CN113024652A - Recombinant expression of human myelin oligodendrocyte glycoprotein and application thereof - Google Patents
Recombinant expression of human myelin oligodendrocyte glycoprotein and application thereof Download PDFInfo
- Publication number
- CN113024652A CN113024652A CN202110213849.7A CN202110213849A CN113024652A CN 113024652 A CN113024652 A CN 113024652A CN 202110213849 A CN202110213849 A CN 202110213849A CN 113024652 A CN113024652 A CN 113024652A
- Authority
- CN
- China
- Prior art keywords
- cells
- mog
- expression
- myelin oligodendrocyte
- oligodendrocyte glycoprotein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses recombinant expression of human myelin oligodendrocyte glycoprotein and application thereof, and particularly provides a preparation method of eukaryotic recombinant MOG protein with high expression quantity, good immunogenicity and high purity. The invention optimizes codons of MOG genes in the process of gene synthesis, optimizes the optimal expression time of 3 different signal peptides and determines the optimal sample collection time of supernatant expression, 10 percent of ethanol is added into a purified buffer solution, the condition of protein aggregation is successfully avoided, and the expressed MOG protein can accurately distinguish positive samples and negative samples of clinical MOG-IgG in the final activity identification of MOG expression.
Description
Technical Field
The invention belongs to the technical field of biological protein recombinant expression, and particularly relates to recombinant expression of human myelin oligodendrocyte glycoprotein and application thereof.
Background
Myelin Oligodendrocyte Glycoprotein (MOG) is an oligodendrocyte membrane-bound glycoprotein and is an important component of central nervous myelin, studies have shown that anti-MOG antibodies play a very important role in the pathological process of Multiple Sclerosis (MS), and experimental au-toimmmune encephalomyelitis (EAE) induced by MOG is an internationally recognized MS animal model at present. Currently, most of MOG used in China is artificially synthesized MOC peptide fragment. The extracellular Ig-like domain (MOG, MOG'8) of MOG contains a plurality of antigen epitopes and has strong immunogenicity.
The invention relates to a preparation method of eukaryotic recombinant MOG protein with high expression level, good immunogenicity and high purity, which clones the extracellular segment of MOG gene by using gene engineering technology, connects to an expression vector and then transforms into host cells to express and purify a large amount of stable MOG protein, and provides a foundation for in vitro diagnosis of MOG antibody.
Disclosure of Invention
The invention aims to provide recombinant expression of human myelin oligodendrocyte glycoprotein and application thereof.
The purpose of the invention can be realized by the following technical scheme:
the recombinant expression of human myelin oligodendrocyte glycoprotein specifically comprises the following steps:
first, recombinant plasmid construction
The Ig-like domain of the recombinant MOG is subcloned into pTT5 with different signal peptide sequences after codon optimization by a gene synthesis technology, and the recombinant MOG plasmid is constructed.
Second, the transfection-grade plasmid is extracted
Third step, transfection
S1, transfection Expi293FTMPre-preparation of cells
S2, transfection Expi293FTMCells
S21, day before transfection (day-1), Expi293FTMCultures were split to a final density of 2.5-3X 106 viable cells/mL and cells were grown overnight;
s22, fresh Expi293 preheated to 37 ℃TMExpression medium the cells in step S21 were diluted to a final density of 3 × 106 viable cells/mL, and the flasks were gently shaken to mix the cells;
s23 preparation of ExpifeacamineTM293/plasmid DNA complex;
s24 incubation of Expifeacylamine prepared in step d of step S23 at room temperatureTM293/plasmid DNA Complex for 10-20 min, then the solution was slowly transferred to the shake flask of step 4, gently shaking the flask during the addition;
s25, in 37 ℃ incubator, 8% CO2Incubating the cells in air on an orbital shaker in a humid environment;
s26, collecting 0.3mL of culture on the third day of transfection, centrifuging at 3000r/min at 4 ℃ for 5min, adding 2 xSDS Loading buffer into the culture medium supernatant, and boiling for 5min in a water bath;
s27, continuously sampling for 4 days, carrying out WB detection on the prepared sample, and verifying the expression condition of the supernatant;
the fourth step, purifying the target protein
(1) Taking 1mL of nickel NTA agarose gel FF prepacked column, using 10mL of 1 PBS, 10% ethanol, pH 7.4;
(2) adding ethanol into the culture supernatant to a final concentration of 10%, filtering with 0.22 μm microporous membrane, loading at 0.5mL/min, collecting eluate, and loading again to allow protein sample to be fully bound to gel;
(3) washing the unadsorbed sample by using 30mL of buffer solution at the flow rate of 1-2 mL/min;
(4) respectively washing unadsorbed samples by using 30mLWashbuffer at the flow rate of 1-2 mL/min;
(5) eluting the target protein with 1mL of Elutionbuffer at a flow rate of 1-2 mL/min;
(6) the column was then washed with 50mL of buffer, filled with 20% ethanol, and blocked for the next use.
As a further embodiment of the present invention, the sequence of the recombinant protein expressed in the first step is:
MASLSRPSLPSCLCSFLLLLLLQVSSSYAGQFRVIGPRHPIRALVGDEVELPCRISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGDQAPEYRGRTELLKDAIGEGKVTLRIRNVRFSDEGGFTCFFRDHSYQEEAAMELKVEDPFYWVSPGHHHHHHHH。
as a further embodiment of the present invention, the sequence of the signal peptide carried by pTT5 is:
MDAMKRGLCCVLLLCGAVFVSG, MDMRVPAQLLGLLLLWLRGARC and the signal peptide of MOG protein itself.
As a further embodiment of the present invention, Expi293F was transfected in step S1TMThe pre-preparation of the cells comprises the following steps:
s11, remove the vial from the liquid nitrogen and rotate in a 37 ℃ water bath for 1 to 2 minutes to rapidly thaw the cells until only a small amount of ice remains.
S12, before the cells were completely thawed, the vials were wiped with 70% ethanol and then purged by opening in a laminar flow hood.
S13, transfer the entire contents of the cryovial to a 125mL polycarbonate, disposable, sterile, vented Erlenmeyer flask containing 20mL of preheated Expi293 using a 1mL pipetteTMExpression medium.
S14, on a track vibration table, under the conditions that the relative humidity is more than or equal to 80 percent and the relative humidity is more than or equal to 8 percent CO2The cells were cultured in a 37 ℃ incubator.
S15, culturing the cells for 3-4 days after thawing, then determining the density and survival rate of the surviving cells, starting passage when the cell viability is equal to or more than 90%, and the density of the viable cells is generally more than 1 × 106 viable cells/mL, and subculturing the cells to 0.3-0.5 × 106 cells/mL.
As a further embodiment of the present invention, Expifeacylamine is prepared in step S23TMThe 293/plasmid DNA compound comprises the following specific steps:
a. gently invert ExpifeacamineTM293 reagent bottle 4-5 times for mixing;
b. using Opti-MEMTMI reducing serum culture medium to dilute plasmid DNA. Mixing by rotating the tube and/or inversion;
c. using Opti-MEMTMI reduction of serum Medium dilution ExpifeacamineTM293 reagent. Mix by rotating the tube 2 to 3 times and incubate at room temperature for 5 minutes before startingPerforming plasmid DNA complexation reaction;
d. diluting ExpifeacmineTM293 reagents were added to the diluted plasmid DNA and the tube was rotated 2 to 3 times for mixing.
As a further embodiment of the invention, the prewash buffer used in the fourth step is 1 × PBS, 10% ethanol, pH 7.4.
As a further embodiment of the invention, the Wash buffer used in the fourth step comprises two of 5mM imidazole, 1 × PBS, 10% ethanol, pH 7.4 and 20mM imidazole, 1 × PBS, 10% ethanol, pH 7.4.
As a further embodiment of the present invention, the Elution buffer in the fourth step comprises 250mM imidazole, 10% ethanol, 0.05% Tween-20.
The invention has the beneficial effects that:
1. the gene of MOG is optimized by codon in the process of gene synthesis, and the optimal sampling time of the supernatant expression is determined by optimizing the optimal expression time of 3 different signal peptides.
2. According to the invention, 10% ethanol is added into the purified buffer solution, so that the condition of protein aggregation is successfully avoided.
3. In the final MOG expression activity identification, the expressed MOG protein can accurately distinguish positive and negative samples of clinical MOG-IgG.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The recombinant expression of human myelin oligodendrocyte glycoprotein specifically comprises the following steps:
first, recombinant plasmid construction
The Ig-like domain of the recombinant MOG is subcloned into pTT5 (with different signal peptides respectively) after codon optimization by a gene synthesis technology to construct a recombinant MOG plasmid.
The expressed recombinant protein sequence is:
MASLSRPSLPSCLCSFLLLLLLQVSSSYAGQFRVIGPRHPIRALVGDEVELPCRISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGDQAPEYRGRTELLKDAIGEGKVTLRIRNVRFSDEGGFTCFFRDHSYQEEAAMELKVEDPFYWVSPGHHHHHHHH
the sequence of the signal peptide is:
MDAMKRGLCCVLLLCGAVFVSG, MDMRVPAQLLGLLLLWLRGARC and the signal peptide of the MOG protein itself;
second, the transfection-grade plasmid is extracted
Third step, transfection
S1, transfection Expi293FTMPre-preparation of cells
S11, remove the vial from the liquid nitrogen and rotate in a 37 ℃ water bath for 1 to 2 minutes to rapidly thaw the cells until only a small amount of ice remains.
S12, before the cells were completely thawed, the vials were wiped with 70% ethanol and then purged by opening in a laminar flow hood.
S13, transfer the entire contents of the cryovial to a 125mL polycarbonate, disposable, sterile, vented Erlenmeyer flask containing 20mL of pre-heated Expi293TM expression medium using a 1mL pipette.
S14, on a track vibration table, under the conditions that the relative humidity is more than or equal to 80 percent and the relative humidity is more than or equal to 8 percent CO2The cells were cultured in a 37 ℃ incubator.
S15, culturing the cells for 3-4 days after thawing, then determining the density and survival rate of the surviving cells, starting passage when the cell viability is equal to or more than 90%, and the density of the viable cells is generally more than 1 × 106 viable cells/mL, and subculturing the cells to 0.3-0.5 × 106 cells/mL.
S2, transfection Expi293FTMCells
S21, day before transfection (day-1), Expi293FTMCultures were split to a final density of 2.5-3X 106 viable cells/mL and cells were grown overnight;
s22, fresh Expi293 preheated to 37 ℃TMExpression Medium the cells in step S21 were diluted to a final density of 3X 106 viable cells/mLGently shake the flask to mix the cells;
s23 preparation of Expifeacylamine according to the instructionsTM293/plasmid DNA complex;
note that: the total plasmid DNA per ml culture volume to be transfected is 1.0. mu.g, suitable for most proteins.
a. Gently invert ExpifeacamineTM293 reagent bottle 4-5 times for mixing;
b. using Opti-MEMTMI reducing serum culture medium to dilute plasmid DNA. Mixing by rotating the tube and/or inversion;
c. using Opti-MEMTMI reduction of serum Medium dilution ExpifeacamineTM293 reagent. Rotating the test tube for 2 to 3 times, mixing, incubating for 5 minutes at room temperature, and then starting plasmid DNA complexation reaction;
d. diluting ExpifeacmineTM293 reagent is added to the diluted plasmid DNA, the tube is rotated 2 to 3 times for mixing;
s24 incubation of Expifeacylamine prepared in step d of step S23 at room temperatureTM293/plasmid DNA Complex for 10-20 min, then the solution was slowly transferred to the shake flask of step 4, gently shaking the flask during the addition;
s25, incubating the cells in air on an orbital shaker in a 37 ℃ incubator in a humid environment of 8% CO 2;
s26, collecting 0.3mL of culture on the third day of transfection, centrifuging at 3000r/min at 4 ℃ for 5min, adding 2 xSDS Loading buffer into the culture medium supernatant, and boiling for 5min in a water bath;
and S27, continuously sampling for 4 days, carrying out WB detection on the prepared sample, and verifying the expression condition of the supernatant.
By detection, on the 4 th day after transfection, the supernatant transfected by the plasmid with the signal peptide of the sequence MDAMKRGLCCVLLLCGAVFVSG has more MOG protein expression;
the fourth step, purifying the target protein
(1) 1mL of nickel NTA Sepharose FF pre-packed column was loaded with 10mL of 1 × PBS, pH 7.4;
(2) in order to completely remove impurities, ethanol is added to the culture supernatant to a final concentration of 10%, a sample of the culture supernatant is filtered through a 0.22-micron-l microporous membrane, then the sample is loaded at 0.5mL/min, the effluent is collected, and the effluent is loaded again to enable the protein sample to be fully bound to the gel;
(3) using 30mL of pre-washing buffer solution to wash the unadsorbed sample, wherein the flow rate is 1-2 mL/min;
(4) respectively washing unadsorbed samples by using 30mLWashbuffer (eluting according to concentration gradient), wherein the flow rate is 1-2 mL/min;
(5) eluting the target protein with 1mL of Elutionbuffer at a flow rate of 1-2 mL/min;
(6) the column was then washed with 50mL of prewash buffer, filled with 20% ethanol and blocked for the next use.
Prewash buffer used in step four: 1 × PBS, 10% ethanol, pH 7.4;
wash buffer 5mM imidazole, 1 × PBS, 10% ethanol, pH 7.4; 20mM imidazole, 10% ethanol, 1 × PBS, pH 7.4; elution buffer 250mM imidazole, 10% ethanol, 0.05% Tween-20.
Activity assay
The MOG protein was diluted to 1. mu.g/mL protein with 0.05M PH9. carbonate coating buffer, 100. mu.l was added to each reaction well of the polystyrene plate overnight at 4 ℃ the next day, the well solutions were discarded, and the wells were washed 1 time with 200. mu.l/well for 1 minute each with washing buffer (PBST).
And (3) sealing: blocking the ELISA plate with 2% BSA at 300. mu.l/well RT 1 h;
sample adding: removing the sealing liquid, drying, washing with washing buffer solution for 1 time (1 min each time) and 200. mu.l/well; cerebrospinal fluid which is positive for clinically detecting MOG-IgG antibody is diluted by 2 times and 4 times into the coated reaction wells, incubated for 1h at 37 ℃, and then washed (meanwhile, blank wells and negative control wells).
Adding an enzyme-labeled antibody: discard the sample and beat dry and wash 6 times 200. mu.l/well with wash buffer for 6 minutes each. Adding 100 mul of enzyme-labeled antibody (HRP-labeled goat anti-human IgG antibody) which is diluted by 1:10000 freshly into each reaction well, and incubating for 1 hour at 37 ℃ (incubation at RT);
adding a substrate solution for color development: discard the sample and wash 6 times 200. mu.l/well with wash buffer for 6 minutes each, add 100. mu.l of the extemporaneously prepared TMB substrate solution to each reaction well for 10 minutes at 37 deg.C (incubation at RT).
And (3) terminating the reaction: 50. mu.l of 2M sulfuric acid was added to each reaction well.
The result shows that the sample for clinically detecting the MOG-IgG antibody positive has higher detection values relative to the negative value and the blank value, which indicates that the expressed and purified MOG antibody has good activity.
The foregoing is merely exemplary and illustrative of the present invention and various modifications, additions and substitutions may be made by those skilled in the art to the specific embodiments described without departing from the scope of the invention as defined in the following claims.
Claims (9)
1. The recombinant expression of human myelin oligodendrocyte glycoprotein is characterized by comprising the following steps:
first, recombinant plasmid construction
The Ig-like domain of the recombinant MOG is subcloned into pTT5 with different signal peptide sequences after codon optimization by a gene synthesis technology, and the recombinant MOG plasmid is constructed.
Second, the transfection-grade plasmid is extracted
Third step, transfection
S1, transfection Expi293FTMPre-preparation of cells
S2, transfection Expi293FTMCells
S21, one day before transfection, Expi293FTMThe culture was divided into 2.5-3X 106Final density of cells/mL and allow cells to grow overnight;
s22, fresh Expi293 preheated to 37 ℃TMExpression Medium the cells in step S21 were diluted to 3X 106Final density of cells/mL, gently shake flask to mix cells;
s23 preparation of ExpifeacamineTM293/plasmid DNA complex;
s24, incubation step at room temperatureExpifeacylamine prepared in step d of S23TM293/plasmid DNA Complex for 10-20 min, then the solution was slowly transferred to the shake flask of step 4, gently shaking the flask during the addition;
s25, in 37 ℃ incubator, 8% CO2Incubating the cells in air on an orbital shaker in a humid environment;
s26, collecting 0.3mL of culture on the third day of transfection, centrifuging at 3000r/min at 4 ℃ for 5min, adding 2 xSDS Loading buffer into the culture medium supernatant, and boiling for 5min in a water bath;
s27, continuously sampling for 4 days, carrying out WB detection on the prepared sample, and verifying the expression condition of the supernatant;
the fourth step, purifying the target protein
(1) 1mL of nickel NTA sepharose FF prepacked column was washed with 10mL of 1 × PBS, 10% ethanol, pH 7.4;
(2) adding ethanol into the culture supernatant sample to a final concentration of 10%; then filtering with 0.22 μm l microporous membrane, loading at 0.5mL/min, collecting eluate, loading again to allow protein sample to be fully bound on the gel;
(3) washing unadsorbed samples by using 30mL of ultrasonic disruption buffer solution at the flow rate of 1-2 mL/min;
(4) respectively washing the unadsorbed samples by using 30mL of Wash buffer at the flow rate of 1-2 mL/min;
(5) eluting the target protein by using 1mL of Elution buffer at the flow rate of 1-2 mL/min;
(6) the column was then washed with 50mL of sonication buffer, filled with 20% ethanol, and blocked for the next use.
2. The recombinant expression of human myelin oligodendrocyte glycoprotein according to claim 1, wherein the recombinant protein sequence expressed in the first step is:
MASLSRPSLPSCLCSFLLLLLLQVSSSYAGQFRVIGPRHPIRALVGDEVELPCRISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGDQAPEYRGRTELLKDAIGEGKVTLRIRNVRFSDEGGFTCFFRDHSYQEEAAMELKVEDPFYWVSPGHHHHHHHH。
3. the recombinant expression of human myelin oligodendrocyte glycoprotein according to claim 2, wherein the sequence of the signal peptide of pTT5 is:
MDAMKRGLCCVLLLCGAVFVSG, MDMRVPAQLLGLLLLWLRGARC and the signal peptide of MOG protein itself.
4. The recombinant expression of human myelin oligodendrocyte glycoprotein according to claim 1, wherein step S1 is performed by transfecting Expi293FTMThe pre-preparation of the cells comprises the following steps:
s11, remove the vial from the liquid nitrogen and rotate in a 37 ℃ water bath for 1 to 2 minutes to rapidly thaw the cells until only a small amount of ice remains.
S12, before the cells were completely thawed, the vials were wiped with 70% ethanol and then purged by opening the vials in a laminar flow hood.
S13, transfer the entire contents of the cryovial to a 125mL polycarbonate, disposable, sterile, vented Erlenmeyer flask containing 20mL of preheated Expi293 using a 1mL pipetteTMExpression medium.
S14, on a track vibration table, under the conditions that the relative humidity is more than or equal to 80 percent and the relative humidity is more than or equal to 8 percent CO2The cells were cultured in a 37 ℃ incubator.
S15, culturing the cells for 3-4 days after thawing, then determining the density and survival rate of the surviving cells, starting passage when the cell viability is equal to or more than 90%, and the density of the viable cells is generally more than 1 × 106 viable cells/mL, and subculturing the cells to 0.3-0.5 × 106 cells/mL.
5. The recombinant expression of human myelin oligodendrocyte glycoprotein according to claim 1, wherein Expifeacamine is prepared in step S23TMThe 293/plasmid DNA compound comprises the following specific steps:
a. gently invert ExpifeacamineTM293 reagent bottle 4-5 times for mixing;
b. using Opti-MEMTMI reducing serum culture medium to dilute plasmid DNA. Mixing by rotating the tube and/or inversion;
c. using Opti-MEMTMI reduction of serum Medium dilution ExpifeacamineTM293 reagent. Rotating the test tube for 2 to 3 times, mixing, incubating for 5 minutes at room temperature, and then starting plasmid DNA complexation reaction;
d. diluting ExpifeacmineTM293 reagents were added to the diluted plasmid DNA and the tube was rotated 2 to 3 times for mixing.
6. The recombinant expression of human myelin oligodendrocyte glycoprotein according to claim 1, wherein the sonication buffer used in step four is 1 × PBS, pH 7.4.
7. The recombinant expression of human myelin oligodendrocyte glycoprotein according to claim 1, wherein the Wash buffer used in step four comprises 5mM imidazole, 1 × PBS, pH 7.4 and 20mM imidazole, 1 × PBS, pH 7.4.
8. The recombinant expression of human myelin oligodendrocyte glycoprotein according to claim 1, wherein the Elution buffer of step four comprises 250mM imidazole, 0.05% Tween-20.
9. Use of human myelin oligodendrocyte glycoprotein recombinantly expressed according to the method of claim 1 for immunodetection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110213849.7A CN113024652A (en) | 2021-02-25 | 2021-02-25 | Recombinant expression of human myelin oligodendrocyte glycoprotein and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110213849.7A CN113024652A (en) | 2021-02-25 | 2021-02-25 | Recombinant expression of human myelin oligodendrocyte glycoprotein and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113024652A true CN113024652A (en) | 2021-06-25 |
Family
ID=76462466
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110213849.7A Withdrawn CN113024652A (en) | 2021-02-25 | 2021-02-25 | Recombinant expression of human myelin oligodendrocyte glycoprotein and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113024652A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115806625A (en) * | 2021-09-15 | 2023-03-17 | 广州百暨基因科技有限公司 | Chimeric antigen receptor with limited expression of T cells and application thereof |
| CN116200414A (en) * | 2022-08-31 | 2023-06-02 | 天津华科泰生物技术有限公司 | Recombinant human myelin oligodendrocyte glycoprotein, and preparation method and application thereof |
| CN117683131A (en) * | 2024-01-24 | 2024-03-12 | 首都医科大学宣武医院 | anti-Myelin Oligodendrocyte Glycoprotein (MOG) antibody and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070117185A1 (en) * | 2005-10-21 | 2007-05-24 | Erhard Kopetzki | Method for the recombinant expression of a polypeptide |
| CN106434684A (en) * | 2016-11-24 | 2017-02-22 | 上海海洋大学 | Scatophagus argus luteinizing hormone (LH) gene, scatophagus argus LH recombinant protein and application |
| CN110606881A (en) * | 2019-08-12 | 2019-12-24 | 陕西脉元生物科技有限公司 | Detection material for anti-MOG autoantibody in human body fluid, preparation method and application |
| CN112375784A (en) * | 2021-01-07 | 2021-02-19 | 北京百普赛斯生物科技股份有限公司 | Method for preparing recombinant novel coronavirus Spike protein |
-
2021
- 2021-02-25 CN CN202110213849.7A patent/CN113024652A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070117185A1 (en) * | 2005-10-21 | 2007-05-24 | Erhard Kopetzki | Method for the recombinant expression of a polypeptide |
| CN106434684A (en) * | 2016-11-24 | 2017-02-22 | 上海海洋大学 | Scatophagus argus luteinizing hormone (LH) gene, scatophagus argus LH recombinant protein and application |
| CN110606881A (en) * | 2019-08-12 | 2019-12-24 | 陕西脉元生物科技有限公司 | Detection material for anti-MOG autoantibody in human body fluid, preparation method and application |
| CN112375784A (en) * | 2021-01-07 | 2021-02-19 | 北京百普赛斯生物科技股份有限公司 | Method for preparing recombinant novel coronavirus Spike protein |
Non-Patent Citations (4)
| Title |
|---|
| FRDBIO: "重组蛋白纯化——可溶性蛋白纯化（NI）", 《百度》 * |
| NCBI: "myelin-oligodendrocyte glycoprotein isoform beta2 precursor [Homo sapiens]", 《GENBANK DATABASE》 * |
| THERMOFISHER: "Expi293™ Expression System: Structural Biology and Inducible Expression Modules USER GUIDE", 《GIBCO》 * |
| 张向阳主编: "《医学分子生物学》", 28 February 2018, 江苏凤凰科学技术出版社 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115806625A (en) * | 2021-09-15 | 2023-03-17 | 广州百暨基因科技有限公司 | Chimeric antigen receptor with limited expression of T cells and application thereof |
| CN115806625B (en) * | 2021-09-15 | 2023-08-04 | 广州百暨基因科技有限公司 | Chimeric antigen receptor expressed by T cell limitation and application thereof |
| CN116200414A (en) * | 2022-08-31 | 2023-06-02 | 天津华科泰生物技术有限公司 | Recombinant human myelin oligodendrocyte glycoprotein, and preparation method and application thereof |
| CN117683131A (en) * | 2024-01-24 | 2024-03-12 | 首都医科大学宣武医院 | anti-Myelin Oligodendrocyte Glycoprotein (MOG) antibody and application thereof |
| CN117683131B (en) * | 2024-01-24 | 2024-04-30 | 首都医科大学宣武医院 | Anti-Myelin Oligodendrocyte Glycoprotein (MOG) antibody and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113024652A (en) | Recombinant expression of human myelin oligodendrocyte glycoprotein and application thereof | |
| WO2021246910A8 (en) | Sars-cov-2 virus rbd recombinant protein and method for producing same | |
| CN110068688B (en) | Lactoferrin competition method nanoflower immunity detection flow chromatography detection card in cow's milk | |
| CN107663235B (en) | The relevant PLA2R recombinant proteins of idiopathic membranous nephropathy and its application | |
| CN110887967B (en) | Method for detecting antibody binding capacity and application thereof | |
| CN112111015B (en) | PLA2R, C1q and THSD7A fusion protein and construction method and application thereof | |
| CN110563840B (en) | Anti-glycocholic acid monoclonal antibody and preparation method thereof | |
| WO2007040242A1 (en) | Isolated human cell, method for obtaining the same and their use | |
| CN114409808A (en) | Aptamer-based targeting chimeras and degradation of tau proteins | |
| JPH0391491A (en) | Production of serum albumin | |
| CN113666988A (en) | Preparation method and application of N-terminal structural domain of novel coronavirus nucleocapsid protein | |
| CN110261620B (en) | Cell immunofluorescence kit for determining human serum phosphotyrosine adaptor protein ShcA antibody and preparation method and application thereof | |
| CN111830261A (en) | Method and kit for rapidly detecting new coronavirus IgG antibody | |
| CN110887961A (en) | Solid phase carrier and kit for antibody screening | |
| CN115947861B (en) | Efficient hybridoma fusion method | |
| WO2024008189A1 (en) | Methods and systems for purifying circular nucleic acids | |
| US20240167006A1 (en) | High production of recombinant protein by making cell hybrids and enriching for a preferred mitochondrial phenotype | |
| An et al. | Activation-induced cytidine deaminase aided in vitro antibody evolution | |
| CN113308491B (en) | Recombinant plasmid and recombinant cell for co-expressing NFAT and human DNAM-1 protein, and construction method and application thereof | |
| CN115819595B (en) | anti-LAG3 nano antibody and preparation method and application thereof | |
| CN117487004B (en) | Monoclonal antibody against coronavirus S protein and application thereof | |
| CN115894675B (en) | New coronavirus SARS-CoV-2 nucleocapsid protein monoclonal antibody and its application | |
| CN112980909A (en) | Method for preparing antibody by hybridoma cell serum-free culture | |
| CN110314414B (en) | Separation and purification method of blood coagulation factor XIII and application thereof | |
| CN117805360A (en) | Functional nucleic acid aptamer screening method combining cell screening and functional screening targeting GPCR (GPCR) protein and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20210625 |
|
| WW01 | Invention patent application withdrawn after publication |