CN111575242A - iPSC-nCoVN cell model for COVID-19 drug screening and establishing and using methods thereof - Google Patents
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Abstract
The invention discloses an iPSC-nCoVN cell model for COVID-19 drug screening and an establishing method and a using method thereof, belonging to the technical field of biological medicines. The cell model is obtained by constructing a vector of pCW-nCoVN through cDNA of nCoVN, and then transfecting iPSC cells after packaging HEK293T cells with lentiviruses. The invention establishes a cell model for COVID-19 drug screening for the first time, has simple and convenient detection method, low operation difficulty, high detection speed and throughput operation, can quickly evaluate whether a tested drug can inhibit the function of nCoVN protein or not in a virus-free safe environment, and reduces or eliminates the influence of the tested drug on the proliferation rate and the pluripotency of iPSC, thereby achieving the purpose of drug screening without safety risk; is used for screening candidate drugs for resisting COVID-19 and provides scientific basis for the treatment of COVID-19.
Description
Technical Field
The invention relates to an iPSC-nCoVN cell model for COVID-19 drug screening and an establishment and use method thereof, belonging to the technical field of biological medicines.
Background
The new medicine for effectively treating COVID-19 has long research and development period, high cost, safety risk and the like, and the most rapid method refers to the pathogenic mechanism of SARS and MERS and selects proper candidate medicines for testing. Therefore, establishing a rapid and accurate COVID-19 drug screening platform is of great significance to epidemic prevention and control work under the current severe situation.
At present, the commonly used drug screening methods include animal experimental screening, computer simulation screening, molecular and cell level screening, and the like. Although the traditional animal experiment screening method has many achievements in drug research and development, the traditional animal experiment screening method has the limitations of high cost, long research and development period, non-large-scale screening and the like. Computer-simulated screening refers to screening effective drugs by simulating the interaction between drug targets and drug structures with a computer, and although the advantages of low cost and high speed exist, the screened drugs often have no or little effect and still need to be further verified through biological experiments. The molecular and cell level screening has the advantages of low cost, high accuracy, high-throughput operation and the like, and is widely applied. Cell level screening is a method which is closer to the real biochemical process in vivo than molecular level screening in the natural state of living cells, so that the method has the most application prospect at present. The conventional cell level research method for COVID-19 drug screening is a mode that SARS-CoV-2 virus strain infects mammalian cells cultured in vitro, but the method relates to experiments such as culture of virus strain, and the like, has safety risk, high requirements on experimental environment and is not beneficial to high-throughput screening.
SARS-CoV-2 is a beta coronavirus, and has genome identity of 79% with SARS-CoV, and its pathogenesis is similar to that of SARS-CoV. Many current studies on SARS-CoV-2 rely on prior work with SARS-CoV. The structural analysis of SARS-CoV genome shows that its coding protein mainly includes spike protein (S), nucleocapsid protein (N), membrane protein (M), envelope protein (E) and so on. Wherein the nucleocapsid protein (N) is the main structural protein of SARS-CoV, can wrap the viral genome into a helical Ribocapsid (RNP), and plays an important role in the self-assembly of the virus. Research shows that the nucleocapsid protein can regulate the division and proliferation of cells, inhibit the generation of I-type interferon and participate in signal pathways such as NF-kappa B, AP-1, TGF-beta and the like. In addition, the nucleocapsid protein has the characteristic of high conservation compared with other SARS-CoV proteins. Thus, the nucleocapsid protein is an ideal tool for coronavirus research.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an iPSC-nCoVN cell model for screening COVID-19 drugs, which is a safe, high-flux and rapid COVID-19 drug screening cell model which does not need to be operated in a P3 laboratory, can be used for screening drugs for inhibiting nucleocapsid protein (N) from damaging pluripotency of human adult stem cells, and can provide experimental basis for drug research and development aiming at new crown pneumonia epidemic situation.
In addition, the invention also provides an establishing method and a using method of the iPSC-nCoVN cell model for COVID-19 drug screening.
In order to solve the technical problems, the invention adopts the following technical scheme:
an establishment method of an iPSC-nCoVN cell model for COVID-19 drug screening comprises the following steps:
s1 construction of lentiviral expression vectors
The cDNA (N end carries puromycin resistance gene) of nucleocapsid protein-nCoVN of SARS-CoV-2 (manufacturer GeneMedi) is subcloned into pCW-Cas9-Blast vector (Addgene, 83481) by using molecular cloning method to replace Cas9 and Blast gene in the original vector, thus obtaining pCW-nCoVN vector.
In the step, GFP genes are simultaneously constructed into a pCW-Cas9-Blast vector as a control;
s2 lentivirus packaging
Inoculating HEK293T cells into a 6-well plate, culturing by using a DMEM culture solution containing 10% fetal calf serum (DMEM culture solution + 10% fetal calf serum), and preparing for transfection when the cell confluency reaches 70% -80%; and co-transfecting HEK293T cells by pCW-nCoVN, pVSVG and psPAX2, continuing culturing after transfection is finished, removing cell residues after centrifugation, and keeping supernatant.
The invention selects pCW-nCoVN, pVSVg and psPAX2 for packaging, increases nCoVN gene compared with the conventional lentivirus, and is pCW-nCoVN nontoxic; pCW-nCoVN and SARS-CoV (toxic) and SARS-CoV-2 (toxic) are used, they share CoV gene, in general laboratory, it can test the property or drug property of pCW-nCoVN to indirectly obtain the property or drug property detection or screening of SARS-CoV-2 (toxic) which can be made in P3 laboratory, and can raise safety.
In the step, 1h before transfection, the original culture solution is discarded, and 2 mL/hole preheated serum-free OptiMEM culture solution is added;
for transfection, pCW-nCoVN (20. mu.g), pVSVg (10. mu.g) (Addge), psPAX2 (15. mu.g) (Addge) were co-transfected into HEK293T cells using Lipofectamine 2000 reagent according to the instructions; after 6h, the culture solution is replaced by a DMEM culture solution containing 10% fetal calf serum and 1% BSA (DMEM culture solution + 10% fetal calf serum + 1% BSA), and the culture is continued for about 60 h;
centrifuging the culture solution at 3000rpm and 4 deg.C for 10min to remove cell residue; the supernatant was filtered through a 0.45 μm low protein binding filter (Millipore Steriflip HV/PVDF) to remove cell debris.
Mixing the virus-containing supernatant obtained by the above centrifugation with 10% sucrose buffer (50mM Tris-HCl, pH7.4, 100mM NaCl, 0.5mM EDTA) at a volume ratio of 4:1, and centrifuging at 10000g and 4 ℃ for 4 hours; discarding the supernatant, draining the centrifuge tube on absorbent paper for 3min, adding 1 × PBS for resuspension, and preserving at-80 deg.C;
s3, transfection iPSC cell
iPSC culture: human induced pluripotent stem cells (ipscs) were seeded on a Matrigel matrix (corning, 354277) -coated plate and then cultured with STEMUP medium (Nissan Chemical Corporation);
the STEMUP medium was changed every two days. ipscs were passaged every 3 days, or when cell cultures reached 80-90% confluence. During passaging, it was washed 1 time with 1 XDPBS (Gibco, 14040133) and then treated in 0.5mM EDTA (Invitrogen, 15575020) diluted with 1 XDPBS (Gibco, 14190144) for 10min at room temperature. The passage ratio is 1: 3-1: 6.
transfection: when the confluency of the cultured iPSC cells reaches 70% -80%, transfection is carried out, and the multiplicity of infection (MOI) is about 0.3-0.5; 24h after transfection, the culture was replaced with fresh STEMUP containing a final concentration of 2. mu.g/mL of polytetracycline hydrochloride (Dox); after 2 days, the culture solution was changed to STEMUP (containing Dox 2. mu.g/mL + puromycin 2. mu.g/mL (puromycin) (InvivoGen)) for screening, and after 2-3 days of screening, about 30% of transformation efficiency was obtained, and individual clones were selected and inoculated into different dishes for culture, thereby obtaining iPSC-nCoVN cell strains.
In addition, the use method of the iPSC-nCoVN cell model for screening the COVID-19 drugs is characterized in that iPSC-nCoVN cells are used as the model, drugs to be tested with proper doses are added into a culture solution, and whether the tested drugs can inhibit the action of nCoVN proteins or not can be rapidly evaluated in a virus-free safe environment through the detection of a cell proliferation rate and a totipotency marker (TRA-1-81), so that the influence of the tested drugs on the proliferation rate and the pluripotency of the iPSC is reduced or eliminated, the effectiveness of the drugs on the COVID-19 is obtained, and the purpose of screening the drugs is achieved.
Compared with the prior art, the invention has the beneficial effects that:
the invention establishes an iPSC-nCoVN cell model for COVID-19 drug screening for the first time, has simple and convenient detection method, low operation difficulty, high detection speed (obtaining a result after 4 days), and flux operation, can quickly evaluate whether a tested drug can inhibit the action of nCoVN protein in a virus-free safe environment, and reduces or eliminates the influence of the tested drug on the proliferation rate and the pluripotency of iPSC, thereby achieving the purpose of drug screening without safety risk; is used for screening candidate drugs for resisting COVID-19 and provides scientific basis for the treatment of COVID-19.
Drawings
FIG. 1 is a schematic diagram of the drug screening of the IPSC-nCoVN cell model of the present invention;
FIG. 2 is a graph showing the expression level of nCoVN gene in iPSC-nCoVN in the DOX group and DMSO control group of the present invention;
FIG. 3 is a graph showing the results of detection of the iPSC-nCoVN pluripotency marker (TRA-1-81) at different induction times (2 days, 4 days, 6 days, 8 days);
FIG. 4 is a graph of the effect of nCoVN on the proliferation rate of iPSC, iPSC-nCoVN, iPSC-GFP.
Detailed Description
In order to make the present invention more clear and intuitive for those skilled in the art, the present invention will be further described with reference to the accompanying drawings.
The invention establishes an iPSC-nCoVN cell model for screening COVID-19 drugs, adds a proper dose of drugs to be tested in a culture solution by taking iPSC-nCoVN cells as the model, can quickly evaluate whether the tested drugs can inhibit the action of nCoVN protein in a virus-free safe environment through the detection of cell proliferation rate and totipotency markers (TRA-1-81), and reduces or eliminates the influence of the proliferation rate and the pluripotency of iPSC, thereby obtaining the effectiveness of the drugs on COVID-19 and achieving the purpose of screening the drugs.
The principle of drug screening of the IPSC-nCoVN cell model of the invention is shown in FIG. 1, and the specific operation of each part is as follows:
example 1: obtaining the IPSC-nCoVN cell strain
1.1 lentivirus expression vector construction:
the nCoVN cDNA (with puromycin resistance gene at the N end) (GeneMedi) is subcloned into pCW-Cas9-Blast vector (Addgene, 83481) by using a conventional molecular cloning method to replace Cas9 and Blast gene in the original vector, so as to obtain pCW-nCoVN vector. Meanwhile, GFP gene was constructed into pCW-Cas9-Blast vector, which was used as a control, and the same treatment as that of pCW-nCoVN vector was carried out.
1.2 Lentiviral packaging
1.2.1 HEK293T cells were seeded into 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum (DMEM + 10% fetal bovine serum), and transfection was performed when the cell confluence reached 70% -80%.
1.2.2 Prior to transfection, 1h, the original medium was discarded and 2 mL/well of pre-warmed serum-free OptiMEM medium was added.
1.2.3 transfection was performed with Lipofectamine 2000 reagent as per the instructions. pCW-nCoVN (20. mu.g), pVSVg (10. mu.g) (Addgene), psPAX2 (15. mu.g) (Addgene) were co-transfected into HEK293T cells.
1.2.4 after 6h, the culture was changed to DMEM containing 10% fetal calf serum and 1% BSA (DMEM + 10% fetal calf serum + 1% BSA).
1.2.5 after further culturing for about 60h, the culture broth was centrifuged at 3000rpm at 4 ℃ for 10min to remove cell debris.
1.2.6 the supernatant was filtered through a 0.45 μm low protein binding filter (Millipore Steriflip HV/PVDF) to remove cellular debris.
1.2.7 the virus-containing culture broth was centrifuged at 10000g and 4 ℃ for 4 hours in a volume ratio of 4:1 with 10% sucrose buffer (50mM Tris-HCl, pH7.4, 100mM NaCl, 0.5mM EDTA). The supernatant was discarded, the tube was drained on absorbent paper for 3min, resuspended in 1 × PBS and stored at-80 ℃.
1.3 transfection of iPSC cells
1.3.1iPSC culture:human Induced Pluripotent Stem Cells (iPSC) DYR0100(ATCC) were seeded on a Matrigel matrix (Corning, 354277) -coated plate, thenPost culture with STEMUP (Nissan Chemical corporation.) STEMUP medium was changed every two days iPSC was passaged every 3 days, or passaged when the cell culture reached 80-90% confluency, washed 1 time with 1 × DPBS (Gibco, 14040133) during passaging, and then treated in 0.5mM EDTA (Invitrogen, 15575020) diluted with 1 × DPBS (Gibco, 14190144) for 10min at room temperature at a passaging ratio of 1: 3-1: 6.
1.3.2 transfection:and (5) carrying out transfection when the confluence degree of the iPSC cells reaches 70-80%. Multiplicity of infection (MOI) is about 0.3-0.5. 24h after transfection, the culture medium was replaced with fresh STEMUP (containing a final concentration of 2. mu.g/mL of tetracycline hydrochloride (Dox)). After 2 days, the culture broth was replaced with STEMUP (containing Dox 2. mu.g/mL + puromycin (puromycin) (InvivoGen)) for selection. After 2-3 days of screening, about 30% conversion efficiency can be obtained. Selecting single clone to inoculate in different dishes for culture, and obtaining the iPSC-nCoVN cell strain.
Example 2: dox induces expression of nCoVN
2.1 inducing:addition of Dox (Sigma, D9891) to STEMUP at a final concentration of 2. mu.g/mL induced nCoVN expression, and Dimethylsulfoxide (DMSO) at a concentration of 2. mu.g/mL in STEMUP was used as a control.
2.2 Total RNA extraction:total RNA from cells was extracted using UNIQ-10Column Trizol Total RNA Isolation Kit (Sangon Biotech, B511321-0100). The samples were treated with DNase I (Sangon Biotech, B618252) for 30min in advance.
2.3 reverse transcription:RNA was Reverse transcribed using the Reverse Transcription kit iScript Reverse Transcription Supermix (Bio-Rad, 1708841).
2.4qPCR assay for nCoVN mRNA expression levels:according to SsoAdvancedTMUniversal
GreenSupermix (Bio-Rad,1725271) instructions, using the PikoReal Real-Time PCR System (ThermoFisher) System, with NAPDH as an internal reference, primers were designed to detect nCoVN expression levels in iPSC and iPSC-nCoVN (Dox-inducible, DMSO-control). The primer sequences are as follows:
nCoVN-RT-F:CATTGGCATGGAAGTCACAC；
nCoVN-RT-R:TCTGCGGTAAGGCTTGAGTT；
GAPDH-RT-F:TGGGTGTGAACCATGAGAAG；
GAPDH-RT-R:GTGTCGCTGTTGAAGTCAGA。
and (4) analyzing results: as shown in fig. 2, the expression level of nCoVN in the Dox-induced group was 267-fold higher than that of the DMSO control group. The result shows that the iPSC-nCoVN cell strain capable of expressing the nCoVN gene at high level is successfully constructed by the method.
Example 3: cell pluripotency assay
3.1Dox induces nCoVN expression:nCoVN gene expression in iPSC-nCoVN was induced by Dox (Sigma, D9891) at a final concentration of 2. mu.g/mL, and samples were taken at 2 days, 4 days, 6 days, and 8 days, respectively, for cell pluripotency determination.
3.2 detection of pluripotency marker (TRA-1-81):
3.2.1 cells were fixed with 4% paraformaldehyde at room temperature for 20min, followed by 3 washes with 1 × PBS. Cells were then permeabilized with PBS containing 0.25% Triton X-100 for 10 minutes at room temperature.
3.2.2 after blocking with blocking buffer (1 XPBS + 10% sheep serum), cells were stained with primary antibody (TRA-1-81, mouse, Invitrogen,14-8883-80) at 4 ℃ overnight with a dilution factor of 1: 250.
3.2.3 cells were washed 3 times with PBS containing 0.1% Triton X-100 and incubated with secondary antibody (Alexa Fluor488 coat anti-mouse) for 1h at 37 ℃.
3.2.4 nuclei were stained with DAPI (4', 6-diamidino-2-phenylindole, 1. mu.g/ml) for 5 min.
3.2.5 were imaged with a laser scanning confocal microscope (Leica) and the images were analyzed with ImageJ.
And (4) analyzing results: as shown in FIG. 3, TRA-1-81 was still expressed in iPSC-nCoVN at 2 days of Dox induction, but TRA-1-81 could not be detected in the cells after 4 days of induction, indicating that the pluripotency of iPSC-nCoVN was lost within 4 days.
Example 4: proliferation Rate detection
4.1Dox induces nCoVN expression:with a final concentration of 2. mu.g/mLDox (Sigma, D9891) simultaneously induces three cells of iPSC, iPSC-GFP and iPSC-nCoVN, and samples are respectively taken at 24h, 42h, 48h, 60h and 72h and used for measuring the cell proliferation rate.
4.2 proliferation Rate determination:the cell counting kit was used to determine the number of viable cells in different groups (iPSC, iPSC-GFP, iPSC-nCoVN) at different time points (24h, 42h, 48h, 60h, 72 h).
And (4) analyzing results: as shown in fig. 4, after Dox induction for 72h, the cell proliferation rate of the iPSC-nCoVN group was significantly reduced compared to the iPSC and iPSC-GFP groups, indicating that nCoVN affects the proliferation of ipscs.
In conclusion, the invention successfully constructs the iPSC-nCoVN cell model. Through verification, Dox can induce iPSC-nCoVN cells to highly express nCoVN protein. Overexpression of nCoVN protein in ipscs significantly affected the proliferation rate of ipscs and caused loss of ipscs pluripotency. The method is characterized in that iPSC-nCoVN cells are taken as a model, a proper dose of a drug to be tested is added into a culture solution, and the detection of a cell proliferation rate and a totipotency marker (TRA-1-81) can quickly evaluate whether the tested drug can inhibit the action of nCoVN protein in a virus-free safe environment, so that the influence of the tested drug on the iPSC proliferation rate and the pluripotency can be reduced or eliminated.
The invention establishes the iPSC-nCoVN cell model for screening the COVID-19 drug for the first time, has simple and convenient detection method, low operation difficulty, high detection speed (obtaining the result after 4 days), flux operation and no safety risk, can be used for screening the anti-COVID-19 candidate drug and provides scientific basis for the treatment of the COVID-19.
The embodiments described above are presented to enable those skilled in the art to make and use the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should make improvements and modifications to the present invention based on the disclosure of the present invention within the protection scope of the present invention.
Claims (8)
1. An establishment method of an iPSC-nCoVN cell model for COVID-19 drug screening is characterized by comprising the following steps:
s1 construction of lentiviral expression vectors
Subcloning cDNA of nucleocapsid protein-nCoVN of SARS-CoV-2 into pCW-Cas9-Blast vector by using molecular cloning method, substituting Cas9 and Blast gene in original vector to obtain pCW-nCoVN vector;
s2 lentivirus packaging
Inoculating HEK293T cells into a 6-well plate, culturing by using DMEM culture solution containing 10% fetal calf serum, and preparing for transfection when the cell confluency reaches 70% -80%; co-transfecting HEK293T cells by pCW-nCoVN, pVSVG and psPAX2, continuously culturing after transfection is finished, removing cell residues after centrifugation, and keeping supernatant;
s3, transfection iPSC cell
iPSC culture: inoculating the iPSC cells on a Matrigel matrix coated plate, and then culturing by using STEMUP;
transfection: carrying out transfection when the confluency of the cultured iPSC cells reaches 70-80%; after 24h of transfection, the culture medium was replaced with fresh STEMUP containing polycyclocycline hydrochloride at a final concentration of 2. mu.g/mL; and after 2 days, replacing the culture solution with STEMUP containing 2 mu g/mL of tetracycline hydrochloride and puromycin for screening, selecting single clone and inoculating the single clone into different dishes for culture after 2-3 days of screening, thus obtaining the iPSC-nCoVN cell strain.
2. The method of claim 1, wherein: in step S2, 2 mL/well of preheated serum-free OptiMEM medium was added 1h before transfection, without discarding the original medium.
3. The method of claim 1, wherein: in the step S2, in the transfection, the Lipofectamine 2000 reagent is used for transfection according to the instruction, and HEK293T cells are co-transfected by 20 ug of pCW-nCoVN vector, 10 ug of pVSVg vector and 15 ug of psPAX2 vector; after 6 hours, the culture medium was replaced with DMEM containing 10% fetal bovine serum and 1% BSA, and the culture was continued for about 60 hours.
4. The method of claim 1, wherein: in step S2, the centrifugation step is to take the culture solution and centrifuge for 10min at 3000rpm and 4 ℃, and then filter the supernatant with 0.45 μm low protein binding filter membrane to remove the cell debris.
5. The method of claim 1, wherein: in the step S2, the method further comprises the steps of mixing the culture solution containing the virus with 10% of sucrose buffer solution according to the volume ratio of 4:1, centrifuging for 4 hours at 10000g and 4 ℃, removing supernatant, draining the centrifuge tube on absorbent paper for 3min, adding 1 XPBS for resuspension, and preserving at-80 ℃ for later use.
6. The method of claim 1, wherein: in step S3, the STEMUP medium was replaced every two days during iPSC culture, washed 1 time with 1 XDPBS during iPSC passage, and then treated with 0.5mM EDTA diluted with 1 XDPBS for another 10min at room temperature.
7. An iPSC-nCoVN cell model for codv-19 drug screening, obtainable by a method according to any one of claims 1 to 6.
8. A method of using the iPSC-nCoVN cell model obtained according to the method of any one of claims 1 to 6, characterized in that: the method is characterized in that an iPSC-nCoVN cell is taken as a model, a proper amount of to-be-tested drug is added into a culture solution, and the effectiveness of the drug on COVID-19 is obtained through the detection of cell proliferation rate and totipotency markers, so that the purpose of screening is achieved.
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