CN106380511A - Method for preparing human sB7-H4 eukaryotic expression protein monomer - Google Patents
Method for preparing human sB7-H4 eukaryotic expression protein monomer Download PDFInfo
- Publication number
- CN106380511A CN106380511A CN201610840603.1A CN201610840603A CN106380511A CN 106380511 A CN106380511 A CN 106380511A CN 201610840603 A CN201610840603 A CN 201610840603A CN 106380511 A CN106380511 A CN 106380511A
- Authority
- CN
- China
- Prior art keywords
- cell
- protein
- culture
- people
- expression
- 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.)
- Pending
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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70532—B7 molecules, e.g. CD80, CD86
-
- 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
-
- 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
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of biotechnology and molecular biologics, and in particular to a method for preparing a human sB7-H4 eukaryotic expression protein monomer. The method for preparing the human sB7-H4 eukaryotic expression protein monomer comprises the following steps of: optimizing codon, inserting human IgFc into an N end as a purification tag, and inserting an artificial leading sequence into the N end of a fusion protein, thereby achieving high-efficiency soluble secretory expression of the protein. The human sB7-H4 eukaryotic expression protein monomer prepared by using the method can be used as an antigen for immunizing animals to prepare an antibody, can be also used as a reference standard product for detecting human sB7-H4, can be also used as a decoy receptor of B7-H4, is adopted for competitive inhibition on a B7-H4 signal path, and can be used for treating immune diseases such as tumor.
Description
Technical field
The present invention relates to biotechnology and technical field of molecular biology are and in particular to people's sB7-H4 eukaryotic expression protein
The preparation method of monomer.
Background technology
Dual signal theory thinks that T cell activation needs antigen signals and costimulatory signal, and the former is known by T cell surface TCR
MHC conjugated antigen peptide in other antigen presenting cell and transmit, the latter interacted by common stimulation/Co inhibitor and its part and
Realize, the integration of two kinds of signals can effective inducing T cell activation.Altogether stimulation/Co inhibitor include large numbers of known and not
The membrane molecule being expressed in antigen presenting cell and T cell surface known, mainly has immunoglobulin superfamily（Ig
Superfamily, IgSF）With TNF superfamily（TNF superfamily）, these molecules are in maintenance immunity of organism balance, decision
T cell immune response direction（Response or tolerance）In play highly important effect, they are referred to as immunologic surveillance molecule（immune
checkpoint molecules）, including excitant immunologic surveillance molecule（stimulatory checkpoint
Molecules, SCPM）With inhibition immunologic surveillance molecule（Inhibitory checkpoint molecules, ICPM）, suppression
Property immunologic surveillance molecule processed plays down regulation to T cell activation, such as CD80/CD86-CTLA4, PD-L1-PD1, B7-H4 and its
Acceptor, B7-H3 and its acceptor, HVEM-BTLA etc., play particularly significant effect, wherein in maintaining the immunologic balance of body periphery
CTLA-4 and PD-1, especially the latter are used widely in oncotherapy as immunotherapy of tumors target spot.
B7-H4（ (B7x, B7S1, VTCN1)）It is a kind of inhibition immunologic surveillance molecule finding for 2003, belong to
The B7-H4 of IgSF, people and mouse has 87% amino acid sequence homologous, and ripe B7-H4 is a kind of cross-film egg of 50 ~ 80kDa
In vain, an IgV area and an IgC area are had, its mRNA can be expressed in lymphoid tissue and non-lymphoid tissue, under normal circumstances at it
Protein is not expressed in immunocyte and non-lymphoid tissue, but inducibility is expressed in BMDC（DC）, monokaryon/huge bites
The cell surfaces such as cell, B cell and T cell.Film combination type B7-H4 and soluble B7-H4-Ig fusion protein all can effectively suppress
T cell activation, its effect is：Suppression T cell（Including CD4+ and CD8+ cell）Propagation and generation IL-2；Blocking T-cell thin
Born of the same parents' cycle；Suppression CTL killing activity.In mouse EAE model, using B7-H4 monoclonal antibody and soluble B7-H4-Ig fusion protein
Block the progress that B7-H4 signal pathway can aggravate disease, but B7-H4 deficient mice only show slight Th1 cytoactive and raises,
And cytotoxic T cell function is constant, point out, B7-H4 is a kind of immunosuppression molecule of T cell activation, although its effect is relatively
Weak, but optimization fine setting effect can be played.Recently, find that B7-H4 is played and the anti-sense of neutrophil leucocyte with Treg immune suppression function
Dye activity is related, such as finds that local expression B7-H4 can suppress the homograft rejecting reaction to islet transplantation for the receptor, and with raising
Treg activity is relevant, treats, using B7-H4-Ig, generation, its mechanism and the suppression that NOD mouse can suppress autoimmune diabetes
Effector T cell infiltration is relevant with improving Treg cytoactive.Also find that human marrow mesenchymal stem cell and bile duct epithelial cell are equal
High expression B7-H4, and the immune suppression function with these cells and Anti-G value in close relations.The above results illustrate B7-H4
Signal pathway all plays negative regulation effect to the function of inherent immunity cell and adaptive immunity cell, is maintaining organismic internal environment
Play an important role in balance.
Recent study finds, B7-H4 in kinds of tumor cells, such as lung cancer, oophoroma, breast cancer, cancer of the stomach, esophagus
The cancer cell surfaces such as cancer, kidney, melanoma are in high expression, the expression of B7-H4 and the parting of tumour, by stages and the close phase of prognosis
Close.And the also high expression of the vascular endothelial cell in tumour and tumor infiltrating macrophage and surface of dendritic cells.We
In previous research work, the patient tumors such as lung cancer, colorectal cancer, breast cancer, cancer of the stomach be have detected using immunohistochemistry technology
The expression of B7-H4 in tissue, finds all high expression B7-H4 of above-mentioned tumor tissues.B7-H4 can also soluble form（soluble
B7-H4, sB7-H4）It is present in blood and other body fluid of people, in kinds of tumors peripheral blood in patients, sB7-H4 level raises,
We establish the ELISA sandwich method of detection sB7-H4 using the pairing of how anti-monoclonal antibody, by more than 600 example difference tumor patients
The detection of peripheral blood, finds that in kinds of tumors peripheral blood in patients, sB7-H4 level raises, and sB7-H4 level and tumour point
Type, by stages all closely related with prognosis.Therefore, B7-H4 is considered as a kind of New-type wide-spectrum tumor markers.
At present, find that B7-H4 is in close relations with the immunologic escape of tumour.Miyatake etc. finds B7- in breast cancer tissue
H4 expression intensity is in inverse ratio with the quantity of wellability CD4+ and CD8+T cell, points out B7-H4 can suppress T cell to tumor tissues
Middle infiltration.And tumor-associated macrophage in tumor microenvironment（tumor associated macrophage, TAM）High table
Reach B7-H4, and constitute negative sense immunological regulation loop with Treg, be conducive to the immunologic escape of tumour, Salceda etc. finds expression
Growth in SCID mice body for the B7-H4 human oophoroma cell line is rapid, and strengthens the Anti-G value of tumour cell；Also send out
Existing, expression B7-H4 can make epithelial cell that vicious transformation occurs, with the propagation of tumour cell, adhesion, transfer and to attack sexual intercourse close
Cut.It is noted that B7-H4 can not be stimulated approach to reverse by B7-CD28 the depression effect of T cell activation altogether, prompt machine
The normal immune system of body by the immunologic escape of the hindrance blocks B7-H4 mediation of itself, thus cannot independently cannot produce
The anti tumor immune response of effect.
The above results explanation B7-H4 participates in mediate tumor immunologic escape, is the target of a preferable immunotherapy of tumors.
Studies have found that lowering B7-H4 expression using siRNA can block tumor immune escape, nearest Sun etc. adopts B7-H4 specificity
ShRNA silence B7-H4 is expressed in lung cancer cell line A549 cell, and this cell is co-cultured with Jurkat cell, and result shows heavy
The expression of silent B7-H4 gene can promote Jurkat cell propagation, suppresses its apoptosis, promotes cell cycle progression and induction IFN-
The generation of γ, IL-10 and IL-2.
In addition to tumour, effect in the immune related diseases such as autoimmune disease, infectious diseases for the B7-H4
Have been reported that.Find that in rheumatoid arthritis patients peripheral blood and joint fluid, sB7-H4 level raises, and with disease severity be in
Positive correlation, in collagen-induced arthritis mouse model, overexpression sB7-H4 and knockout B7-H4 gene all lead to disease serious
Degree increases, and points out sB7-H4 can block the signal transmission of B7-H4 as Receptor Competition agent.In addition high expression in islet cells
B7-H4 can suppress the activation of autoreactive T cell, thus protecting islet cells it was found that B7-H4 and diabetes, transplanting
The correlations such as rejection.We are in conventional research it was also found that B7-H4 genetic modification can make Mice Islet Cells in diabetes
Time-to-live in mouse body is obviously prolonged；Soluble B7-H4-Ig by lowering IL-2, IFN-γ, IL-4 and can raise IL-
10 expression come to suppress ConA induction hepatic injury.Effect in anti-infectious immunity for the B7-H4 also has been reported that, domestic scholars find
In chronic hepatitis B and liver failure patient's liver organization, CD3+T cell, macrophage, bile duct cell and endothelial cell are high
Expression B7-H4, finds lung's sense that B7-H4 deficient mice leads to acute streptococcus in acute meningitis from Streptococcus pneumoniae mouse model
Dye has preferable tolerance.
Above result of study prompting：（1）B7-H4 is a kind of broad-spectrum tumor mark, can be used for the primary dcreening operation of tumour；（2）B7-
H4 is the promising target of tumour, treating immune relative disease；（3）SB7-H4 monomer can block its signal as Decoy receptor and lead to
Road, for releasing the inhibitory action to T cell, can activate body's immunity.
At present although there being the paper report of eucaryon and prokaryotic expression people's sB7-H4 albumen both at home and abroad, but, existing skill
In art, the structure about sB7-H4 protein eukaryotic expression system lacks high efficiency, and, expressed protein is not reported
People's sB7-H4 protein monomer of eukaryotic expression can be obtained by subsequent treatment.
Content of the invention
Present invention aims to the deficiencies in the prior art, provide people the system of sB7-H4 eukaryotic expression protein monomers
Preparation Method.
To achieve these goals, the present invention adopts the following technical scheme that：
There is provided the preparation method of people's sB7-H4 eukaryotic expression protein monomers, it comprises the following steps：
It comprises the following steps：
Step one, gene order determination, codon optimization and gene chemical synthesis：Select people's sB7-H4 gene order, by full genome
The mode of synthesis synthesizes expression people's sB7-H4 full-length gene；Using people IgF c as label, and insert FLAG/EK protease enzyme
Enzyme site, manual signal peptide is inserted in front end, and according to the characteristic of carrier and expression cell, codon is optimized；With
PcDNA3.1 is expressed in 293-T cell as expression vector；
Step 2, vector construction and plasmid extraction：
（1）Gene chemical synthesis：People sB7-H4 extracellular functional areas full-length gene to the synthesized expression of step one, adds at 5 ' ends
EcoRI restriction enzyme site, 3 ' ends add HindIII restriction enzyme site, connect in pcDNA3.1 carrier after purpose fragment double digestion；
（2）Purpose fragment and empty carrier double digestion：EcoRI and HindIII is added to carry out respectively in purpose fragment and empty carrier
Double digestion reacts；
（3）Purpose fragment is connected with carrier：Use T4 DNA ligase to connect purpose fragment and vector plasmid after double digestion, from
Configure coupled reaction liquid in heart pipe, blown and beaten with pipettor and mix ligation reaction, and carry out normal-temperature reaction certain time；
（4）Transformed competence colibacillus cell Trans1 T1；
（5）Identification：Carry out the PCR identification of positive bacterium colony, then the positive bacterium solution being detected by bacterium solution PCR is sequenced, will
Step（3）People's sB7-H4 full-length gene order that the sequence obtaining in purpose fragment and carrier Connection Step is synthesized with step one enters
Row comparative analysis, determines whether it is genes of interest；
（6）Endotoxin-free plasmid extraction：According to E.Z.N.A. Endo-free Plasmid Maxi Kit（Omega）Reagent
Box specification carries out endotoxin-free plasmid extraction, and Cord blood；
Step 3, cell culture and transfection：
（1）Cell recovery；
（2）Cell culture；
（3）Cell transient transfection；
Step 4, protein expression and purification：
（1）Protein expression and identification：SB7-H4 protein is expressed in 293-T and Chinese hamster ovary celI, and to expressed sB7-
H4 protein is identified；
（2）Proteolytic cleavage；
（3）Protein purification.
In technique scheme, described step 2 vector construction and plasmid extraction step, described（2）Purpose fragment and zero load
In body double digestion step, described double digestion reaction system is：Purpose fragment/empty carrier 2 μ g,EcoRI 1 μL、HindIII 1
μL、10X FastDigest Buffer 3 μL、ddH2O 30 μL；And incubate 1 h at 37 DEG C, then record 1.5% agar
Sugared gel carries out electrophoresis, reclaims purpose fragment, measures concentration.
In technique scheme, described step 2 vector construction and plasmid extraction step, described（3）Purpose fragment and carrier
In Connection Step, the time of normal-temperature reaction is 30min, and reaction system is：Purpose fragment digestion products 3 μ L, empty carrier digestion are produced
Thing 5 μ L, 10X T4 DNA Ligase Buffer 1 μ L, T4 DNA Ligase 1 μ L.
In technique scheme, described step 2 vector construction and plasmid extraction step, described（4）Transformed competence colibacillus cell
Trans1 T1 comprises the following steps：
A, from -80 DEG C of refrigerators take out competent cell, be placed in thawed on ice；
The competent cell taking 50 μ L under B, germ-free condition is placed in the centrifuge tube of 1.5 mL after sterilizing；
C, addition 10 μ L connection products, place 30 minutes in ice；
D, place 30 seconds at 42 DEG C（Heat shock）, centrifuge tube must not be shaken；
E, fast transfer centrifuge tube place 2～3 min in ice；
F, addition 200 μ L LB fluid nutrient mediums, piping and druming mixes, and coats on LA Agar Plating, is inverted culture for 37 DEG C
12～16 h.
In technique scheme, described step 2 vector construction and plasmid extraction step, described（5）In authentication step, sun
Property bacterium colony PCR identification concretely comprise the following steps：Picking single bacterium colony is inoculated in LA nutrient solution, 37 DEG C of shaking table 220 rpm cultures
Take 1 μ L bacterium solution to enter performing PCR amplification as template after 4h, then record 1.5% Ago-Gel, take 5 μ L PCR primer to carry out electricity
Swimming detection.
In technique scheme, described step 3 cell culture and transfection procedure, described（1）The concrete steps of cell recovery
As follows：
A, water-bath is preheated to 37 DEG C, irradiates the superclean bench table top of 30min with 75% alcohol wipe ultraviolet；
B, it is well placed sterilized centrifuge tube, suction pipe and blake bottle in superclean bench in order, take out cryopreservation tube, rapidly will
Cryopreservation tube is put in the water-bath having been warmed up and is thawed rapidly, and will constantly shake, and so that the liquid in pipe is melted rapidly,
When also there is little by little no thawing in cryopreservation tube, take out, wipe the outer wall of cryopreservation tube with cotton ball soaked in alcohol, then take into super-clean bench
Interior；
C, prepare cell suspension, cell be transferred in a 15ml centrifuge tube, drop by drop add preheated culture medium,
Simultaneously while rocking centrifuge tube；The amount adding culture medium will reach more than 10ml；5 min are centrifuged with 800rpm；Suck supernatant, so
Use 1ml culture medium re-suspended cell afterwards；Cell suspension is distributed in culture dish, culture dish is put into 37 DEG C and contains 5%CO2Culture
Culture in case, change time of liquid by cell settlement speed conditions depending on.
In technique scheme, described step 3 cell culture and transfection procedure, described（2）The concrete steps of cell culture
As follows：
A, water-bath is preheated to 37 DEG C, irradiates the superclean bench table top of 30min with 75% alcohol wipe ultraviolet, ultra-clean
It is well placed sterilized centrifuge tube, suction pipe, blake bottle in order in workbench；
B, taking-up Tissue Culture Flask, sterile working；Open bottle cap, sop up old nutrient solution；With PBS washed cell one to secondary；
C, the appropriate trypsin-EDTA solution of addition, rinse cell ware bottom, sop up trypsin-EDTA solution, put into 37 DEG C of trainings
Foster case 1-3 minute, observes under inverted microscope, when cell will be to be separated and when assuming round shaped grain shape, patting culture bottle wall makes greatly
Part cell detachment, adds the fresh culture containing serum in right amount to terminate trypsin effect；
D, inhaled up and down with suction pipe and put for several times to break up cell mass, after being mixed evenly, supply 3n（N is to pass on a bottle number）Ml cultivates
Base, is transferred in new blake bottle according to dilution ratio；Cell suspension is distributed in culture dish, culture dish is put into 37 DEG C and contains 5%
CO2Incubator in culture, change time of liquid by vitro growth rates situation depending on.
In technique scheme, described step 3 cell culture and transfection procedure, described（3）Cell transient transfection concrete
Step is as follows：
A, day before transfection, 2 × 105Individual cell is seeded on 6 orifice plates, 2ml complete medium, and before transfection, cell confluency reaches
80-90%；
B, 100 ul serum free mediums add 3 ug plasmids, soft mixing, mix lipofectamine reagent, use 100ul
Serum free medium dilutes 2 ul lipofectamine reagent, gently mixes, and room temperature places 5min；
C, by the plasmid having diluted and the mixing of lipofectamine reagent, soft mix, room temperature places 20min, to form matter
Grain-lipofectamine compound；
D, 200 ul plasmid-lipofectamine compounds are added in the cell hole containing 800ul serum free medium, back and forth
Softly rock Tissue Culture Plate；Cell at 37 DEG C, 5% CO2In incubator, suck transfection media after culture 5-6h, change no
Blood serum medium；
E, different time take cell culture supernatant detection protein expression situation.
In technique scheme, described step 4 protein expression and purification step, described（1）Protein expression and identification step
Suddenly it is specifically divided into following two steps：
A, protein expression：293-T and Chinese hamster ovary celI are incubated in free serum culture respectively, cell transfecting the previous day carries out sub-bottle,
Collect cell culture supernatant within 2 ~ 6 days after transfected plasmids；
B, Identification of Fusion Protein：Cell supernatant is centrifuged, takes supernatant, using SDS-PAGE and Western blotting inspection
Survey the expression of destination protein, and calculate estimation expressing quantity.
In technique scheme, described step 4 protein expression and purification step, described（2）Proteolytic cleavage step is concrete
For：Supernatant collection, is filtered after centrifugation, and filtrate, through Protein A purification, then carries out enzyme using EK protease to fusion protein
Cut；
Described（3）Protein purification steps are specially：Mixed system after digestion by Protein A remove enzyme, Fc fragment and
There is no digested recombinant protein, collect the destination protein flowing out, carry out dialysis in PBS.
The present invention compared with prior art, has the beneficial effects that：
（1）The preparation method of people's sB7-H4 eukaryotic expression protein monomers that the present invention provides, is excellent by carrying out to codon
Change, and in N-terminal insertion people IgFc as purification tag, and the N-terminal in fusion protein inserts artificial homing sequence, realizes albumen
Highly-soluble secreting, expressing.
（2）The preparation method of people's sB7-H4 eukaryotic expression protein monomers that the present invention provides, inserts homing sequence in N-terminal
Realize PE expression；Insertion human IgG1's Fc sequence is conducive to the extraction and purification of albumen, also can reduce albumen to greatest extent
The generation of aggressiveness；Insert the impact to destination protein when IgG1Fc sequence can control digestion to greatest extent in the N-terminal of sB7-H4.
（3）The preparation method of people's sB7-H4 eukaryotic expression protein monomers that the present invention provides, by existing protein
Through protein A column purification after expressing in 293-T cell, and through EK protease digestion, obtain highly purified people's sB7-H4 eukaryotic expression
Protein monomers, this people's sB7-H4 eukaryotic expression protein monomers be used as antigen-immunized animal prepare antibody it is also possible to
In the normative reference product as detection people sB7-H4, it is also used as the Decoy receptor of B7-H4, Competitive assays B7-H4 believes simultaneously
Number path, for the treatment of the immunity diseases such as tumour.
Specific embodiment
In order that technical problem solved by the invention, technical scheme and beneficial effect become more apparent, below in conjunction with
Embodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only in order to explain
The present invention, is not intended to limit the present invention.
The preparation method of people's sB7-H4 eukaryotic expression protein monomers, it comprises the following steps：
It comprises the following steps：
Step one, gene order determination, codon optimization and gene chemical synthesis：Select people's sB7-H4 gene order, by full genome
The mode of synthesis synthesizes expression people's sB7-H4 full-length gene；Using people IgF c as label, and insert FLAG/EK protease enzyme
Enzyme site, manual signal peptide is inserted in front end, and according to the characteristic of carrier and expression cell, codon is optimized；With
PcDNA3.1 is expressed in 293-T cell as expression vector；
Step 2, vector construction and plasmid extraction：
（1）Gene chemical synthesis：People sB7-H4 extracellular functional areas full-length gene to the synthesized expression of step one, adds at 5 ' ends
EcoRI restriction enzyme site, 3 ' ends add HindIII restriction enzyme site, connect in pcDNA3.1 carrier after purpose fragment double digestion；
（2）Purpose fragment and empty carrier double digestion：EcoRI and HindIII is added to carry out respectively in purpose fragment and empty carrier
Double digestion reacts；
（3）Purpose fragment is connected with carrier：Use T4 DNA ligase to connect purpose fragment and vector plasmid after double digestion, from
Configure coupled reaction liquid in heart pipe, blown and beaten with pipettor and mix ligation reaction, and carry out normal-temperature reaction certain time；
（4）Transformed competence colibacillus cell Trans1 T1；
（5）Identification：Carry out the PCR identification of positive bacterium colony, then the positive bacterium solution being detected by bacterium solution PCR is sequenced, will
Step（3）People's sB7-H4 full-length gene order that the sequence obtaining in purpose fragment and carrier Connection Step is synthesized with step one enters
Row comparative analysis, determines whether it is genes of interest；
（6）Endotoxin-free plasmid extraction：According to E.Z.N.A. Endo-free Plasmid Maxi Kit（Omega）Reagent
Box specification carries out endotoxin-free plasmid extraction, and Cord blood；
Step 3, cell culture and transfection：
（1）Cell recovery；
（2）Cell culture；
（3）Cell transient transfection；
Step 4, protein expression and purification：
（1）Protein expression and identification：SB7-H4 protein is expressed in 293-T and Chinese hamster ovary celI, and to expressed sB7-
H4 protein is identified；
（2）Proteolytic cleavage；
（3）Protein purification.
Specifically, described step 2 vector construction and plasmid extraction step, described（2）Purpose fragment and empty carrier double digestion
In step, described double digestion reaction system is：Purpose fragment/empty carrier 2 μ g,EcoRI 1 μL、HindIII 1 μL、10X
FastDigest Buffer 3 μL、ddH2O 30 μL；And incubating 1 h at 37 DEG C, the Ago-Gel then recording 1.5% enters
Row electrophoresis, reclaims purpose fragment, measures concentration.
Specifically, described step 2 vector construction and plasmid extraction step, described（3）Purpose fragment and carrier Connection Step
In, the time of normal-temperature reaction is 30min, and reaction system is：Purpose fragment digestion products 3 μ L, empty carrier digestion products 5 μ L,
10X T4 DNA Ligase Buffer 1 μL、T4 DNA Ligase 1 μL.
Specifically, described step 2 vector construction and plasmid extraction step, described（4）Transformed competence colibacillus cell Trans1
T1 comprises the following steps：
A, from -80 DEG C of refrigerators take out competent cell, be placed in thawed on ice；
The competent cell taking 50 μ L under B, germ-free condition is placed in the centrifuge tube of 1.5 mL after sterilizing；
C, addition 10 μ L connection products, place 30 minutes in ice；
D, place 30 seconds at 42 DEG C（Heat shock）, centrifuge tube must not be shaken；
E, fast transfer centrifuge tube place 2～3 min in ice；
F, addition 200 μ L LB fluid nutrient mediums, piping and druming mixes, and coats on LA Agar Plating, is inverted culture for 37 DEG C
12～16 h.
Specifically, described step 2 vector construction and plasmid extraction step, described（5）In authentication step, positive bacterium colony
What PCR identified concretely comprises the following steps：Picking single bacterium colony is inoculated in LA nutrient solution, takes 1 μ L after 37 DEG C of shaking table 220 rpm culture 4h
Bacterium solution enters performing PCR amplification as template, then records 1.5% Ago-Gel, takes 5 μ L PCR primer to carry out electrophoresis detection.
Specifically, described step 3 cell culture and transfection procedure, described（1）The comprising the following steps that of cell recovery：
A, water-bath is preheated to 37 DEG C, irradiates the superclean bench table top of 30min with 75% alcohol wipe ultraviolet；
B, it is well placed sterilized centrifuge tube, suction pipe and blake bottle in superclean bench in order, take out cryopreservation tube, rapidly will
Cryopreservation tube is put in the water-bath having been warmed up and is thawed rapidly, and will constantly shake, and so that the liquid in pipe is melted rapidly,
When also there is little by little no thawing in cryopreservation tube, take out, wipe the outer wall of cryopreservation tube with cotton ball soaked in alcohol, then take into super-clean bench
Interior；
C, prepare cell suspension, cell be transferred in a 15ml centrifuge tube, drop by drop add preheated culture medium,
Simultaneously while rocking centrifuge tube；The amount adding culture medium will reach more than 10ml；5 min are centrifuged with 800rpm；Suck supernatant, so
Use 1ml culture medium re-suspended cell afterwards；Cell suspension is distributed in culture dish, culture dish is put into 37 DEG C and contains 5%CO2Culture
Culture in case, change time of liquid by cell settlement speed conditions depending on.
Specifically, described step 3 cell culture and transfection procedure, described（2）The comprising the following steps that of cell culture：
A, water-bath is preheated to 37 DEG C, irradiates the superclean bench table top of 30min with 75% alcohol wipe ultraviolet, ultra-clean
It is well placed sterilized centrifuge tube, suction pipe, blake bottle in order in workbench；
B, taking-up Tissue Culture Flask, sterile working；Open bottle cap, sop up old nutrient solution；With PBS washed cell one to secondary；
C, the appropriate trypsin-EDTA solution of addition, rinse cell ware bottom, sop up trypsin-EDTA solution, put into 37 DEG C of trainings
Foster case 1-3 minute, observes under inverted microscope, when cell will be to be separated and when assuming round shaped grain shape, patting culture bottle wall makes greatly
Part cell detachment, adds the fresh culture containing serum in right amount to terminate trypsin effect；
D, inhaled up and down with suction pipe and put for several times to break up cell mass, after being mixed evenly, supply 3n（N is to pass on a bottle number）Ml cultivates
Base, is transferred in new blake bottle according to dilution ratio；Cell suspension is distributed in culture dish, culture dish is put into 37 DEG C and contains 5%
CO2Incubator in culture, change time of liquid by vitro growth rates situation depending on.
Specifically, described step 3 cell culture and transfection procedure, described（3）The concrete steps of cell transient transfection are such as
Under：
A, day before transfection, 2 × 105Individual cell is seeded on 6 orifice plates, 2ml complete medium, and before transfection, cell confluency reaches
80-90%；
B, 100 ul serum free mediums add 3 ug plasmids, soft mixing, mix lipofectamine reagent, use 100ul
Serum free medium dilutes 2 ul lipofectamine reagent, gently mixes, and room temperature places 5min；
C, by the plasmid having diluted and the mixing of lipofectamine reagent, soft mix, room temperature places 20min, to form matter
Grain-lipofectamine compound；
D, 200 ul plasmid-lipofectamine compounds are added in the cell hole containing 800ul serum free medium, back and forth
Softly rock Tissue Culture Plate；Cell at 37 DEG C, 5% CO2In incubator, suck transfection media after culture 5-6h, change no
Blood serum medium；
E, different time take cell culture supernatant detection protein expression situation.
Specifically, described step 4 protein expression and purification step, described（1）Protein expression is specifically divided with authentication step
For following two steps：
A, protein expression：293-T and Chinese hamster ovary celI are incubated in free serum culture respectively, cell transfecting the previous day carries out sub-bottle,
Collect cell culture supernatant within 2 ~ 6 days after transfected plasmids；
B, Identification of Fusion Protein：Cell supernatant is centrifuged, takes supernatant, using SDS-PAGE and Western blotting inspection
Survey the expression of destination protein, and calculate estimation expressing quantity.
Specifically, described step 4 protein expression and purification step, described（2）Proteolytic cleavage step is specially：Supernatant
Liquid is collected, and is filtered, filtrate, through Protein A purification, then carries out digestion using EK protease to fusion protein after centrifugation；
Described（3）Protein purification steps are specially：Mixed system after digestion by Protein A remove enzyme, Fc fragment and
There is no digested recombinant protein, collect the destination protein flowing out, carry out dialysis in PBS.
Finally it should be noted that above example is only in order to illustrating technical scheme, rather than the present invention is protected
The restriction of shield scope, although having made to explain to the present invention with reference to preferred embodiment, those of ordinary skill in the art should
Work as understanding, technical scheme can be modified or equivalent, without deviating from the reality of technical solution of the present invention
Matter and scope.
Claims (10)
1. people sB7-H4 eukaryotic expression protein monomers preparation method it is characterised in that：It comprises the following steps：
Step one, gene order determination, codon optimization and gene chemical synthesis：Select people's sB7-H4 gene order, by full genome
The mode of synthesis synthesizes expression people's sB7-H4 full-length gene；Using people IgF c as label, and insert FLAG/EK protease enzyme
Enzyme site, manual signal peptide is inserted in front end, and according to the characteristic of carrier and expression cell, codon is optimized；With
PcDNA3.1 is expressed in 293-T cell as expression vector；
Step 2, vector construction and plasmid extraction：
（1）Gene chemical synthesis：People sB7-H4 extracellular functional areas full-length gene to the synthesized expression of step one, adds at 5 ' ends
EcoRI restriction enzyme site, 3 ' ends add HindIII restriction enzyme site, connect in pcDNA3.1 carrier after purpose fragment double digestion；
（2）Purpose fragment and empty carrier double digestion：EcoRI and HindIII is added to carry out respectively in purpose fragment and empty carrier
Double digestion reacts；
（3）Purpose fragment is connected with carrier：Use T4 DNA ligase to connect purpose fragment and vector plasmid after double digestion, from
Configure coupled reaction liquid in heart pipe, blown and beaten with pipettor and mix ligation reaction, and carry out normal-temperature reaction certain time；
（4）Transformed competence colibacillus cell Trans1 T1；
（5）Identification：Carry out the PCR identification of positive bacterium colony, then the positive bacterium solution being detected by bacterium solution PCR is sequenced, will
Step（3）People's sB7-H4 full-length gene order that the sequence obtaining in purpose fragment and carrier Connection Step is synthesized with step one enters
Row comparative analysis, determines whether it is genes of interest；
（6）Endotoxin-free plasmid extraction：According to E.Z.N.A. Endo-free Plasmid Maxi Kit（Omega）Reagent
Box specification carries out endotoxin-free plasmid extraction, and Cord blood；
Step 3, cell culture and transfection：
（1）Cell recovery；
（2）Cell culture；
（3）Cell transient transfection；
Step 4, protein expression and purification：
（1）Protein expression and identification：SB7-H4 protein is expressed in 293-T and Chinese hamster ovary celI, and to expressed sB7-
H4 protein is identified；
（2）Proteolytic cleavage；
（3）Protein purification.
2. people's sB7-H4 eukaryotic expression protein monomers according to claim 1 preparation method it is characterised in that：Described
Step 2 vector construction and plasmid extraction step, described（2）In purpose fragment and empty carrier double digestion step, described double digestion is anti-
The system is answered to be：Purpose fragment/empty carrier 2 μ g,EcoRI 1 μL、HindIII 1 μL、10X FastDigest Buffer 3
μL、ddH2O 30 μL；And incubate 1 h at 37 DEG C, the Ago-Gel then recording 1.5% carries out electrophoresis, reclaims purpose fragment,
Measurement concentration.
3. people's sB7-H4 eukaryotic expression protein monomers according to claim 1 preparation method it is characterised in that：Described
Step 2 vector construction and plasmid extraction step, described（3）In purpose fragment and carrier Connection Step, the time of normal-temperature reaction is
30min, reaction system is：Purpose fragment digestion products 3 μ L, empty carrier digestion products 5 μ L, 10X T4 DNA Ligase
Buffer 1 μL、T4 DNA Ligase 1 μL.
4. people's sB7-H4 eukaryotic expression protein monomers according to claim 1 preparation method it is characterised in that：Described
Step 2 vector construction and plasmid extraction step, described（4）Transformed competence colibacillus cell Trans1 T1 comprises the following steps：
A, from -80 DEG C of refrigerators take out competent cell, be placed in thawed on ice；
The competent cell taking 50 μ L under B, germ-free condition is placed in the centrifuge tube of 1.5 mL after sterilizing；
C, addition 10 μ L connection products, place 30 minutes in ice；
D, place 30 seconds at 42 DEG C（Heat shock）, centrifuge tube must not be shaken；
E, fast transfer centrifuge tube place 2～3 min in ice；
F, addition 200 μ L LB fluid nutrient mediums, piping and druming mixes, and coats on LA Agar Plating, is inverted culture for 37 DEG C
12～16 h.
5. people's sB7-H4 eukaryotic expression protein monomers according to claim 1 preparation method it is characterised in that：Described
Step 2 vector construction and plasmid extraction step, described（5）In authentication step, the concretely comprising the following steps of the PCR of positive bacterium colony identification：
Picking single bacterium colony is inoculated in LA nutrient solution, takes 1 μ L bacterium solution to enter performing PCR as template after 37 DEG C of shaking table 220 rpm culture 4h
Amplification, then records 1.5% Ago-Gel, takes 5 μ L PCR primer to carry out electrophoresis detection.
6. people's sB7-H4 eukaryotic expression protein monomers according to claim 1 preparation method it is characterised in that：Described
Step 3 cell culture and transfection procedure, described（1）The comprising the following steps that of cell recovery：
A, water-bath is preheated to 37 DEG C, irradiates the superclean bench table top of 30min with 75% alcohol wipe ultraviolet；
B, it is well placed sterilized centrifuge tube, suction pipe and blake bottle in superclean bench in order, take out cryopreservation tube, rapidly will
Cryopreservation tube is put in the water-bath having been warmed up and is thawed rapidly, and will constantly shake, and so that the liquid in pipe is melted rapidly,
When also there is little by little no thawing in cryopreservation tube, take out, wipe the outer wall of cryopreservation tube with cotton ball soaked in alcohol, then take into super-clean bench
Interior；
C, prepare cell suspension, cell be transferred in a 15ml centrifuge tube, drop by drop add preheated culture medium,
Simultaneously while rocking centrifuge tube；The amount adding culture medium will reach more than 10ml；5 min are centrifuged with 800rpm；Suck supernatant, so
Use 1ml culture medium re-suspended cell afterwards；Cell suspension is distributed in culture dish, culture dish is put into 37 DEG C and contains 5%CO2Culture
Culture in case, change time of liquid by cell settlement speed conditions depending on.
7. people's sB7-H4 eukaryotic expression protein monomers according to claim 1 preparation method it is characterised in that：Described
Step 3 cell culture and transfection procedure, described（2）The comprising the following steps that of cell culture：
A, water-bath is preheated to 37 DEG C, irradiates the superclean bench table top of 30min with 75% alcohol wipe ultraviolet, ultra-clean
It is well placed sterilized centrifuge tube, suction pipe, blake bottle in order in workbench；
B, taking-up Tissue Culture Flask, sterile working；Open bottle cap, sop up old nutrient solution；With PBS washed cell one to secondary；
C, the appropriate trypsin-EDTA solution of addition, rinse cell ware bottom, sop up trypsin-EDTA solution, put into 37 DEG C of trainings
Foster case 1-3 minute, observes under inverted microscope, when cell will be to be separated and when assuming round shaped grain shape, patting culture bottle wall makes greatly
Part cell detachment, adds the fresh culture containing serum in right amount to terminate trypsin effect；
D, inhaled up and down with suction pipe and put for several times to break up cell mass, after being mixed evenly, supply 3n（N is to pass on a bottle number）Ml cultivates
Base, is transferred in new blake bottle according to dilution ratio；Cell suspension is distributed in culture dish, culture dish is put into 37 DEG C and contains 5%
CO2Incubator in culture, change time of liquid by vitro growth rates situation depending on.
8. people's sB7-H4 eukaryotic expression protein monomers according to claim 1 preparation method it is characterised in that：Described
Step 3 cell culture and transfection procedure, described（3）The comprising the following steps that of cell transient transfection：
A, day before transfection, 2 × 105Individual cell is seeded on 6 orifice plates, 2ml complete medium, and before transfection, cell confluency reaches 80-
90%；
B, 100 ul serum free mediums add 3 ug plasmids, soft mixing, mix lipofectamine reagent, use 100ul
Serum free medium dilutes 2 ul lipofectamine reagent, gently mixes, and room temperature places 5min；
C, by the plasmid having diluted and the mixing of lipofectamine reagent, soft mix, room temperature places 20min, to form matter
Grain-lipofectamine compound；
D, 200 ul plasmid-lipofectamine compounds are added in the cell hole containing 800ul serum free medium, back and forth
Softly rock Tissue Culture Plate；Cell at 37 DEG C, 5% CO2In incubator, suck transfection media after culture 5-6h, change no
Blood serum medium；
E, different time take cell culture supernatant detection protein expression situation.
9. people's sB7-H4 eukaryotic expression protein monomers according to claim 1 preparation method it is characterised in that：Described
Step 4 protein expression and purification step, described（1）Protein expression and authentication step are specifically divided into following two steps：
A, protein expression：293-T and Chinese hamster ovary celI are incubated in free serum culture respectively, cell transfecting the previous day carries out sub-bottle,
Collect cell culture supernatant within 2 ~ 6 days after transfected plasmids；
B, Identification of Fusion Protein：Cell supernatant is centrifuged, takes supernatant, using SDS-PAGE and Western blotting inspection
Survey the expression of destination protein, and calculate estimation expressing quantity.
10. people's sB7-H4 eukaryotic expression protein monomers according to claim 1 preparation method it is characterised in that：Institute
State step 4 protein expression and purification step, described（2）Proteolytic cleavage step is specially：Supernatant collection, is carried out after centrifugation
Filter, filtrate, through Protein A purification, then carries out digestion using EK protease to fusion protein；
Described（3）Protein purification steps are specially：Mixed system after digestion by Protein A remove enzyme, Fc fragment and
There is no digested recombinant protein, collect the destination protein flowing out, carry out dialysis in PBS.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610840603.1A CN106380511A (en) | 2016-09-22 | 2016-09-22 | Method for preparing human sB7-H4 eukaryotic expression protein monomer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610840603.1A CN106380511A (en) | 2016-09-22 | 2016-09-22 | Method for preparing human sB7-H4 eukaryotic expression protein monomer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106380511A true CN106380511A (en) | 2017-02-08 |
Family
ID=57936104
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610840603.1A Pending CN106380511A (en) | 2016-09-22 | 2016-09-22 | Method for preparing human sB7-H4 eukaryotic expression protein monomer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106380511A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114081941A (en) * | 2021-11-19 | 2022-02-25 | 上海奢旭企业管理中心(有限合伙) | Preparation method of B7-H4 protein and application of B7-H4 protein in preparation of medicine for resisting excessive immune response or resisting cytokine storm |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611340A (en) * | 2015-01-08 | 2015-05-13 | 山西医科大学 | Recombinant human CC16 gene, construction of eukaryotic expression vector of CC16 gene, and purification of recombinant protein |
-
2016
- 2016-09-22 CN CN201610840603.1A patent/CN106380511A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611340A (en) * | 2015-01-08 | 2015-05-13 | 山西医科大学 | Recombinant human CC16 gene, construction of eukaryotic expression vector of CC16 gene, and purification of recombinant protein |
Non-Patent Citations (3)
| Title |
|---|
| XIAOJIE WANG，ET AL: "B7-H4 Treatment of T Cells Inhibits ERK, JNK, p38, and", 《PLOS ONE》 * |
| 姜碧: "分泌型mB7-H4-hIg 重组融合蛋白定量", 《中国优生与遗传杂志》 * |
| 邵联波: "《人B7-H4-Fc融合蛋白的制备及B7-H4 受体表达的检测》", 《 中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114081941A (en) * | 2021-11-19 | 2022-02-25 | 上海奢旭企业管理中心(有限合伙) | Preparation method of B7-H4 protein and application of B7-H4 protein in preparation of medicine for resisting excessive immune response or resisting cytokine storm |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106755088A (en) | A kind of autologous CAR T cells preparation method and application | |
| US11932872B2 (en) | Dual chimeric antigen receptor-t cell which can be regulated, construction method therefor and use thereof | |
| CN106591363A (en) | Preparation method of universal heterologous CAR-T cells and application | |
| CN107109421A (en) | CAR expression vectors and CAR expression T cells | |
| CN107236762A (en) | A kind of method that minicircle dna transfecting T cells prepare clinical grade CAR T cell preparations | |
| CN113416260B (en) | Claudin18.2-targeted specific chimeric antigen receptor cell and preparation method and application thereof | |
| CN105296431A (en) | Tumor binding specific gamma delta TCR gene modified alpha beta T cell and cancer suppression application thereof | |
| CN109652380B (en) | CAR-T cell based on base editing targeting Lewis Y and preparation method and application thereof | |
| US8075882B2 (en) | Adoptive immune cells for tumor vaccines | |
| CN113896801B (en) | Chimeric antigen receptor cell targeting human Claudin18.2 and NKG2DL, and preparation method and application thereof | |
| WO2022262130A1 (en) | Macrophage specific chimeric antigen receptor, controllable polarized monocyte/macrophage expressing receptor, preparation method therefor and application thereof | |
| CN103784950A (en) | Preparation method of breast cancer-specific epitope polypeptide-loaded dendritic cell vaccine and kit thereof | |
| CN106834354A (en) | The preparation method and purposes of a kind of enhanced targeting immunocyte group of modification | |
| KR20220097984A (en) | Manufacturing method for making T cells expressing chimeric antigen receptor | |
| CN108341881A (en) | Chimeric antigen receptor and its expressing gene with safety switch, the NK cells of its modification and application | |
| CN110423767A (en) | Express Chimeric antigen receptor CAR gene and the application of solubility PD-1 | |
| WO2023104171A1 (en) | Chimeric antigen receptor t cell secreting bispecific antibodies, preparation method therefor and use thereof | |
| EA012520B1 (en) | Process for producing cytotoxic lymphocytes | |
| CN105218682A (en) | Through tumor therapeutic agent and method for making and the purposes of the transformation of IL-12/CD62L fusion rotein | |
| CN102532269B (en) | Dominant sequence of delta 1 chain complementary determining region (CDR) 3 in gamma delta T lymphocytes, and T cell receptor (TCR) transfected cells and application thereof | |
| CN106380511A (en) | Method for preparing human sB7-H4 eukaryotic expression protein monomer | |
| CN104524560A (en) | Dendritic cell tumor vaccine and preparation method and application thereof | |
| CN114703142B (en) | Human induced pluripotent stem cell, construction method and application thereof | |
| CN110331132A (en) | A method of external extensive induced NK cell amplification | |
| CN105732814A (en) | Human-mouse chimeric monoclonal antibody for human von willebrand factor A3 region as well as preparation method and application of human-mouse chimeric monoclonal antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170208 |
|
| RJ01 | Rejection of invention patent application after publication |