CN104711292A - Method for developing membrane expression PD-1 stable transfected cells - Google Patents
Method for developing membrane expression PD-1 stable transfected cells Download PDFInfo
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Abstract
The invention is suitable for the technical field of biologics and provides a method for developing membrane expression PD-1 stable transfected cells. The method comprises the following steps: carrier pLVX-PD-1-IRES-ZsGreen1 developing; virus packaging; and stable transfected cell development. According to the invention, slow viruses are packaged to re-infect eukaryocytes, and the re-infected eukaryocytes are sorted by a flow cytometry to obtain cell line capable of stably expressing PD-1 antigens, so that surface expression antigens are combined in the monoclonal antibody research and development process to quickly screen monoclonal antibody production lines with high appetency, and accordingly, lots of the monoclonal screening time is saved and much screening cost is lowered.
Description
Technical field
The invention belongs to biological technical field, what particularly relate to film expression people PD-1 albumen surely turns strain cell construction method.
Background technology
Apoptosis albumen 1(Programmed cell death protein1, hereinafter referred to as PD-1) be the albumen being also CD279 genes encoding by PDCD1 gene, for one of epicyte protein member in immunoglobulin superfamily, it is the I type membranin be typically made up of 268 amino acid.PD-1 is one of T cell regulatory factor in CD28/CTLA4 family, and its structure comprises the IgV structural domain outside born of the same parents, thereafter followed by end in a membrane spaning domain and born of the same parents.PD1 obtains in the T cell hybridoma of apoptosis, is named as programmed death-1 acceptor because it is relevant with apoptosis.Acceptor PD-1 in PD-L1 and its T cell interacts, play an important role in immunne response negative regulation, this molecule has distribution expression pattern widely, fastens higher expression at some tumour cells, and much research all shows that its Immune escaping mechanism in tumour is correlated with.The microenvironment of tumor locus can the expression of PD-L1 on inducing tumor cell, and wide model is expressed, and the PD-L1 of expression is conducive to generation and the growth of tumour, the apoptosis of inducing antitumor T cell.Therefore effectively can grow by Tumor suppression by blocking PD-L1/PD-1 signal path, the new treatment plan to tumor immune escape can be designed according to this, strengthening the lethal effect of T cell to tumour.
In antineoplastic immune, usually follow tumor specific T cells activation, step that T cell increment, tumor-infiltrated and T cell memory response are strengthened.Research shows, the PD-L1 that APCs in tumour cell and tumor microenvironment expresses all can through the activation of PD-1/PD-L1 signal path Tumor suppression T cells with antigenic specificity, lower the tumor immune response of T cell mediation, intervene PD-1/PD-L1 signal and be expected to the New Policy becoming immunotherapy of tumors.
At present, build stable expression cell line and have two kinds of methods:
1) antibiotic-screening: the ultimate principle of the structure stable expression cell line of this kind of method is cloned into by foreign DNA to have on the carrier of certain resistance, target gene is built up on the plasmid with antibiotic-screening mark, carrier, being transfected into host cell by modes such as calcium phosphate transfection, liposome transfection or electricity turn and being incorporated in host chromosome, screens with the microbiotic mark contained by carrier.The resistance screening mark of the most frequently used carrier for expression of eukaryon has Liu Suanyan NEOMYCIN SULPHATE (neomycin), Totomycin (hygromycin) and tetracycline (puromycin), replace Liu Suanyan NEOMYCIN SULPHATE to carry out selective screening with long G418, screening obtains the cell strain of the target protein that Absorbable organic halogens is expressed.But its screening time is long, and be limited to adopted rotaring transfecting mode and corresponding clone, can not the primary cell of transfection long-term cultivation and the cell of difficult transfection will be not suitable for adopting in this way for some, and the height turning right efficiency directly affects the probability integrated and occur, and false positive incidence is high, cell easily develops immunity to drugs in long-term Antibiotic medium, and subsequent cell Screening and Identification workload is large.
2) screen after slow virus infection: slow virus (Lentivirus) carrier is by HIV-1(human immune deficiency I C-type virus C) based on the gene therapy vector that grows up, distinguish general retroviral vector, it all has infection ability to somatoblast and Unseparated Cell.Lentiviral vectors is with carrying out high efficiency packaging, transfection stable integration enters the sequential element in the genome of target cell, and goal gene participates in the gene recombination of cell, forms genetic stability material, makes goal gene Absorbable organic halogens long-term expression in cell.Can effectively infect polytype cells such as neuronal cell, liver cell, myocardial cell, tumour cell, endotheliocyte, stem cell in infection ability, thus reach good gene therapy effect.After slow virus infected cell can very fast by goal gene random integration in genome, the advantages such as integration efficiency is high, and false positive is few.And its selection markers thing extends to the non-antibiotic marker thing of all kinds of fluorescent protein labeling, as EGFP, RFP, YFP, ZsGreen1 etc., flow cytometer can be easily passed through carry out identifying and screening, without the need to adding streaming antibody in follow-up screening.
Summary of the invention
What the object of the present invention is to provide a kind of film expression people PD-1 albumen surely turns strain cell construction method, is intended to solve the problem that prior art monoclonal antibody production strain false positive incidence is high, avidity is not high, mono-clonal screening time long and screening cost is high.
What the present invention was achieved in that a kind of film expression people PD-1 albumen surely turns strain cell construction method, comprises the steps:
The primer pLVX-PD-1-F of design containing restriction enzyme site EcoRI and the primer pLVX-PD-1-R containing restriction enzyme site BamHI, and the DNA sequence dna of described medicine and PD-1 carries out pcr amplification, collects amplified production;
Slow virus packaging plasmid carrier pLVX-IRES-ZsGreen1 and described amplified production are carried out enzyme respectively and cut process, obtain pLVX-IRES-ZsGreen1(EcoRI+BamHI respectively) and PD-1(EcoRI+BamHI) digestion products; Described pLVX-IRES-ZsGreen1(EcoRI+BamHI) with PD-1(EcoRI+BamHI) digestion products be connected after conversion processing, by converted product increase after carry out plasmid extraction process, obtain the carrier pLVX-PD-1-IRES-ZsGreen1 of film expression PD-1;
By the carrier pLVX-PD-1-IRES-ZsGreen1 of described film expression PD-1 and viral packaging plasmid pMD2.G and PsPAX
2cotransfection, in eukaryotic cell, carries out virus packaging, monitors simultaneously to virus titer;
When virus titer infects described eukaryotic cell with MOI=2-10, add the polybrene assistance virus that final concentration is 5-8ug/ml; After the described eukaryotic cell infecting virus is normally gone down to posterity, choose fluorescencepositive cell and carry out sorting, and the rear frozen process of amplification is carried out to the cell of sorting, obtain the cell strain of stably express PD-1.Film expression people PD-1 albumen provided by the invention surely turn strain cell construction method, by packaging slow virus infection of eukaryotic cells again, after fluorescent screening, obtain the cell strain that Absorbable organic halogens expresses PD-1 antigen.Owing to the slow virus of packaging further having been infected eukaryotic cell in the method, make its antigen rapid screening can expressed by mating surface in monoclonal antibody R&D process to the high PD-1 cell strain of avidity; And employing carries the carrier of fluorescin ZsGreen and then surely turns the structure of strain, make to become more simple and convenient, a large amount of saving mono-clonal screening time and screening cost in the screening process of PD-1 cell strain.
Accompanying drawing explanation
Fig. 1 be embodiment of the present invention film expression people PD-1 albumen surely turn strain cell construction method flow diagram;
The pcr amplification product electrophorogram of the DNA of the PD-1 that Fig. 2 embodiment of the present invention provides, wherein, M represents Marker, and "-" represents negative control, and 1-2 is the PCR primer of the DNA of PD-1;
Fig. 3 is that the pLVX-PD-1-IRES-ZsGreen1 vector construction enzyme that the embodiment of the present invention provides cuts qualification electrophorogram, and wherein, M represents Marker; 1-4 is pLVX-PD-1-IRES-ZsGreen1 mono-clonal EcoRI+BamHI double digestion;
Fig. 4 be the embodiment of the present invention provide protein expression qualification in plasmid-transfected cells fluorogram;
Fig. 5 is the Western Blot protein expression figure that the embodiment of the present invention provides, and wherein, 1 is 293T cell cytosol, and 2 is 293T cytolemma extract, and 3 is ZsGreen cell cytosol; 4 is ZsGreen cytolemma extract, and 5 is PD-1 cell cytosol; 6 is PD-1 cytolemma extract;
Fig. 6 be the embodiment of the present invention provide virus packaging time cell transfecting fluorogram;
Fig. 7 is the virus infection fluorogram of the virus titer that provides of embodiment of the present invention when measuring, and wherein, a is 5 μ l virus infection English hat figure, and b is 0.5 μ l virus infection fluorogram;
Fig. 8 is the malicious titer analysis streaming figure that the embodiment of the present invention provides, wherein, and the malicious titer analysis streaming figure of a to be malicious titer analysis streaming figure, the b of 5 μ l virus-infected wells be 0.5 μ l virus-infected wells;
Fig. 9 be the embodiment of the present invention provide surely turn strain cell construction time virus infection fluorogram;
Figure 10 is the stable expression cell strain sorting streaming figure that the embodiment of the present invention provides.
Embodiment
In order to make the technical problem to be solved in the present invention, technical scheme and beneficial effect clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
What embodiments provide a kind of film expression people PD-1 albumen surely turns strain cell construction method, comprises the steps, as shown in Figure 1:
S01. carrier construction pLVX-PD-1-IRES-ZsGreen1: the primer pLVX-PD-1-F of design containing restriction enzyme site EcoRI and the primer pLVX-PD-1-R containing restriction enzyme site BamHI, and the DNA sequence dna of described medicine and PD-1 carries out pcr amplification, collects amplified production;
Slow virus packaging plasmid carrier pLVX-IRES-ZsGreen1 and described amplified production are carried out enzyme respectively and cut process, obtain pLVX-IRES-ZsGreen1(EcoRI+BamHI respectively) and PD-1(EcoRI+BamHI) digestion products; Described pLVX-IRES-ZsGreen1(EcoRI+BamHI) with PD-1(EcoRI+BamHI) digestion products be connected after conversion processing, by converted product increase after carry out plasmid extraction process, obtain the carrier pLVX-PD-1-IRES-ZsGreen1 of film expression PD-1;
S02. virus packaging: by the carrier pLVX-PD-1-IRES-ZsGreen1 of described film expression PD-1 and viral packaging plasmid pMD2.G and PsPAX
2cotransfection, in eukaryotic cell, carries out virus packaging, monitors simultaneously to virus titer;
S03. surely turn the structure of strain cell: when virus titer infects described eukaryotic cell with MOI=2-10, add the polybrene assistance virus that final concentration is 5-8ug/ml; After the described eukaryotic cell infecting virus is normally gone down to posterity, choose fluorescencepositive cell and carry out sorting, and the rear frozen process of amplification is carried out to the cell of sorting, obtain the cell strain of stably express PD-1.
Particularly, above-mentioned steps S01 specifically comprises the steps:
S011. in order to carrier construction pLVX-PD-1-IRES-ZsGreen1, in the embodiment of the present invention, need to build primer pLVX-PD-1-F and pLVX-PD-1-R with the DNA sequence dna (sequence table title 1) of the PD-1 of NCBI announcement, the gene order of described primer pLVX-PD-1-F and primer pLVX-PD-1-R respectively containing restriction enzyme site EcoRI and restriction enzyme site BamHI.
Find through contriver's repetition test, as preferred embodiment, the sequence of described primer pLVX-PD-1-F and primer pLVX-PD-1-R is respectively:
PLVX-PD-1-F:5'-CCGGAATTCATGCAGATCCCACAGGCG-3 ' (sequence table title 2),
PLVX-PD-1-R:5'-CGCGGATCCTCAGAGGGGCCAAGAGCAGT-3'(sequence table title 3).
When described primer pLVX-PD-1-F and primer pLVX-PD-1-R is respectively above-mentioned sequence, both ensure that meanwhile, the DNA sequence dna that can complete following PD-1 more efficiently carried out pcr amplification containing restriction enzyme site gene fragment in gene order.
S012. the DNA sequence dna of described primer pLVX-PD-1-F and primer pLVX-PD-1-R and PD-1 is carried out pcr amplification.Particularly, by described primer pLVX-PD-1-F and pLVX-PD-1-R ddH
2o dissolves, and makes its concentration be 18-22mM, utilizes round pcr to increase.
As preferred embodiment, in the step of above-mentioned carrier construction pLVX-PD-1-IRES-ZsGreen1, the amplification system that the DNA sequence dna of described PD-1 carries out pcr amplification process is:
The method that the DNA sequence dna of described PD-1 carries out pcr amplification process is:
First stage: 95-98 DEG C/2-3min;
Subordinate phase: 95-98 DEG C/25-35s, 58-62 DEG C/25-35s, 68-74 DEG C/25-35s, 30-35 circulation altogether;
Phase III: 68-74 DEG C/8-12min, 10 DEG C of preservations.
The amplification system of above-mentioned pcr amplification process and amplification method, effectively can improve the pcr amplification efficiency of the DNA sequence dna of PD-1.
The pcr amplification product that impurity is few in order to obtain, purity is high, need be separated above-mentioned pcr amplification product, purifying, and described separation method adopts 1.5% agarose gel electrophoresis.Embodiment of the present invention use glue recovery test kit carries out pcr amplification product and carries out purifying recovery, the sample 25-35 μ l ddH after purified
2o reclaims from post.In the embodiment of the present invention, described glue reclaims the glue recovery test kit that test kit can adopt this area conventional, preferred embodiment of doing, and described glue recycling step employing Transgen article No. is that the glue of EG101-02 reclaims test kit specification sheets.In order to carry out yield and purity test to recovery sample, concentration and OD can be carried out to the PCR primer after recovery
260/280test.
S013. in order to improve the screening time of PD-1 cell strain, improve screening efficiency and accuracy, the embodiment of the present invention selects the plasmid pLVX-IRES-ZsGreen1 carrying fluorescin ZsGreen1 as the carrier building PD-1 cell strain.The design of this carrier, effectively improves the screening efficiency surely turned in strain cell processes building PD-1 albumen, meanwhile, to the mensuration non-false positive phenomenon of fluorescin ZsGreen1, effectively improves the accuracy rate of screening.
In order to obtain carrier pLVX-PD-1-IRES-ZsGreen1, needing that enzyme is carried out to the pcr amplification product of plasmid vector pLVX-IRES-ZsGreen1 and PD-1 provided and cutting process.
It is as follows that the enzyme of described plasmid vector pLVX-IRES-ZsGreen1 cuts system:
It is as follows that the PCR primer enzyme of described PD-1 cuts system:
The endonuclease reaction system of the pcr amplification product of above-mentioned plasmid vector pLVX-IRES-ZsGreen1 and PD-1 is placed in respectively after on 37 DEG C of constant-temperature metal baths, enzyme cuts 3.5-4.5h, use 1.5% agarose gel electrophoresis, digestion products glue is recycled, and with 25-35 μ l ddH
2dNA fragmentation on O wash-out post, calculates glue and reclaims yield.Described glue reclaims the glue recovery test kit that test kit can adopt this area conventional, preferred embodiment of doing, and described glue recycling step employing Transgen article No. is that the glue of EG101-02 reclaims test kit specification sheets.
S014. by pLVX-IRES-ZsGreen1(EcoRI+BamHI obtained above), PD-1(EcoRI+BamHI), connect, obtain carrier pLVX-PD-1-IRES-ZsGreen1.Particularly, the linked system of described connection pLVX-IRES-ZsGreen1 and PD-1 is as follows:
Above-mentioned linked system is connected on 20-25 DEG C of constant-temperature metal bath transformed competence colibacillus cell after 1-2h, as preferred embodiment, described competent cell can select competent cell DH5 α.As specific embodiment, described step of converting is as follows: draw 10 μ l connection products and add in the 100 μ l DH5 α competent cells thawed on ice, 20min is placed on ice gently after mixing, 42 DEG C of heat shock 45s, add 500 μ lLB substratum, constant temperature culture 45min in 37 DEG C after placing 2min on ice, the centrifugal 1min of rear 6000rpm, abandon 500 μ l supernatants, coat after utilizing residue about 100 μ l liquid gravity treatment thalline in the LB flat board containing Ampr, spend the night in 37 DEG C of incubators to be inverted and cultivate.
Whether correct in order to identify DNA whether successful connection, the DNA sequence dna of the foreign gene embedded in described carrier pLVX-PD-1-IRES-ZsGreen1, as preferred embodiment, in the embodiment of the present invention, the product after above-mentioned connection transforms is identified.Particularly, authentication method is: the mono-clonal on picking Ampr LB flat board, be seeded to during LB liquid culture connects, sterile manner is adopted to take out a small amount of bacterium liquid for plasmid extraction after incubated overnight, plasmid extraction method can adopt this area to commonly use plasmid extraction technology, as preferred embodiment, described plasmid extraction employing Omega article No. is the plasmid extraction method in the specification sheets of D6943-02.
The plasmid obtained through above-mentioned plasmid extraction process is carried out enzyme and cuts authentication process, it is as follows that its enzyme cuts identification system:
Above-mentioned enzyme is cut identification system enzyme on 37 DEG C of constant-temperature metal baths and cut the detection of 3-5h rear electrophoresis, the sample that the embodiment of the present invention is chosen through above-mentioned process detects.
S015. in order to obtain a large amount of carrier pLVX-PD-1-IRES-ZsGreen1, need the bacterium enlarged culturing of plasmid be contained to above-mentioned and carry out plasmid extraction.Particularly:
By the microbionation containing plasmid of above-mentioned correct structure to containing in the LB nutrient solution of Ampr, carry out constant-temperature table overnight incubation, amplification plasmid.As preferred embodiment, shaking table temperature is 37 DEG C, and revolution is 250rpm.
The bacterium cultivated by above-mentioned shaking table carries out centrifugal treating, goes to precipitate pellet fraction.
The thalline vibrator of above-mentioned acquisition is broken up cell, under oscillation condition, progressively adds TE solution make cell be in complete resuspended state.The mixed solution of described TE solution to be final concentration the be EDTA of Tris-HCl, 50mmol/l of 25mmol/l, pH is 8.0.Add in above-mentioned resuspension fluids NaOH-SDS solution fully mix after cooling process, NaOH, the weight percent of described NaOH-SDS solution to be final concentration be 0.2mol/l are the mixed solution of 1%SDS.As preferred embodiment, the described type of cooling is preferably put and is placed on ice, and the treatment time is 8-12min.The 2.5-3.5mol/l pH adding precooling under oscillation condition is the KAC solution of 4.5-5, then carries out centrifugal treating, gets supernatant liquor, and centrifugal condition is preferably: at 4 DEG C, the centrifugal 15-20min of 10000rpm.The Pre-cooling Mode of described KAC solution can adopt the conventional type of cooling, as optimal way, KAC solution is placed 15-20min on ice.
Supernatant liquor will be added in the supernatant liquor of above-mentioned acquisition and Virahol volume ratio is the Virahol of 2:1, mixing, put after room temperature places 15 ~ 30min and carry out centrifugal treating.After abandoning supernatant, add the abundant dissolution precipitation of TE Buffer of body 1.5ml.And add the NH that volume ratio is the 10mol/l of 1:3
4aC mixes, and carries out centrifugal, get supernatant liquor after cooling process.As preferred embodiment, described cooling process can adopt the mode being placed on and placing 15-25min on ice.After the centrifugal 10min of 12000rpm, in above-mentioned supernatant liquor, add the dehydrated alcohol that volume ratio is 2:1, under-80 DEG C of environment, place 20-30min, after centrifugal treating, abandon its supernatant liquor.Precipitation carried out washing rear recentrifuge process with 1ml70% ethanol, after repeated washing 1-2 time, precipitation left and taken by reject supernatant liquor.As preferred embodiment, above-mentioned centrifugal condition is: at 4 DEG C, the centrifugal 15-20min of 12000rpm.
With the abundant dissolution precipitation of TE Buffer, add RNase A and make its final concentration be 10ug/ml, after 15-25min is placed in 37 DEG C of water-baths, add the NaCl solution that concentration is 5mol/l, mixing; Add 30%PEG6000 (m/V)-1.5mol/l NaCl solution again, mixing; Put after placing 30min on ice and carry out centrifugal treating.By the supernatant liquor reject after centrifugal, add 5mol/l NaCl with after the complete dissolution precipitation of TE Buffer, mixing, then to add volume ratio be 25:24:1 phenol-chloroform-isoamyl alcohol, mixing, gets upper strata aqueous phase after centrifugal treating.In above-mentioned aqueous phase, add phenol, mixing, gets upper strata aqueous phase after centrifugal treating.The dehydrated alcohol putting-80 DEG C of refrigerator precoolings is in advance added, mixing in above-mentioned aqueous phase; Centrifugal after putting-80 DEG C of refrigerators placement 15min, by 70% washing with alcohol precipitation also centrifugal treating.The preferred 15000rpm of above-mentioned centrifugal treating in this step, centrifugation time can regulate according to centrifugal effect.Centrifugal to 6000rpm after abandoning supernatant, take out sample loading gun and draw 70% alcohol, put into Bechtop 2 ~ 5min and get final product seasoning.In above-mentioned dry thing, add TE dissolve, and measure concentration with Nanodrop, and to carry out being diluted to ultimate density according to concentration be 1ug/ μ l.
In the embodiment of the present invention, in order to confirm the exactness of foreign gene in carrier pLVX-PD-1-IRES-ZsGreen1 further, and the carrying out smoothly of protein expression, protein expression qualification can be carried out to the plasmid extracted in above-mentioned S015 step.Concrete, operate as follows:
Endotoxic extracting plasmid transfection eukaryotic cell is removed in utilization, described eukaryotic cell can adopt eukaryotic cell conventional in this area, as preferred embodiment, described eukaryotic cell selects 293T cell, after transfection 68-76h, collecting cell carries out Protein Extraction, transfection time is preferably 72h, then carries out Western Blot and identifies protein expression.Concrete steps are as follows:
Plasmid transfection: utilize calcium phosphate transfection to carry out cell transfecting, day before transfection carries out passage is 60-80% to cytogamy degree next day; Before transfection, 3.5-4.5h uses instead without dual anti-DMEM+10%FBS substratum, and rotaring redyeing system is made up of A liquid and B liquid.The time used instead without dual anti-DMEM+10%FBS substratum is preferably 4h.
Wherein, A liquid is made up of 50 μ l calcium phosphate solution A, and calcium phosphate solution A is 2 parts of HEPES buffering salts, and pH is 7.05, and its HEPES buffering salt is composed as follows:
HEPES 50mM，
NaCl 280mM，
Na
2HPO
41.5mM。
Calcium phosphate reagent B liquid is composed as follows:
Calcium phosphate solution B 5 μ l,
pLVX-PD-1-IRES-ZsGreen1 4μg，
DdH
2o mends to 50 μ l.
Wherein, calcium phosphate solution B liquid is 2.5M CaCl
2.
Utilize calcium phosphate transfection method transfection 4ug control vector pLVX-IRES-ZsGreen1 and protein expression vector pLVX--PD-1IRES-ZsGreen1 respectively, blank is set simultaneously; After transfection 24h, change containing dual anti-DMEM substratum, and carry out with fluorescent microscope observation transfection efficiency of taking pictures.37 DEG C of CO are put into by changing the sample after substratum
2continue in incubator to cultivate 46-50h, preferably cultivate 48h, blow afloat adherent layer gently with liquid-transfering gun, and move to collecting cell in centrifuge tube.
Protein Extraction: by centrifugal treating after the cells rinsed with PBS of collection, after reject supernatant liquor, add PBS re-suspended cell respectively, PMSF proteinase inhibitor that concentration is 100mM, in-80 DEG C and 37 DEG C of multigelations three times, each 10-15min, then the centrifugal 5-10min of 3000rpm is carried out at, get supernatant liquor and be labeled as cytosol, precipitate with after RIPA on ice cracking 15-20min, the centrifugal 15min of 14000 × g, supernatant is moved in new EP pipe, be labeled as film extract.
SDS protein electrophoresis: configure the separation gel of 12% concentration and the concentrated glue of 5% concentration, thickness is that 1.5mm is for subsequent use; In above-mentioned cytosol and film extract, add the Loading Buffer of 5 times of volumes respectively, 100 DEG C are boiled sample 8-12min, centrifugal rear loading 35-45 μ l, after 80V electrophoresis 15min, adjust voltage to 120V electrophoresis 1h.As preferred embodiment, described in boil the sample time be 10min, centrifugal condition is the centrifugal 5min of 12000rpm, and applied sample amount is every hole 40 μ l.
Western Blot: put into transferring film Buffer after being soaked by pvdf membrane anhydrous methanol together with filter paper etc., glue is laid in and is lined with on the filter paper of sponge corresponding to negative pole, pvdf membrane is covered on glue, after removing bubble between the two, filter paper and sponge in covering, clamping " sandwich " type transfer plate, puts into transfer groove, fills it up with 100V transferring film after transferring film Buffer; Put into the PBST solution containing 5% skim-milk after transferring film terminates, room temperature is closed; Abandon confining liquid, the anti-PD-1 antibody at room temperature of rabbit adding 1:1200 dilution hatches 1.5-2h, PBST washing 2-4 time; Abandon waste liquid, add the anti-incubated at room 1-1.2h of Goat anti-Rabbit bis-of 1:8000 dilution, with PBST washing 2-4 time; Add ECL exposure.
In above-mentioned steps S02, by the carrier pLVX-PD-1-IRES-ZsGreen1 of PD-1 correct for the described film expression obtained and viral packaging plasmid pMD2.G and PsPAX
2carry out the process of virus packaging, concrete steps are as follows:
Passage: plasmid transfection goes down to posterity eukaryotic cell in Tissue Culture Dish the day before yesterday, makes the degrees of fusion on transfection same day reach 60%-80%.Eukaryotic cell can adopt eukaryotic cell conventional in this area.As preferred embodiment, described eukaryotic cell selects T293 cell.3-5h before transfection, be preferably 4h substratum is changed into half volume without dual anti-DMEM+10%FBS substratum;
Cell transfecting: utilize calcium phosphate transfection method transfection transfectional cell, two centrifuge tubes on transfection tense marker, write Buffer A and Buffer B respectively, and wherein Buffer A pipe directly adds 1.25ml calcium phosphate A liquid, and Buffer B manages composed as follows:
Add in Buffer A pipe by dropwise in Buffer B pipe, add after leaving standstill 15-25min by the rear room temperature of bubble method mixing and change in the culture dish supernatant of liquid, 5%CO is put in mixing back and forth gently
237 DEG C of incubators in continue cultivate; After transfection 24h, change liquid become perfect medium and observation transfection efficiency of taking pictures under fluorescent microscope; After transfection 48h, first time collects virus liquid; And add fresh substratum; After transfection 72h collect second time virus liquid, filter rear 4 DEG C for subsequent use, 0.45um syringe needle filter is preferably used in described filtration.
Viral concentration: the centrifuge tube that virus liquid is housed is carried out centrifugal treating, centrifugal treating condition optimization be at 4 DEG C in the centrifugal 2h of 25000rpm, abandon supernatant, with the resuspended virus liquid of the PBS of precooling, be put in-80 DEG C for subsequent use.
In order to obtain in above-mentioned virus liquid virus concentration, virus titer mensuration need be carried out to virus liquid.Particularly, slow virus infection is gone down to posterity in eukaryotic cell to six orifice plate the day before yesterday, each sample at least uses holes to be used for gradient infection slow virus, and cell quantity when blank control wells is set for counting infection, after the cytogamy degree infecting the viral same day reaches 50%-70%, respectively at adding 5 μ l and 0.5 μ l virus liquid in the holes be arranged in parallel, and add simultaneously final concentration be 5-8ug/ml polybrene assist virus better infect target cell, liquid is changed virus-free and without the perfect medium of polybrene after cultivating 4-6h in 37 DEG C of 5%CO2 incubators, fluorescence microscope virus infection efficiency after continuation cultivation 24h.Flow cytometer showed infection rate is carried out to this infection hole.Again according to the cell infection rate of low cytometric analysis according to following formulae discovery virus titer:
Cell count × cell infection rate/viral the volume (μ l) × 10 on virus titer (TU/ml)=infection same day
3
After measured, the infection rate of 0.5 μ l PD-1 slow virus can reach 14.5%, and according to initial cell value, the virus titer obtaining PD-1 as calculated can be 2.61 × 10
8tU/ml.
In embodiment of the present invention step S03, according to the virus titer result of monitoring, when virus titer infects 293T cell with MOI=2-10, adding final concentration is the infection that the polybrene of 5-8ug/ml assists virus, infects fluorescent microscope after 24h and to take pictures observation efficiency of infection.Can carry out positive cell sorting according to fluorescin ZsGreen intensity after treating normally to go down to posterity, described passage number can adjust according to specific experiment situation, preferably goes down to posterity three times.In the present embodiment, owing to carrying ZsGreen in carrier, therefore, flow cytometer can be adopted to carry out airflow classification positive cell to the virus carrying fluorescin ZsGreen.In the embodiment of the present invention, according to the special carrier selected, flow cytometer is adopted to carry out fluidic cell screening, without the need to adding the ancillary components such as microbiotic, avoid the aftertreatment trouble that culturing process causes due to microbiotic generation resistance, and it screens the positive cell non-false positive phenomenon appearance obtained, the selection result accurately and reliably; In addition, screening efficiency is high, has saved a large amount of time costs.Choose fluorescencepositive cell and carry out sorting, the frozen cell strain being required stably express PD-1 after gained cell amplification.
The embodiment of the present invention builds the stable expression cell line of film expression PD-1 albumen by the mode of infection of eukaryotic cells again after packaging slow virus, this clone energy stabilizing membrane surface expression PD-1 obtained, owing to have passed through the process of packaging slow virus infection of eukaryotic cells again, the correct conformation this PD-1 being had express in human cell, it has more close to the structure of native protein, make monoclonal antibody directly can be attached to surface of cell membrane, thus can be used for the anti-antibody of this albumen of effectively qualification and the binding ability of PD-1.The foundation of the method is intended to the PD-1 albumen of film expression people, make it have the correct conformation expressed in human cell, thus make in mono-clonal triturating, can by use natural expression conformation the strain of PD-1 overexpressing cell screening monoclonal antibody, with screen there is high-affinity antibody for clinical development monoclonal antibody drug.
Simultaneously, when utilizing the slow virus of band ZsGreen fluorescence to carry out the structure of stable expression cell line can fast by goal gene random integration in genome, integration efficiency is high, false positive is few, not easily lose in passage process, and without the need to adding the advantages such as microbiotic, the separation of positive cell can be carried out easily by fluorescent microscope or flow cytometer, and without the need to buying specific antibody, save a large amount of screening time, human cost and goods and materials cost to a certain extent.
As preferred embodiment, above-mentioned solution selects the solution of fresh configuration.
The following solutions used in the embodiment of the present invention, can be obtained by following method configuration:
PBS: take 7.9g NaCl respectively, 0.2g KCl, 1.44g Na
2hPO
4with 1.8g K
2hPO
4, be dissolved in 800ml distilled water, by the pH value to 7.4 of HCl regulator solution, last adding distil water is settled to 1L.Be stored in 4 DEG C of refrigerators.
5mol/l NaCl: dissolve 292g NaCl with 800ml distilled water, be settled to 1L.High pressure steam sterilization after packing, room temperature preservation.
0.5mol/l Tris-HCl pH8.0: dissolve 60.5g Tris alkali with 800ml distilled water, adds concentrated hydrochloric acid and adjusts pH to 8.0.After solution should be made to be chilled to room temperature, finally can set up pH value.Add water and be settled to 1L.High pressure steam sterilization after packing.
0.5mol/l EDTA (pH8.0): 186.1g bis-water disodium ethylene diamine tetraacetate (EDTA-Na2H2O) is added in 800ml water, vigorous stirring on magnetic stirring apparatus.About need 20g NaOH particle by the pH value of NaOH regulator solution to 8.0(, be settled to 1L.High pressure steam sterilization after packing.EDETATE DISODIUM need add NaOH the pH value of solution is adjusted to close to 8.0 time, just can dissolve.
The preparation of 10mol/l NaOH:10mol/l NaOH relates to highly exothermic reactions, may cause breaking of Glass Containers.Take special care to prepare this solution with plastic beaker.400g NaOH particle is slowly added in 800ml water, limit edged continuously stirring.As a kind of preventive measures, beaker is placed on ice.After particle dissolves completely, be settled to 1L with water.Room temperature preservation in plastic containers, need not be degerming.
10%SDS solution (m/V): sodium laurylsulfonate (SDS) is also known as sodium lauryl sulphate.With 900ml aqueous solution 200g electrophoresis level SDS, be heated to 68 DEG C and contribute to dissolving by magnetic stirrer.If needed, add several dense HCl adjust ph to 7.2, be settled to 1L with water.Room temperature preservation, need not be degerming, not high pressure steam sterilization.
10mol/l ammonium acetate solution: with 800ml water dissolution 770g ammonium acetate, be settled to 1L with water.Filtration sterilization, or, be the water dissolution 77g ammonium acetate of room temperature by 70ml temperature, be settled to 100ml, degerming with 0.22um frit, room temperature preservation in the bottle being contained in sealing.Ammonium acetate decomposes in the hot water, and the solution containing ammonium acetate can not high pressure steam sterilization.
3mol/l potassium acetate solution: take in potassium acetate particle 294g to 300ml water, stirs and makes it dissolve, about need Glacial acetic acid 400ml by Glacial acetic acid adjust ph to 4.8(), be settled to 1L with water.Room temperature preservation, can not autoclaving.
TE Buffer(10mM Tris-HCl pH7.6,1mM EDTA pH8.0): take 12.1g Tris alkali in 800ml water, by concentrated hydrochloric acid adjust ph to 7.6; Then add 2ml0.5mol/l EDTA pH8.0, be settled to 1L with water.
30%PEG6000 (m/V)-1.5mol/l NaCl solution: dissolve 30g PEG6000 with 30ml distilled water, stirring and dissolving gently, then add 30ml5mol/l NaCl, add water and be settled to 100ml.Degerming with 0.22um frit, room temperature preservation.
LB (Luria-Bertani) substratum: prepare often liter of substratum, should add in 950ml deionized water:
Tryptone 10g
Yeast extract 5g
NaCl 10g
Shake container is until solute dissolves.By 10mol/l NaOH (about 0.1ml) adjust ph to 7.0, be settled to 1L with deionized water, high pressure steam sterilization 20min.
Be described below in conjunction with specific embodiment.
Embodiment 1 film expression people PD-1 albumen surely turn strain cell construction method
S11. carrier construction pLVX-PD-1-IRES-ZsGreen1:
The primer pLVX-PD-1-F of design containing restriction enzyme site EcoRI and the primer pLVX-PD-1-R containing restriction enzyme site BamHI, the sequence of described primer pLVX-PD-1-F and primer pLVX-PD-1-R is respectively:
pLVX-PD-1-F：5'-CCGGAATTCATGCAGATCCCACAGGCG-3’，
pLVX-PD-1-R：5'-CGCGGATCCTCAGAGGGGCCAAGAGCAGT-3'。
The DNA sequence dna of described medicine and PD-1 is carried out pcr amplification, collects amplified production.Wherein before pcr amplification, by described primer pLVX-PD-1-F and pLVX-PD-1-R ddH
2o dissolves, and makes its concentration be 20mM, utilizes round pcr to increase.The amplification system that the DNA sequence dna of described PD-1 carries out pcr amplification process is:
The method that the DNA sequence dna of described PD-1 carries out pcr amplification process is:
First stage: 98 DEG C/2min;
Subordinate phase: 98 DEG C/30s, 60 DEG C/30s, 72 DEG C/30s, totally 30 circulations;
Phase III: 72 DEG C/10min, 10 DEG C of preservations.
Above-mentioned pcr amplification product adopts 1.5% agarose gel electrophoresis, and as shown in Figure 2, wherein M represents Marker to its electrophoresis result, and "-" represents negative control, and 1-2 is the PCR primer sample of PD-1.Use glue recovery test kit to carry out pcr amplification product and carry out purifying recovery, the 30 μ l ddH of the sample after purified
2o reclaims from post.Recovery sample concentration is 71.3ng/ μ l, OD
260/280be 1.96.
Enzyme is carried out to the pcr amplification product of plasmid vector pLVX-IRES-ZsGreen1 and PD-1 and cuts process.
It is as follows that the enzyme of described plasmid vector pLVX-IRES-ZsGreen1 cuts system:
It is as follows that the PCR primer enzyme of described PD-1 cuts system:
The endonuclease reaction system of the pcr amplification product of above-mentioned plasmid vector pLVX-IRES-ZsGreen1 and PD-1 is placed in respectively after enzyme cuts 4h on 37 DEG C of constant-temperature metal baths, uses 1.5% agarose gel electrophoresis, digestion products glue is recycled, and with 30 μ l ddH
2dNA fragmentation on O wash-out post, it is as follows that glue reclaims yield:
pLVX-IRES-ZsGreen1（EcoRI+BamHI）17.1ng/μl，PD-1（EcoRI+BamHI）33.6ng/μl。
By pLVX-IRES-ZsGreen1(EcoRI+BamHI obtained above), PD-1(EcoRI+BamHI), connect, obtain carrier pLVX-PD-1-IRES-ZsGreen1.The linked system of described connection pLVX-IRES-ZsGreen1 and PD-1 is as follows:
Above-mentioned linked system is connected on 22 DEG C of constant-temperature metal baths transformed competence colibacillus cell DH5 α after 1.5h.Described step of converting is as follows: draw 10 μ l connection products and add in the 100 μ l DH5 α competent cells thawed on ice, 20min is placed on ice gently after mixing, 42 DEG C of heat shock 45s, 500 μ lLB substratum are added after placing 2min on ice, the centrifugal 1min of constant temperature culture 45min in 37 DEG C, rear 6000rpm, abandons 500 μ l supernatants, coat after utilizing residue about 100 μ l liquid gravity treatment thalline in the LB flat board containing Ampr, spend the night in 37 DEG C of incubators to be inverted and cultivate.
Product after above-mentioned connection transforms is identified.Particularly, authentication method is: the mono-clonal on picking Ampr LB flat board, be seeded to during LB liquid culture connects, sterile manner is adopted to take out a small amount of bacterium liquid for plasmid extraction after incubated overnight, plasmid extraction method can adopt this area to commonly use plasmid extraction technology, as preferred embodiment, described plasmid extraction employing Omega article No. is the plasmid extraction method in the specification sheets of D6943-02.
The plasmid obtained through above-mentioned plasmid extraction process is carried out enzyme and cuts authentication process, it is as follows that its enzyme cuts identification system:
Above-mentioned enzyme is cut identification system enzyme on 37 DEG C of constant-temperature metal baths to cut 4h rear electrophoresis and detect, as shown in Figure 3, and the Arbitrary Samples that picking is wherein numbered in 1-4 checks order detected result.
By the microbionation containing plasmid of above-mentioned correct structure to containing in the LB nutrient solution of Ampr, carry out constant-temperature table overnight incubation, amplification plasmid.Shaking table temperature is 37 DEG C, and revolution is 250rpm.
The bacterium cultivated by above-mentioned shaking table carries out centrifugal treating, goes to precipitate pellet fraction.The condition of described centrifugal treating is at 4 DEG C, the centrifugal 5min of 6000rpm.
Cell broken up by the thalline vibrator obtained, and progressively adds TE solution and make cell be in complete resuspended state under oscillation condition.The mixed solution of described TE solution to be final concentration the be EDTA of Tris-HCl, 50mmol/l of 25mmol/l, pH is 8.0.Add in above-mentioned resuspension fluids NaOH-SDS solution fully mix after cooling process, NaOH, the weight percent of described NaOH-SDS solution to be final concentration be 0.2mol/l are the mixed solution of 1%SDS.As preferred embodiment, the described type of cooling is preferably put and is placed on ice, and the treatment time is 10min.Under oscillation condition, add the KAC solution that the 3mol/l pH placing 15min precooling is on ice 4.8, then carry out centrifugal treating, get supernatant liquor, centrifugal condition is: at 4 DEG C, the centrifugal 15min of 10000rpm.
The Virahol of 14ml will be added in the supernatant liquor of above-mentioned acquisition, mixing, put after room temperature places 15 ~ 30min and carry out centrifugal treating.After abandoning supernatant, add the abundant dissolution precipitation of TE Buffer of 1.5ml.And add the NH of 10mol/l
4aC mixes, and carries out centrifugal, get supernatant liquor after cooling process.As preferred embodiment, described cooling process can adopt the mode being placed on and placing 20min on ice.In above-mentioned supernatant liquor, add 4ml dehydrated alcohol, under-80 DEG C of environment, place 30min, after centrifugal treating, get its supernatant liquor.Precipitation carried out washing rear recentrifuge process with 1ml70% ethanol, after repeated washing 1-2 time, precipitation left and taken by reject supernatant liquor.Above-mentioned centrifugal condition is: at 4 DEG C, the centrifugal 15min of 12000rpm.
With the abundant dissolution precipitation of TE Buffer, add 10 μ l RNase A (final concentration: 10ug/ml), after 20min is placed in 37 DEG C of water-baths, add the NaCl solution that concentration is 5mol/l, mixing; Add 30%PEG6000 (m/V)-1.5mol/l NaCl solution again, mixing; Put after placing 30min on ice and carry out centrifugal treating.By the supernatant liquor reject after centrifugal, add 5mol/l NaCl with after the complete dissolution precipitation of TE Buffer, mixing, then to add volume ratio be 25:24:1 phenol-chloroform-isoamyl alcohol, mixing, gets upper strata aqueous phase after centrifugal treating.In above-mentioned aqueous phase, add phenol, mixing, gets upper strata aqueous phase after centrifugal treating.The dehydrated alcohol putting-80 DEG C of refrigerator precoolings is in advance added, mixing in above-mentioned aqueous phase; Centrifugal after putting-80 DEG C of refrigerators placement 15min, by 70% washing with alcohol precipitation also centrifugal treating.In this step, above-mentioned centrifugal treating is 15000rpm.Centrifugal to 6000rpm after abandoning supernatant, take out sample loading gun and draw 70% alcohol, put into Bechtop 2 ~ 5min and get final product seasoning.In above-mentioned dry thing, add TE dissolve, and measure concentration with Nanodrop, and to carry out being diluted to ultimate density according to concentration be 1ug/ μ l.
In the embodiment of the present invention, in order to confirm the exactness of foreign gene in carrier pLVX-PD-1-IRES-ZsGreen1 further, and the carrying out smoothly of protein expression, protein expression qualification can be carried out to the plasmid extracted in above-mentioned steps.Concrete, operate as follows:
Endotoxic extracting plasmid transfection 293T cell is removed in utilization, and after transfection 72h, collecting cell carries out Protein Extraction, then carries out Western Blot and identifies protein expression.Concrete steps are as follows:
Plasmid transfection: utilize calcium phosphate transfection to carry out cell transfecting, day before transfection carries out passage is 60-80% to cytogamy degree next day; Before transfection, 4h uses instead without dual anti-DMEM+10%FBS substratum, and rotaring redyeing system is made up of A liquid and B liquid.The time used instead without dual anti-DMEM+10%FBS substratum is preferably 4h.
Wherein, A liquid is made up of 50 μ l calcium phosphate solution A, and calcium phosphate solution A is 2 parts of HEPES buffering salts, and pH is 7.05, and its HEPES buffering salt is composed as follows:
HEPES 50mM，
NaCl 280mM，
Na
2hPO
41.5mM; Calcium phosphate reagent B liquid is composed as follows:
Calcium phosphate solution B 5 μ l,
pLVX-PD-1-IRES-ZsGreen1 4μg，
DdH
2o mends to 50 μ l.
Wherein, calcium phosphate solution B liquid is 2.5M CaCl
2.
Utilize calcium phosphate transfection method transfection 4ug control vector pLVX-IRES-ZsGreen1 and protein expression vector pLVX--PD-1IRES-ZsGreen1 respectively, blank is set simultaneously; After transfection 24h, change containing dual anti-DMEM substratum, and carry out with fluorescent microscope observation transfection efficiency of taking pictures, result as shown in Figure 4.37 DEG C of CO are put into by changing the sample after substratum
2continue in incubator to cultivate 46-50h, preferably cultivate 48h, blow afloat adherent layer gently with liquid-transfering gun, and move to collecting cell in centrifuge tube.
Protein Extraction: by centrifugal treating after the cells rinsed with PBS of collection, after reject supernatant liquor, add PBS re-suspended cell respectively, PMSF proteinase inhibitor that concentration is 100mM, in-80 DEG C and 37 DEG C of multigelations three times, each 10min, then the centrifugal 5min of 3000rpm is carried out at, get supernatant liquor and be labeled as cytosol, precipitate with after RIPA on ice cracking 15min, the centrifugal 15min of 14000 × g, supernatant is moved in new EP pipe, be labeled as film extract.
SDS protein electrophoresis: configure the separation gel of 12% concentration and the concentrated glue of 5% concentration, thickness is that 1.5mm is for subsequent use; In above-mentioned cytosol and film extract, add the Loading Buffer of 5 times of volumes respectively, 100 DEG C are boiled sample 8-12min, centrifugal rear loading 35-45 μ l, after 80V electrophoresis 15min, adjust voltage to 120V electrophoresis 1h.As preferred embodiment, described in boil the sample time be 10min, centrifugal condition is the centrifugal 5min of 12000rpm, and applied sample amount is every hole 40 μ l.
Western Blot: pvdf membrane anhydrous methanol is soaked after 10s and put into transferring film Buffer together with filter paper etc., glue is laid in and is lined with on the filter paper of sponge corresponding to negative pole, pvdf membrane is covered on glue, after removing bubble between the two, filter paper and sponge in covering, clamping " sandwich " type transfer plate, puts into transfer groove, fills it up with 100V transferring film 90min after transferring film Buffer; Put into the PBST solution containing 5% skim-milk after transferring film terminates, room temperature closes 1h; Abandon confining liquid, the anti-PD-1 antibody at room temperature of rabbit adding 1:1200 dilution hatches 1.5h, PBST washing 2-4 time, each 5min; Abandon waste liquid, add the anti-incubated at room 1h of Goat anti-Rabbit bis-of 1:8000 dilution, with PBST washing 2-4 time, each 5min; Add ECL exposure, its result as shown in Figure 5.Fig. 5 shows, transfection has energy normal expression in the carrier energy 293T cell of PD-1, and the cell of 293T blanc cell and transfection zero load does not express PD-1, and PD-1 has methylating in various degree in 293T cell, therefore the PD-1 albumen of different methylation can be detected.
S12. virus packaging: by the carrier pLVX-PD-1-IRES-ZsGreen1 of described film expression PD-1 and viral packaging plasmid pMD2.G and PsPAX
2cotransfection is in eukaryotic cell, and carry out virus packaging, monitor virus titer, concrete steps are as follows simultaneously:
Passage: plasmid transfection goes down to posterity T293 cell in Tissue Culture Dish the day before yesterday, makes the degrees of fusion on transfection same day reach 60%-80%.Before transfection 4h substratum is changed into half volume without dual anti-DMEM+10%FBS substratum;
Cell transfecting: utilize calcium phosphate transfection method transfection transfectional cell, two centrifuge tubes on transfection tense marker, write Buffer A and Buffer B respectively, and wherein Buffer A pipe directly adds 1.25ml calcium phosphate A liquid, and Buffer B manages composed as follows:
Add in Buffer A pipe by dropwise in Buffer B pipe, add after leaving standstill 20min by the rear room temperature of bubble method mixing and change in the culture dish supernatant of liquid, 5%CO is put in mixing back and forth gently
237 DEG C of incubators in continue cultivate; After transfection 24h, change liquid become perfect medium and observation transfection efficiency of taking pictures under fluorescent microscope, as shown in Figure 6; After transfection 48h, first time collects virus liquid; And add fresh substratum; After transfection 72h collect second time virus liquid, with after 0.45um syringe needle frit 4 DEG C for subsequent use.
Viral concentration: the centrifuge tube that virus liquid is housed is carried out centrifugal treating, centrifugal treating condition optimization be at 4 DEG C in the centrifugal 2h of 25000rpm, abandon supernatant, with the resuspended virus liquid of the PBS of precooling, be put in-80 DEG C for subsequent use.
In order to obtain in above-mentioned virus liquid virus concentration, virus titer mensuration need be carried out to virus liquid.Particularly, slow virus infection is gone down to posterity in eukaryotic cell to six orifice plate the day before yesterday, each sample at least uses holes to be used for gradient infection slow virus, and cell quantity when blank control wells is set for counting infection, after the cytogamy degree infecting the viral same day reaches 50%-70%, respectively at adding 5 μ l and 0.5 μ l virus liquid in the holes be arranged in parallel, and add simultaneously final concentration be 5-8ug/ml polybrene assist virus better infect target cell, liquid is changed virus-free and without the perfect medium of polybrene after cultivating 4-6h in 37 DEG C of 5%CO2 incubators, fluorescence microscope virus infection efficiency after continuation cultivation 24h, as shown in Figure 7.After showing to add 0.5 μ l virus liquid according to the fluorescent microscope result of Fig. 7, its infection rate is at 10-80%, therefore carries out flow cytometer showed infection rate to this infection hole, and its infection rate is 14.5%, and it the results are shown in Figure 8.Again according to the cell infection rate of low cytometric analysis according to following formulae discovery virus titer:
Cell count × cell infection rate/viral the volume (μ l) × 10 on virus titer (TU/ml)=infection same day
3
After measured, the infection rate of 0.5 μ l PD-1 slow virus can reach 14.5%, and according to initial cell value, the virus titer obtaining PD-1 as calculated can be 2.61 × 10
8tU/ml.
S13. the structure of strain cell is surely turned: this is according to the virus titer result of monitoring, when virus titer infects 293T cell with MOI=5, adding final concentration is the infection that the polybrene of 5-8ug/ml assists virus, and infect fluorescent microscope after 24h and to take pictures observation, efficiency of infection as shown in Figure 9.Treat normally to go down to posterity after three times, adopt flow cytometer to carry out airflow classification positive cell to the virus carrying fluorescin ZsGreen.Airflow classification result can be as shown in Figure 10.Figure 10 shows, positive cell rate is about 84%.Does is choosing fluorescence intensity? cell carry out sorting, the frozen cell strain being required stably express PD-1 after gained cell amplification.
In the embodiment of the present invention, the following meaning of writing a Chinese character in simplified form is as follows:
Plasmid: plasmid;
BSA: bovine serum albumin
Buffer: damping fluid;
Polybrene: polybrene;
ZsGreen: green fluorescent protein;
MOI: infection multiplicity;
Template: template;
Phusion:PCR increases enzyme;
Thermo T4Buffer:Thermo damping fluid;
The T4 ligase enzyme of Thermo T4Ligase:Thermo;
Ampr: anti-penbritin;
Loading Buffer: sample loading buffer.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. film expression people PD-1 albumen surely turn a strain cell construction method, comprise the steps:
The primer pLVX-PD-1-F of design containing restriction enzyme site EcoRI and the primer pLVX-PD-1-R containing restriction enzyme site BamHI, and the DNA sequence dna of described medicine and PD-1 carries out pcr amplification, collects amplified production;
Slow virus packaging plasmid carrier pLVX-IRES-ZsGreen1 and described amplified production are carried out enzyme respectively and cut process, obtain pLVX-IRES-ZsGreen1(EcoRI+BamHI respectively) and PD-1(EcoRI+BamHI) digestion products; Described pLVX-IRES-ZsGreen1(EcoRI+BamHI) with PD-1(EcoRI+BamHI) digestion products be connected after conversion processing, by converted product increase after carry out plasmid extraction process, obtain the carrier pLVX-PD-1-IRES-ZsGreen1 of film expression PD-1;
By the carrier pLVX-PD-1-IRES-ZsGreen1 of described film expression PD-1 and viral packaging plasmid pMD2.G and PsPAX
2cotransfection, in eukaryotic cell, carries out virus packaging, monitors simultaneously to virus titer;
When virus titer infects described eukaryotic cell with MOI=2 ~ 10, add the polybrene assistance virus that final concentration is 5-8ug/ml; After normally being gone down to posterity by the described eukaryotic cell infecting virus, (cell carries out sorting, and carries out the rear frozen process of amplification to the cell of sorting, obtains the cell strain of stably express PD-1 to choose fluorescent positive.
2. film expression people PD-1 albumen as claimed in claim 1 surely turn strain cell construction method, it is characterized in that, in the step of carrier construction pLVX-PD-1-IRES-ZsGreen1, described primer pLVX-PD-1-F sequence is: 5'-CCGGAATTCATGCAGATCCCACAGGCG-3 '; Described primer pLVX-PD-1-R sequence is: 5'-CGCGGATCCTCAGAGGGGCCAAGAGCAGT-3'.
3. film expression people PD-1 albumen as claimed in claim 1 surely turn strain cell construction method, it is characterized in that, surely turn in the step of strain structure described, described fluorescence intensity carries out detection acquisition by using flow cytometer to carry fluorescin ZsGreen to virus.
4. the film expression people PD-1 albumen as described in as arbitrary in claims 1 to 3 surely turn strain cell construction method, it is characterized in that, in the step of described carrier construction pLVX-PD-1-IRES-ZsGreen1, the amplification system that the DNA sequence dna of described PD-1 carries out pcr amplification process is:
5. the film expression people PD-1 albumen as described in as arbitrary in claims 1 to 3 surely turn strain cell construction method, it is characterized in that, in the step of described carrier construction pLVX-PD-1-IRES-ZsGreen1, the method that the DNA sequence dna of described PD-1 carries out pcr amplification process is:
First stage: 95-98 DEG C/2-3min;
Subordinate phase: 95-98 DEG C/25-35s, 58-62 DEG C/25-35s, 68-74 DEG C/25-35s, 30-35 circulation altogether;
Phase III: 68-74 DEG C/8-12min, 10 DEG C of preservations.
6. the film expression people PD-1 albumen as described in as arbitrary in claim 1-3 surely turn strain cell construction method, it is characterized in that, in the step of described carrier construction pLVX-PD-1-IRES-ZsGreen1, describedly slow virus packaging plasmid carrier pLVX-IRES-ZsGreen1 is carried out enzyme that enzyme cuts to cut system as follows:
7. the film expression people PD-1 albumen as described in as arbitrary in claim 1-3 surely turn strain cell construction method, it is characterized in that, in the step of described carrier construction pLVX-PD-1-IRES-ZsGreen1, it is as follows that the PCR primer enzyme of the DNA of described PD-1 cuts system:
8. the film expression people PD-1 albumen as described in as arbitrary in claim 1-3 surely turn strain cell construction method, it is characterized in that, in the step of described carrier construction pLVX-PD-1-IRES-ZsGreen1, the linked system connecting pLVX-IRES-ZsGreen1 and PD-1 is as follows:
9. the film expression people PD-1 albumen as described in as arbitrary in claim 1-3 surely turn strain cell construction method, it is characterized in that, before carrying out described plasmid extraction process, also comprise and mini-scale plasmid extracting, sampling qualification, order-checking process are carried out to converted product, wherein, the identification system of described authentication process is:
10. the film expression people PD-1 albumen as described in as arbitrary in claim 1-3 surely turn strain cell construction method, it is characterized in that, after plasmid extraction process is carried out to described converted product, also comprise to extracting to plasmid carry out protein expression authentication process, the step of described protein expression authentication process by extracting to plasmid remove transfecting eukaryotic cells after intracellular toxin, after transfection 48-72h, collecting cell carries out Protein Extraction, uses Western Blot to identify protein expression to extract proteins.
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Application publication date: 20150617 |