CN108823170A - A kind of construction method of the cell model for anti-oxidation medicine screening based on protein Misfolding - Google Patents
A kind of construction method of the cell model for anti-oxidation medicine screening based on protein Misfolding Download PDFInfo
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Abstract
The invention belongs to cell biology, for protein misfolding at present can not real-time quantitative is assessed in active somatic cell defect, a kind of construction method of cell model for anti-oxidation medicine screening based on protein Misfolding is provided.Construct the wild of fluorescent protein labeling and mutation reporter geneCOMPGene Lentiviral Vector, the packaging virus in human embryo kidney (HEK) 293T cell, HeLa cell of transduceing, puromycin screening obtain the cell model for stablizing expression COMP albumen.The drug screening cell model with world level, autonomous innovation is established, key parameter can not only be provided for protein misfolding drug screening study on the industrialization from now on, provides related science foundation to solve the sustainability research of major disease.Cell model has the characteristics that high chromosomal integration degree, high fluorescent, stabilization characteristics of genetics and sensitive to anti-oxidation medicine, is a kind of model of ideal research protein Misfolding drug screening.
Description
Technical field
The invention belongs to cell biologies, and in particular to a kind of to be used for antioxidant drug based on protein Misfolding
The construction method of the cell model of object screening.
Background technique
Protein is the executor of all functions organism Nei.The intracorporal any function of people, from catalytic chemistry be reacted to
Imperial foreign aggression is all the result of protein effect.Albumen must be folded accurately into as correct three-dimensional structure, could be in the mankind
Function is executed in cell.In recent years find that the false folding of protein can lead to some diseases.The false folding and disease of protein
The relationship of disease has become the new study frontier of molecular biology.Construct a kind of simple, safe and efficient drug screening cell model
It is the neurodegenerative disease such as alzheimer's disease, Parkinson's disease and constitutional bone disease, phenylpropyl alcohol for solving current prevalence in the world
The bottleneck and difficult point of the protein misfoldings disease prevention and cure such as Ketonuria.
In drug screening process, it is also a most important link that the model construction of medicine sieve model, which is initial, for
The efficiency and effect of new drug discovery play a crucial role.High flux screening (high-throughput screening,
), it HTS is the high efficiency large-scale medicine screening occurred under the promotion of the subjects such as combinatorial chemistry, genomics, pharmacology
Technology.HTS be broadly divided into screening based on simple target (target protein is isolated and purified, and is carried out under extracellular environment) and
Screening based on cell model.Screening based on simple target (extracellular) action target and must act on target knowing for sure
Albumen just can be carried out under conditions of capable of isolating and purifying, and the drug screening mechanism of action carried out on this model is clear, target list
One.Screening based on cell is carried out on cell model, be difficult to separate especially suitable for functional protein, biochemical analysis cannot
The complicated target of completion, the high flux screening based on cell model are the main screening models of high flux screening from now on.
Multiple epiphyseal dysplasia（MED）It is a kind of rare heredity osteochondrodysplasia disease, due to cartilage
Extracellular matrix protein（For example, cartilage oligo-substrate protein；COMP）And it is complete caused by the defect of cell membrane transporter albumen etc.
Body skeleton development is abnormal, belongs to one of disease caused by the false folding of protein.The intracorporal COMP albumen meeting of normal person
It is secreted into extracellular, exercises normal function, and for MED patient,COMPGene mutates, the COMP proteinosis of mutation
On the rough surfaced endoplasmic reticulum (RER) of cartilage cell, makes the albumen of false folding in the online bulk deposition of endoplasm, be unable to normal secretions and turn
Fortune causes endoplasmic reticulum stress response, and the multiplication rate of the apoptosis and cell that eventually lead to cartilage cell reduces, to induce an illness
Generation.
It is current the study found thatCOMP D469del In the mouse model of mutation, antioxidant and anti-inflammatory medicaments can be effective
Reduce the accumulation of the upper COMP albumen of cartilage cell, especially acetylsalicylic acid（Aspirin）And resveratrol medicament, thus
The skeleton development for having succoured mutant mice is abnormal, and the mouse model of the mutation will lead to serious pseudoachondroplasia
（PSACH）, but for researchCOMPThere is also certain limitations for MED disease caused by being mutated.
Summary of the invention
The present invention for protein misfolding at present can not in active somatic cell real-time quantitative assess defect, provide one
The construction method of cell model for anti-oxidation medicine screening of the kind based on protein Misfolding.
The present invention is realized by following technical solution：A kind of screening for anti-oxidation medicine based on protein Misfolding
Cell model construction method, reported using molecular biology gene recombination technology building fluorescent protein labeling wild and mutation
Accuse geneCOMPGene Lentiviral Vector, the packaging virus in human embryo kidney (HEK) 293T cell, HeLa cell of transduceing, puromycin screening
Obtain the cell model for stablizing expression COMP albumen.
Specific step is as follows：
（1）Human embryo kidney (HEK) 293T cell is transfected with the slow virus carrier of carrying antibiotics resistance gene, reporter gene, target gene,
Obtain the lentiviral particle of resistant gene and reporter gene；
（2）Use step（1）The lentiviral particle of acquisition infects HeLa Cells, and antibiotic-screening obtains cell model；
Wherein：The slow virus carrier is HIV-1 carrier system, including three kinds of plasmids：Express the Plvx-IRES- of reporter gene
Puro plasmid, packaging plasmid psPAX2, envelope protein plasmid pMD2.G, wherein Plvx-IRES-Puro plasmid contains puromycin
Resistant gene and ampicillin resistance gene, packaging plasmid psPAX2 include the gag gene of inhibition of HIV, envelope protein
Grain pMD2.G includes the VSV-G gene in herpe simplex source；The antibiotic is puromycin；The reporter gene iseGFP-COMPFusion；The target gene is wild typeCOMPGene and saltant typeCOMPGene p.D401N.
Specifically screening appraisal procedure is：With anti-oxidation medicine function cells model, 485 nm of excitation wavelength, launch wavelength
The fluorescence intensity that eGFP is detected under the conditions of 530 nm, measures GFP content, with the absorbance and cell culture medium of cell pyrolysis liquid
The ratio of absorbance indicate the delay situation of COMP albumen in the cell, expression wild type and prominent is stablized by check and evaluation
ModificationCOMPThe content of COMP albumen in the cell model of gene, assesses the effect of anti-oxidation medicine.
The anti-oxidation medicine is acetylsalicylic acid ASA and resveratrol Res, and wherein the concentration of acetylsalicylic acid is 75
MM, the concentration of resveratrol are 100 μM, and the time of drug effect cell model is for 24 hours.
The present invention establishes a set ofCOMPThe cell model of single base mutation utilizes antioxidant acetylsalicylic acid and white Chenopodiaceae
Reed alcohol function cells model can effectively assess the function and effect of antioxidant by measuring the fluorescence intensity of eGFP.
The present invention is based at present during drug screening using misfolding cause the COMP albumen of constitutional bone disease as
Study target, for protein misfolding at present can not in active somatic cell real-time quantitative assess defect, using molecular biosciences
Gene recombination technology building is learned containing the wild of the fluorescent protein labeling that can be used for real-time quantitative trace detection and mutation report base
It makes it stable expression because then recombinant vector being transferred in the cell line of in vitro culture respectively as selection markers and obtains drug sieve
Select cell model.Sepectrophotofluorometer technology is used to can change egg to those as the quantitative detection means of molecular level
White matter folds and the small molecule of secretion level is screened on a cellular level, using functional response as screening index, establishes one
A drug screening cell model with world level, autonomous innovation, carries out the research of protein misfolding disease medicament screening.
The present invention can not only provide key parameter for protein misfolding drug screening study on the industrialization from now on, and to solve China
The sustainability research of major disease provides related science foundation.The cell model has high chromosomal integration degree, Gao Ying
Luminous intensity, stabilization characteristics of genetics and the feature sensitive to anti-oxidation medicine are a kind of ideal research protein Misfolding medicines
The model of object screening.
Detailed description of the invention
Fig. 1 is virus expression carrier Plvx-IRES-Puro schematic diagram；Fig. 2 is slow virus packaging plasmid psPAX2 signal
Figure；Fig. 3 is slow virus envelope protein plasmid pMD2.G schematic diagram；Fig. 4 is the positive rate of flow cytometry cell model；Figure
5 be the comparision contents of wild type and saltant type COMP；Fig. 6 be acetylsalicylic acid function cells model optimum concentration and it is best when
Between；Fig. 7 is the optimum concentration and Best Times of resveratrol function cells model.
Specific embodiment
The present inventor by long-term research and screening, construct a kind of pair of anti-oxidation medicine it is sensitive to stablize expression slow
The cell model for the COMP gene that viral vectors carries.The cell model is a kind of mould of ideal research anti-oxidation medicine screening
Type.
The structure for the cell model for anti-oxidation medicine screening that the present invention provides a kind of based on protein Misfolding
Construction method.A kind of human cervical carcinoma cell mould of the new antibiotics resistance gene and reporter gene for expressing slow virus carrier carrying
Type can be used for anti-oxidation medicine screening.
The present invention passes through buildingCOMPGene Lentiviral Vector, the packaging virus in 293T cell, HeLa cell of transduceing are fast
Purine mycin screens the cell model for stablizing expression COMP albumen, followed by anti-oxidation medicine acetylsalicylic acid and resveratrol
Function cells model, by assessment mutant cell in COMP content, obtain anti-oxidation medicine most suitable activity and when
Between.
Embodiment 1：Pack lentiviral particle
Slow virus system includes three kinds of plasmids：Express the Plvx-IRES-Puro plasmid of reporter gene（Fig. 1）, packaging plasmid
psPAX2（Fig. 2）, envelope protein plasmid pMD2.G（Fig. 3）.Wherein Plvx-IRES-Puro plasmid contains puromycin-resistant base
Cause and ampicillin resistance gene, packaging plasmid psPAX2 include the gag gene of inhibition of HIV, encode the main knot of virus
Structure albumen, envelope protein plasmid pMD2.G include the VSV-G gene in herpe simplex source, provide the required packet of virus packaging
Memebrane protein.
Reporter gene building：It is template with the known plasmid there are also eGFP-COMP fusion, carries out polymerase chain reaction
Target fragment should be expanded（TAKARA company）, kit recycling target fragment（Shanghai Sheng Gong bioengineering Co., Ltd）, digestion
Target fragment is connected with carrier using T4 DNA Ligase, and ambient temperature overnight, connection product is used for transformation experiment, constructs completion
For plasmid after bacterium solution PCR and digestion identification, Shanghai Sheng Gong bioengineering Co., Ltd carries out sequencing identification.
The packaging of slow virus：It transfects preceding 24 h to be laid on 293T cell in the culture bottle of 25 T, extremely to cell adherent growth
80% or so, it is replaced with the culture solution of the DMEM of antibiotic-free（10% fetal calf serum）；B. sterile 1.5 mL EP pipe is taken to be added
233.5 6 Transfection Reagent of μ L, FuGENE of Opti-MEM, 16.5 μ L, soft to mix, incubation at room temperature 5
Min, separately take sterile 1.5 mL EP pipe be added 250 μ L of Opti-MEM, 0.5 μ g of pMD2.G plasmid, 2.5 μ g of psPAX2 plasmid,
2.5 μ g of recombinant plasmid COMP-Plvx-IRES-Puro and the transfection reagent after being incubated for, soft to mix, incubation at room temperature 20
It after min, is even added in the culture bottle of 25 T and shakes up, in 37 DEG C（5% CO2）Incubator in cultivate；C.48 it is received after h
Collect cell culture supernatant, fresh culture solution is added, continues to cultivate；Continue to collect cell culture supernatant after 72 h, by two
The supernatant of secondary collection is centrifuged 10 min in 3000 g, and sedimentation cell fragment collects supernatant, with 0.45 μM of filter filtration sterilization,
It is saved backup after packing in -80 DEG C；
Embodiment 2：Viral transduction HeLa cell
Slow virus containing recombinant C OMP in the supernatant of collection utilizes the further transduction HeLa cell of virus after packaging, screening
The cell strain of stable transfection, concrete operations are as follows：
HeLa cell is laid in 6 orifice plates by preceding 24 h that transduces, and to cell adherent growth to 80% or so, is sucked old culture medium, is added
Enter the culture medium of fresh MEM（10% fetal calf serum, 1% Penicillin-Streptomycin）1.8 mL, recombinant C OMP
200 μ L and Hexadimethrine bromide of slow virus（Transfect reinforcing agent）1.6 μ L, after soft mixing, in 37 DEG C
It is cultivated in the incubator of 5% CO2；
The culture medium containing virus is sucked after 24 h, is replaced fresh 2 mL of culture medium, is continued to cultivate；
Culture medium is replaced after 48 h, Puromycin is added while fresh 2 mL of culture medium is added（2 μg/mL）；
Every 3-4 days later one subcultures of replacement（Containing puro）, until screening stable monoclonal cell.
By flow cytometry, the positive rate of the HeLa cell after transduction reaches 99% or more（Fig. 4）, cell model
It can be used for the drug screening research in later period.
Embodiment 3：Utilize the intracellular COMP content of sepectrophotofluorometer assessment cell model
In the HeLa cell line of stable transfection, COMP albumen and eGFP are amalgamation and expressions, it is possible to pass through containing for detection GFP
Amount to reflect indirectly the content of COMP albumen.
We utilize Cary Eclipse Fluorescence Spectrophotometer（Agilent
Technologies）The content of GFP is detected, initial stage exploration discovery GFP works as 485 nm of excitation wavelength, when 530 nm of launch wavelength,
It can most reflect the relative intensity of fluorescence of GFP.
We collect the cell and cell culture fluid of wild type and saltant type, after the dilution of Tris-EDTA solution, utilize
Cary Eclipse Fluorescence Spectrophotometer（Agilent Technologies）Measure GFP fluorescence
Intensity, interpretation of result indicate the variation feelings of intracellular COMP using the ratio of GFP in intracellular GFP and cell culture fluid
Condition.
The result shows that：The GFP content ratio of saltant type COMP is apparently higher than wild type COMP, illustrates saltant type COMP thin
The content of GFP intracellular（That is the content of COMP）Height, mutation COMP albumen are detained in the cell（Fig. 5）.
Embodiment 4：Using anti-oxidation medicine function cells model, the content of COMP in mutant cell is assessed
Cell is uniformly laid in 24 orifice plates by 24 h before drug-treated（20×104 cell/well）, to cell adherent growth
To 80% or so, the culture solution of fresh serum-free is replaced, with different time gradients or various concentration drug-treated cell,
After cell processing, the total protein of cell culture fluid and cell is collected, the 100 μ L of cell culture fluid of collection is taken to use later
Tris-EDTA（PH8.0）10 times of dilution, takes 50 μ L Tris-EDTA of cell pyrolysis liquid（PH8.0）20 times of dilution；Use Cary
Eclipse Fluorescence Spectrophotometer（Agilent Technologies）Measure GFP content（Excitation
485 nm of wavelength, 530 nm of launch wavelength）, with the ratio of the absorbance of cell pyrolysis liquid and the absorbance of cell culture medium come table
Show the delay situation of COMP albumen in the cell, whether has an impact to the COMP of delay after illustrating drug-treated with this, every group
Three independent repetitions are tested, significant difference is analyzed with SPSS17.
Through textual research, in the mouse model of COMP mutation, antioxidant acetylsalicylic acid（ASA）And resveratrol
（Res）Delay, the death of reduction cartilage cell and the proliferation of recovery cartilage cell of mutation COMP in the cell can be reduced
[Posey K L, et al. Antioxidant and anti-inflammatory agents mitigate
pathology in a mouse model of pseudoachondroplasia. Hum Mol Genet, 2015, 24
(14), 3918-28.], so we utilize the COMP cell model of our foundation of both drug-treateds.
1. utilizing acetylsalicylic acid（ASA）Cell model is handled, setting concentration is followed successively by 1,5,10,25,50,75,100mM
24 h of acetylsalicylic acid drug-treated COMP cell model after, utilize Cary Eclipse Fluorescence
Spectrophotometer（Agilent Technologies）Detect the content of GFP, as a result as shown in Figure 6A, various concentration
ASA drug influence is nearly free from wild type COMP cell, and for saltant type COMP cell, when low concentration, into the cell
GFP content increase, with the increase of concentration, intracellular GFP content starts to reduce after 10 mM, in 75 mM and 100
When mM, the content of intracellular GFP tends to be steady, and shows fine to saltant type COMP cell when drug concentration is in 75-100 mM
Function and effect.
In order to further explore the optimum time of ASA function cells, we choose the ASA function cells model of 75 mM, if
Different time gradients is set, as a result such as Fig. 6 B, as time went on, ASA drug is obvious when acting on 12 h to wild cell
It reduces, changes later unobvious, it may be possible to since drug has certain facilitation to the secretion of albumen in 12 h, and be mutated
Cell is in drug effect 12 h and 24 h, hence it is evident that reduces, i.e., intracellular delay is reduced, and variation later is risen
It is high, the results showed that：When ASA 24 h of drug effect of 75 mM, there are good function and effect to the cell of mutation.
2. we handle cell model using antioxidant resveratrol, exploration drug effect cell is most suitable dense first
Degree.After 48 h of Res drug-treated COMP cell model of various concentration is arranged in we, Cary Eclipse is utilized
Fluorescence Spectrophotometer（Agilent Technologies）Detect the content of GFP.As a result such as Fig. 7 A
Shown, the Res drug of various concentration is nearly free from influence to wild type COMP cell, and for saltant type COMP cell, it is low
When concentration, the content of intracellular GFP is increased, but with the increase of concentration, 25 μM of GFP intracellular later contain
Amount starts to reduce, and after 100 μM, the content of intracellular GFP tends to be steady, and shows when drug concentration is at 100 μM to prominent
Modification COMP cell has good function and effect.
In order to further explore the optimum time of Res function cells, we choose 100 μM of Res function cells model,
Different time gradients is set, as a result as shown in Figure 7 B, as time went on, Res drug is unobvious to wild cytosis,
And the cell being mutated is in 24 h of drug effect, hence it is evident that it reduces, i.e., intracellular delay is reduced, and variation later is unobvious,
The result shows that：When 100 μM of Res 24 h of drug effect, there are good function and effect to the cell of mutation.
The present invention relates to many aspects done elaboration as above.However, it should be understood that without departing from spirit of that invention
Under the premise of, those skilled in the art can carry out equivalent change and modification to it, and the change and modification equally fall into the application
The coverage area of appended claims.
Claims (4)
1. a kind of construction method of the cell model for anti-oxidation medicine screening based on protein Misfolding, feature exist
In：Using the wild and mutation reporter gene of molecular biology gene recombination technology building fluorescent protein labelingCOMPGene is sick slowly
Poisonous carrier, the packaging virus in human embryo kidney (HEK) 293T cell, HeLa cell of transduceing, puromycin screening, which obtains, stablizes expression COMP egg
White cell model.
2. a kind of cell model for anti-oxidation medicine screening based on protein Misfolding according to claim 1
Construction method, it is characterised in that：Steps are as follows：
（1）Human embryo kidney (HEK) 293T cell is transfected with the slow virus carrier of carrying antibiotics resistance gene, reporter gene, target gene,
Obtain the lentiviral particle of resistant gene and reporter gene；
（2）Use step（1）The lentiviral particle of acquisition infects HeLa Cells, and antibiotic-screening obtains cell model；
Wherein：The slow virus carrier is HIV-1 carrier system, including three kinds of plasmids：Express the Plvx-IRES- of reporter gene
Puro plasmid, packaging plasmid psPAX2, envelope protein plasmid pMD2.G, wherein Plvx-IRES-Puro plasmid contains puromycin
Resistant gene and ampicillin resistance gene, packaging plasmid psPAX2 include the gag gene of inhibition of HIV, envelope protein
Grain pMD2.G includes the VSV-G gene in herpe simplex source；The antibiotic is puromycin；The reporter gene iseGFP-COMPFusion；The target gene is wild typeCOMPGene and saltant typeCOMPGene p.D401N.
3. the cell model for anti-oxidation medicine screening based on protein Misfolding constructed in claims 1 or 2 exists
Screen the application in anti-oxidation medicine, it is characterised in that：Specifically screening appraisal procedure is：With anti-oxidation medicine function cells mould
Type detects the fluorescence intensity of eGFP under the conditions of 485 nm of excitation wavelength, 530 nm of launch wavelength, measures GFP content, uses cell
The ratio of the absorbance of the absorbance and cell culture medium of lysate indicates the delay situation of COMP albumen in the cell, passes through
Check and evaluation stablizes expression wild type and saltant typeCOMPThe content of COMP albumen in the cell model of gene assesses antioxidant drug
The effect of object.
4. the cell model for anti-oxidation medicine screening according to claim 3 based on protein Misfolding is sieving
Select the application in anti-oxidation medicine, it is characterised in that：The anti-oxidation medicine is acetylsalicylic acid ASA and resveratrol Res,
The concentration of middle acetylsalicylic acid is 75 mM, and the concentration of resveratrol is 100 μM, and the time of drug effect cell model is for 24 hours.
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| CN111411126A (en) * | 2020-03-27 | 2020-07-14 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | G3BP1 recombinant lentiviral vector and application thereof |
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