing the solubility of an expression product of FGF-17 protein obtained at different temperatures according to an embodiment of the present invention.
- a SDS-PAGE electrophoresis analysis chart wherein reference numeral 1 is a protein standard marker; reference numeral 2 is a result of inducing expression at 37 ° C; and reference numeral 3 is an induced expression at 37 ° C.
- the reference numeral 4 is the result of the supernatant portion after the crushing at 37 ° C
- the reference numeral 5 is the expression after the induction at 37 ° C, and after purification
- the result of the supernatant fraction after the crushing is the result of the precipitated portion after the induction was induced at 16 ° C and after the purification
- the reference numeral 7 is the result of the induced expression at 16 ° C. .
- reference numeral 4 is a SDS-PAGE electrophoresis analysis diagram of denaturation and renaturation of inclusion body FGF-17 protein according to an embodiment of the present invention, wherein reference numeral 1 is a protein standard marker; and reference numeral 2 is a denaturation treatment upon purification.
- the result of the supernatant portion; the reference numeral 3 is the result of the precipitated portion subjected to the denaturation treatment at the time of purification; and the reference numeral 4 is the result of the supernatant portion at the time of renaturation after denaturation.
- reference numeral 1 is a protein standard marker
- reference numeral 2 is a result of penetrating the affinity column
- Reference numerals 3-7 are the results of elution of different concentrations of sodium chloride
- reference numeral 1 is a protein standard marker
- reference numeral 2 is a supernatant portion after fragmentation after purification.
- Results; reference numerals 3-5 are the results of elution of different concentrations of sodium chloride;
- reference numeral 7 is a SDS-PAGE electrophoresis analysis diagram of a soluble affinity ion exchange column chromatography according to an embodiment of the present invention, wherein reference numeral 1 is a protein standard marker; and reference numeral 2 is an affinity column elution peak. Results; reference numerals 3-5 are the results of elution of different concentrations of sodium chloride;
- Figure 8 is a graph showing the proliferative biological activity of FGF-17 protein against NIH 3T3 cells by MTT assay according to an embodiment of the present invention, wherein the uppermost two are produced by the FGF-17 protein obtained according to the production method of the present invention. Value-added activity, which is much greater than the value-added activity produced by the standard.
- DNA molecule refers to a polymeric form of deoxyribonucleotides (thymine, cytosine, adenine or guanine) that is a major component of the chromosome and a material for the gene. This term refers only to the primary and secondary structure of the molecule and does not limit any specific tertiary form. This term includes double-stranded DNA found especially in linear DNA molecules, viruses, chromosomes, plasmids. The structure discussed here, according to the customary given, is only the 5' of the DNA justice chain. Sequence of 3' directions.
- Vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked, one type of vector being a "plasmid” and a plasmid being a circular double stranded DNA loop to which other DNA fragments can be ligated.
- Another type of vector is a viral vector that can join other DNA fragments to the viral genome.
- Certain vectors integrate into the host cell genome and are replicated along with the host genome.
- certain vectors are capable of directing the expression of a gene to which they are operably linked, and such expression vectors generally used are in the form of plasmids.
- "plasmid” and “vector” can be used interchangeably.
- Recombinant vector refers to an expression vector to which a gene has been ligated.
- “recombinant plasmid” and “recombinant vector” can be used interchangeably.
- host includes not only prokaryotes, but also eukaryotes such as yeast, plant and animal cells.
- Reverse complementation in the present invention refers to a nucleotide sequence associated by the principle of base pairing.
- sequence “5'-A-T-G-3'” is inversely complementary to the sequence "5'-C-A-T-3'".
- Primer also known as the primer. Is a small piece of single-stranded DNA or RNA, as a starting point for DNA replication, as a starting point for the extension of each polynucleotide strand during nucleic acid synthesis, on the 3'-OH of the primer
- the nucleotides are synthesized as a diester chain, so the 3'-OH of the primer must be free.
- Primers are needed because DNA polymerase can only add new nucleotides to an existing DNA strand in DNA synthesis. Primers are artificially synthesized two-stage oligonucleotide sequences, one primer complementary to one DNA template strand at one end of the region of interest and the other primer complementary to another DNA template strand at the other end of the region of interest.
- a strand carrying a nucleotide sequence encoding amino acid information of a protein on a DNA is called a sense strand, which is also called a coding strand.
- the other strand nucleotide sequence is complementary to the sense strand and is called the antisense strand.
- a primer complementary to the sense strand is generally referred to as an upstream primer, and a primer complementary to the antisense strand is referred to as a downstream primer.
- Codon optimization In gene expression studies, researchers pay attention to the selection of appropriate expression vectors and host systems, and whether the genes themselves are optimally matched to the vector and host system.
- the optimal expression of the gene can be achieved by redesigning and synthesizing the gene, such as eliminating rare codons, optimizing the codons, minimizing the secondary structure, and adjusting the GC content.
- Another object of the invention is to provide expression vectors and host cells for use in the methods.
- the present invention firstly performs primer digestion and codon optimization of the native sequence of FGF-17 according to E. coli preference, and design primer pairs, wherein the upstream primer is as shown in SEQ ID No. 2, and the downstream primer is as SEQ. ID No.3 is shown.
- the rhFGF-17 sequence was further amplified by PCR.
- the present invention provides a combination of an optimized expression vector and a host cell, a pET3a expression vector and a corresponding BL21(DE3) plysS host cell, and is constructed by comparing the time, temperature, and inducer concentration. Expressed strain.
- the present invention provides a method for producing recombinant human fibroblast growth factor-17, and explores the induced expression of a protein at 16 to 37 ° C, and performs fragmentation verification, and finally, at 37 ° C, FGF 17 is mainly composed of inclusion bodies. The form is present, while at 16 ° C a considerable soluble FGF 17 protein is obtained.
- the expression level of the inclusion body of FGF-17 can reach more than 30% of the total bacterial protein, and the soluble fraction can reach more than 20% of the total bacterial protein; the bacterial yield reaches 10g/L fermentation. Above the liquid;
- Inclusion body part high expression level and not easy to be degraded during crushing and purification; further establishment of inclusion body washing, denaturation renaturation and column chromatography method, simplifying purification step, improving protein and activity yield, protein
- the yield is up to 8mg/g E. coli; the soluble protein fraction, the protein yield reaches 1mg/g E. coli; the protein purity is higher than 95%;
- a method for measuring FGF-17 activity was established.
- Biological activity is an important indicator reflecting the amount and efficacy of recombinant proteins.
- the present invention establishes an activity assay method for promoting proliferation of FGF-17 by using NIH 3T3 cell line. The results showed that FGF17 had a significant value-adding effect on NIH3T3. Compared with the standard FGF17 protein, the activity was better. Compared with the blank group, there was a significant difference, showing good repeatability and reliability.
- the coding sequence primer of rhFGF-17 was designed without changing the amino acid sequence, and Specific restriction sites NdeI and BamHI were introduced.
- the nucleotide sequence of rhFGF-17 was obtained by PCR amplification.
- the obtained product was ligated to the T vector (pEASY T1 simple vector) at 16 ° C, and the ligated product was digested with NdeI and BamHI for 4 h at 37 ° C to recover rhFGF-
- the 17 gene fragment was ligated with Solution I ligase at 16 ° C overnight to construct a recombinant expression vector (see Fig. 1, Fig. 2), and the resulting expression vector pET3a gene fragment was ligated with NdeI and BamHI.
- the ligation product was transformed into E.
- coli or BL21(DE3)plysS positive clones were selected on LB plates containing vector resistance and subjected to plasmid double digestion to confirm that the expression sequences were correctly inserted into the vector.
- the gene sequencing company was commissioned to perform forward and reverse sequence analysis to confirm that the cloned sequence was identical to the designed sequence.
- the shake flask culture was carried out, and the expression of the target protein was determined to be about 25% of the total protein after induction, and a positive reaction was detected by Western Blot, and the expressed recombinant foreign protein was confirmed to be FGF-17.
- the stability test of engineering bacteria was carried out for 50 times, and the plasmid stability, structural stability and expression stability were tested.
- the engineered strain inserts the target gene into the induced pET series expression vector containing the Lac operon, and then transforms the E. coli host strain compatible with the expression vector, and induces expression by IPTG.
- tryptone, yeast powder or the like is used as a nitrogen source
- glucose or the like is a carbon source
- phosphate is used as a buffer system
- sodium chloride is used as a basic medium; and by controlling conditions (pH, temperature, dissolution)
- the regulation of oxygen, number of revolutions, etc. significantly increased the yield and expression level of the cells.
- the FGF-17 engineering strain was inoculated into the LB medium at 1:50-100 to obtain the first-generation bacteria, cultured for 8 h to 12 h, and the A 600 was about 1.2-2.0, and inoculated at a ratio of 1:100-200.
- the culture is carried out in LB medium containing phosphate buffer, cultured for 2 h to 4 h, until A 600 reaches 0.6 to 0.8, at a temperature of 16 ° C and 37 ° C, the final concentration of the inducer (IPTG) is 0.4 mM and 1 mM, time.
- Induction induction was carried out at 16 to 30 h and 4 to 6 h, respectively, and the number of rotations of the incubator was 180 rpm to 200 rpm.
- the cells were cryopreserved at a low temperature and centrifuged at -20 ° C. The samples were subjected to SDS-PAGE and the expression was measured (see Figure 3).
- the precipitate after centrifugation was taken, and 20-fold amount of washing buffer (20 mM Tris-HCl, 1 mM EDTA-2Na, 0.2 M NaCl, 1% Triton X-100, pH 7.5) was added and thoroughly stirred and washed, and after 30 minutes, it was centrifuged at 20000 rpm. The supernatant was discarded at 4 ° C for 25 min and the precipitate was collected.
- washing buffer 20 mM Tris-HCl, 1 mM EDTA-2Na, 0.2 M NaCl, 1% Triton X-100, pH 7.5
- washing buffer (20 mM Tris-HCl, 1 mM EDTA-2Na, 0.2 M NaCl, 1% Triton X-100, pH 7.5) was added and thoroughly stirred and washed, and after 30 minutes, centrifuged at 20000 rpm, 4 ° C, After 25 min, the supernatant was discarded, and the precipitate was collected, and washed once with a washing buffer (20 mM Tris-HCl, 0.1 M NaCl, pH 7.5) to obtain inclusion bodies.
- inclusion bodies was weighed, 25 times the amount (g/V) of lysis buffer (20 mM Tris-HCl, 8 M urea, pH 7.5), fully suspended, and dissolved overnight at room temperature under magnetic stirring. The precipitate was centrifuged at 20000 rpm, 4 ° C, and 25 min, and an inclusion body solution was obtained.
- the inclusion body solution was taken and dialyzed to a urea concentration of 4 M under magnetic stirring at 4 ° C. Then, the dialyzed solution was slowly dropped into 3 volumes of refolding buffer (20 mM Tris-HCl, pH 7.5) to urea. The final concentration is 1M, and the renaturation process is about 12h ⁇ 20h. After renaturation, centrifuge at 20000 rpm, 4 ° C, 25 min, take the supernatant, which is a refolding solution. The sample was assayed for protein content and its purity was determined by silver staining.
- Buffer solution Solution I: 20 mM Tris-HCl (pH 7.0-7.5) + 25 mM NaCl
- FGF-17 sample peaks were collected, protein content was determined, and the purity was determined by SDS-PAGE combined with silver staining.
- Buffer solution Solution I: 20 mM Tris-HCl (pH 7.0-7.5) + 25 mM NaCl
- FGF-17 sample peaks were collected, protein content was determined, and the purity was measured by SDS-PAGE.
- the biological activity of genetically engineered recombinant protein is an important indicator reflecting the quality and efficacy of the drug. Therefore, establishing a stable and sensitive method for measuring the activity is of great significance for the production and quality detection of the product, and is also an important indicator for guiding the clinical dosage.
- the present invention is based on the specific function of FGF-17 to promote the mitosis of epithelial and mesenchymal cells.
- the cell proliferation method is used to select mouse embryonic fibroblasts (NIH 3T3 cells) as target cells, and the biological activity of the protein in vitro is detected by MTT colorimetry. .
- NIH 3T3 cells grew slowly in starvation medium; while rhHGF-17 protein was added to starvation medium, NIH 3T3 cells grew significantly faster.
- the absorbance was measured at 570 nm using a fully automatic microplate reader with a reference wavelength of 630 nm.
- the results showed that the purified FGF-17 protein had a significant proliferative effect on NIH 3T3 cells at a concentration of about 30 ng/ml. Compared with the FGF-17 standard, the biological activity was better and there was no statistical difference (see Figure 6).
- the present invention provides a method for producing a recombinant human fibroblast growth factor-17 protein, which comprises at least the following steps:
- Step 1 The native sequence of human fibroblast growth factor-17 protein FGF-17 is recombined and amplified according to the preference of E. coli, and the recombinant human fibroblast growth factor-17 protein rhFGF-17 gene is obtained, and the base sequence thereof is as follows. SEQ ID NO: 1 and the amino acid sequence thereof is shown in SEQ ID No. 4;
- Step 2 The recombinant rhFGF-17 gene is ligated into an E. coli expression plasmid, as shown in FIG. 1 , to form a recombinant expression vector, and then the recombinant expression vector is transformed into an E. coli host strain to obtain an FGF-17 protein expression engineering bacteria; as well as
- Step 3 cultivating the expression engineering bacteria, followed by purification
- the cell lysis buffer of the following concentration components is used: 45-55 mM Tris, 280-320 mM sodium chloride NaCl, 1.5-2.5 mM ethylenediaminetetraacetic acid DTA 0.1 to 0.3 M sucrose, and 0.8 to 1.2% by volume of polyethylene glycol octylphenyl ether Triton X-100, 0.1 to 0.3% sodium deoxycholate, 5% to 10% glycerol, It is 0.8 to 1.2% of phenylmethylsulfonyl fluoride PMSF; and the pH of the cell lysis buffer is adjusted to be between 7.3 and 7.49.
- Example 1 Cell lysis buffer uses the following components:
- Tris 45 mM Tris, 280 mM sodium chloride NaCl, 1.5 mM ethylenediaminetetraacetic acid DTA, 0.1 M sucrose, and 0.8% by volume of polyethylene glycol octyl phenyl ether Triton X- 100, which is 0.1% sodium deoxycholate, 5% glycerol, and 0.8% phenylmethylsulfonyl fluoride PMSF.
- the pH is adjusted to 7.45:
- composition and pH of the cell lysis buffer have a significant effect on the final activity.
- Example 2 Cell lysis buffer uses the following components:
- Example 3 Cell lysis buffer uses the following components:
- Tris 50 mM Tris, 300 mM sodium chloride, 2 mM ethylenediaminetetraacetic acid DTA, 0.2 M sucrose, and 1% by volume of polyethylene glycol octyl phenyl ether Triton X-100 It is 0.2% sodium deoxycholate, 7.5% glycerol, and 1% phenylmethylsulfonyl fluoride PMSF. The pH is adjusted to 7.4:
- the FGF-17 protein cultured by the present invention has higher biological activity than the naturally-growing protein in the human body, as shown in FIG. In Fig. 10, the second line from the bottom to the top is a naturally growing protein, and the third and fourth lines are the FGF-17 protein cultured in the present invention, and it is apparent that the activity is much higher than that of the naturally growing protein.
- the culture of the expression engineering bacteria comprises: culturing the soluble FGF-17 protein at 16 ° C, and The inclusion body FGF-17 protein was cultured at 37 ° C as shown in FIG.
- the E. coli expression plasmid is pET3a, as shown in FIG. 1, wherein the position of the dicerase is displayed, and the Escherichia coli host strain is BL21 (DE3) plysS.
- the cell lysis buffer comprises: 50 mM Tris, 300 mM sodium chloride, 2 mM ethylenediamine. Tetraacetic acid DTA, 0.2 M sucrose, and 1% by volume of polyethylene glycol octylphenyl ether Triton X-100, 0.2% sodium deoxycholate, 5%-10% glycerol, 1% Benzylsulfonyl fluoride PMSF; and the pH of the cell lysis buffer was adjusted to 7.4.
- the culturing the expression engineering bacteria comprises the following steps:
- the FGF-17 protein expression engineering bacteria were inoculated to the first generation LB medium at a ratio of 1:50-100 to obtain the first generation bacteria; the first generation bacteria were cultured for 8h to 12h, and the bacteria concentration A 600 reached 1.2. When it is about 2.0, it is inoculated into a second-generation LB medium containing phosphate buffer at a ratio of 1:100 to 200;
- Example 5 The cultivation process uses the following parameters:
- the FGF-17 protein expression engineering bacteria were inoculated into the first generation LB medium at a ratio of 1:100 to obtain the first generation bacteria; the first generation bacteria were cultured for 12 hours, and when the bacteria concentration A 600 reached 2.0 or so, press 1: The ratio of 200 was inoculated into a second generation LB medium containing phosphate buffer for culture;
- the FGF-17 protein expression engineering bacteria were inoculated into the first generation LB medium at a ratio of 1:130 to obtain the first generation bacteria; the first generation bacteria were cultured for 20 hours (transition culture, which is unfavorable for the second generation culture), and the bacteria were treated.
- concentration A 600 reaches about 4.0, the cells are inoculated into a second-generation LB medium containing phosphate buffer at a ratio of 1:200;
- the temperature is 16 ° C, and the time is 16 to 30 h.
- the temperature is 37 ° C, and the time is 4-6 h.
- the obtained FGF-17 protein is frozen at -20 °C.
- the first generation medium includes: tryptone 10g / L, yeast powder 5g / L, and sodium chloride 10g / L;
- the second generation medium includes: tryptone 10g/L, yeast powder 10g/L, dipotassium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 1g/L, sodium chloride 4g/ L, and glucose 5g / L;
- the second generation medium includes: tryptone 10g/L, yeast powder 4g/L, dipotassium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 1g/L, and sodium chloride. 10g/L.
- Example 6 The culture medium used the following medium:
- the first generation medium includes: tryptone 10g / L, yeast powder 5g / L, and sodium chloride 10g / L;
- the second generation medium includes: tryptone 10g/L, yeast powder 10g/L, dipotassium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 1g/L, sodium chloride 4g/ L, and glucose 5g / L;
- the second generation medium includes: tryptone 10g/L, yeast powder 4g/L, dipotassium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 1g/L, and sodium chloride. 10g/L.
- the culture medium uses the following medium:
- the first generation medium includes: tryptone 13g / L, yeast powder 8g / L, and sodium chloride 5g / L;
- the second generation medium includes: tryptone 13g / L, yeast powder 7g / L, dipotassium hydrogen phosphate 6g / L, potassium dihydrogen phosphate 3g / L, sodium chloride 8g / L, and glucose 10g / L;
- the method for producing the recombinant human fibroblast growth factor-17 protein if a soluble FGF-17 protein is obtained, a 25-fold g/V cell lysis buffer is added, wherein glycerol is fully suspended in 5%, and then low-temperature ultrasound is performed. Broken, wherein the power is 300w, the vibration frequency output is 40%; then the ultrasound is stopped for 5s for 5s, the total ultrasound is 10mins;
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Abstract
提供了一种重组人成纤维细胞生长因子-17的生产方法,包括:将人成纤维细胞生长因子-17(FGF-17)的编码核酸序列按照大肠杆菌偏好进行重组和扩增;将重组后的rhFGF-17基因连接至表达质粒构建成重组表达载体,然后将该重组表达载体转化入大肠杆菌宿主菌,得到FGF-17蛋白表达工程菌;以及对该工程菌进行培养,并纯化FGF-17。
Description
本发明涉及基因工程领域,具体涉及重组人成纤维细胞生长因子-17的生产方法。
成纤维细胞生长因子(Fibroblast Growth Factor,FGF)是一类广泛存在于多种组织中的多肽细胞生长因子,目前已发现23个成员,分子量为16kD~34kD,其中心区域含有高度同源性(30%~70%)的大约120个氨基酸序列,对来源于中胚层和神经外胚层的多种类型细胞具有广泛的生物学活性。FGFs在体内含量甚微但分布广泛,各个因子调控着一系列的发育过程,包括脑的发育、肢体的分化和躯干的形成。大多数FGFs成员与胚胎发育、组织损伤修复、肿瘤的发生、发展和转移等等有密切关系,具有可观的应用价值。
FGF-17属FGF8亚家族(FGF8、FGF17、FGF18),与其他FGF成员一样,均属于旁分泌因子,而在前列腺癌和肝癌中,FGF17还以自分泌方式参与其中。在胚胎发育过程中的组织和器官形成中起重要作用。1998年,日本科学家Masamitsu Hoshikawa等人从大鼠胚胎中分离得到一种能够编码含有216个氨基酸的FGF17的cDNA,利用转染了含cDNA的杆状病毒的High Five昆虫细胞有效得到了大鼠FGF17蛋白。科学家发现FGF17mRAN在大鼠胚胎脑组织峡部及隔膜的神经上皮优先表达。说明FGF17对于胚胎脑组织发育至关重要。人FGF17有两种亚型,FGF17a和b,b为主要形式,本实验中要表达的人FGF17也就是FGF17b。人FGF17基因定位于染色体8p21.3,共有3个外显子,6个突变位点,由216个氨基酸组成,与其他已发现的家族成员之间具有30%~70%的同源性,人FGF-17与老鼠的氨基酸序列具有93%的高度同源性。人FGF17分子量约为25kD,而本次实验设计中切除了信号肽,只获取成熟蛋白部分序列,最终蛋白分子量为22.6kD,等电点10.43,为碱性蛋白。结构上,FGF17与FGF8、FGF18相似,属同一家族成员,其同源性分别为60%和50%,均具有信号肽序列,采用大肠杆菌表达时需将其切除。
FGF17对脑组织神经系统正常发育意义重大。FGF17是脑组织结构正常发育的关键因子,其表达和同家族的FGF8出现部分重叠,两者在MHB区域(mid-hindbrain,中后脑)和皮质层扮演重要角色。Cholfin等研究发现FGF17是小鼠额叶皮层分区的重要参与部分,
敲除FGF17会导致背侧额叶皮层缩小以及额叶皮层投射到皮层下靶部位程度的减少;Jingsong Xu等以FGF17敲除小鼠为模型发现无论是出生后两天的幼鼠还是成年小鼠,在解剖学上无异常且呈现健康状态,而经过组织学检测却发现其中脑尾端、下丘脑和小脑蚓部出现显著的组织缺失现象,即发育异常。(Hoshikawa et al.Biochem Biophys Res Commun.1998,244:187–191;Maruoka et al.Mech Dev,1998,74(1-2):175–177;Reifers et al.Mech Dev,2000,99(1-2):39–49;Cholfin et al.Proc Natl Acad Sci U S A,2007,104(18):7652–7657;Xu J et al.Development,2000,127(9):1833-1843;)
FGF17在研究神经精神性疾病方面具有潜在价值。针对这点,要涉及FGF17于染色体上的位置。2009年的一篇报道综述了染色体8p为神经精神病学易感位点,与神经精神性紊乱(精神分裂症、自闭症)、神经退行性疾病(阿尔茨海默病、帕金森)、卡尔曼综合征等密切联系;8p21-23是脑胼胝体发育不全的关键位点,后者和认知缺陷、抽搐、自闭症以及精神分裂症密切相关。FGF17基因位于染色体8p21.3,从而对于研究上述病症意义重大。Zanni等人经过研究案例来探讨FGF17与小脑发育和疾病的关系,该案例中,患者伴有严重的生长发育迟缓、癫痫以及DWM(Dandy–Walker malformation,第四脑室孔闭塞综合征,也称非交通性脑积水)症状,检测发现患者体内染色体8p21.2-p21.3距始端2.3Mb处有缺失现象,也即FGF17基因有所缺损,而分析患者的血液淋巴细胞和皮肤成纤维细胞发现FGF17表达水平明显降低,这是关于人类小脑畸形与FGF17基因转录下调的首次报道;Scearce-Levie等研究发现FGF17-/-小鼠社会行为出现异常,表现为幼鼠发声减少,成年鼠社会互动缺陷,并发现在人体中,FGF17信号通路的干扰可能会导致一系列神经精神类疾病,比如精神分裂症和自闭症,而这些异常行为可能由于FGF17缺失的额叶皮层结构中Fos基因的激活减少所致,然而相关的神经循环和信号通路机制依然所知甚少,需待进一步探究。目前对于神经精神方面的疾病治疗仍有不足和很大的空间,从而对于涉及在内的FGF17基因的研究具有很大的前景。(Tabarés-Seisdedos et al.Mol Psychiatry.2009,14(6):563-589;Paul et al.Nat Rev Neurosci,2007,8(4):287-299;Zanni et al.Neurogenetics,2011,12(3):241-245;Scearce-Levie et al.Genes Brain Behav,2008,7(3):344-354)
FGF17在骨骼方面的研究具有重要价值。可能与FGF17作用于受体FGFR3有关。FGFR3在骨骼发育中起重要作用,是软骨细胞中主要表达的受体,其激活突变会导致一系列骨骼发育缺陷性疾病,如软骨发育不全(ACH)、季肋发育不全(HCH)和致死性骨发育不全(TD),持续性活化骨细胞中FGFR3会导致侏儒症。Krejci P等人利用免疫组化方法检
测到在正常人类生长板软骨中有FGF17蛋白的表达,且水平和FGF19一致,随后在细胞增殖实验中发现FGF17与FGF1同等水平抑制RCS(大鼠软骨瘤软骨细胞)的软骨细胞增殖,随着FGF17浓度加大,抑制作用愈明显。低浓度时,FGF17通过与肝素联用显著抑制RCS增殖,而单独的FGF17抑制效果不明显,检测发现FGF17能够诱使ERK信号通路磷酸化。从而可知FGF17参与激活软骨细胞中FGFR3信号通路,诱使ERK磷酸化,抑制软骨瘤细胞的生长,而要想达到显著抑制作用需要有肝素的配合使用。(Passos-Bueno et al.Hum Mutat,1999,14(2):115-125;Naski et al.Front.Biosci,1998,3:d781-794;Naski et al.Nat Genet,1996,13(2):233-237;Shiang et al.Cell,1994,78(2):335-342;Krejci et al.Pediatr Res,2007,61(3):267-272.)
FGF17对于肿瘤发生、发展和转移的影响。和其他成纤维细胞生长因子一样,FGF17也是一种潜在的致癌因子。FGF17主要通过与细胞表面的酪氨酸激酶受体FGFR3c、FGFR4、FGFR2c和FGFR1c发生特异性结合(FGFR3c>FGFR4>FGFR2c>FGFR1c),通过MAPK途径引发DNA合成以及促进细胞增殖,且在促进DNA合成时,FGF17诱导有丝分裂的活性是FGF8的两倍;Jingsong Xu等人将携带有FGF17a和FGF17b(人FGF17的主要表达形式)基因的载体转入NIH3T3细胞中,一个星期之后,含有FGF17b的细胞40%出现肿瘤化,两个星期之后,所有细胞全部形成肿瘤,而含有FGF17a的细胞依然成正常状态;Polnaszek和Heer等人均检测FGF17在前列腺癌细胞中出现过表达,尤其DU145细胞系中FGF17表达水平较高,Heer等还发现在高度分化的前列腺癌细胞中FGF17mRNA以4倍于BPH(良性前列腺增生)的水平上调。Polnaszek等从根治性前列腺切除手术标本的癌细胞中检测到癌细胞和正常上皮细胞中均有类似水平的FGF17的表达(由于癌细胞密度大,所以总体而言,FGF17在肿瘤组织浓度偏高),表达量相对于FGF7偏低(FGF7为28ng/g,FGF17为0.4~0.5ng/g);关于乳腺癌,目前依然没有足够的证据证明FGF17在乳腺癌细胞中有表达,不过Meijer等人在三苯氧胺治疗乳腺癌中发现组织中的受体FGFR4与其配体FGF17的信号传导造成了三苯氧胺抵抗,这一通路的发现对于内分泌治疗乳腺癌无效的患者进行靶向治疗提供了指导价值。Gauglhofer等人研究人肝癌细胞(HCC,HepG2 and Hep3B)发现包括FGF17在内的FGF8家族的表达出现上调,而此现象可能与wnt通路下调有关。当饥饿或添加模拟低氧药物甲磺酸去铁胺进行肝癌细胞培养时,发现FGF8家族的表达显著增加,饥饿培养的肝癌细胞,在添加了FGF8亚家族因子后,细胞凋亡减少,而这个与ERK1/2和核糖体蛋白S6的磷酸化有关,研究发现无血清时,pERK和pS6下调,pGSK3β上调,细胞存活减弱,当添加FGF17后,pERK
上调,肝癌细胞增多。目前已知FGF17与肿瘤密切相关,因此将其作为肿瘤诊断和治疗的靶分子具有广阔的前景。(Zhang X et a.J Biol Chem,2006,281(23):15694–15700;Murray et al.Curr Cancer Drug Targets,2009,9(5):639–651;Reifers et al.Mech Dev,2000,99(1–2):39–49;Xu J et al.Mech Dev,1999,83(1-2):165-78;Polnaszek et al.Prostate,2004,60(1):18-24;Heer et al.J Pathol,2004,204(5):578-586;Meijer et al.Mol Cancer Res,2006,4(6):379-86;Gauglhofer et al.Hepatology,2011,53(3):854-864.)
由于FGF-17对于脑组织、神经系统、骨骼及肿瘤等方面有着重要意义,其研究和临床应用前景广阔,日益受到科研界的关注。FGF-17作为一种新型生长因子,目前国内外相关报道比较少。本申请专利,通过基因优化、表达载体和宿主菌的筛选,进而获得大肠杆菌的高效表达菌株;优化培养条件和菌液破碎方法以加大可溶蛋白的比例;优化包涵体洗涤、变性和复性方法,得到浓度较高且纯度较好的包涵体蛋白;通过阳离子交换柱层析(HiTrapTM SP HP)、肝素柱亲和层析(Heparin Sepharose CL-6B),最终获得蛋白纯度超过95%且具有较高生物学活性的重组人FGF-17蛋白,为进一步开展FGF-17相关作用机制研究和新药研发奠定了基础。
然而,目前FGF17蛋白表达水平低,同时很难制备具有高活性的蛋白。这些已经构成了FGF18基础研究和临床应用的瓶颈问题。在生产FGF17蛋白表达时,通常采用PH值为7.5的细胞裂解缓冲液,培养时的温度也未经严格控制,由此导致最终的培养纯度和浓度都不够高。
发明内容
本发明在前期研究基础上,构建了在以大肠杆菌表达质粒为载体的FGF-17细胞表达系统,主要改进了其培养方法和纯化方法,以及对培养温度进行优化。
因此,本发明的目的之一是提供一种重组人成纤维细胞生长因子-17蛋白的生产方法,解决了FGF-17蛋白生产浓度和纯度较低的问题;
本发明的另一个目的是提供一种能够培养出可溶性FGF-17蛋白和包涵体FGF-17蛋白两种形式的蛋白的方法。
为此,本发明提供的技术方案为:
一种重组人成纤维细胞生长因子-17蛋白的生产方法,至少包括以下步骤:步骤一、将天然序列的人成纤维细胞生长因子-17蛋白FGF-17按照大肠杆菌偏好进行重组和扩增,得到重组人成纤维细胞生长因子-17蛋白rhFGF-17基因,其碱基序列如SEQ ID NO:1所示;
步骤二、将重组后的rhFGF-17基因连接至大肠杆菌表达质粒,构成重组表达载体,然后将重组表达载体转化进入大肠杆菌宿主菌,得到FGF-17蛋白表达工程菌;以及
步骤三、对表达工程菌进行培养,之后进行纯化;
其中,在纯化过程中,采用如下浓度组分的细胞裂解缓冲液:45-55mM的三羟甲基氨基甲烷Tris,280~320mM的氯化钠NaCl,1.5~2.5mM的乙二胺四乙酸DTA,0.1~0.3M的蔗糖,以及体积百分比为0.8~1.2%的聚乙二醇辛基苯基醚TritonX-100,为0.1~0.3%的去氧胆酸钠,为5%-10%甘油,为0.8~1.2%的苯甲基磺酰氟PMSF;且调节所述细胞裂解缓冲液的PH值介于7.3~7.49之间。
优选的是,所述的重组人成纤维细胞生长因子-17蛋白的生产方法中,所述对表达工程菌进行培养包括:在16℃的条件下培养出可溶性FGF-17蛋白,而在37℃的条件下培养出包涵体FGF-17蛋白。
优选的是,所述的重组人成纤维细胞生长因子-17蛋白的生产方法中,所述大肠杆菌表达质粒为pET3a,且所述大肠杆菌宿主菌为BL21(DE3)plysS。
优选的是,所述的重组人成纤维细胞生长因子-17蛋白的生产方法中,所述细胞裂解缓冲液中,包含:50mM的三羟甲基氨基甲烷Tris,300mM的氯化钠NaCl,2mM的乙二胺四乙酸DTA,0.2M的蔗糖,以及体积百分比为1%的聚乙二醇辛基苯基醚TritonX-100,为0.2%的去氧胆酸钠,为5%-10%甘油,为1%的苯甲基磺酰氟PMSF;且调节所述细胞裂解缓冲液的PH值为7.4。
优选的是,所述的重组人成纤维细胞生长因子-17蛋白的生产方法中,所述对表达工程菌进行培养包括以下步骤:
1)将FGF-17蛋白表达工程菌以1:50~100的比例接种至第一代LB培养基中得到第一代菌;将第一代菌培养8h~12h,待菌浓度A600达到1.2~2.0左右时,按1:100~200的比例接种到含有磷酸盐缓冲液的第二代LB培养基中进行培养;
2)再培养2h~4h,待菌浓度A600达到0.6~0.8左右时,温度为15~38℃,诱导剂IPTG终浓度为0.3~0.5mM和0.8~1.2mM,时间为4~30h,培养箱转数为180rpm~200rpm,以进行诱导表达;以及
3)进行离心操作,将得到的FGF-17蛋白在-15℃以下冻存。
优选的是,所述的重组人成纤维细胞生长因子-17蛋白的生产方法中,如果得到可溶性FGF-17蛋白,则在所述步骤2)中,所述温度为16℃,且所述时间为16~30h;而如果得到包涵体FGF-17蛋白,则在所述步骤2)中,所述温度为37℃,且所述时间为4~6h。
优选的是,所述的重组人成纤维细胞生长因子-17蛋白的生产方法中,在所述步骤3)中,得到的FGF-17蛋白在-20℃冻存。
优选的是,所述的重组人成纤维细胞生长因子-17蛋白的生产方法中,在所述步骤1)中,第一代培养基中包括:胰蛋白胨10g/L,酵母粉5g/L,和氯化钠10g/L;
如果得到可溶性FGF-17蛋白,则第二代培养基中包括:胰蛋白胨10g/L,酵母粉10g/L,磷酸氢二钾3g/L,磷酸二氢钾1g/L,氯化钠4g/L,和葡萄糖5g/L;而
如果得到包涵体FGF-17蛋白,则第二代培养基中包括:胰蛋白胨10g/L,酵母粉4g/L,磷酸氢二钾3g/L,磷酸二氢钾1g/L,和氯化钠10g/L。
优选的是,所述的重组人成纤维细胞生长因子-17蛋白的生产方法中,在培养包涵体FGF-17蛋白时,仅使用肝素柱进行纯化,而不使用离子交换柱。
优选的是,所述的重组人成纤维细胞生长因子-17蛋白的生产方法中,如果得到可溶性FGF-17蛋白,则加入25倍量g/V的细胞裂解缓冲液,其中甘油为5%充分悬浮,然后低温超声破碎,其中功率为300w,振频输出为40%;然后超声5s停5s,总计超声10mins;
镜检在可见视野范围内无完整细胞结构的大肠杆菌后,以20000rpm离心,4℃,25mins,收集上清,留沉淀。
本发明的有益效果为:
本发明构建了以大肠杆菌表达质粒为载体的FGF-17大肠杆菌宿主细胞来生产FGF-17的方法,主要对于细胞裂解缓冲液的浓度组分进行了优化,并且也对培养温度进行了优化,最终导致产品的纯度和浓度获得极大的提高。
本发明还对培养过程和纯化过程的众多参数进行了优化和规定,最终获得高浓度和高纯度的可溶性蛋白和包涵体蛋白。
图1为根据本发明的一个实施例的大肠杆菌表达质粒PET-3a的结构图,其中下划线处为酶切点;
图2为根据本发明的一个实施例的对FGF-17蛋白进行诱导表达SDS-PAGE后结果的电泳分析图,其中附图标记1为蛋白标准品marker,附图标记2-5为在37℃的条件下诱导表达后的结果,附图标记6-9为在16℃的条件下诱导表达后的结果,附图标记10为诱导前的结果;
图3为根据本发明的一个实施例的FGF-17蛋白在不同温度下得到的表达产物的可溶
性SDS-PAGE电泳分析图,其中附图标记1为蛋白标准品marker;附图标记2为在37℃的条件下诱导表达后的结果;附图标记3为在37℃的条件下诱导表达后,并经过纯化时破碎后沉淀部分的结果;附图标记4为在37℃的条件下破碎后上清部分的结果;附图标记5为在37℃的条件下诱导表达后,并经过纯化时破碎后上清部分的结果;附图标记6为在16℃的条件下诱导表达后,并经过纯化时破碎后沉淀部分的结果;附图标记7为在16℃的条件下诱导表达后的结果。
图4为根据本发明的一个实施例的包涵体FGF-17蛋白变性和复性的SDS-PAGE电泳分析图,其中附图标记1为蛋白标准品marker;附图标记2为经过纯化时变性处理的上清部分的结果;附图标记3为经过纯化时变性处理的沉淀部分的结果;附图标记4为变性后又进行复性时上清部分的结果。
图5为根据本发明的一个实施例的包涵体亲和肝素柱层析SDS-PAGE电泳分析图,其中附图标记1为蛋白标准品marker;附图标记2为穿出亲合柱的结果;附图标记3-7为不同浓度氯化钠洗脱的结果;
图6为根据本发明的一个实施例的可溶体亲和肝素柱层析SDS-PAGE电泳分析图,其中附图标记1为蛋白标准品marker;附图标记2为经过纯化时破碎后上清部分的结果;附图标记3-5为不同浓度氯化钠洗脱的结果;
图7为根据本发明的一个实施例的可溶体亲和离子交换柱层析SDS-PAGE电泳分析图,其中附图标记1为蛋白标准品marker;附图标记2为亲和柱洗脱峰的结果;附图标记3-5为不同浓度氯化钠洗脱的结果;以及
图8为根据本发明的一个实施例的MTT法测定FGF-17蛋白对NIH 3T3细胞的促增殖生物学活性图,其中最上面两条为根据本发明的生产方法得到的FGF-17蛋白产生的增值活性,其远远大于标准品产生的增值活性。
根据本发明,可采用本领域技能中的常规分子生物学、微生物学、细胞学和DNA重组技术。如果出现于本文下来术语的定义如下。
“DNA分子”指脱氧核糖核苷酸(胸腺嘧啶、胞嘧啶、腺嘌呤或鸟嘌呤)的聚合形式,是染色体主要组成成分,同时也是组成基因的材料。这一术语只指分子的一级和二级结构,不限制其任何具体的三级形式。本术语包括特别是在线性DNA分子、病毒、染色体、质粒中国发现的双链DNA。这里讨论的结构,按照习惯上给出的只是DNA正义链的5'到
3'方向的序列。
“载体”是指能够转运与其连接的另一个核酸的核酸分子,一种类型的载体是“质粒”,质粒是其他的DNA片段可与其连接的环状双链DNA环。另一类型的载体是病毒载体,其可将其他的DNA片段连接至病毒基因组。某些载体整合至宿主细胞基因组中,并得以与宿主基因组一起复制。并且,某些载体能指导与其可操作连接的基因的表达,一般使用的这样的表达载体为质粒形式。在本发明中,可交互使用“质粒”和“载体”。
“重组载体”是指已连接了基因的表达载体。在本发明中,可交互使用“重组质粒”和“重组载体”。
本文中的术语“宿主”不仅包括原核生物,也包括真核生物如酵母、植物和动物细胞。
本发明中的“反向互补”,指通过碱基配对原则关联的核苷酸序列。例如,序列“5’-A-T-G-3’”与序列“5’-C-A-T-3’”反向互补。
引物,又名引子。是一小段单链DNA或RNA,作为DNA复制的起始点,在核酸合成反应时,作为每个多核苷酸链进行延伸的出发点而起作用的多核苷酸链,在引物的3’-OH上,核苷酸以二酯链形式进行合成,因此引物的3’-OH,必须是游离的。之所以需要引物是因为在DNA合成中DNA聚合酶仅仅可以把新的核苷酸加到已有的DNA链上。引物是人工合成的两段寡核苷酸序列,一个引物与感兴趣区域一端的一条DNA模板链互补,另一个引物与感兴趣区域另一端的另一条DNA模板链互补。DNA上携带有编码蛋白质氨基酸信息的核苷酸序列的链称为正义链,又称编码链。另一条链核苷酸序列与正义链互补,称为反义链。一般将与正义链互补的一个引物成为上游引物,与反义链互补的一个引物称为下游引物。
密码子优化:在基因表达研究中,研究者比较注意选择合适的表达载体和宿主系统,以及基因本身是否与载体和宿主系统为最佳匹配。基因的最佳化表达可以通过对基因的重新设计和合成来实现,如消除稀有密码子而利用最佳化密码子,二级结构最小化,调整GC含量等。
下面结合附图和实施例对本发明做进一步的详细说明,以令本领域技术人员参照说明书文字能够据以实施。
本发明的目的是提供一种简便且高效的生产FGF-17的方法,包括包涵体和可溶两部分。
本发明的目的还在于提供含有编码FGF-17基因的载体或质粒。
本发明的另一目的是提供用于该方法的表达载体和宿主细胞。
为实现上述目的,本发明首先将FGF-17的天然序列按照大肠杆菌偏好性进行信号肽切除和密码子优化,设计引物对,其中上游引物如SEQ ID No.2所示,而下游引物如SEQ ID No.3所示。再通过PCR扩增得到rhFGF-17序列。同时本发明提供了筛选优化的表达载体和宿主细胞的组合,pET3a表达载体和对应的BL21(DE3)plysS宿主细胞,并通过诱导的时间、温度、诱导剂浓度多方因素的摸索比较构建了高效稳定表达的菌株。
进一步,本发明提供了一种生产重组人成纤维细胞生长因子-17的方法,,摸索16~37℃蛋白的诱导表达,进行破碎验证,最终得出在37℃时,FGF 17主要以包涵体形式存在,而在16℃时得到可观的可溶性FGF 17蛋白。
首先,对于包涵体部分,通过优化包涵体处理条件,最后通过柱层析分离纯化得到高纯度和活性的rhFGF-17。具体包括如下步骤:
(1)通过改良种子培养基对工程菌进行活化和扩增,接种至2L发酵瓶中通过优化诱导时间、诱导剂浓度和转数等高效表达外源蛋白FGF-17;
(2)优化裂解溶液并通过超声仪器进行菌体破碎,低温高速离心弃上清收沉淀;优化包涵体清洗、变性及复性方法与步骤;
(3)通过柱层析进行分离纯化,具体包括:阳离子交换柱层析(SP Sepharose Fast Flow)、肝素柱层析(Heparin Sepharose CL-6B)。
其次,对于可溶蛋白部分,具体操作如下:
(1)通过改良培养基成分和含量,低温(16℃)诱导并对诱导剂浓度、时间和转数予以优化;
(2)改变裂解液成分,超声破碎,低温高速离心,得上清液中的可溶部分;
(3)通过柱层析分离纯化,同包涵体部分。
本发明具有以下优点:
(1)通过对FGF-17核苷酸序列进行优化及适宜的表达载体和宿主细胞的组合,获得高效稳定表达的工程菌株;
(2)通过优化培养方法,可使FGF-17的包涵体部分表达水平达到菌体总蛋白的30%以上,可溶部分达到菌体总蛋白的20%以上;菌体产量达10g/L发酵液以上;
(3)包涵体部分,表达量高且破碎与纯化过程中不易被降解;进一步建立了包涵体清洗、变性复性和柱层析方法,简化了纯化步骤,提高了蛋白及活性收率,蛋白收率达8mg/g大肠杆菌;可溶蛋白部分,蛋白收率达到1mg/g大肠杆菌;蛋白纯度均高于95%以上;
(4)建立了FGF-17活性测定方法。生物学活性是反映重组蛋白质量及功效的重要指
标,本发明建立了用NIH 3T3细胞株进行FGF-17促增殖作用的活性测定方法。实验结果表明FGF17对NIH3T3有明显的促增值作用,对照标准FGF17蛋白,活性更优,与空白组比较,有显著性差异,显示出良好的重复性和可靠性。
实施例1 FGF-17蛋白高效表达工程菌的构建
根据GenBank中公布的人FGF17天然序列(登录号:AY358869.1)和氨基酸序列,按照大肠杆菌密码子偏好性进行优化,在不改变氨基酸序列的条件下,设计rhFGF-17的编码序列引物,并引入特异性酶切位点NdeⅠ和BamHⅠ。通过PCR扩增的方法获得rhFGF-17的核苷酸序列,所得产物与T载体(pEASY T1 simple vector)16℃连接,连接产物用NdeI和BamHI在37℃下双酶切4h,回收得rhFGF-17基因片段,再与用NdeI和BamHI双酶切同处理并回收所得的表达载体pET3a基因片段用Solution I连接酶16℃连接过夜,构建重组表达载体(见图1、图2)。连接产物转化进入大肠杆菌或BL21(DE3)plysS,在含有载体抗性的的LB平板上挑选阳性克隆并进行质粒双酶切鉴定,证实表达序列已正确插入载体中。委托基因测序公司进行正向、逆向序列分析测定,证实克隆序列与设计序列完全一致。进行摇瓶培养,诱导后测定目的蛋白表达量占总蛋白25%左右,并进行免疫印迹(Western Blot)检测呈现阳性反应,证实所表达的重组外源蛋白为FGF-17。进行工程菌稳定性试验,连续传代50次,检测其质粒稳定性、结构稳定性和表达稳定性,并证实获得了高效表达、遗传稳定的FGF-17工程菌株,为下一步试验奠定了良好的基础。本工程菌将目的基因插入含Lac操纵子的诱导的pET系列表达载体中,再转化与表达载体相容的大肠杆菌宿主菌,利用IPTG进行诱导表达。
实施例2 FGF-17蛋白较高密度培养方法的建立
在本发明中,以胰蛋白胨、酵母粉等为氮源,葡萄糖等为碳源,磷酸盐为缓冲体系组成,并辅以氯化钠作为基础培养基;并通过控制条件(pH、温度、溶解氧、转数等)的调节显著提高了菌体产量及表达水平。具体为:将FGF-17工程菌株以1:50~100接种至LB培养基中得到第一代菌,培养8h~12h,待A600达到1.2~2.0左右,按1:100~200的比例接种到含有磷酸盐缓冲液的LB培养基中进行培养,培养2h~4h,待A600达到0.6~0.8左右,以温度16℃和37℃、诱导剂(IPTG)终浓度为0.4mM和1mM、时间分别为16~30h和4~6h、培养箱转数180rpm~200rpm分别进行诱导表达,低温高速离心,菌体-20℃冻存,样品进行SDS-PAGE检测并测定表达量(见图3)。
表1 培养基组成
实施例3 FGF-17蛋白纯化方法的建立
1、包涵体的处理
(1)菌体破碎
将菌体反复冻融后,以1:25比例充分悬浮于细胞裂解缓冲液(20mM Tris-HCl,1mM EDTA-2Na,0.2M NaCl,1%Triton X-100,0.2%去氧胆酸钠,pH7.5),进行低温超声破碎,功率300w,振频输出40%,超声5s停5s,超声10min,镜检在可见视野范围内无完整细胞结构的大肠杆菌后,20000rpm离心,4℃,25min,弃上清,收集沉淀。
(2)包涵体清洗
取上述离心后的沉淀,加入20倍量洗涤缓冲液(20mM Tris-HCl,1mM EDTA-2Na,0.2M NaCl,1%Triton X-100,pH7.5)充分搅拌洗涤,30min后,20000rpm离心,4℃,25min,弃上清,收集沉淀。再取沉淀,加入20倍量洗涤缓冲液(20mM Tris-HCl,1mM EDTA-2Na,0.2M NaCl,1%Triton X-100,pH7.5)充分搅拌洗涤,30min后,20000rpm离心,4℃,25min,弃上清,收集沉淀,再用洗涤缓冲液(20mM Tris-HCl,0.1M NaCl,pH7.5)洗涤一次,得包涵体。
(3)包涵体变性,如图4所示。
称取适量包涵体,加入25倍量(g/V)溶解缓冲液(20mM Tris-HCl,8M脲,pH7.5),充分悬浮,并在磁力搅拌条件下室温溶解过夜。20000rpm离心,4℃,25min,离心弃沉淀物,得包涵体溶解液。
(4)包涵体复性,如图4所示。
取包涵体溶解液,在4℃磁力搅拌条件下,先透析到脲浓度为4M,然后将透析后溶液缓慢滴入3倍体积复性缓冲液(20mM Tris-HCl,pH7.5),至脲终浓度为1M,复性过程约12h~20h。复性完毕,20000rpm离心,4℃,25min,取上清液,即为复性液。样品测定蛋白含量,并利用银染法检测其纯度。
(5)柱层析纯化--亲和层析
层析介质:Heparin Sepharose CL-6B
缓冲溶液:溶液I:20mM Tris-HCl(pH7.0~7.5)+25mM NaCl
溶液II:20mM Tris-HCl(pH7.0~7.5)+2.0M NaCl
平衡:用溶液A平衡3~5个柱体积。
上样:将离子交换柱层析收集的FGF-17蛋白溶液除盐后上样。
清洗:上样后用3~5个柱体积的溶液I清洗层析柱,至基线平衡。
梯度:清洗后用20~30个柱体积将溶液B从0%调节至100%。
收集:收集FGF-17样品峰,测定蛋白含量,并利用SDS-PAGE结合银染检测其纯度。
以上(见图5)。
2、可溶蛋白处理,见图6和图7。
(1)菌体破碎
将菌体反复冻融后,加入25倍量(g/V)细胞裂解缓冲液(20mM Tris,300mM Nacl1%TritonX-100,1mM EDTA,0.2%去氧胆酸钠,5%甘油,1%PMSF,PH=7.4)充分悬浮,然后低温超声破碎,功率300w,振频输出40%,超声5s停5s,超声10min,镜检在可见视野范围内无完整细胞结构的大肠杆菌后,20000rpm离心,4℃,25min,收集上清,留沉淀。
(2)沉淀洗涤
将沉淀以1:10(g:ml)比例悬浮于洗涤缓冲液(20mM Tris,300mM Nacl,1%TritonX-100,1mM EDTA,5%甘油,PH=7.5)充分搅拌洗涤30min后,20000rpm离心,4℃,25min,收集上清。
(3)柱层析纯化
a.阳离子交换
层析介质:SP-Sepharose FF
缓冲溶液:溶液I:20mM Tris-HCl(pH7.0~7.5)+25mM NaCl
溶液II:20mM Tris-HCl(pH7.0~7.5)+2.0M NaCl
平衡:用溶液I平衡3~5个柱体积。
上样:将复性后的蛋白溶液上样。
清洗:上样后用3~5个柱体积的溶液I清洗层析柱,至基线平衡。
梯度:清洗后用20~30个柱体积将溶液B从0%升调节至100%。
收集:收集FGF-17样品峰,测定蛋白含量,并利用SDS-PAGE检测其纯度。
b.亲和层析
同包涵体。
实施例4 FGF-17蛋白细胞活性检测方法建立
基因工程重组蛋白的生物学活性是反映药物质量及功效的重要指标,因此建立稳定、灵敏的活性测定方法对于产品的生产及质量检测具有重要意义,同时也是指导临床用药剂量的重要指标。本发明根据FGF-17特异性促进上皮和间质细胞有丝分裂的功能,采用细胞增殖法,选择小鼠胚胎成纤维细胞(NIH 3T3细胞)为靶细胞,结合MTT比色法检测蛋白体外生物学活性。实验表明,在饥饿培养基中,NIH 3T3细胞生长缓慢;而在饥饿培养基中添加rhFGF-17蛋白后,NIH 3T3细胞生长明显加快。采用全自动酶标仪以630nm为参比波长,于570nm处测定吸光度。结果显示,纯化的FGF-17蛋白在约30ng/ml浓度左右时,对NIH 3T3细胞具有显著的促增殖作用。与FGF-17标准品相比,生物学活性更优,无统计学差异(见图6)。
具体而言,本发明提供了一种重组人成纤维细胞生长因子-17蛋白的生产方法,其特征在于,至少包括以下步骤:
步骤一、将天然序列的人成纤维细胞生长因子-17蛋白FGF-17按照大肠杆菌偏好进行重组和扩增,得到重组人成纤维细胞生长因子-17蛋白rhFGF-17基因,其碱基序列如SEQ ID NO:1所示,而其氨基酸序列如SEQ ID No.4所示;
步骤二、将重组后的rhFGF-17基因连接至大肠杆菌表达质粒,如图1所示,构成重组表达载体,然后将重组表达载体转化进入大肠杆菌宿主菌,得到FGF-17蛋白表达工程菌;以及
步骤三、对表达工程菌进行培养,之后进行纯化;
其中,在纯化过程中,采用如下浓度组分的细胞裂解缓冲液:45-55mM的三羟甲基氨基甲烷Tris,280~320mM的氯化钠NaCl,1.5~2.5mM的乙二胺四乙酸DTA,0.1~0.3M的蔗糖,以及体积百分比为0.8~1.2%的聚乙二醇辛基苯基醚TritonX-100,为0.1~0.3%的去氧胆酸钠,为5%-10%甘油,为0.8~1.2%的苯甲基磺酰氟PMSF;且调节所述细胞裂解缓冲液的PH值介于7.3~7.49之间。
实例一:细胞裂解缓冲液采用如下成分:
45mM的三羟甲基氨基甲烷Tris,280mM的氯化钠NaCl,1.5mM的乙二胺四乙酸DTA,0.1M的蔗糖,以及体积百分比为0.8%的聚乙二醇辛基苯基醚TritonX-100,为0.1%的去氧胆酸钠,为5%甘油,为0.8%的苯甲基磺酰氟PMSF。PH值调整为7.45:
试验表明,纯化得到的FGF-17蛋白活性为:
浓度ng/ml | 活性OD600 |
250 | 0.733 |
62.5 | 0.713 |
15.625 | 0.675 |
反例一:细胞裂解缓冲液采用如下成分:
20mM Tris-HCl,1mM EDTA-2Na,0.2M NaCl,1%Triton X-100,0.2%去氧胆酸钠,pH7.5。PH值调整为7.5,其余情况完全相同。
试验表明,纯化得到的FGF-17蛋白活性为:
浓度ng/ml | 活性OD600 |
250 | 0.621 |
62.5 | 0.601 |
15.625 | 0.538 |
申请人发现,细胞裂解缓冲液的组分和PH值对最终活性影响很大。
实例二:细胞裂解缓冲液采用如下成分:
55mM的三羟甲基氨基甲烷Tris,320mM的氯化钠NaCl,2.5mM的乙二胺四乙酸DTA,0.3M的蔗糖,以及体积百分比为1.2%的聚乙二醇辛基苯基醚TritonX-100,为0.3%的去氧胆酸钠,为10%甘油,为1.2%的苯甲基磺酰氟PMSF。PH值调整为7.3:
试验表明,纯化得到的FGF-17蛋白活性为:
浓度ng/ml | 活性OD600 |
250 | 0.741 |
62.5 | 0.729 |
15.625 | 0.700 |
实例三:细胞裂解缓冲液采用如下成分:
50mM的三羟甲基氨基甲烷Tris,300mM的氯化钠NaCl,2mM的乙二胺四乙酸DTA,0.2M的蔗糖,以及体积百分比为1%的聚乙二醇辛基苯基醚TritonX-100,为0.2%的去氧胆酸钠,为7.5%甘油,为1%的苯甲基磺酰氟PMSF。PH值调整为7.4:
试验表明,纯化得到的FGF-17蛋白活性为:
浓度ng/ml | 活性OD600 |
250 | 0.774 |
62.5 | 0.730 |
15.625 | 0.711 |
并且,通过本发明培养出来的FGF-17蛋白,其生物活性高于人体中自然生长的蛋白,具体请见图10。在图10中,从下向上第二条线是自然生长的蛋白,而第三条和第四条是本发明培养出来的FGF-17蛋白,显然其活性远高于自然生长的蛋白。
其中一种情况是,所述的重组人成纤维细胞生长因子-17蛋白的生产方法中,所述对表达工程菌进行培养包括:在16℃的条件下培养出可溶性FGF-17蛋白,而在37℃的条件下培养出包涵体FGF-17蛋白,如图3所示。
这个温度条件相当重要,是申请人通过大量实验而的出来的结论,具体请见下列实验结果:
实例四:
试验表明,纯化得到的FGF-17蛋白的活性为(其中以浓度为30ng/ml为例,此时活性最高):
温度 | 浓度30ng/ml时的活性OD600 |
16℃ | 0.761 |
37℃ | 0.760 |
38℃ | 0.714 |
15℃ | 0.681 |
17℃ | 0.662 |
36℃ | 0.657 |
上述测试均是在除温度不同,而其余条件相同的情况下做的实验。
所述的重组人成纤维细胞生长因子-17蛋白的生产方法中,所述大肠杆菌表达质粒为pET3a,如图1所示,其中显示出来双切酶的位置,且所述大肠杆菌宿主菌为BL21(DE3)plysS。
所述的重组人成纤维细胞生长因子-17蛋白的生产方法中,所述细胞裂解缓冲液中,包含:50mM的三羟甲基氨基甲烷Tris,300mM的氯化钠NaCl,2mM的乙二胺四乙酸DTA,0.2M的蔗糖,以及体积百分比为1%的聚乙二醇辛基苯基醚TritonX-100,为0.2%的去氧胆酸钠,为5%-10%甘油,为1%的苯甲基磺酰氟PMSF;且调节所述细胞裂解缓冲液的PH值为7.4。
所述的重组人成纤维细胞生长因子-17蛋白的生产方法中,所述对表达工程菌进行培养包括以下步骤:
1)将FGF-17蛋白表达工程菌以1:50~100的比例接种至第一代LB培养基中得到第一代菌;将第一代菌培养8h~12h,待菌浓度A600达到1.2~2.0左右时,按1:100~200的比例接种到含有磷酸盐缓冲液的第二代LB培养基中进行培养;
2)再培养2h~4h,待菌浓度A600达到0.6~0.8左右时,温度为15~38℃,诱导剂IPTG终浓度为0.3~0.5mM和0.8~1.2mM,时间为4~30h,培养箱转数为180rpm~200rpm,以进行诱导表达,如图2所示;以及
3)进行离心操作,将得到的FGF-17蛋白在-15℃以下冻存。
实例五:培养过程采用如下参数:
将FGF-17蛋白表达工程菌以1:100的比例接种至第一代LB培养基中得到第一代菌;将第一代菌培养12h,待菌浓度A600达到2.0左右时,按1:200的比例接种到含有磷酸盐缓冲液的第二代LB培养基中进行培养;
2)再培养4h,待菌浓度A600达到0.8左右时,温度为37℃,诱导剂IPTG终浓度为0.5mM和1.2mM,时间为30h,培养箱转数为200rpm,以进行诱导表达;以及
3)进行离心操作,将得到的FGF-17蛋白在-20℃冻存。
试验表明,纯化得到的FGF-17蛋白活性为:
浓度ng/ml | 活性OD600 |
250 | 0.701 |
62.5 | 0.727 |
15.625 | 0.709 |
在上述参数中,时间和浓度的组合配置非常关键,如果换其它配置,则实验结果如下:
反例二:培养过程采用如下参数:
将FGF-17蛋白表达工程菌以1:130的比例接种至第一代LB培养基中得到第一代菌;将第一代菌培养20h(过渡培养,对第二代培养不利),待菌浓度A600达到4.0左右时,按1:200的比例接种到含有磷酸盐缓冲液的第二代LB培养基中进行培养;
2)再培养2h,待菌浓度A600达到0.8左右时,温度为40℃,诱导剂IPTG终浓度为0.5mM和1.2mM,时间为30h,培养箱转数为200rpm,以进行诱导表达;以及
3)进行离心操作,将得到的FGF-17蛋白在-20℃冻存。
试验表明,纯化得到的FGF-17蛋白活性为:
浓度ng/ml | 活性OD600 |
250 | 0.558 |
62.5 | 0.603 |
15.625 | 0.572 |
可见,如果修改第一次和第二次培养的参数,会产生不良后果。
所述的重组人成纤维细胞生长因子-17蛋白的生产方法中,如果得到可溶性FGF-17蛋白,则在所述步骤2)中,所述温度为16℃,且所述时间为16~30h;而如果得到包涵体FGF-17蛋白,则在所述步骤2)中,所述温度为37℃,且所述时间为4~6h。
温度与时间的结合,也是本申请人的发现,其决定了蛋白的溶解度。
所述的重组人成纤维细胞生长因子-17蛋白的生产方法,在所述步骤3)中,得到的FGF-17蛋白在-20℃冻存。
所述的重组人成纤维细胞生长因子-17蛋白的生产方法,在所述步骤1)中,
第一代培养基中包括:胰蛋白胨10g/L,酵母粉5g/L,和氯化钠10g/L;
如果得到可溶性FGF-17蛋白,则第二代培养基中包括:胰蛋白胨10g/L,酵母粉10g/L,磷酸氢二钾3g/L,磷酸二氢钾1g/L,氯化钠4g/L,和葡萄糖5g/L;而
如果得到包涵体FGF-17蛋白,则第二代培养基中包括:胰蛋白胨10g/L,酵母粉4g/L,磷酸氢二钾3g/L,磷酸二氢钾1g/L,和氯化钠10g/L。
实例六:培养过程采用如下培养基:
第一代培养基中包括:胰蛋白胨10g/L,酵母粉5g/L,和氯化钠10g/L;
如果得到可溶性FGF-17蛋白,则第二代培养基中包括:胰蛋白胨10g/L,酵母粉10g/L,磷酸氢二钾3g/L,磷酸二氢钾1g/L,氯化钠4g/L,和葡萄糖5g/L;而
如果得到包涵体FGF-17蛋白,则第二代培养基中包括:胰蛋白胨10g/L,酵母粉4g/L,磷酸氢二钾3g/L,磷酸二氢钾1g/L,和氯化钠10g/L。
试验表明,纯化得到的FGF-17蛋白活性为:
浓度ng/ml | 活性OD600 |
1.953 | 0.561(可溶体)、0.571(包涵体) |
3.906 | 0.603(可溶体)、0.597(包涵体) |
31.25 | 0.652(可溶体)、0.665(包涵体) |
在上述参数中,时间和浓度的组合配置非常关键,如果换其它配置,则实验结果如下:
反例三:培养过程采用如下培养基:
第一代培养基中包括:胰蛋白胨13g/L,酵母粉8g/L,和氯化钠5g/L;
第二代培养基中包括:胰蛋白胨13g/L,酵母粉7g/L,磷酸氢二钾6g/L,磷酸二氢钾3g/L,氯化钠8g/L,和葡萄糖10g/L;
试验表明,在其余条件不变的情况下,纯化得到的FGF-17蛋白活性为:
浓度ng/ml | 活性OD600 |
1.953 | 0.505 |
3.906 | 0.574 |
31.25 | 0.532 |
所述的重组人成纤维细胞生长因子-17蛋白的生产方法中,在培养包涵体FGF-17蛋白时,仅使用肝素柱进行纯化,而不使用离子交换柱。如图5所示。这样不但能够节约成本,还能得到更好的纯化效果。
所述的重组人成纤维细胞生长因子-17蛋白的生产方法,如果得到可溶性FGF-17蛋白,则加入25倍量g/V的细胞裂解缓冲液,其中甘油为5%充分悬浮,然后低温超声破碎,其中功率为300w,振频输出为40%;然后超声5s停5s,总计超声10mins;
镜检在可见视野范围内无完整细胞结构的大肠杆菌后,以20000rpm离心,4℃,25mins,收集上清,留沉淀。
这是可溶性蛋白的独特处理方式,其要点在于间歇性超声,即超声5s停5s,总计超声10mins。这样能够提高纯化后蛋白的活性。
Claims (10)
- 一种重组人成纤维细胞生长因子-17蛋白的生产方法,其特征在于,至少包括以下步骤:步骤一、将天然序列的人成纤维细胞生长因子-17蛋白FGF-17按照大肠杆菌偏好进行重组和扩增,得到重组人成纤维细胞生长因子-17蛋白rhFGF-17基因,其碱基序列如SEQ ID No.1所示;步骤二、将重组后的rhFGF-17基因连接至大肠杆菌表达质粒,构成重组表达载体,然后将重组表达载体转化进入大肠杆菌宿主菌,得到FGF-17蛋白表达工程菌;以及步骤三、对表达工程菌进行培养,之后进行纯化;其中,在纯化过程中,采用如下浓度组分的细胞裂解缓冲液:45-55mM的三羟甲基氨基甲烷Tris,280~320mM的氯化钠NaCl,1.5~2.5mM的乙二胺四乙酸DTA,0.1~0.3M的蔗糖,以及体积百分比为0.8~1.2%的聚乙二醇辛基苯基醚TritonX-100,为0.1~0.3%的去氧胆酸钠,为5%-10%甘油,为0.8~1.2%的苯甲基磺酰氟PMSF;且调节所述细胞裂解缓冲液的PH值介于7.3~7.49之间。
- 如权利要求1所述的重组人成纤维细胞生长因子-17蛋白的生产方法,其特征在于,所述对表达工程菌进行培养包括:在16℃的条件下培养出可溶性FGF-17蛋白,而在37℃的条件下培养出包涵体FGF-17蛋白。
- 如权利要求1所述的重组人成纤维细胞生长因子-17蛋白的生产方法,其特征在于,所述大肠杆菌表达质粒为pET3a,且所述大肠杆菌宿主菌为BL21(DE3)plysS。
- 如权利要求1所述的重组人成纤维细胞生长因子-17蛋白的生产方法,其特征在于,所述细胞裂解缓冲液中,包含:50mM的三羟甲基氨基甲烷Tris,300mM的氯化钠NaCl,2mM的乙二胺四乙酸DTA,0.2M的蔗糖,以及体积百分比为1%的聚乙二醇辛基苯基醚TritonX-100,为0.2%的去氧胆酸钠,为5%-10%甘油,为1%的苯甲基磺酰氟PMSF;且调节所述细胞裂解缓冲液的PH值为7.4。
- 如权利要求1所述的重组人成纤维细胞生长因子-17蛋白的生产方法,其特征在于,所述对表达工程菌进行培养包括以下步骤:1)将FGF-17蛋白表达工程菌以1:50~100的比例接种至第一代LB培养基中得到第一代菌;将第一代菌培养8h~12h,待菌浓度A600达到1.2~2.0左右时,按1:100~200的比例接种到含有磷酸盐缓冲液的第二代LB培养基中进行培养;2)再培养2h~4h,待菌浓度A600达到0.6~0.8左右时,温度为15~38℃,诱导剂IPTG终浓度为0.3~0.5mM和0.8~1.2mM,时间为4~30h,培养箱转数为180rpm~200rpm,以进行诱导表达;以及3)进行离心操作,将得到的FGF-17蛋白在-15℃以下冻存。
- 如权利要求5所述的重组人成纤维细胞生长因子-17蛋白的生产方法,其特征在于,如果得到可溶性FGF-17蛋白,则在所述步骤2)中,所述温度为16℃,且所述时间为16~30h;而如果得到包涵体FGF-17蛋白,则在所述步骤2)中,所述温度为37℃,且所述时间为4~6h。
- 如权利要求5所述的重组人成纤维细胞生长因子-17蛋白的生产方法,其特征在于,在所述步骤3)中,得到的FGF-17蛋白在-20℃冻存。
- 如权利要求5所述的重组人成纤维细胞生长因子-17蛋白的生产方法,其特征在于,在所述步骤1)中,第一代培养基中包括:胰蛋白胨10g/L,酵母粉5g/L,和氯化钠10g/L;如果得到可溶性FGF-17蛋白,则第二代培养基中包括:胰蛋白胨10g/L,酵母粉10g/L,磷酸氢二钾3g/L,磷酸二氢钾1g/L,氯化钠4g/L,和葡萄糖5g/L;而如果得到包涵体FGF-17蛋白,则第二代培养基中包括:胰蛋白胨10g/L,酵母粉4g/L,磷酸氢二钾3g/L,磷酸二氢钾1g/L,和氯化钠10g/L。
- 如权利要求2所述的重组人成纤维细胞生长因子-17蛋白的生产方法,其特征在于,在培养包涵体FGF-17蛋白时,仅使用肝素柱进行纯化,而不使用离子交换柱。
- 如权利要求4所述的重组人成纤维细胞生长因子-17蛋白的生产方法,其特征在于,如果得到可溶性FGF-17蛋白,则加入25倍量g/V的细胞裂解缓冲液,其中甘油为5%充分悬浮,然后低温超声破碎,其中功率为300w,振频输出为40%;然后超声5s停5s,总计超声10mins;镜检在可见视野范围内无完整细胞结构的大肠杆菌后,以20000rpm离心,4℃,25mins,收集上清,留沉淀。
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CN117838835A (zh) * | 2024-02-01 | 2024-04-09 | 暨南大学 | 成纤维细胞生长因子17蛋白和/或其激活剂在制备治疗阿尔兹海默病药物中的应用 |
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CN107012150A (zh) * | 2017-05-12 | 2017-08-04 | 温州医科大学 | 重组人成纤维细胞生长因子‑16的克隆、表达和应用及其生物学活性的测定方法 |
CN107217069B (zh) * | 2017-05-31 | 2020-06-26 | 何伟 | 原核表达载体及rbFGF-2表达方法与工程菌和应用 |
CN111100795A (zh) * | 2019-12-27 | 2020-05-05 | 深圳康泰生物制品股份有限公司 | 表达手足口病疫苗抗原的重组汉逊酵母细胞破碎缓冲液及其制备方法与应用 |
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