SgRNA recombinant vector of pBD2 gene and preparation method of pBD2 gene knockout cell line

Abstract

The invention relates to the technical field of gene engineering, and relates to a construction method of sgRNA with a pBD2 gene knocked out and a construction method of a pig intestine epithelial cell line with the pBD2 gene knocked out. According to a pBD2 gene sequence, sgRNA capable of targeting the gene is designed and synthesized, plasmids of a CRISPR / Cas9 system containing the sgRNA are constructed by using a molecular cloning and recombination technology, the plasmids are transfected into a pig small intestine epithelial cell line, gene editing is performed on a specific position of the pBD2 gene, and a stable monoclonal cell line with the pBD2 gene knocked out is obtained through puromycin drug screening. The homozygous pBD2 gene knockout cell strain obtained by the method is used as a cell model and can be used for exploring the effects of the pBD2 gene in the aspects of immunoregulation, sphingolipid metabolism, cell cycle and the like of small intestine cells.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and relates to a method for constructing a knockout cell line. Background Art

[0002] pBD2 (porcine beta-defensin 2) is an antimicrobial peptide secreted by mammalian pigs. It has good antibacterial and immunomodulatory activities, and is not easy to induce microbial resistance. In addition, it has the advantages of stable structure, good water solubility, high temperature resistance, no immunogenicity, and no drug residue. It is an ideal substitute for antibiotics. Studies have shown that pBD2 has a strong killing effect on clinically isolated intestinal pathogens and drug-resistant bacteria, and pBD2 reduces the inflammatory response caused by LPS and Escherichia coli through pathways such as NF-κB. Adding pBD2 to feed weaned piglets can improve intestinal health, reduce the number of harmful bacteria in the cecum, increase the number of beneficial bacteria such as lactobacillus, reduce the diarrhea rate of weaned piglets, and has a good growth-promoting effect. Using the defense peptides secreted by pigs themselves can improve the immunity and disease resistance of pigs, prevent and treat pig diseases, and promote healthy breeding.

[0003] CRISPR / Cas9 (tandem intergenic repeats / CRISPR-associated protein 9) is one of the most efficient, simplest and lowest-cost technologies in existing gene editing and gene modification, and has become the mainstream gene editing system today. The CRISPR / Cas9 system is mainly composed of single-stranded guide RNA (sgRNA) and restriction endonuclease Cas9. Under the guidance of sgRNA, the Cas protein cuts the target site and then uses non-homologous end-linking mechanism or recombination repair mechanism to self-repair, so that the latter base is inserted or deleted, resulting in frameshift mutation or structural damage of the gene, thereby achieving gene knockout. The plasmid vector of the CRISPR / Cas9 system can express the Cas9 enzyme in mammalian systems and carry a targeting sequence of about 20 nucleotides, that is, the gRNA sequence. By transfecting the recombinant plasmid expressing Cas9 and the sgRNA targeting the gene into mammalian cells, the knockout of a specific gene can be finally achieved. However, there are still some problems with CRISPR / CAS9 technology. The CRISPR / Cas9 system may cut at non-target sites, resulting in off-target effects. Delivering the CRISPR / Cas9 system into cells is one of the key steps to achieve gene editing. Current delivery methods all have certain limitations. Viral vectors may trigger an immune response and have certain restrictions on cell types; non-viral vectors may face problems such as low delivery efficiency and high cytotoxicity. When choosing a delivery method, it is necessary to comprehensively consider factors such as the specific experimental purpose, cell type, editing efficiency, and safety. In addition, gene editing efficiency may be affected by multiple factors such as cell type, gene location, and sgRNA design. Therefore, the design and selection of sgRNA requires a lot of analysis. In addition, there is no unified and effective method for how to effectively transfect recombinant plasmids expressing the CRISPR / Cas9 system into cells. More effective and safe methods need further development. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for preparing a sgRNA recombinant vector of the pBD2 gene and a method for preparing a cell line for knocking out the pBD2 gene.

[0005] The technical solution of the present invention is achieved in this way: In the first aspect, a method for preparing a sgRNA recombinant vector of the pBD2 gene is based on CRISPR-Cas9 technology to specifically target and knock out the sgRNA of the pBD2 gene, and the steps are: (1) Design sgRNA based on the sequence of the pBD2 gene; (2) The sgRNA in step (1) is added with the linker sequence -CACCG-, and the annealing primer is designed. The annealed primer is connected to the linear vector (lentiCRISPR v2 plasmid) after enzyme digestion to construct the sgRNA recombinant vector. The sgRNA recombinant vector is transformed into the competent cell TOP10, and the sgRNA recombinant vector of the pBD2 gene is screened; (3) The sgRNA recombinant vector of step (2) is transformed and screened to obtain the sgRNA recombinant vector of the pBD2 gene.

[0006] In the above step (1), the sgRNA is sgRNA1, sgRNA2 or sgRNA3, the nucleotide sequence of sgRNA1 is shown as SEQ ID No.1, the nucleotide sequence of sgRNA2 is shown as SEQ ID No.2, and the nucleotide sequence of sgRNA3 is shown as SEQ ID No.3.

[0007] In the above step (2), the nucleotide sequence of the forward primer used for annealing sgRNA1 is shown in SEQ ID No.4, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.5; the nucleotide sequence of the forward primer used for annealing sgRNA2 is shown in SEQ ID No.6, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.7; the nucleotide sequence of the forward primer used for annealing sgRNA3 is shown in SEQ ID No.8, and the nucleotide sequence of the reverse primer is shown in SEQ ID No.9.

[0008] The conditions for mixing the forward primer and the reverse primer are as follows: incubate at 95°C for 10 min, then decrease from 95°C to 85°C at a rate of 2°C per second, and decrease from 85°C to 25°C at a rate of 0.1°C per second.

[0009] In a second aspect, the present invention provides a porcine pBD2 gene targeting vector, which is a sgRNA recombinant vector based on the CRISPR-Cas9 system, and the sgRNA action site is located on exon 2 of the porcine pBD2 gene.

[0010] In a third aspect, the present invention provides a method for knocking out the pBD2 gene in a porcine intestinal epithelial cell line using the CRISPR-CAS9 system, which comprises designing and synthesizing sgRNA targeting the porcine pBD2 gene based on the porcine pBD2 gene sequence, and then constructing a CRISPR-Cas9 targeting vector containing the sgRNA, which is transferred into porcine intestinal epithelial cells to obtain porcine intestinal epithelial cells with the pBD2 gene knocked out.

[0011] Wherein, the nucleotide sequence of the sgRNA is shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3.

[0012] Preferably, the DNA sequence of the sgRNA action site is as shown in SEQ ID No.2.

[0013] In a fourth aspect, the present invention provides a porcine intestinal epithelial cell line with a pBD2 gene knockout, which is prepared by transfecting porcine intestinal epithelial cells with a recombinant plasmid packaged by a lentivirus to obtain gene-edited monoclonal cells, namely, a porcine intestinal epithelial cell line with a pBD2 gene knockout.

[0014] The present invention has the following beneficial effects: 1. The porcine intestinal epithelial cell line provided by the present invention can be used to study the function of pBD2 protein and the related pathways regulated by it. The constructed pBD2 gene knockout porcine intestinal epithelial cell line can be used as a cell model or animal model for studying immune regulation, sphingolipid metabolism, cell cycle and other aspects.

[0015] 2. According to the functional region of the pBD2 gene, the present invention designs multiple sgRNAs and conducts experimental verification, screens out sgRNAs with high efficiency, strong specificity and the highest gene editing efficiency, and provides a basis for the establishment of pBD2 gene knockout cell lines.

[0016] 3. The present invention utilizes the CRICPR / Cas9 system to construct a pBD2 gene knockout IPEC-J2 cell line, which edits the pBD2 gene at the DNA level and completely silences the expression of the pBD2 protein, providing a practical tool for the study of the pBD2 gene function.

[0017] 4. The present invention can be used for targeted knockout of the pBD2 gene. The designed sgRNA has high gene editing efficiency and uses lentiviral packaging to transfect cells, thereby improving the transfection efficiency of the recombinant plasmid. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 These are the sequencing results of the successful construction of three different sgRNA recombinant plasmids.

[0020] Figure 2 The results of T7 restriction enzyme digestion identification of the PCR products of the pBD2 gene in three pBD2 gene knockout cell lines.

[0021] Figure 3These are the sequencing results of PCR of the pBD2 gene in three cell lines with stable knockout of the pBD2 gene.

[0022] Figure 4 is the number of differentially expressed genes between IPEC-J2 and IPEC-J2pBD2 / KO.

[0023] Figure 5 KEGG enrichment analysis of up-regulated differentially expressed genes in IPEC-J2 and IPEC-J2pBD2 / KO.

[0024] Figure 6 KEGG enrichment analysis of differentially down-regulated genes in IPEC-J2 and IPEC-J2pBD2 / KO. DETAILED DESCRIPTION

[0025] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0027] The present invention provides a method for preparing pBD2 gene knockout pig intestinal epithelial cells based on CRISPR-Cas9 technology. The pBD2 gene is edited by CRISPR-Cas9 system, and the target site is located in the second exon of the pBD2 gene. The pBD2 gene can be effectively knocked out to obtain pBD2 gene knockout pig intestinal epithelial cells. The knockout cells can be used to explore the immunoregulatory effect of the pBD2 gene on small intestinal cells.

[0028] The present invention provides a method for knocking out the pig pBD2 gene using CRISPR-Cas9 technology, the method comprising: 1) The pBD2 gene transcript sequence (NCBI database Gene ID: 404699) includes two exons. The mature peptide sequence of pBD2 is located in the second exon and starts from the second exon of pBD2. http: / / crispr.mit.edu ) predicted the PAM sequence NGG that meets the requirements of spCas9. Designed three sgRNAs, named sgRNA1, sgRNA2, and sgRNA3, and packaged the recombinant plasmids with lentivirus.

[0029] 2) Design PCR primers for verification including the second exon: F: ACAGACAGCGTTGTATGG; R: ATACTTCACTTGGCCTGTG, and the amplified product size is 531 bp.

[0030] 3) The recombinant plasmid obtained in step 1) was used to transfect porcine intestinal epithelial cells, and cell DNA was extracted. PCR amplification was performed using the specific primers obtained in step 2). sgRNA1, sgRNA2, and sgRNA3 had a certain knockout efficiency.

[0031] 4) Using the transfected cells obtained in step 3), select sgRNA2 with the highest gene editing efficiency to screen monoclonal cells, and obtain monoclonal cells by limiting dilution method. Extract genomic DNA of monoclonal cells, amplify pBD2 gene by PCR, and then sequence.

[0032] The sequencing results were compared with the pBD2 genome in NCBI, and a total of three cell lines with stable knockout of the pBD2 gene were obtained.

[0033] In a specific embodiment of the present invention, the three sgRNAs in step 1) are designed for the second exon of the porcine pBD2 gene: preferably, the three sgRNA sequences are: In the present invention, the specific primers for verifying whether the three sgRNAs function respectively in step 2) are: F: ACAGACAGCGTTGTATGG; R: ATACTTCACTTGGCCTGTG, and the amplified product size is 531 bp.

[0034] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example: The present invention relates to preparing pBD2 gene knockout porcine small intestinal epithelial cells using CRISPR-Cas9 technology, and the main methods included are: 1. Design of sgRNA based on porcine pBD2 gene and preparation of pBD2 gene knockout vector 1. Annealing primers According to the porcine pBD2 gene sequence (NCBI database Gene ID: 404699), starting from the second exon of pBD2, the online website ( http: / / crispr.mit.edu ) predicted sgRNAs that met the requirements of spCas9. The sequences of the three sgRNAs are shown in SEQ ID NOs: 1-3, respectively.

[0036] The present invention adds an additional base to the 5' end of the sgRNA sequence described in the above technical solution, the linker is preferably CACCG, and the nucleotide sequence of the inserted fragment is shown in SEQ ID No. 4, 6, 8, specifically as follows: The primer annealing reaction system and reaction conditions are as follows: Forward primer (100µM) 5μL Reverse primer (100µM) 5μL Total system 10μL Mix well, incubate at 95℃ for 10 min, then decrease from 95℃ to 85℃ at a rate of 2℃ per second. Then decrease from 85℃ to 25℃ at a rate of 0.1℃ per second. Then take the primer mixture and mix it according to the following composition: Mixed primer 5 μL 10× T4 DNA Ligase Reaction Buffer 1μL PNK 0.5 μL Ultrapure water 3.5 μL Total system 10μL Mix well and incubate at 37℃ for 30min.

[0037] 2. Plasmid digestion The plasmid digestion system and reaction conditions are as follows: BsmB1 restriction enzyme 1 μL lentiCRISPR v2 plasmid 2 μg 10×Nebuffer 3 μL Ultrapure water 24 μL Mix well and incubate at 37°C for 3h. Perform agarose gel electrophoresis on the above enzyme digestion products and recover the vector fragment according to the instructions of the agarose gel DNA recovery kit.

[0038] 3. Connect The annealed primer and the recovered linear vector fragment were connected. The connection system and reaction conditions were as follows: Vector fragment 2μL Annealed primer 6.5 μL 10×T4 DNA Ligase Buffer 1μL T4 DNA Ligase 0.5μL Total system 10μL Mix well and incubate at 16°C overnight.

[0039] 4. Competent cell transformation and screening Take the above-constructed sgRNA-lentiCRISPR v2 recombinant plasmid and add it to the competent cell TOP10, mix thoroughly, and place in an ice bath for 30 min. Incubate at 42 ℃ for 45 s, and quickly place in an ice bath for incubation for 3 min. Then add 500 μL LB liquid culture medium, mix thoroughly, place on a 37 ℃ shaker, and shake and culture at 150 rpm for 45 min to revive the bacteria. Take 100 μL of bacterial solution and evenly spread it on LB solid culture medium containing ampicillin. Place the culture plate in a constant temperature incubator and incubate at 37 ℃ overnight. Pick a single clone colony and inoculate it in LB liquid culture medium containing ampicillin for culture. When the bacteria grow to the logarithmic phase, send the bacterial solution to Sangon for sequencing. The sequencing results show that the sequences of the three sgRNAs are completely consistent with the design, such as Figure 1 As shown in the figure, the sgRNA-lentiCRISPRv2 recombinant plasmid was constructed, and the sequencing results showed that the sequences of the three sgRNAs were completely consistent with the design. The sgRNA recombinant plasmid was successfully constructed.

[0040] 2. pBD2 gene knockout in porcine intestinal epithelial cells 1. Transfect cells with recombinant plasmid HEK293T / 17 cells were cultured in 25 mL cell culture flasks until 50% confluence. The cells were divided into four groups for transfection, namely control, sgRNA1, sgRNA2 and sgRNA3. 0.5 μg of lentiviral packaging plasmid pMD2.G, 1.5 μg of pspax2 and 2 μg of recombinant plasmid were taken, mixed thoroughly, and added to 100 μL Opti-MEM medium and allowed to stand for 5 min; in addition, 32 μg of FuGENEHD liposomes were taken and added to 400 μL Opti-MEM medium. The solution containing the plasmid and the solution containing the liposome were mixed and allowed to stand at room temperature for 15 min. The transfection reagent was added to the culture flask and cultured for 8-10 h. The culture medium was replaced with a culture medium containing 10% serum and cultured for 48 h. The virus-containing culture medium was collected, centrifuged at 1000 rpm for 5 min, and the supernatant was taken. IPEC-J2 cells were inoculated in a 6-well plate and cultured until the cells were 50% confluent. The collected four groups of virus solutions were used to infect cells respectively. After 48 h of culture, the medium was replaced with 1640 medium containing 3 μg / mL puromycin and 10% serum. The medium was changed every other day and cultured continuously until all cells in the control group were observed to die. The puromycin concentration of the other three groups of cells was reduced to 1 μg / mL. One-third of the cells were taken to extract the cell genome, and the remaining cells were cultured and subcultured.

[0041] 2. Cell genomic DNA extraction Wash the collected cells thoroughly with pBS, centrifuge at 12000 rpm for 1 min, discard the supernatant, add 200 μL solution A, and oscillate to mix. Add 20 μL RNase A (10 mg / mL) to the suspension and place at 55°C for 15 min. Add 20 μL proteinase K (10 mg / mL), mix thoroughly by inversion, and digest in a 55°C water bath until the sample is completely digested and the liquid is clear and viscous. Add 200 μL volume solution B and mix thoroughly by inversion. Add 200 μL anhydrous ethanol, mix thoroughly, and then drip into the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, add 600 μL rinsing solution (containing anhydrous ethanol) to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid. Repeat once. Place at room temperature to remove the residual rinsing solution in the adsorption column. Place the adsorption column in a clean centrifuge tube, drop 50-200 μL of elution solution preheated in a 65 ℃ water bath into the center of the adsorption membrane, place at room temperature for 5 minutes, and centrifuge at 12,000 rpm for 2 minutes. High-quality genomic DNA can be obtained.

[0042] 3. Gene editing efficiency detection According to the defensin gene sequence in NCBI, primers F including the second exon were designed: ACAGACAGCGTTGTATGG; R: ATACTTCACTTGGCCTGTG, and the amplified product size was 531 bp. Using the extracted cell genomic DNA as a template, PCR amplification was performed using a high-fidelity polymerase, and then gel recovery was performed. Take 200 ng of PCR product, NEbuffer21μL, and ultrapure water to 10μL. Place it on the PCR instrument for annealing. The annealing program is as described in the sgRNA primer annealing program above. Add T7 endonuclease to the annealed product and incubate at 37℃ for 1 h. The enzyme digestion product was analyzed by polyacrylamide electrophoresis. Gel configuration: 30% acrylamide + 4M urea 1.5 mL, 5×TBE 2.25 mL, 10% APS 0.125 mL, TEMED 5 μL, water 6 mL. Mix well and pour into the gel. The samples to be tested were added to DNA loading buffer, electrophoresed at 120 V for 40 min, and stained with TBE buffer containing SYBR nucleic acid dye for 40 min. The gel imaging system was used to take pictures. The grayscale was analyzed using Image J software.

[0043] The results showed that both the control and knockout cells had obvious bands at position 530, and T7 endonuclease had a cutting effect on all three experimental groups, but not on the control group, indicating that there were gene mutations at these targets. Grayscale analysis using Image j software showed that the gene editing efficiencies of sgRNA1, sgRNA2, and sgRNA3 were 35%, 45%, and 5%, respectively (Figure 2 Figure 2 as shown).

[0044] 4. Screening of monoclonal knockout cell lines T7 enzyme digestion was used to screen out the sgRNA2 with the highest gene editing activity, and monoclonal cells were obtained by limiting dilution. The genomic DNA of the monoclonal cells was extracted, and the pBD2 gene was amplified by PCR and then sequenced. The sequencing results were compared with the pBD2 genome in NCBI, and a total of three cell lines with stable knockout of the pBD2 gene were obtained, such as Figure 3 The knockout monoclonal cell line was saved and named IPEC-J2 pBD2 / KO cell.

[0045] 3. Effects of pBD2 gene knockout on gene expression To investigate the function of pBD2 in porcine intestinal epithelial cells, wild-type IPEC-J2 cells and pBD2 knockout IPEC-J2 cells were used to investigate the function of pBD2 in porcine intestinal epithelial cells. pBD2 / KO Transcriptome analysis of cells was performed, and FDR < 0.01 and Fold Change > 2 were used as the screening criteria for differentially expressed genes. A total of 2964 differentially expressed genes were found, including 1627 up-regulated genes and 1337 down-regulated genes ( Figure 4 KEGG enrichment analysis was performed on the up-regulated genes and down-regulated genes respectively. The results showed that the up-regulated differentially expressed genes were mainly enriched in lysosome, sphingolipid metabolism, osteoclast differentiation, proteoglycans in cancer, RAS signaling pathway and MAPK signaling pathway. Figure 5Down-regulated genes were mainly enriched in ribosome biogenesis in eukaryotes, Fanconi anemia pathway, cell cycle, DNA replication, pyrimidine metabolism, spliceosome, RNA transport, mismatch repair, and base excision repair. These biological processes are mainly related to metabolism and repair of DNA molecules. Figure 6 ). This indicates that pBD2 has a wider range of biological functions under normal cell growth conditions.

[0046] Lysosomes are membrane-delimited organelles in animal cells and are the main digestive compartments in cells. Lysosomes contain a variety of hydrolases, and cells transport macromolecules into lysosomes and degrade them through endocytosis, phagocytosis, and autophagy. Studies have shown that defensins inhibit cell autophagy and increase the number of intracellular pathogens cleared by cells. These studies have confirmed that defensins are related to lysosomal function. However, it is unclear whether defensins work by regulating hydrolases in lysosomes.

[0047] Sphingolipids are a class of complex compounds with sphingosine as the skeleton. Sphingolipids and their metabolites are not only important structural molecules that constitute the cell membrane, but also participate in many important signal transduction processes such as regulating cell growth, differentiation, aging and programmed cell death, which enable cells to produce various biological functions. In this study, after the pBD2 gene was knocked out, the sphingolipid metabolism process was enhanced. Whether pBD2 functions through the sphingolipid metabolism process still needs further study.

[0048] Osteoclasts (OC) are differentiated from monocytes in bone marrow hematopoietic stem cells. They are mainly found in the periosteum and are the only cells that have bone absorption. When the bone absorption capacity of osteoclasts is too strong, it will cause osteolytic diseases such as osteoporosis. The results of this study show that pBD2 has an inhibitory effect on osteoclast differentiation.

[0049] The MAPK pathway is involved in many important cellular physiological / pathological processes, such as cell growth, differentiation, adaptation to environmental stress, and inflammatory response. Especially the immune regulation pathway. After pBD2 gene knockout, the MAPK signaling pathway was abnormally activated, which indicates that pBD2 has an inhibitory effect on the MAPK signaling pathway.

[0050] In cells with pBD2 gene knockout, the downregulated differentially expressed genes were mainly enriched in ribosome biosynthesis, cell cycle, DNA replication, transport and repair functions, indicating that pBD2 may have the function of promoting protein synthesis, synthesis and modification of DNA substances.

[0051] The cell cycle is an important process in life activities and is closely related to cell proliferation and apoptosis. Studies have shown that defensins affect cell proliferation and apoptosis by regulating the cell cycle. Knockout of the pBD2 gene leads to inhibition of the cell cycle process of intestinal epithelial cells, indicating that pBD2 has the effect of promoting epithelial cell proliferation.

[0052] In summary, the function of pBD2 in host cells may be mainly achieved by regulating the MAPK signaling pathway, osteoclast differentiation, sphingolipid metabolism, lysosomes, cell cycle and spliceosomes.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a sgRNA recombinant vector of the pBD2 gene, characterized in that: The steps are: (1) Design sgRNA based on the sequence of the pBD2 gene; (2) Designing primer pairs for the sgRNA in step (1), annealing and mixing the primer pairs and connecting them with the linear vector to obtain the sgRNA recombinant vector; (3) The sgRNA recombinant vector of step (2) is transformed and screened to obtain the sgRNA recombinant vector of the pBD2 gene.

2. The method for preparing the sgRNA recombinant vector of the pBD2 gene according to claim 1, characterized in that: In step (1), the sgRNA is sgRNA1, sgRNA2 or sgRNA3, the nucleotide sequence of sgRNA1 is shown as SEQ ID No.1, the nucleotide sequence of sgRNA2 is shown as SEQ ID No.2, and the nucleotide sequence of sgRNA3 is shown as SEQ ID No.

3.

3. The method for preparing the sgRNA recombinant vector of the pBD2 gene according to claim 2, characterized in that: In the step (2), the primer pair used for annealing sgRNA1 includes a forward primer nucleotide sequence as shown in SEQ ID No.4 and a reverse primer nucleotide sequence as shown in SEQ ID No.5; the primer pair used for annealing sgRNA2 includes a forward primer nucleotide sequence as shown in SEQ ID No.6 and a reverse primer nucleotide sequence as shown in SEQ ID No.7; the primer pair used for annealing sgRNA3 includes a forward primer nucleotide sequence as shown in SEQ ID No.8 and a reverse primer nucleotide sequence as shown in SEQ ID No.

9.

4. The method for preparing the sgRNA recombinant vector of the pBD2 gene according to claim 3, characterized in that: The conditions for mixing the forward primer and the reverse primer are: incubation at 95°C for 10 min, decreasing from 95°C to 85°C by 2°C per second, and decreasing from 85°C to 25°C by 0.1°C per second.

5. An sgRNA recombinant vector of the pBD2 gene prepared by the method according to any one of claims 1 to 4.

6. A method for preparing a knockout cell line using the sgRNA recombinant vector according to claim 5, characterized in that: The steps are: co-transfecting the sgRNA recombinant vector described in claim 5 into HEK293T / 17 cells, performing lentiviral packaging, collecting the virus-containing supernatant, then infecting pig epithelial cells, subculturing the cells, detecting the gene editing efficiency, screening the cell line with the highest gene editing efficiency, and obtaining a pig intestinal epithelial cell line with the pBD2 gene knocked out.

7. The method for preparing a knockout cell line according to claim 6, characterized in that: The co-transfection system includes a sgRNA recombinant vector, a pMD2.G vector and a pspax2 plasmid.

8. The method for preparing a knockout cell line according to claim 6, characterized in that: The added mass ratio of the sgRNA recombinant vector, pMD2.G vector and pspax2 plasmid is 4:1:

3.

9. The method for preparing a knockout cell line according to claim 6, characterized in that: The porcine intestinal epithelial cells are IPEC-J2.

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