CN117802097A - sgRNA for knocking out ISG15 gene, ISG15 gene-deleted bovine kidney cell and application thereof - Google Patents
sgRNA for knocking out ISG15 gene, ISG15 gene-deleted bovine kidney cell and application thereof Download PDFInfo
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Abstract
The invention relates to the field of gene editing, in particular to sgRNA for knocking out an ISG15 gene, a bovine kidney cell with the ISG15 gene deleted and application thereof. The invention provides an sgRNA for knocking out an ISG15 gene, which can efficiently knock out the ISG15 gene. At the same time, the invention also provides a cattle kidney cell MDBKISG15 with the ISG15 gene deleted ‑/‑ The cell can be used for researching the influence of ISG15 genes on virus replication, a related regulation mechanism or a related path, improvement of virus yield and the like. Moreover, the cell can be used as an ideal cell model for antiviral research and can also be shownSignificantly increasing the viral titer of the virus grown on the cell line.
Description
Technical Field
The invention relates to the field of gene editing, in particular to sgRNA for knocking out an ISG15 gene, a bovine kidney cell with the ISG15 gene deleted and application thereof.
Background
Interferon-stinum ridge 15 (ISG 15) was originally found in 1979 in IFN-treated cells, and this protein was shown to be in the 17kD precursor form at the beginning of synthesis, but was subsequently hydrolyzed to a 15kD protein, hence the designation "15kD protein". The innate immune response is the first line of defense against invading pathogens that can be perceived through host pattern recognition receptors. Type I IFN from the infected cells after release and infected cells and adjacent cells IFN alpha/beta receptor binding, thereby playing the role of autocrine and paracrine. Binding of secreted IFN to its receptor activates downstream signaling, ultimately resulting in the expression of hundreds of IFN-stimulated genes (ISGs), with ISG15 acting as one of the most strongly induced, fastest ISG proteins, playing an important role therein. ISG15 is an important regulator in the natural immune pathway of antiviral only the most important class found in vertebrates, and has important roles in regulating immune response, signal transduction pathway, antiviral, regulating viral replication, evading host natural immunity, etc.
MDBK cells are commonly used for proliferation and culture of bovine viruses and are widely used in the production of bovine viral vaccines. Therefore, the cell line for efficiently propagating the virus is constructed, and has important effect on researching the interaction between the virus and a host and improving the production efficiency of the vaccine.
However, no ISG15 knockout cell line has been reported. It can be seen that it is important to find a method capable of editing ISG15 gene with high efficiency, thereby obtaining a knockout cell line for knocking out ISG15 gene.
Disclosure of Invention
The invention aims to provide a sgRNA for knocking out an ISG15 gene, a bovine kidney cell with the ISG15 gene deleted and application thereof, so as to solve the problems in the prior art. The sgRNA can knock out the ISG15 gene with high efficiency.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides an sgRNA for knocking out an ISG15 gene, which comprises sgRNA1 and sgRNA2;
the sgRNA1 comprises an upstream primer shown as SEQ ID NO.1 and a downstream primer shown as SEQ ID NO. 2;
the sgRNA2 comprises an upstream primer shown as SEQ ID NO.3 and a downstream primer shown as SEQ ID NO. 4.
The invention provides an ISG15 gene knockout recombinant vector, which comprises the sgRNA.
The invention provides a recombinant lentivirus for knocking out an ISG15 gene, which is obtained by rescuing co-transfected cells of the recombinant vector and an auxiliary plasmid.
Preferably, the helper plasmids are the psPAX2 helper plasmid and the pMD2.0G helper plasmid;
at the time of co-transfection, the mass ratio of the recombinant vector, the psPAX2 helper plasmid and the pMD2.0G helper plasmid is 3:2:1.
The invention provides a CRISPR-Cas9 system for knocking out an ISG15 gene, which comprises the sgRNA.
The invention provides application of the sgRNA, the recombinant vector or the recombinant lentivirus in knocking out ISG15 genes in cells.
The invention provides a bovine kidney cell MDBKISG15 with ISG15 gene deletion -/- The bovine kidney cell MDBK ISG15 -/- The preservation number of the catalyst is CGMCC No.45617.
The invention provides the cattle kidney cell MDBKISG15 -/- The construction method of (1) comprises the following steps:
transfecting the recombinant lentivirus into MDBK cells to obtain IMDBKISG15 -/- 。
The invention provides the aboveBovine kidney cell MDBKISG15 -/- Use in promoting BVDV replication.
The invention provides the cattle kidney cell MDBKISG15 -/- Use in BVDV vaccine research. The invention discloses the following technical effects:
the invention provides an sgRNA for knocking out an ISG15 gene, which can knock out a target gene with high efficiency.
Moreover, the invention utilizes CRISPR/Cas9 gene editing technology to construct the cattle kidney cell MDBKISG15 with ISG15 gene deletion for the first time -/- The method is used for researching the influence of the ISG15 gene (accession number: NM_ 174366.1) on virus replication, and the related regulation mechanism or passage, the improvement of virus yield and the like. The cell can be used as an ideal cell model for antiviral research, can also remarkably improve the virus titer of the virus growing on the cell line and promote BVDV replication, and can be applied to BVDV vaccine research and survival; meanwhile, compared with technical means such as gene silencing, interference and the like, the CRISPR/Cas9 gene knockout technology is more effective, and is beneficial to researching related functions and application of ISG15 proteins.
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 embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of PCR identification of a recombinant plasmid of sgRNA, wherein M is Marker,1 is negative control, 2 is negative control, 3 is sgRNA1,4 is sgRNA2, and 5 is sgRNA2;
FIG. 2 shows the result of the expansion culture of positive monoclonal cells, wherein the left side is the initial growth stage of single positive clone cells, the right side is the later growth stage of single positive clone cells, and the magnification is 40 times;
FIG. 3 shows the results of ISG15 gene sequencing after positive monoclonal cell expansion;
FIG. 4 shows an ISG15 knockout cell line (bovine kidney cell MD)BKISG15 -/- ) The Western-blot verification result of (2);
FIG. 5 shows BVDV in wild type MDBK and bovine kidney cell MDBKISG15 -/- Replication in cell lines;
FIG. 6 shows BVDV in wild type MDBK and bovine kidney cell MDBKISG15 -/- Titer changes in cell lines.
Biological preservation information
Bovine kidney cell MDBKISG15 -/- : the microbial strain is preserved in China general microbiological culture collection center (CGMCC) for 5 months and 25 days in 2023, the preservation address is the North Chen Silu No.1, 3 of the Korean area of Beijing, and the preservation number is CGMCC No.45617.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
Construction of 1MDBK cell ISG15 Gene knockout cell line
1.1sgRNA design
The bovine ISG15 gene sequence (GeneID: 281871) was searched and determined with reference to http:// www.ncbi.nlm.nih.gov/website. The target gene sgrnas for the ISG15 protein were designed using http:// crispr. Mit. Edu/two pairs of sgrnas and their complementary strands were designed per protein, and the cleavage sites of BbsI were added separately, the details of the two pairs of sgrnas are given in table 1 below. sgRNA sequence synthesis and sequencing was done by the company of biosciences, inc.
TABLE 1sgRNA sequences
1.2 LenkiCRISPR-V2-GFP vector linearization
The vector was subjected to cleavage reaction using Bbs1 at 37℃for 30min. The linearized LenkiCRISPR-V2-GFP vector after cleavage was recovered by reference to the Tiangen purification kit and the concentration of the linearized vector was determined using a nucleic acid protein meter.
1.3 Synthesis of double-stranded sgRNA
The sgRNA oligonucleotide strand and its complementary strand were synthesized artificially, dimers were formed by drop PCR, and the drop PCR system is shown in table 2.
TABLE 2 drop PCR System
| Composition of the components | Dosage of |
| Forward oligo | 5μL |
| Reverse oligo | 5μL |
| DNA annealing Buffer (5×) | 10μL |
| RNase-Free-H 2 O | Make up 50 mu L |
| Totals to | 50μL |
Note that: the nucleotide sequences of the forward oligo are shown as SEQ ID NO.1 and SEQ ID NO.3, and are specifically CACCG ATCCTGGTGAGGAACGACAA, CACCGCTTCAGCTCGGATACCGTCA; the nucleotide sequences of the reverse oligo are shown as SEQ ID NO.2 and SEQ ID NO.4, and are specifically AAACTTGTCGTTCCTCACCAGGATC, AAACTGACGGTATCCGAGCTGAAGC.
PCR reaction procedure: 95℃for 5min, 0.1℃every 8s, to 25℃for a total of 700 cycles, 4℃hold.
1.4 ligation of sgRNA dimer to Linear vector
The linearized Lenigirspr-V2-GFP vector and the landing PCR product were reacted at 37 ℃ for 30min at a ligation ratio of 1:13 to give a ligation product, the ligation system is shown in table 3.
Table 3 connection system
1.5 transformation of recombinant plasmids
Adding the connection product into TOP10 competent cells, mixing, ice-bathing for 30min, water-bathing at 42 ℃ for 90s, ice-bathing for 2min, adding 700 μl of antibiotic-free LB culture solution, and culturing at 37 ℃ for 60min at 180 r/min; centrifuging the bacterial liquid at 4000rpm at normal temperature for 3min, and discarding 600 mu L of supernatant; the residual bacterial liquid is evenly coated on an LB agar plate containing 100 mug/mL of ampicillin after being resuspended; incubated overnight at 37℃until milky colonies developed.
1.6 PCR identification of recombinant plasmids
Designing a synthetic vector identification primer PG according to the LenigirSPR-V2-GFP vector sequence, picking a single positive colony, respectively carrying out colony PCR identification by using the primer PG and sgRNA as the upstream and downstream, and recording a PCR system as shown in the following table 4, wherein a template is used as a negative control, the amplified fragment length is about 200bp (the result is shown in figure 1), then carrying out sequencing, and carrying out seed preservation on a plasmid with correct sequence, wherein the plasmid with correct sequence is recorded as a recombinant plasmid LenigirSPR-V2-GFP.
The nucleotide sequence of PG is shown as SEQ ID NO.5, and specifically comprises the following steps: GCCTATTTCCCATGATTC.
TABLE 4PCR System
| Composition of the components | Dosage of |
| PG | 1μL |
| sgRNA | 1μL |
| PrimeSTARMaxPremix | 12.5μL |
| Template | 1μL |
| RNase-Free-H 2 O | Make up 25. Mu.L |
| Totals to | 25μL |
Note that: the nucleotide sequences of the sgRNA are shown as SEQ ID NO.2 and SEQ ID NO.4, and are specifically AAACTTGTCGTTCCTCACCAGGATC, AAACTGACGGTATCCGAGCTGAAGC.
The reaction condition is 98 ℃ for 5min;98℃for 30s, 55℃for 30s, 72℃for 30s (35 cycles), 72℃for 10min,12 ℃.
1.7 packaging of recombinant lentiviruses
Laying 293T cells in 75T cell culture flasks at 37℃in 5% (V/V) CO 2 Culturing in an incubator for 12-24h, and carrying out transfection when the cell fusion degree is 80-90%. The recombinant plasmid LentiCRISPR-V2-GFP was transfected with helper plasmids psPAX2 and pMD2.0G in a mass ratio of 3:2:1 in amounts of 15. Mu.g, 10. Mu.g and 5. Mu.g, respectively. The plasmid is added into 2mL of serum-free DMEM culture medium, gently mixed, added with 30 mu L of HP DNA transfection reagent, kept stand at room temperature for 20min after being mixed, cells are replaced by serum-free DMEM culture medium before transfection, and then the mixed transfection complex is evenly dripped into a 75T cell culture flask. After incubation at 37℃for 6h, 10mL of fresh medium (DMEM medium with 10% FBS) was replaced, and after incubation at 37℃for 72h, the supernatant was harvested and the cell culture supernatant was collectedTransfer to a 15mL sterile centrifuge tube in an ultra clean bench and centrifuge at 6000rpm for 20min at 4 ℃.
1.8 concentration of lentiviruses
Filtering the supernatant with 0.45 μm filter membrane, mixing the supernatant with Lentivirus Precipitation Solution at a volume ratio of 4:1, standing at 4deg.C for 45min, centrifuging for 45min at 7000 Xg to obtain white precipitate, re-suspending the precipitate with PBS (1/100 of the volume of the supernatant), and packaging at-80deg.C.
1.9 lentiviral transduction
MDBK cells were prepared in 6 well plates and transduced until the cells fused to 70%. Firstly, PBS is used for washing cells, a DMEM culture medium without antiserum is used for preparing polybrene (infectious agent) with the final concentration of 6 mug/mL, slow virus liquid is slowly melted on ice, 100 mug of slow virus liquid and 3mL of DMEM are added into each hole, after 6 hours, the whole culture medium is changed into the whole culture medium, the cells are collected for 72-96 hours for flow sorting, and the single cells with green fluorescence are sorted out and are subjected to expansion culture by a 96-well plate, and the result is shown in figure 2. As can be seen from FIG. 2, cells with green fluorescence were successfully selected, and the cells were cultured in an expanded manner to obtain cell lines.
1.10 Gene knockout MDBK cell DNA sequencing and western blot detection
Designing primers on the upstream and downstream of sgRNA by taking the genomic DNA sequence of the screened MDBK cells as a template, and F:5'-ATGGGCGGGGACCTGACG-3' (SEQ ID NO. 6), R:5'-CCCACCCCGAAGACGTAGA-3' (SEQ ID NO. 7), sequencing the PCR products, and evaluating the shearing efficiency according to the sequencing result, the result is shown in FIG. 3. The results showed that the cells all exhibited distinct overlapping peaks, indicating efficient editing in the amplified fragment. After IFN-alpha stimulation for 16h, cell proteins were collected using wild MDBK cells as a control and ISG15 mab (purchased from Senta Cruz Biotechnology company) as primary antibody and beta-actin as an internal reference, and Westernblot verification was performed. As shown in FIG. 4, the gene knockout cell line KO1 strain did not express the ISG15 gene, indicating successful gene knockout.
Effect of ISG15 knockout MDBK cell line on Bovine Viral Diarrhea Virus (BVDV) replication
Knocking out MDBK cell line and wild MDB from screened ISG15 geneWhen the K cell line was cultured to a confluency of about 90%, cells (MOI=0.5) were inoculated with BVDV (laboratory preservation NM21 strain, genbank accession number: OP 792028), and after 1h incubation, the virus solution was discarded and fresh DMEM medium containing 3.5% HBS was supplemented. Supernatant and cell samples were collected at 24h, 36h and 48h of infection, respectively, using qPCR and TCID, respectively 50 BVDV replication was detected. RNA was extracted and reverse transcribed into cDNA, and the copy number of BVDV5' UTR gene was detected by fluorescent quantitative PCR, and beta-actin was used as an internal reference, and the primer sequences were shown in Table 5. The reaction system: 2 XTBgreen 10. Mu.L, each of the primers 0.8. Mu.L, template 2. Mu.L, and ultrapure water were used to make up the system to 20. Mu.L. Reaction conditions: 95 ℃ for 30s;95 ℃ for 5s; 30s at 60 ℃ for 40 cycles; the dissolution profile conditions were 95℃for 10s and 0.5℃per 5s rise from 65℃to 90 ℃.
TABLE 5 primer sequences
The fluorescent quantitative results are shown in fig. 5, and the mRNA level of the ISG15 gene knockout MDBK cells is significantly up-regulated compared with that of the wild type cells after BVDV inoculation.
TCID 50 The results are shown in FIG. 6, in which BVDV was inoculated at 24h and 48h after the ISG15 gene was knocked out of MDBK, as compared with the wild type MDBK cells.
Taken together, obtaining ISG15 knockout MDBK cells by gene editing techniques can significantly enhance BVDV replication. Therefore, the constructed ISG15 gene deletion cell line can be used for improving BVDV titer and can be applied to BVDV vaccine production.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. An sgRNA for knocking out ISG15 gene, comprising sgRNA1 and sgRNA2;
the sgRNA1 comprises an upstream primer shown as SEQ ID NO.1 and a downstream primer shown as SEQ ID NO. 2;
the sgRNA2 comprises an upstream primer shown as SEQ ID NO.3 and a downstream primer shown as SEQ ID NO. 4.
2. A recombinant vector for knocking out ISG15 gene, comprising the sgRNA of claim 1.
3. The recombinant lentivirus for knocking out the ISG15 gene is obtained by rescuing the recombinant vector and auxiliary plasmid cotransfected cells according to claim 2.
4. The recombinant lentivirus of claim 3, wherein the helper plasmids are a psPAX2 helper plasmid and a pmd2.0g helper plasmid;
at the time of co-transfection, the mass ratio of the recombinant vector, the psPAX2 helper plasmid and the pMD2.0G helper plasmid is 3:2:1.
5. A CRISPR-Cas9 system for knocking out an ISG15 gene, comprising the sgRNA of claim 1.
6. Use of the sgRNA of claim 1, the recombinant vector of claim 2 or the recombinant lentivirus of claim 3 or 4 for knocking out the ISG15 gene in a cell.
7. Bovine kidney cell MDBK ISG15 with ISG15 gene deleted -/- Characterized in that the bovine kidney cell MDBK ISG15- / The preservation number is CGMCC No.45617.
8. The bovine kidney cell MDBK ISG15 of claim 7 -/- Is characterized by comprising the following steps:
transfection of the recombinant lentivirus of claim 3 into MDBK cells to obtain IMDBK ISG15 -/- 。
9. The bovine kidney cell MDBK ISG15 of claim 7 -/- Use in promoting BVDV replication.
10. The bovine kidney cell MDBK ISG15 of claim 7 -/ -use in BVDV vaccine research.
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