CN115896035B - Vero cell strain applied to virus amplification, construction method and application thereof - Google Patents
Vero cell strain applied to virus amplification, construction method and application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The application discloses a Vero cell strain applied to virus amplification, a construction method and application thereof. The Vero cell strain is formed by knocking out two gene fragments of STAT1 and EMX2 in the genome of the Vero cell strain. Compared with the prior art, the method has the advantages that by knocking out the STAT1 and the EMX2 in the Vero cell strain, the titer of the virus can be remarkably improved when the method is applied to amplification of porcine epidemic diarrhea virus and the like, and the method has wide application prospects in preparation of virus live vaccines and inactivated vaccines.
Description
Technical Field
The application relates to a Vero cell strain applied to virus amplification, in particular to a Vero cell strain applied to virus amplification, a construction method and application thereof, for example, application in virus amplification, especially porcine epidemic diarrhea virus amplification, and belongs to the technical field of biology.
Background
Vero cells, also known as green monkey kidney cells, are an aneuploidy Vero (generic name: chlorocebus) kidney cell line, isolated from the kidney epithelium of normal adult Vero in 1962, university of japan kilo, an Cunmei and obtained. Vero cells are continuous aneuploidy cell lines, meaning that their chromosome number is abnormal, whereas as continuous cell lines Vero cells can undergo many division cycles without aging. The interferon secretion function of Vero cells is defective, and unlike normal mammalian cells, they do not secrete interferon alpha/beta when infected with viruses. However, they still have receptors for interferon- α/β, so that they can still react when recombinant interferon is added to their culture medium. Vero cells are currently widely used in the study of the molecular mechanism of viral infection, in the production of vaccines and recombinant proteins, and are considered as ideal cell models for the cultivation of influenza vaccines and the study of the molecular mechanism of viral infection.
Vero cells can be widely infected with influenza virus, porcine epidemic diarrhea, simian vacuole virus, measles virus, rubella virus, arthropod-borne virus, adenovirus and the like; later found to be also susceptible to bacterial toxins, including diphtheria toxins, thermolabile enterotoxins, shigella toxins, and the like.
Porcine Epidemic Diarrhea Virus (PEDV) belongs to the genus alfa coronavirus among coronaviruses. PEDV is one of the important pathogens causing diarrhea in pigs, and pigs of all ages are susceptible, and the harm of piglets is greatest. The current prevention and treatment means mainly comprise vaccine immunization, and clinically used vaccines mainly comprise inactivated vaccines and attenuated vaccines. Inactivated vaccines are one of the main vaccines, and there is a great demand for viruses. However, the proliferation titer of the porcine epidemic diarrhea virus in the prior art is not high enough, and the cost is high.
Porcine epidemic diarrhea virus can replicate on Vero cells. Amplification of porcine epidemic diarrhea virus by Vero cells is a major way of producing porcine epidemic diarrhea vaccine. Although interferon expression in Vero cells has problems with less effect on replication after viral infection, there are still a number of genes in the cell that have adverse effects on viral replication.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a Vero cell strain applied to virus amplification, a construction method and application thereof, and the cell strain obtained by the invention improves the proliferation titer of PEDV and obviously reduces the cost.
In order to achieve the above object, the present application adopts the technical scheme that:
in one aspect, the present application provides a Vero cell line for viral expansion by knocking out two nucleotide fragments of STAT1 and EMX2 within the gene sequence of the Vero cell line.
In one embodiment, the Vero cell line used for virus amplification is formed by knocking out two nucleotide fragments of STAT1 and EMX2 in the gene sequence of the Vero cell line.
In another aspect, the present application provides a method for constructing a Vero cell line for virus expansion, comprising: obtaining a Vero cell gene sequence through amplification sequencing, carrying out gene knockout on one or both of STAT1 and EMX2 in the Vero cell gene by adopting a gene editing technology, and obtaining the Vero cell strain applied to virus amplification through cloning, screening and purifying.
Preferably, STAT1 and EMX2 can be co-knocked out, and then subjected to cloning, screening and purification to obtain the Vero cell strain applied to virus amplification.
In one embodiment, the construction method specifically includes:
(1) Finding out a target point of a Vero cell STAT1 gene, and designing corresponding targeting sgRNAs, wherein the targets comprise sgRNA-STAT1-F and sgRNA-STAT1-R;
(2) PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 vector was digested with Bbs I endonuclease, followed by cleavage with sgRNA-STAT1-F
And a double-stranded molecule formed after annealing the sgRNA-STAT1-R sequence is connected to obtain a knockout plasmid PX459 (pSpCas 9 (BB) -2A-Puro-sgRNA) V2.0;
(3) And (3) transfecting the knockout plasmid PX459 (pSpCas 9 (BB) -2A-Puro-sgRNA) V2.0 into Vero cells, carrying out pressurized screening on puromycin, and carrying out monoclonalization, and obtaining a Vero cell strain of which STAT1 is applied to virus amplification through PCR verification.
The sequence of the target point, the sequence of the gene knockout sgRNA designed for STAT1 (SEQ ID NO: nw_ 023666040.1) in the Vero cell gene is as follows: sgRNA-STAT1-F: caccgCGTAATCTTCAGGTATGACC and sgRNA-STAT1-R: aaacGGTCATACCTGAAGATTACGc.
(4) Finding a target point of the Vero cell EMX2 gene, and designing corresponding targeting sgRNAs, including sgRNA-EMX2-F and sgRNA-STAT2-R;
(5) The PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 vector was digested with Bbs I, and then ligated to a double-stranded molecule formed by annealing the sgRNA-EMX2-F and sgRNA-EMX2-R sequences to obtain a knockout plasmid PX459 (pSpCas 9 (BB) -2A-Puro-sgRNA) V2.0;
(6) And (3) transfecting the knockout plasmid PX459 (pSpCas 9 (BB) -2A-Puro-sgRNA) V2.0 into Vero cells from which the STAT1 gene has been knocked out, carrying out pressurized screening on puromycin, and carrying out monoclonalization, thus obtaining the Vero cell strain applied to virus amplification through PCR verification.
The sequence of the target point, the sequence of the gene knockout sgRNA designed for EMX2 (SEQ ID NO: NW_ 023666048.1) in Vero cell genes is as follows: sgRNA-EMX2-F: CACCGTAGGGGCGTCTACTCCAACC and sgRNA-EMX2-R: AAACGGTTGGAGTAGACGCCCCTAC.
In one embodiment, the gene editing technique comprises a CRISPR/Cas9 gene editing technique.
In the present invention, the growth solution is RPMI1640 medium containing 10% of fetal bovine serum, and the maintenance solution is serum-free V-LSM medium.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) The STAT1 and EMX2 gene knockout method based on the CRISPR/Case9 technology designs a section of specific sgRNA to recognize STAT1 and EMX2 genes, and finally causes abnormal expression of the genes, wherein the provided Vero cell STAT1 and EMX2 knockout method has strong practicability.
(2) The method can also be used for specific knockout of other genes, and has the advantages of high efficiency, economy and the like.
(3) The invention provides important basis for researching STAT1 and EMX2 genes, developing novel anti-PEDV drugs and searching effective prevention and control methods for porcine diarrhea.
(4) According to the invention, virus titer is measured through a PEDV virus-grafting experiment of Vero-STAT1-EMX2-KO cells, and it is determined that STAT1 and EMX2 genes have the effect of inhibiting PEDV replication.
(5) The cell line for knocking out the STAT1 and the EMX2, which is established by the invention, improves the proliferation titer of the PEDV, obviously reduces the cost, provides a basis for exploring the cell line with high replication level of the PEDV, has an important effect for further researching the proliferation of the PEDV, and has a wide application prospect in the production of PEDV vaccines.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the structure of PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 vector, wherein BbsI cleavage site is located at both 245bp and 267 bp;
FIG. 2 shows the results of virus titer determinations by harvesting cell supernatants for propagation of PEDV using wild-type Vero cells and Vero-STAT1-EMX2-KO cells, respectively, with time after virus challenge (hpi) on the abscissa and log of PEDV virus titer level (TCID) on the ordinate 50 /ml)。
Detailed Description
The technical solutions of the present application are further described in detail below by way of examples and with reference to the accompanying drawings. However, the examples selected are merely illustrative of the present application and do not limit the scope of the present application. The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
EXAMPLE 1 construction of Vero-STAT1-EMX2-KO cells
1. sgRNA design and preparation
The exon sequences of the African green monkey genome STATU gene sequence (SEQ ID NO: NW_ 023666040.1) and the EMX2 gene sequence (SEQ ID NO: NW_ 023666048.1) registered in GenBank were searched for. STAT1 and EMX2 gene targets were screened through the sgRNA design website (http:// crispr. Mit. Edu). Designing an sgRNA sequence targeting the target, adding CACCG at the 5' end to obtain an sgRNA-STAT1-F and an sgRNA-EMX2-F sequence, adding AAAC at the 5' end and adding C at the 3' end to obtain an sgRNA-STAT1-R and an sgRNA-EMX2-R sequence.
sgRNA-STAT1-F：CACCGCGTAATCTTCAGGTATGACC
sgRNA-STAT1-R：AAACGGTCATACCTGAAGATTACGC
sgRNA-EMX2-F：CACCGTAGGGGCGTCTACTCCAACC
sgRNA-EMX2-R：AAACGGTTGGAGTAGACGCCCCTAC
2. Recombinant plasmid construction
1. Linearization carrier
The PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 vector map is shown in FIG. 1. PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 vector was cut singly with Bbs I endonuclease. And (3) enzyme cutting system: restriction enzyme Bbs I1. Mu.L, PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 plasmid 1. Mu.g, 10 XNE Buffer 2. Mu.L, ddH2O make up to 20. Mu.L. The reaction procedure of the enzyme digestion is as follows: the enzyme digestion is carried out for 1h at 37 ℃ and is carried out for 30min by heat treatment at 65 ℃ to terminate the enzyme digestion. The digested product was subjected to electrophoresis using 1% agarose gel, and the target band was excised and subjected to gel recovery using a gel recovery kit to recover DNA.
2. Annealing of sgRNA fragments
The sgRNA-STAT1-F fragment and the sgRNA-STAT1-R, the sgRNA-EMX2-F and the sgRNA-EMX2-R fragments were annealed to form a double stranded sgRNA molecule having cohesive ends at both ends, respectively. Annealing system: 10×T Polynucleotide Kinase (T4 PNK) Reaction Buffer 2 μ L, sgRNA-DBN1-F (100 uM) 1 μ L, sgRNA-DBN1-R (100 uM) 1 μL, PNK enzyme 1 μL, ddH2O make up to 10uL. Setting a reaction program in a PCR instrument at 20 ℃ for 30min, cooling to 25 ℃ after 5min at 95 ℃ with a cooling speed of 5min -1 。
3. Connection
The sgRNA duplex formed after annealing was ligated with Bbs I single cleavage product of PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 vector plasmid. The connection system is as follows: 1. Mu.L of the 200-fold dilution of the annealed product, 1.5. Mu.L of PX459 (pSpCas 9 (BB) -2A-Puro) V2.0 cleavage product, 1. Mu.L of T4 DNA Ligase Bufier, 0.5. Mu.L of T4 DNA library, and a total system of 10. Mu.L; the ligation system was placed in an electronic incubator and either overnight at 16℃or at room temperature for 1.5h.
4. Transformation
Taking out Trans5α chemically competent cells from-80deg.C, immediately inserting into ice for thawing, adding connection product when thawing, and incubating on ice for 30min; taking out, placing in a water bath at a temperature of 42 ℃ for heat shock for 45s, placing back on ice, standing for 2min, adding 500uL of LB culture medium, and culturing at a temperature of 37 ℃ for 1h at 200 rpm. 200uL, 50uL and 10uL of culture solution are respectively sucked, added to LB (100 mu g/mL ampicillin) culture dishes containing ampicillin resistance, uniformly coated by a turning bar and cultured in a incubator at 37 ℃ for 12 hours.
5. Screening of colonies of interest
And selecting single colony for sequencing and identifying, further amplifying and culturing the identified correct strain, and extracting plasmids for sequencing by the engine biological company. The correct recombinant plasmids for the sequencing alignment were designated PX459 (pSpCas 9 (BB) -2A-Puro-STAT1 sgRNA) V2.0 and PX459 (pSpCas 9 (BB) -2A-Puro-EMX2 sgRNA) V2.0.
3. Knockout of Vero cell-targeted STAT1 and EMX-2 genes
Vero cells were cultured in high-sugar DMEM complete medium containing 8% neo-calf serum and 1% diabodies (penicillin, streptomycin). Vero cells were grown at 1X 10 6 The wells/well were seeded with 6-well plates. When the cell confluency reached 70%, recombinant plasmid PX459 (pSpCas 9 (BB) -2A-Puro-STAT1 sgRNA) V2.0 was transfected into Vero cells using Lipofectamine3000 kit. After 8h of transfection, the medium was changed to medium containing 8ug/mL puromycin for pressure screening. The culture was changed once on the third day, and the culture was continued until the 6 th day of the pressure screening, the cells were digested with pancreatin, diluted to 10 cells/mL using the limiting dilution method, and plated in 96-well plates at 100uL per well. And observing and marking the hole of the single cell, and timely replenishing. When the cells grow to have the confluence of 50-70%, performing pancreatin digestion, and performing primary pore passageAfter that, expansion culture of 96, 24, 12 well plates was gradually performed. After the cells are transferred into a 12-well plate, a part of the cells are taken to determine whether the cells are monoclonal cell strains knocking out STAT1 genes by a PCR method. The cells selected were Vero-STAT1-KO cells.
Vero-STAT1-KO cells were cultured in high-sugar DMEM complete medium containing 8% neo-calf serum and 1% diabody (penicillin, streptomycin). Vero cells were grown at 1X 10 6 The wells/well were seeded with 6-well plates. When the cell confluency reached 70%, recombinant plasmid PX459 (pSpCas 9 (BB) -2A-Puro-EMX2 sgRNA) V2.0 was transfected into Vero-STAT1-KO cells using Lipofectamine3000 kit. After 8h of transfection, the medium was changed to medium containing 8ug/mL puromycin for pressure screening. The culture was changed once on the third day, and the culture was continued until the 6 th day of the pressure screening, the cells were digested with pancreatin, diluted to 10 cells/mL using the limiting dilution method, and plated in 96-well plates at 100uL per well. And observing and marking the hole of the single cell, and timely replenishing. When the cells grow to 50% -70% of confluence, pancreatin digestion is carried out, primary pore passage is carried out, and then expansion culture of 96, 24 and 12 pore plates is gradually carried out. After transferring the cells into a 12-well plate, a part of the cells are taken to determine whether the cells are monoclonal cell strains knocking out the EMX2 gene by a PCR method. The cells selected were Vero-STAT1-EMX2-KO cells.
EXAMPLE 2 Vero-STAT1-EMX2-KO cell PEDV replication level study
After obtaining the Vero-STAT1-EMX2-KO cell line, in order to investigate the effect of the cells on the proliferation of PEDV, a virus-grafting experiment was performed on wild-type Vero cells and Vero-STAT1-EMX2-KO cells. In T-25 square flasks, 1X 10E6 Vero cells or Vero-STAT1-EMX2-KO cells were seeded per flask. Culturing for 48h, washing cells with PBS, inoculating PEDV at MOI=0.1 dose, adding 5ml of virus and maintenance solution (VLSM containing 15ug/ml pancreatin) per bottle, culturing for 24h, harvesting culture supernatant, and performing virus titer log (TCID) 50 /ml) detection.
TCID 5o The specific operation steps of the detection method are as follows: vero cells were plated in 96-well plates and the cultured cells were just confluent with monolayers. The virus culture was washed 3 times with PBS and diluted 8 gradients with maintenance solution (high-sugar DMEM complete medium containing 15ug/mL pancreatin) (10 -1 -10 -8 ) 24 replicate wells were set per gradient and 100ul per well was added to a 96-well plate, while a control well with only maintenance fluid was set. Culturing in 5% CO2 cell incubator at 37deg.C, and recording cytopathy every day until the cells are not diseased. The number of diseased cells was counted and the TCID50 of the virus was calculated by Reed-Muench two-step method, and the results are shown in FIG. 2.
It should be understood that the above embodiments are merely for illustrating the technical concept and features of the present application, and are intended to enable those skilled in the art to understand the content of the present application and implement the same according to the content of the present application, not to limit the protection scope of the present application. All equivalent changes or modifications made in accordance with the spirit of the present application are intended to be included within the scope of the present application.
Claims (2)
1. A Vero cell strain applied to virus amplification is characterized in that: it is formed by knocking out STAT1 and EMX2 nucleotide fragments in the gene sequence of Vero cell strain.
2. Use of Vero cell lines for virus amplification according to claim 1 for the production of live or inactivated vaccines against porcine epidemic diarrhea virus.
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