CN109486771B - CHO cell strain construction method for high expression SOD3 and SOD3 protein purification method - Google Patents
CHO cell strain construction method for high expression SOD3 and SOD3 protein purification method Download PDFInfo
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- CN109486771B CN109486771B CN201811638520.XA CN201811638520A CN109486771B CN 109486771 B CN109486771 B CN 109486771B CN 201811638520 A CN201811638520 A CN 201811638520A CN 109486771 B CN109486771 B CN 109486771B
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Abstract
The invention discloses a construction method of a CHO cell strain with high expression SOD3 and a purification method of SOD3 protein, wherein the construction method comprises the following steps: firstly, constructing a lentiviral vector plasmid for expressing the SOD3 gene; secondly, transfecting a 293T cell with a lentiviral vector plasmid and a packaging plasmid psPAX2 and pMD2.0G together, and packaging into a lentivirus carrying an SOD3 coding gene; and thirdly, infecting CHO cells with lentiviruses carrying SOD3 coding genes, screening by puromycin to obtain a CHO cell group expressing SOD3 protein, further screening the CHO cells expressing SOD3 protein after the puromycin screening by a limiting dilution method and a Western blot method to obtain a stable SOD3+ CHO cell strain, and performing fermentation culture and protein separation and purification to obtain the SOD 3. The invention can produce SOD3 protein with biological activity in large scale.
Description
Technical Field
The invention belongs to the technical field of cell biology, and relates to a construction method of a CHO cell strain with high expression of SOD3 and a purification method of SOD3 protein.
Background
Superoxide Dismutase (SOD for short) is an important antioxidant enzyme in organisms, and is widely distributed in various organisms such as animals, plants, microorganisms and the like. In animal bodies, SOD can be divided into three types, wherein SOD1 is a dimeric protein containing copper (Cu) and zinc (Zn) metal prosthetic groups, also called Cu, Zn-SOD, mainly exists in cytoplasm, and other existing parts also include mitochondrial membrane space (inter membrane space), nucleus, lysosome and peroxisome; SOD2 is a tetrameric protein containing manganese (Mn) metal prosthetic group, also called Mn-SOD, existing in mitochondrial matrix; SOD3 is a tetrameric protein containing copper (Cu) zinc (Zn) metal prosthetic group, and is mainly present on extracellular matrix and cell surface (99% of SOD 3), and a small part is present in blood plasma and extracellular fluid, also known as ecSOD-SOD (extracellular SOD).
The body can generate a large amount of active oxygen (reactive oxygen) during aerobic respirationgen species (ROS)) (e.g.1O2，O2 -Hydroxyl radical (. OH) and hydroperoxyl radical (HO)2) Hydrogen peroxide (H)2O2) And lipid peroxide (ROOH), NO and O2 -Strong oxidant formed after action, namely peroxynitroso ONOO-) Intracellular/extracellular low levels of ROS are indispensable in many vital phenomena (e.g. intracellular signalling, the body's defense reactions against microbes, etc.). Under normal conditions, the generation and the elimination of ROS in a body are balanced, and when the ROS is excessively generated and cannot be eliminated in time, the excessive ROS can damage biological macromolecules such as protein, nucleic acid and lipid, so that the normal physiological and biochemical functions of the ROS are influenced, and the cells are aged and killed. SOD can be obtained by catalyzing O2-Carrying out disproportionation reaction to generate hydrogen peroxide and oxygen, and converting the hydrogen peroxide into water under the action of catalase, glutathione peroxidase and peroxide reductase, thereby reducing or eliminating the super O2-And the oxidative or peroxidative reactions induced by metabolites or derivatives thereof, such as cell membrane damage and destruction; inactivation of serum protease; the damage of DNA causes the occurrence and accumulation of cell mutation, etc.
SOD3 has different expression in different tissues among different species, but SOD3 is generally highly expressed in blood vessels, lungs, kidneys and uterus; expression of SOD3 was weaker in the heart than in the above tissues. In vascular tissue, SOD3 is synthesized primarily by vascular smooth muscle cells and fibroblasts; in atherosclerotic tissues, inflammatory cells may also synthesize SOD 3. SOD3 is secreted by the above cells, and then binds to Heparin Sulfate Proteoglycan (HSPGs), collagen and fibulin-5 to fix extracellular matrix and endothelial cell surface, and is taken into endothelial cells by endocytosis to exert anti-ROS effect. The important physiological function of SOD3 can be reflected in some diseases, for example, in some human species, the 213 th arginine of SOD3 protein is replaced by glycine (R213G), resulting in a decrease in binding of SOD3 to heparan sulfate, and thus a decrease in tissue binding and an increase in plasma SOD 3. Juul et al, in a prospective study of 9188 people from Copenhagen, found that the risk of ischemic heart disease in the R213G heterozygote was significantly increased; the over-expression of SOD3 can inhibit tumor angiogenesis and tumor invasion and infiltration; in a rat hind limb ischemia model, SOD3 over-expressed in ischemic hind limb tissues can activate AP1 and CRE transcription factors through a Ras-Mek-Erk pathway and increase the expression of Vascular Endothelial Growth Factor (VEGF) -A and cyclin D1, thereby promoting cell proliferation, improving the metabolic function of damaged tissues and accelerating the healing of the damaged tissues.
In conclusion, the special function of SOD can make it have good preventing and curing effect for senility, inflammation, tumor and tumor induced by superoxide anion free radical after chemotherapy and radiotherapy, radiation disease, autoimmune disease, digestive system disease (chronic gastritis, atrophic gastritis, etc.), respiratory system disease (asthma, bronchitis, etc.), cardio-cerebral ischemia-reperfusion syndrome, cataract, dermatosis (contact dermatitis, solar dermatitis, chloasma, etc.), burn, etc. and the SOD also has good curative effect for some subhealth state. Therefore, the SOD3 protein with biological activity has the biological effect of ROS resistance in various diseases, and has great application prospect.
Disclosure of Invention
The invention provides a construction method of a CHO cell strain with high expression of SOD3 and a purification method of SOD3 protein, and aims to produce SOD3 protein with biological activity in a large scale.
The purpose of the invention is realized by the following technical scheme:
a construction method of a CHO cell strain with high expression of SOD3 comprises the following steps:
firstly, connecting the nucleotide sequence of SOD3 to a PUC57 cloning vector to construct a lentiviral vector plasmid pCDH-CMV-MCS-EF1-SOD3-Puro for expressing SOD3 gene;
secondly, transfecting 293T cells with a lentiviral vector plasmid pCDH-CMV-MCS-EF1-SOD3-Puro and packaging plasmids psPAX2 and pMD2.0G together, and packaging into a lentivirus carrying an SOD3 encoding gene;
thirdly, infecting CHO cells with lentivirus carrying SOD3 coding gene, obtaining CHO cell group expressing SOD3 protein after puromycin screening, and further using limiting dilution method to further screen the table after puromycin screeningScreening CHO cells which reach SOD3 protein to obtain stable SOD3+CHO cell line.
A method for purifying SOD3 protein comprises the following steps:
firstly, connecting the nucleotide sequence of SOD3 to a PUC57 cloning vector to construct a lentiviral vector plasmid pCDH-CMV-MCS-EF1-SOD3-Puro for expressing SOD3 gene;
secondly, transfecting 293T cells with a lentiviral vector plasmid pCDH-CMV-MCS-EF1-SOD3-Puro and packaging plasmids psPAX2 and pMD2.0G together, and packaging into a lentivirus carrying an SOD3 encoding gene;
infecting CHO cells with lentivirus carrying SOD3 coding genes, screening by puromycin to obtain a CHO cell group expressing SOD3 protein, and further screening the CHO cells expressing SOD3 protein after the puromycin screening by a limiting dilution method and a Western blot method to obtain a stable SOD3+ CHO cell strain;
fourthly, the obtained SOD3+ CHO cell strain is amplified, separated and purified by the secretory SOD3 protein, namely: and carrying out ultrafiltration, ammonium sulfate sedimentation, ion exchange chromatography and gel chromatography purification on the obtained secretory protein of the SOD3+ CHO cell strain to obtain the SOD3 protein in a pure enzyme form.
In the invention, the specific steps of infecting the CHO cell with the lentivirus carrying the SOD3 coding gene are as follows: 293T cells release lentivirus carrying SOD3 coding genes, supernatant is collected, and CHO cells are infected with lentivirus liquid after ultra-high speed centrifugal concentration.
In the present invention, the method for screening CHO cells is as follows: a CHO cell group integrated with SOD3 is screened by a selection medium containing puromycin, then a 96-well plate is paved by a limiting dilution method, a positive CHO cell strain capable of efficiently expressing SOD3 protein is further screened by Dot blot (Dot blot) and Western blot (Western blot), and the obtained positive CHO cell strain is subjected to suspension fermentation culture.
The invention has the following advantages:
the invention constructs a CHO cell strain for stably and efficiently expressing SOD3 and establishes a method for separating and purifying SOD3 in a cell culture solution of SOD 3. The invention aims to produce SOD3 protein with biological activity in large scale, which is characterized in that it is expressed by eukaryotic system, thus having post-translational modification (such as protein space folding, glycosylation modification, etc.) of secretory protein of eukaryotic cell, thus having the disadvantage of no biological activity of protein expressed differently from prokaryotic (such as colibacillus, yeast) due to lack of post-translational modification.
Drawings
FIG. 1 is a backbone plasmid map of the lentiviral vector pCDH-CMV-MCS-EF 1-Puro;
FIG. 2 is a diagram of SOD3 DNA gel electrophoresis;
FIG. 3 is a gel electrophoresis diagram of pCDH-CMV-MCS-EF1-Puro vector and SOD3 after enzyme digestion and enzyme ligation to obtain pCDH-CMV-MCS-EF1-SOD3-Puro lentiviral vector plasmid, and after enzyme digestion again, M: marker, 1: pCDH-CMV-MCS-EF1-Puro vector and SOD 3;
FIG. 4 is a microscopic image of a CHO cell before and after infection with a virus, a before infection with a lentivirus and b after infection with a lentivirus;
FIG. 5 is a flow diagram of lentivirus-sensitive CHO cells;
FIG. 6 is a Dot blot screening diagram of a CHO cell strain 96-well plate expressing SOD 3;
FIG. 7 is a Western blot diagram of high expression CHO cell lines (A6, B4, E7, H10);
FIG. 8 shows the monitoring of cell density and viability in a 3L shake flask of SOD3+ CHO cell line;
FIG. 9 shows the monitoring of the expression level of SOD3 as a target protein in a 3L shake flask of a SOD3+ CHO cell strain;
FIG. 10 shows the enzymatic analysis of SOD3 at different temperatures;
FIG. 11 shows the thermal stability analysis of SOD 3;
FIG. 12 is an enzymatic analysis of SOD3 at different pH conditions;
FIG. 13 is an analysis of the ability of SOD3 to resist hydrolytic enzymes;
fig. 14 shows the results of anti-apoptotic flow analysis of SOD3, a: control a431 cells; b: pretreatment with SOD3 protein, then H2O2Post-treatment a431 cells; c: h2O2A431 cells after treatment.
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings, but not limited thereto, and any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be covered by the protection scope of the present invention.
Example 1: the SOD3 gene is inserted into a lentiviral expression vector pCDH-CMV-MCS-EF1-Puro
The gene sequence of SOD3 is derived from Human, and is shown in SEQ ID NO.1 of the sequence table. The invention entrusts Beijing Hua Dageney technology Limited company to synthesize SOD3 gene, and connects to PUC57 cloning vector for amplification sequencing.
1. Designing a primer: the gene sequence (SEQ ID NO.1) of SOD3 was analyzed using sequence analysis software pDRAW32, restriction sites EcoRI and NotI were suitable for gene cloning, and could be matched with the multiple cloning site of pCDH-CMV-MCS-EF1-Puro vector (FIG. 1), and the primers at both ends were designed as follows:
EcoRI-upstream primer: CGGAATTCATGCTGGCGCTACTGTGTTCC, respectively;
NotI-downstream primer: TTGCGGCCGCTCAGCGCAGTTCCCACCACT are provided.
2. And (3) PCR amplification: after synthesis of the gene fragments and primers, PCR was performed in the system shown in Table 1(Toyobo KOD Fx enzyme).
TABLE 1
| 2 XPCR buffer for KOD Fx | 25μL |
| 2mM dNTP | |
| 10mM primer | |
| 1 | 1.5 |
| 10mM primer | |
| 2 | 1.5μL |
| SOD3 structural gene | 1μL(50ng/μl) |
| KOD FX | 1μL |
| H2O | 10μL |
| Total volume | 50μL |
3. PCR procedure: see table 2.
TABLE 2
| Step 1: 94 |
2 minutes |
| Step 2: 98 |
10 seconds |
| And step 3: 50 |
30 seconds |
| And 4, step 4: 68 |
1 minute, to step 2, 5 cycles |
| And 5: 98 |
10 seconds |
| Step 6: 60 |
30 seconds |
| And 7: 68 |
1 minute, to step 5, 30 cycles |
| And 8: 68 |
10 minutes |
| And step 9: 4 |
1 hour |
4. 10uL of the PCR product was electrophoresed (see FIG. 2). The remaining PCR product (40. mu.l) was subjected to gel electrophoresis, and the gel (Biomiga, cat: DC-3511-01) was recovered, and after quantification of the concentration of the PCR fragment by comparison with the brightness of DNA marker, the digestion was carried out.
5. Enzyme digestion: the digestion reaction system is shown in Table 3.
TABLE 3
Carrying out water bath at 37 ℃, and carrying out enzyme digestion for 1-2 hours.
6. 10uL of the cleavage product was electrophoresed (see FIG. 3). The remaining PCR product (40. mu.l) was subjected to gel electrophoresis, and the gel (Biomiga, cat. No.: DC-3511-01) was recovered and subjected to the manufacturer's instructions.
7. And (3) connection reaction:
according to the mole number of the carrier n: fragment n mole number 1: 3 (i.e.:
) The ligation system was formulated (Table 4).
TABLE 4
| pCDH-CMV-MCS-EF1-Puro vector | 2.3uL(100ng) |
| SOD3 | 1.2uL |
| H2O | 1.5uL |
| Solution I (Takara) | 5uL |
| 10uL |
Adding the components into a 1.5mL centrifuge tube, and fully and uniformly mixing; water bath at 22 ℃ for 30 minutes.
8. Conversion, ampicillin coated plates: adding the ligation product into 50 mu L of competence, flicking, and standing for 30 minutes; water bath at 42 ℃ for 90 seconds; standing on ice for 5 minutes; adding 500 mu L of LB without antibiotics, and activating for 1 hour at 37 ℃; centrifuging at 4000rpm for 5 minutes, removing the supernatant, adding 50 microliter of antibiotic-free LB, resuspending by a sample adding gun, and coating an ampicillin resistant plate; incubate at 37 ℃ overnight.
9. Selecting and cloning, shaking bacteria: a negative control is arranged during the ligation reaction, and the number of clones in the plate added with the pCDH-CMV-MCS-EF1-SOD3-Puro fragment is obviously more than that of the negative control clones without the fragment when observed on the second day, so the ligation is judged to be successful. Randomly picking 6 clones, and shake bacteria for amplification.
10. Bacterial liquid PCR: taking 1 mul of bacterial liquid, carrying out PCR by using EcoRI-upstream primer and NotI-downstream primer, picking positive bacteria, and extracting plasmid from corresponding bacterial liquid.
11. Plasmid extraction: the Biomiga plasmid extraction kit (cat # PD1220-02) was used for plasmid extraction according to the manufacturer's instructions.
12. Sequencing: 3 plasmids with higher concentration are selected from the positive plasmids, the sequencing result is compared with SEQ ID NO.1 and is completely correct, and the lentiviral vector plasmid pCDH-CMV-MCS-EF1-SOD3-Puro for expressing the SOD3 gene is obtained.
Example 2 packaging of pCDH-CMV-MCS-EF1-Puro-SOD3 lentivirus
1. A50 mL centrifuge tube was pre-warmed with 8mL fresh complete medium, wiped dry, and placed in a biosafety cabinet. And taking out the frozen 293T cells from the liquid nitrogen tank, quickly placing the cells into a 37 ℃ water bath, quickly shaking, and completely dissolving the cell suspension within 1-2 min as far as possible. The cell suspension was slowly added dropwise to the above 8ml of preheated fresh medium, centrifuged at 1500rpm for 5 min. The supernatant was removed and 1mL of fresh complete medium was added to resuspend the cell pellet and then added to a T25 flask supplemented with 8mL of medium. Smoothly adding 5% CO at 37 deg.C2And 95% relative humidity. And (5) regularly observing the cell morphology and the confluence, carrying out passage according to a ratio of 1:5 when the confluence reaches 90%, and expanding the cell number to the required cell number.
2. One day before packaging the virus, cells were trypsinized, 1X 106～1×107Cells/dish were seeded on 10cm culture dishes (NEST).
3. When cells are transfected, the shuttle plasmid pCDH-CMV-MCS-EF1-SOD3-Puro needs to be co-transfected with the packaging plasmids psPAX2 and pMD2.0G. 5. mu.g of pCDH-CMV-MCS-EF1-SOD3-Puro, 3.75. mu.g of psPAX2 plasmid and 1.25. mu.g of pMD2.0G plasmid were used. For transfection, the mixture of the three plasmids was added to 500. mu.l MEM medium, 25. mu.l Lipofectamine 2000 reagent was added to 500. mu.l MEM medium in another microcentrifuge tube, then the diluted transfection reagent was added dropwise onto the diluted plasmid, mixed well, centrifuged, and left to stand at room temperature for 20 minutes, during which time the complete medium (DMEM + 10% FBS) of 293T cells in a 10cm dish was replaced with MEM serum-free medium preheated to 37 ℃, and CO was placed2Incubator flatAnd (5) balancing the pH value. After 20 minutes, the mixture of plasmid and transfection reagent was added to a 10cm petri dish, gently shaken 10 times, mixed well, and CO was added2And (5) culturing an incubator. Fresh complete medium (DMEM + 10% FBS) was replaced after 6 hours.
4. After 3 days of cell transfection, the virus was harvested, the supernatant of 10 ml/dish virus-containing medium was transferred into a 50ml centrifuge tube, 4 ℃, 1250rpm, 5 minutes to remove dead 293T cells, then the virus fluid was transferred into a high speed centrifuge tube, trimmed, concentrated by centrifugation (25, 000rpm (82, 700g), 4 ℃, 2 hours) using a super high speed centrifuge (Beckman SW28), the supernatant was discarded, the viral particles were resuspended in 0.9% physiological saline, gently blown and mixed, aliquoted, frozen at-80 ℃, and the titer of the remaining portion of virus was determined.
Example 3: CHO cell infected by lentivirus and screening, amplifying and culturing of infected CHO cell
The host cell used in the present invention is a serum-free domesticated CHO cell, and the culture medium thereof is a CD CHO medium (Gibco, Cat No. 10743001).
Screening for stably transfected CHO cell populations integrated into SOD3 with puromycin (puromycin); then, carrying out low-density plate laying and Western blot detection and screening to obtain a stable positive CHO cell strain with the highest SOD3 protein expression quantity; and (3) performing fermentation culture on the screened CHO cell strain which stably expresses the SOD3 protein and is positive. The specific implementation is as follows:
1. by 2X 106One well, 2 ml/well, CHO cells were inoculated in 6-well plates, and then infected overnight with pCDH-CMV-MCS-EF1-SOD3-Puro virus solution at MOI of 10.
2. The day after infection, each well was supplemented with 2ml of fresh medium.
3. On the third day of infection, CHO cells are proliferated vigorously and have high cell density, 2ml of fresh culture medium is added into each hole, and on the fourth day of infection, the cells in each hole are transferred into a T25 culture bottle and supplemented with a proper amount of culture medium to ensure that the cell density is 1 multiplied by 106Per ml; on the fifth day of infection, the cells in the above culture flasks were packed at 2X 1062 ml/well, inoculating to 6-well plate, adding puromycin at final concentration of 1. mu.g/ml, 2. mu.g/ml, 3. mu.g/ml for positive cell screening, repeating three wells per concentration. Then, observing CHO cells in each hole added with puromycin with final concentration of 1 mug/ml, 2 mug/ml and 3 mug/ml every day, if cell death is obvious, transferring the cell suspension into a 15ml centrifuge tube, 1250rpm, centrifuging for 5 minutes, discarding supernatant, after 2ml fresh culture medium is re-suspended, adding puromycin with corresponding concentration again to continue positive cell screening, and discarding cells in a hexawell plate without obvious change of puromycin. And repeating the screening process, and adding puromycin for screening for 5-7 days. After the sixth day of screening, the surviving and well-conditioned CHO strain was kept after the highest dose of puromycin had been added and further expanded in T25 flasks.
4. Determining the positive rate of the SOD3 molecule expressed in CHO cells after screening: 2X 10 CHO cells selected in 3 above were used6And (4) respectively. Centrifugation, washing with flow-through wash 2 times, fixing and membrane-breaking CHO cells, dividing into two tubes, resuspending the cells with 100. mu.l of flow-through wash, labeling one tube with rabbit anti-human SOD3 antibody (FITC direct standard antibody), and adding isotype control antibody to the other tube for flow detection. The results are shown in FIG. 5.
5. Dot blot screening CHO cell strain expressing SOD3
SOD3-CHO cells after the puromycin screening were seeded in a 96-well plate by limiting dilution method. And (3) performing Dot blot screening when the cells grow on a 96 plate until the cell confluency reaches 80 percent: using an NC membrane or a PVDF membrane (soaking methanol for 1 minute and then washing with distilled water for 5 minutes by shaking), taking 10 mul of culture medium supernatant after cells are removed by centrifugation from each well according to the inoculation sequence of a 96-well plate, adding a corresponding amount of 5x Loading buffer, and boiling for 5 min; spotting the membrane, 5. mu.L/spot, setting negative control (empty culture medium, supernatant of untransfected CHO cell culture medium) and positive control (A12 well, 2. mu.g recombinant human SOD3 protein)，cat.h00006649-Q01Abnova) and marked with a ball-point pen; air-drying the membrane, placing the membrane in 5% skimmed milk powder/TBST confining liquid, and sealing for lh by a shaking table at room temperature; washing with TBST for 1-2 times; 1, 800 rabbit anti-human SOD3 primary antibody (prepared by 5% skimmed milk powder/TBST) is incubated for 2 hours at room temperature; TBST washing for three times, each time for 10 min; adding goat anti-rabbit IgG secondary antibody-HRP diluent (prepared by TBST) into the mixture according to the ratio of 1: 1000, and incubating for 1 h; TBST washing for three times, each time for 10 min; ECL development (fig. 6).
6. Western blot semi-quantitative detection of SOD3 protein in supernatant of CHO cell strain culture medium with high expression of SOD3
And collecting the CHO cells (four holes of A6, B4, E7 and H10) with high expression SOD3 screened by the Dot blot, transferring the CHO cells into a 24-hole plate for continuous culture, and continuing transferring the CHO cells into a 6-hole plate for amplification culture until the confluency reaches 90%. And taking the culture solution supernatant of each well for Western blot detection. Sample treatment: after determination of total protein concentration from cell supernatants (cells in 6-well plates) derived from expanded cells from the above four wells (four wells a6, B4, E7, H10), 25 μ g of equal mass equal volume load: adding a corresponding amount of Loading buffer, and boiling for 5 min; SDS-PAGE electrophoresis; transferring membrane, after electrophoresis, carefully peeling off the gel, packaging with filter paper, fiber pad and nitrocellulose membrane in the correct order, and soaking in transfer buffer solution at-20 deg.C for 40 min; loading a transfer electrophoresis box, putting a rotor and an ice box, connecting a power supply, and transferring for 1h on a magnetic stirrer at a constant voltage of 100V; removing the transfer device after the transfer is finished, removing the film, marking the front side and the back side (the side where the film and the glue are adhered is the front side), adding confining liquid, and standing overnight at 4 ℃; washing the membrane for 1-2 times by TBST; diluting the primary antibody diluent with TBST, and incubating for 1h on a horizontal shaking table at room temperature; TBST washing for three times, each time for 10 min; diluting the secondary antibody with TBST, and incubating for 1h on a horizontal shaking table at room temperature; TBST was washed three times for 10min each, and ECL developed (FIG. 7).
Example 4: 3L Shake flask fed-batch culture of Stable cell lines
And (3) transferring the CHO cell strain E7 with high expression of SOD3 further screened by Dot blot and Western blot from a six-hole plate to a T75 culture bottle for continuous amplification culture to a certain quantity.
3L shake flask fed-batch culture: using CHO fermentation special medium CDM4CHOTM80mL of seed solution of CHO cell line highly expressing SOD3 (HyCloneCat No: SH30558) was prepared at a density of 4.0X 106one/mL. The rotation speed was 110rpm, the pH was controlled to 7.2, and the culture temperature was 37 ℃. Adding into 3L shake flask, counting every day, observing cell state, and supplementing CDM4CHOTMAdjusting the cell density to 2.0 × 106Stopping adding CDM4CHO until cell culture solution reaches 1LTMContinuing to culture, measuring the content of residual sugar every day until the cell density reaches 6-7 multiplied by 106Per mLThen, the temperature is reduced, and the culture medium Cell Boost5 is fedTM(HyClone, Cat No:: SH30865.2,) the culture was continued with the sugar content kept at 3 g/L. The results showed that the cell density doubled daily for the first three days in large shake flasks and reached as high as 6.4X 10 for the third day6cell/mL (figure 8), the cell density is not changed greatly when the temperature is reduced after three days, the cells begin to produce the target protein in large quantity, the cell death rate is increased in the later period of fermentation, but the target protein is accumulated all the time, and the final protein concentration can reach 90mg/L (figure 9).
Example 5: purification of recombinant SOD3 protein
The specific purification steps are as follows:
and (2) centrifuging the fermentation culture cell suspension prepared in the embodiment 4 at 4000rpm for 10min to remove cell fragments, taking the supernatant as a crude enzyme solution, performing ultrafiltration by using an external pressure type hollow fiber ultrafiltration membrane with the molecular weight cutoff of 6000Da to remove small molecular impurities in the crude enzyme solution, and concentrating by 3-5 times.
Placing the concentrated solution of the crude enzyme solution in an ice bath, slowly adding ammonium sulfate to 85% while stirring, centrifuging at 13000rpm for 15min, taking a precipitate, dissolving again by using a buffer solution, placing the precipitate in a dialysis bag with the molecular weight cutoff of 6000Da, taking 20mM Tris-HCl as an external dialysis solution with the pH value of 8.0 and 20mM, dialyzing for 12-16 h at 4 ℃, replacing the external dialysis solution once every 4h, concentrating the internal dialysis solution by using a vacuum rotary evaporator after dialysis, freeze-drying, and placing in a low-temperature refrigerator at-20 ℃ for later use.
20mg of the lyophilized powder obtained above was put into a centrifuge tube, and 2ml of 50mM Tris-HCl buffer solution (pH 8.0) was added to dissolve it sufficiently, and then applied to a TOSOH Toyopearl EDAE-650C anion column. The method comprises the steps of firstly balancing a column by using 50mM Tris-HCl buffer solution with pH8.0, then feeding a sample, eluting 5 column volumes by using a 0-0.8 mol/L NaCl gradient prepared by the same buffer solution, wherein the flow rate is 1ml/min, and collecting by using a partial collector, wherein each tube contains 3 ml. The solution in the collection tube was then assayed for SOD3 viability and protein electrophoretic analysis.
The collected SOD3 was further purified by SuperdeX75 gel chromatography: the active peak after ion exchange separation was collected, concentrated, desalted, lyophilized, dissolved in 20mM PBS buffer at pH7.0, and applied to Superdex75HPLC column. The column was equilibrated with pH7.0, 20mM PBS buffer, loaded, eluted with pH7.0, 20mM PBS buffer for 1.5 column volumes at a flow rate of 0.25ml/min, collected by peak, and the collected samples were assayed for SOD3 activity and subjected to protein electrophoresis.
After the purification, the specific activity of SOD3 was increased from 237U/mg of the crude enzyme solution to 3447U/mg of the pure enzyme, the purification fold was 14.5, and the yield was 13.8. (enzyme activity unit is defined in the following way, in 1ml of reaction solution, the enzyme quantity which can inhibit the pyrogallol autoxidation rate by 50% per minute is defined as one enzyme activity unit, 325nm, and 0.030OD/min is one enzyme activity unit.)
Example 6: enzymatic Properties analysis of recombinant SOD3
Determination of optimum reaction temperature and analysis of thermal stability: performing enzymatic reaction in a phosphate (pH7.0) buffer system at different temperatures (30-80 ℃). The thermal stability study is to treat the mixture for 10-60 min at different temperatures and then to determine the enzyme activity. The optimum reaction temperature of SOD3 was 40 ℃ as shown in FIG. 10. Keeping the temperature within the range of 30-50 ℃ for 60min, and keeping the residual enzyme activity at more than 85%. The temperature is kept at 60 ℃ for 30min, the residual enzyme activity is about 80 percent, and the SOD3 has better thermal stability (figure 11).
Determination of optimum pH: the SOD3 prepared in example 4 was subjected to enzymatic reactions at different pH to determine its optimum pH. The buffer solution is a wide range of buffer solutions (citric acid, potassium dihydrogen phosphate, boric acid, sodium hydroxide, barbital) with a pH of 2.0-10.0. The pH adaptation results of SOD3 in buffers with different pH values at 40 ℃ show that the optimum pH value of SOD3 is 7.0-8.0 (FIG. 12). SOD3 enzyme solutions were treated in buffers of different pH values at room temperature for 60min, and the residual enzyme activity was determined to investigate the pH stability of SOD 3. The results show that the residual activity of SOD3 is above 90% at pH4.0-9.0. This indicates that SOD3 has good pH stability.
Analysis of the ability of SOD3 to resist hydrolytic enzymes: 0.05ml of trypsin (0.1mg/ml, prepared with pH7.0PBS buffer) and pepsin (0.1mg/ml, prepared with p H2.0 glycine-HCL buffer) were added to each SOD3 enzyme solution, treated at 37 ℃ for 30-240 min, and measured for SOD3 activity after dilution. After being treated by trypsin for 240min, the residual enzyme activity of SOD3 is still maintained at 100%, and no obvious loss exists; after 240min of pepsin treatment, the residual enzyme activity of SOD3 is about 65%, which indicates that SOD3 has better anti-protease hydrolysis capability (figure 13).
Functional analysis of the SOD3 enzyme:
a431 cells were arranged at 1X 105The cells were inoculated in 24-well plates and prepared for apoptosis when the confluency of cells reached 80%: washing each well cell with PBS twice, replacing with fresh serum-free DMEM medium, adding the prepared SOD3 protein at concentration of 10 μmol/L, 1 μmol/L, 0.1 μmol/L, and placing in CO2Incubate for 2 hours in incubator, wash with PBS 2 times, replace fresh 10% serum/DMEM medium and add 400. mu. M H2O2Cells from each well were harvested 6 hours after treatment and flow assayed for ANNEXIN V and PI to determine the apoptosis rate of A431 cells, as shown in FIG. 14.
The results show that 10. mu. mol/L SOD can effectively protect H2O2The resulting apoptosis of A431 cells (control A431 cell apoptosis rate of 6.27%; pretreatment with SOD3 protein, H)2O2The apoptosis rate of A431 cells after treatment is 20.49%; h2O2The apoptosis rate of A431 cells after treatment is 35.91 percent, the flow-type result of A431 cells pretreated by 1 mu mol/L and 0.1 mu mol/L SOD3 protein is not shown), and the prepared SOD3 protein is proved to have biological function.
<110> Shenzhen Shenqi Biotech Limited
<120> construction method of CHO cell strain with high expression SOD3 and purification method of SOD3 protein
<160>1
<210> 1
<211> 723
<212> DNA
<213> SOD3 Gene sequence
<400> 1
atgctggcgc tactgtgttc ctgcctgctc ctggcagccg gtgcctcgga cgcctggacg 60
ggcgaggact cggcggagcc caactctgac tcggcggagt ggatccgaga catgtacgcc 120
aaggtcacgg agatctggca ggaggtcatg cagcggcggg acgacgacgg cgcgctccac 180
gccgcctgcc aggtgcagcc gtcggccacg ctggacgccg cgcagccccg ggtgaccggc 240
gtcgtcctct tccggcagct tgcgccccgc gccaagctcg acgccttctt cgccctggag 300
ggcttcccga ccgagccgaa cagctccagc cgcgccatcc acgtgcacca gttcggggac 360
ctgagccagg gctgcgagtc caccgggccc cactacaacc cgctggccgt gccgcacccg 420
cagcacccgg gcgacttcgg caacttcgcg gtccgcgacg gcagcctctg gaggtaccgc 480
gccggcctgg ccgcctcgct cgcgggcccg cactccatcg tgggccgggc cgtggtcgtc 540
cacgctggcg aggacgacct gggccgcggc ggcaaccagg ccagcgtgga gaacgggaac 600
gcgggccggc ggctggcctg ctgcgtggtg ggcgtgtgcg ggcccgggct ctgggagcgc 660
caggcgcggg agcactcaga gcgcaagaag cggcggcgcg agagcgagtg caaggccgcc 720
tga 723
Claims (9)
1. A CHO cell strain construction method of high expression SOD3 is characterized in that the method comprises the following steps:
firstly, connecting the nucleotide sequence of SOD3 shown in SEQ ID NO.1 to a PUC57 cloning vector to construct a lentiviral vector plasmid pCDH-CMV-MCS-EF1-SOD3-Puro for expressing SOD3 gene;
secondly, transfecting 293T cells with a lentiviral vector plasmid pCDH-CMV-MCS-EF1-SOD3-Puro and packaging plasmids psPAX2 and pMD2.0G together, and packaging into a lentivirus carrying an SOD3 encoding gene;
and thirdly, infecting CHO cells with lentiviruses carrying SOD3 coding genes, screening by puromycin to obtain a CHO cell group expressing SOD3 protein, and further screening the CHO cells expressing SOD3 protein after the puromycin screening by a limiting dilution method to obtain a stable SOD3+ CHO cell strain.
2. The method for constructing CHO cell line highly expressing SOD3 according to claim 1, wherein primers are designed as follows in the amplification sequencing:
EcoRI-upstream primer: CGGAATTCATGCTGGCGCTACTGTGTTCC, respectively;
NotI-downstream primer: TTGCGGCCGCTCAGCGCAGTTCCCACCACT are provided.
3. The method for constructing CHO cell line with high expression of SOD3 according to claim 1, wherein the slow virus carrying SOD3 encoding gene is infected into CHO cells by the following steps: 293T cells release lentivirus carrying SOD3 coding genes, supernatant is collected, and CHO cells are infected with lentivirus liquid after ultra-high speed centrifugal concentration.
4. The method for constructing CHO cell line highly expressing SOD3 according to claim 1, wherein the method for screening CHO cells comprises the following steps: a selection medium containing puromycin is used for screening a CHO cell group integrated with SOD3, then a 96-well plate is paved by a limiting dilution method, a positive CHO cell strain capable of efficiently expressing SOD3 protein is further screened by a dot hybridization method and a protein immunoblotting method, and the obtained positive CHO cell strain is subjected to suspension fermentation culture.
5. A method for purifying SOD3 protein, which is characterized by comprising the following steps:
firstly, connecting the nucleotide sequence of SOD3 shown in SEQ ID NO.1 to a PUC57 cloning vector to construct a lentiviral vector plasmid pCDH-CMV-MCS-EF1-SOD3-Puro for expressing SOD3 gene;
secondly, transfecting 293T cells with a lentiviral vector plasmid pCDH-CMV-MCS-EF1-SOD3-Puro and packaging plasmids psPAX2 and pMD2.0G together, and packaging into a lentivirus carrying an SOD3 encoding gene;
infecting CHO cells with lentivirus carrying SOD3 coding genes, screening by puromycin to obtain a CHO cell group expressing SOD3 protein, and further screening the CHO cells expressing SOD3 protein after the puromycin screening by a limiting dilution method and a Western blot method to obtain a stable SOD3+ CHO cell strain;
and fourthly, amplifying the obtained SOD3+ CHO cell strain, separating and purifying the secretory SOD3 protein to obtain the SOD3 protein in a pure enzyme form.
6. The method for purifying SOD3 protein according to claim 5, wherein in the amplification sequencing, primers are designed as follows:
EcoRI-upstream primer: CGGAATTCATGCTGGCGCTACTGTGTTCC, respectively;
NotI-downstream primer: TTGCGGCCGCTCAGCGCAGTTCCCACCACT are provided.
7. The SOD3 protein purification method of claim 5, wherein the slow virus carrying the gene encoding SOD3 is infected into CHO cells by the following steps: 293T cells release lentivirus carrying SOD3 coding genes, supernatant is collected, and CHO cells are infected with lentivirus liquid after ultra-high speed centrifugal concentration.
8. The SOD3 protein purification method of claim 5, wherein the method for screening CHO cells comprises: a selection medium containing puromycin is used for screening a CHO cell group integrated with SOD3, then a 96-well plate is paved by a limiting dilution method, a positive CHO cell strain capable of efficiently expressing SOD3 protein is further screened by a dot hybridization method and a protein immunoblotting method, and the obtained positive CHO cell strain is subjected to suspension fermentation culture.
9. The SOD3 protein purification method according to claim 5, wherein the four steps of separating and purifying comprise the following steps: and carrying out ultrafiltration, ammonium sulfate sedimentation, ion exchange chromatography and gel chromatography purification on the obtained secretory protein of the SOD3+ CHO cell strain to obtain the SOD3 protein in a pure enzyme form.
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