Construction method and application of HepG2.215 cell line capable of stably knocking out ifitm1 gene

Abstract

The invention relates to a construction method and application of a HepG2.215 cell line capable of stably knocking out an ifitm1 gene, the HepG2.215 cell line capable of knocking out the human ifitm1 gene is obtained based on a CRISPR-Cas9 editing technology, sgRNA targeting the human ifitm1 gene is scientifically and reasonably constructed, then the sgRNA is cloned to a LentiCRISPRV2-PURO plasmid with a Cas9 gene, the plasmid is transfected into the HepG2.215 cell line, a CRISPR-Cas9 system is utilized to achieve gene silencing, and the human ifitm1 gene is stably knocked out. And finally obtaining a positive cell strain through a drug screening method, thereby obtaining the human-derived ifitm1 knockout HepG2.215 cell line. After the IFITM1 is knocked out, HBV in HepG2.215 cells is obviously increased, so that the IFITM1 knocked out HepG2.215 is used for researching innate immunity related diseases of host anti-HBV viruses and the like, a new thought is provided for research and development of antiviral target drugs, and the IFITM1 knocked out HepG2.215 has relatively high application research value and huge social and economic benefits.

Description

Technical Field

[0001] The invention relates to the field of biological cell technology, and in particular to a method for constructing a HepG2.215 cell line with a stably knocked-out ifitm1 gene and an application thereof. Background Art

[0002] Interferons (IFNs) are the first line of defense against invading microorganisms or pathogens, and their stimulated interferon-induced transmembrane proteins (IFITMs) contain IFITM1, 2, 3, 5, and 10 in humans. IFITM1, IFITM2, and IFITM3 are antiviral restriction factors against non-enveloped and enveloped RNA and DNA viruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), influenza A virus (IAV), hepatitis C virus (HCV), dengue virus, vesicular stomatitis virus (VSV), human immunodeficiency virus type I (HIV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV). Human hepatitis B virus (HBV) is a double-stranded DNA virus in the Hepadnaviridae family, and its infection can cause several liver diseases, including acute hepatitis, fulminant hepatic failure, chronic hepatitis, cirrhosis, and hepatocellular carcinoma. It has been reported that HBV can evade the host's natural immune response by preventing the induction of IFNs, but there is little research on how IFITM1 regulates the host's resistance to HBV infection.

[0003] The HBV life cycle is a complex process consisting of five steps: viral entry, covalently closed circular DNA (cccDNA) biosynthesis, progeny nucleocapsid production, virion formation, and egress. HBV replication is not directly cytotoxic to cells, and the host immune response in infected hepatocytes is the main determinant of hepatocyte damage and HBV pathogenesis. Innate immunity is important in controlling viral spread immediately after infection and initiates the effective development of adaptive immune responses. Type I interferons can be directly triggered by viral replication through the detection of viral RNA or DNA and induce a large number of effectors working in concert to achieve a fully functional antiviral state.

[0004] In addition, the construction of a cell line based on CRISPR-Cas9 targeted knockout of the human ifitm1 gene has not been reported in the relevant field. Therefore, the present invention focuses on revealing the regulatory mechanism of ifitm1's antiviral innate immunity, using ifitm1 knockout HepG2.215 cells to study its role in the host's anti-HBV innate immune defense system, and also provides a new idea for the development of anti-HBV target drugs. Summary of the invention

[0005] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a method and application of a HepG2.215 cell line for knocking out the ifitm1 gene based on the CRISPR-Cas9 system, which can effectively use the ifitm1 knocked-out HepG2.215 cells to study the role of ifitm1 in the host's antiviral innate immune defense system.

[0006] To achieve the above purpose, the technical solution provided by the present invention is:

[0007] A method for constructing a HepG2.215 cell line with a stable knockout of the ifitm1 gene comprises the following steps:

[0008] (1) Download the human ifitm1 full gene sequence (Gene ID: 8519) from the Genebank database, identify its CDS region (exons), and perform GG(N) on the CDS region using DNA Star software. 18 NGG or CC(N) 18 Search for the NCC motif, use the found motif as the positive strand of the sgRNA, add CACCG to the 5' end of the positive strand, add AAAC to the 5' end of the negative strand, and add C to the 3' end to synthesize a single-stranded sgRNA;

[0009] (2) The double-stranded LentiCRISPRV2 vector was digested with BsmBI endonuclease. The digestion system was: 2 μg of template plasmid, 1 μL of Fast Digest BsmBI, 1 μL of Fast AP, 2 μL of 10× Fast Digest Buffer, supplemented with ddH2O to 20 μL system, mixed and incubated at 37°C for 30 min, and then subjected to 1% agarose gel electrophoresis to recover the linearized vector after digestion;

[0010] (3) Add 3 μL of forward primer and 3 μL of reverse primer to 20 μL system with ddH2O, mix well and place in PCR instrument, and perform the procedure at 95°C for 10 min, 90°C for 3 min, 85°C for 3 min, 80°C for 3 min, with a gradient of 5°C, 25°C for 3 min, and 4°C hold. After the procedure is completed, take out the sample and dilute the annealed oligonucleotide into sterile water at a ratio of 1:200. If the clone is to be constructed on the same day, it can be temporarily stored at 4°C, otherwise it can be stored at -20°C.

[0011] (4) Insert the sgRNA prepared in step (3) into the multiple cloning site of the LentiCRISPRV2 expression plasmid vector to obtain a recombinant vector that simultaneously expresses the Cas9 protein gene and the targeting sgRNA sequence; that is, 100-200 ng of the linearized pLentiCRISPRv2 vector is connected at 16°C for 6 hours, and the connection system is: 10 μL of the linearized vector (gel recovery product), 2 μL of the inserted fragment (annealed oligo), 1 μL of T4 Ligase, 2 μL of 10×T4 Ligase Buffer and 5 μL of sterile double distilled water were mixed evenly, placed on a 16°C PCR instrument for 1 hour or placed in a 4°C refrigerator overnight for 12-15 hours; the ligation product was obtained, and then the ligation product was slowly transferred to the competent DH5α, and the positive clones were screened on an ampicillin-resistant plate and a single clone was picked, inoculated with liquid ampicillin-resistant LB medium, and the plasmid was extracted using a plasmid extraction kit, and the extracted plasmid was sequenced to verify whether it was the correct recombinant plasmid. If the sequencing results were 100% consistent, it proved that the recombinant CRISPR-Cas9-ifitm1-sgRNA plasmid was successfully constructed;

[0012] (5) Prepare HepG2.215 cells in 6-well plates. When the cell density grows to 85%-95%, transfect each well with 4 μg of CRISPR-Cas9-ifitm1-sgRNA1, CRISPR-Cas9-ifitm1-sgRNA2, and CRISPR-Cas9-ifitm1-sgRNA3 plasmids respectively. Set the untransfected cells as the negative control. After 6 hours of transfection, replace the medium with 10% fetal bovine serum. After 24 hours, discard the original medium and replace it with 10% medium containing 4 μg / mL puromycin for screening. After that, replace it with fresh 10% growth medium containing 2 μg / mL puromycin every 48 hours. After all the cells in the negative control group die, replace it with 10% fetal bovine serum medium without drugs, collect the remaining surviving cells for cell lysis, verify the knockout effect, and finally mark them as IFITM1KO-1, IFITM1KO-2, and IFITM1KO-3.

[0013] Furthermore, the

[0014] The nucleotide sequence 5′→3′ of ifitm1-sgRNA1 is 5′-TGATCACGGTGGACCTTGGA-3′;

[0015] The nucleotide sequence 5′→3′ of ifitm1-sgRNA2 is 5′-CTTCTTGAACTGGTGCTGTC-3′;

[0016] The nucleotide sequence 5′→3′ of ifitm1-sgRNA3 is 5′-GGATGAATCCAATGGTCATG-3′.

[0017] Furthermore, the function of the protein encoded by the ifitm1 gene in the host cell is lost by gene targeting technology; the double-stranded sgRNA obtained in the step (3) is gel-recovered and purified using an oligonucleotide recovery kit, and the purified double-stranded sgRNA is recovered and subjected to the next step.

[0018] Furthermore, the sequencing identification method in step (4) is to perform agarose gel electrophoresis on the extracted plasmid after bacterial solution PCR, and send the bacterial solution corresponding to the positive band for sequencing. If the sequencing results are 100% consistent, it is confirmed that CRISPR-Cas9-ifitm1-sgRNA1, 2, 3 are successfully constructed.

[0019] The invention discloses an application of a method for constructing a HepG2.215 cell line with ifitm1 gene knocked out based on CRISPR-Cas9 editing technology, which is used for the study of diseases related to host's natural immunity against hepatitis B virus.

[0020] Furthermore, the method is further applied in the host's natural immune defense system against hepatitis B virus.

[0021] Furthermore, this method is further applied to the development of anti-HBV target drugs.

[0022] The present invention provides a construction method of a HepG2.215 cell line with ifitm1 gene knocked out based on CRISPR-Cas9 editing technology and its application, which is used for research on diseases related to host natural immunity against hepatitis B virus, etc., verifies its application in the host's natural immune defense system against hepatitis B virus, provides a new idea for the development of anti-hepatitis B virus target drugs, and has great application research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a structural diagram of the recombinant vector LentiCRISPRV2 of the present invention;

[0024] Figure 2 This is a design diagram of the sgRNA primer of the present invention;

[0025] Figure 3 It is the ifitm1sgRNA sequence information diagram of the present invention;

[0026] Figure 4 This is a diagram showing the sequencing results of the sgRNA recombinant plasmid of the present invention;

[0027] Figure 5 It is the IFITM1 protein expression diagram in HepG2.215 and IFITM1 KO-1, 2 and 3HepG2.215 cells of the present invention;

[0028] Figure 6 It is a diagram for detecting cell morphology and proliferation rate in HepG2.215 and IFITM1 KO-1, 2 and 3HepG2.215 cells of the present invention;

[0029] Figure 7 This is a bar graph showing that the IFITM1 of the present invention inhibits HBV infection; DETAILED DESCRIPTION

[0030] Here, exemplary embodiments will be described in detail, and unless otherwise indicated, the embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. Instead, they are only examples consistent with some aspects of the present disclosure as detailed in the appended claims.

[0031] The specific implementation of the present invention is as follows:

[0032] A method for constructing a HepG2.215 cell line with ifitm1 gene knockout based on the CRISPR-Cas9 system, comprising the following steps:

[0033] (1) Design of gRNA and synthesis of oligonucleotide chains: The human ifitm1 full gene sequence (Gene ID: 8519) was downloaded from the Genebank database, and its CDS region (exon) was identified. The CDS region was GG(N)-encoded using DNA Star software. 18 NGG or CC(N) 18 The NCC motif was searched and the found motif was used as the positive strand of the sgRNA, and CACCG was added to the 5' end of the positive strand, AAAC was added to the 5' end of the negative strand, and C was added to the 3' end to synthesize the single-stranded sgRNA. The oligonucleotide chain was synthesized by Henan Shangya Biotechnology Co., Ltd. The nucleotide sequence of the sgRNA is shown in Table 1.

[0034] (2) Enzyme digestion of double-stranded LentiCRISPRV2 vector: The double-stranded LentiCRISPRV2 vector was digested with BsmBI endonuclease (Fluentex Biotechnologies Co., Ltd.) using the following digestion system: 2 μg of template plasmid, 1 μL of Fast Digest BsmBI, 1 μL of Fast AP, 2 μL of 10× Fast Digest Buffer, supplemented with ddH2O to make up to 20 μL of the system. After mixing, the system was incubated at 37°C for 30 min, and then the linearized vector was recovered by 1% agarose gel electrophoresis.

[0035] (3) Annealing of single-stranded sgRNA primers: Anneal the synthesized single-stranded sgRNA primers to synthesize double-stranded sgRNA with sticky ends. The method is to add 3 μL of forward primer and 3 μL of reverse primer to 20 μL of system with ddH2O, mix well and place in a PCR instrument at 95℃ for 10 min, 90℃ for 3 min, and 85℃ for 15 min.

[0036] 3min, 80℃3min, with a gradient of 5℃ each, 25℃3min, 4℃hold. After the program is completed, take out the sample and dilute the annealed oligonucleotide in sterile water at a ratio of 1:200. If you want to construct clones on the same day, you can temporarily store it at 4℃, otherwise store it at -20℃.

[0037] (4) Connecting sgRNA annealed fragment and vector: Insert the sgRNA prepared in step (3) into the multiple cloning site of the LentiCRISPRV2 expression plasmid vector to obtain a recombinant vector that simultaneously expresses the Cas9 protein gene and the targeting sgRNA sequence. That is, connect 100-200ng of the pLentiCRISPRv2 vector after linearization at 16°C for 6h. The connection system is: 10μL of linearized vector (gel recovery product), 2μL of inserted fragment (annealed oligo), 1μL of T4 Ligase, 2μL of 10×T4 Ligasebuffer and 5μL of sterile double distilled water, mix evenly, place on a 16°C PCR instrument for 1h or place in a 4°C refrigerator overnight for 12-15h. The ligation product was obtained, and then the ligation product was slowly transferred to the competent DH5α, and the positive clones were screened on the ampicillin resistance plate and the single clone was picked, and the liquid ampicillin resistance LB medium was inoculated, and the plasmid was extracted using a plasmid extraction kit. The extracted plasmid was sequenced to verify whether it was the correct recombinant plasmid. If the sequencing results were 100% consistent, it proved that the recombinant CRISPR-Cas9-ifitm1-sgRNA plasmid was successfully constructed;

[0038] (5) Verification of knockout effect of recombinant CRISPR-Cas9-ifitm1-sgRNA: Prepare HepG2.215 cells in 6-well plates. When the cell density grows to 85%-95%, transfect each well with 4 μg of CRISPR-Cas9-ifitm1-sgRNA1, CRISPR-Cas9-ifitm1-sgRNA2, and CRISPR-Cas9-ifitm1-sgRNA3 plasmids respectively. Set the untransfected cells as negative control. After 6 hours of transfection, replace with 10% fetal bovine serum culture medium. After 24 hours, discard the original culture medium and replace with 10% culture medium containing 4 μg / mL puromycin for screening. After that, replace with fresh 10% growth medium containing 2 μg / mL puromycin every 48 hours. After all the cells in the negative control group die, replace with 10% fetal bovine serum culture medium without drugs, collect the remaining surviving cells for cell lysis, verify the knockout effect, and finally mark them as IFITM1 KO-1, IFITM1 KO-2 and IFITM1 KO-3.

[0039] Furthermore, the

[0040] The nucleotide sequence 5′→3′ of ifitm1-sgRNA1 is 5′-TGATCACGGTGGACCTTGGA-3′;

[0041] The nucleotide sequence 5′→3′ of ifitm1-sgRNA2 is 5′-CTTCTTGAACTGGTGCTGTC-3′;

[0042] The nucleotide sequence 5′→3′ of ifitm1-sgRNA3 is 5′-GGATGAATCCAATGGTCATG-3′ (as shown in Table 1 ).

[0043]

[0044] Table 1: Nucleotide sequences of sgRNA

[0045] Furthermore, the function of the protein encoded by the ifitm1 gene in the host cell is lost by gene targeting technology. The double-stranded sgRNA obtained in step (3) is gel-recovered and purified using an oligonucleotide recovery kit, and the purified double-stranded sgRNA is recovered and subjected to the next step.

[0046] Furthermore, the sequencing identification method in step (4) is to perform agarose gel electrophoresis on the extracted plasmid after bacterial solution PCR, and send the bacterial solution corresponding to the positive band for sequencing. If the sequencing results are 100% consistent, it proves that CRISPR-Cas9-ifitm1-sgRNA1, 2, 3 are successfully constructed.

[0047] Taking the above specific implementation mode as an example, the specific experimental data of the present invention are as follows:

[0048] 1. Materials and Methods

[0049] Material

[0050] Cell lines and plasmids

[0051] The HepG2.215 cell line was maintained by the laboratory, the lenticCRISPRv2 vector was purchased from Suzhou Quanyang Biotechnology Co., Ltd., and the DH5α competent cells were made and maintained by the laboratory.

[0052] Experimental reagents

[0053] The restriction endonuclease BsmBI was purchased from NEB, USA. The transfection reagent Lipo8000 Reagent and agarose were purchased from Invitrogen. The gel recovery kit was purchased from Beijing Kangwei Century Biotechnology Co., Ltd., fetal bovine serum and DMEM medium were purchased from Gibco. High-fidelity (pfu) DNA polymerase was purchased from Beijing Bomade Biotechnology Co., Ltd., plasmid extraction kit and blood / cell / tissue genomic DNA extraction kit were purchased from Beijing Tiangen Biotechnology Co., Ltd., reverse transcription kit was purchased from Biosharp, RNA extraction kit was purchased from Nanjing Novozyme Biotechnology Co., Ltd., 2×HQSYBR fluorescence quantification kit was purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd., genomic DNA purification kit was purchased from Thermo Fisher, USA, mung bean nuclease was purchased from NEB, USA, and CCK8 kit was purchased from Shanghai Biotech.

[0054] Primer synthesis and sequencing of PCR products

[0055] The synthesis of all primers and sequencing of PCR products were completed by Henan Shangya Biotechnology Co., Ltd.

[0056] Experimental methods

[0057] See the above examples.

[0058] Verification of knockout effect of recombinant plasmid

[0059] Prepare a 6-well plate of HepG2.215 cells. When the cell density grows to about 90%, transfect each well with 4 μg of recombinant plasmid expressing sgRNA, and set the transfected cells as negative control. After 6 hours of transfection, replace with 10% fetal bovine serum growth medium. After 24 hours, discard the original culture medium and replace with 10% culture medium containing 4 μg / mL puromycin for screening. After that, replace with fresh 10% growth medium containing 2 μg / mL puromycin every 8 hours. After all the cells in the negative control group die, replace with 10% growth medium without drugs, collect some of the remaining surviving cells for cell lysis, and detect the expression of IFITM1 in HepG2.215 cells by Western blot, and mark them as IFITM1 KO-1, IFITM1 KO-2 and IFITM1 KO-3. 2. Summary of the invention

[0061] Screening and identification of HepG2.215 cell lines with stable knockout of ifitm1 gene

[0062] Prepare HepG2.215 cells in 6-well plates. When the cell density grows to 85%-95%, transfect each well with 4 μg of recombinant plasmid expressing sgRNA. Set untransfected cells as negative controls. After 6 hours of transfection, replace with medium containing 10% fetal bovine serum. After 24 hours, discard the original medium and replace with 10% fetal bovine serum DMEM medium containing 4 μg / mL puromycin for screening. After that, replace with fresh 10% growth medium containing 2 μg / mL puromycin every 48 hours. After all the cells in the negative control group die, replace with 10% medium without drugs. After 7-10 days, expand the culture when the cells grow to a sufficient density, collect the cells for Western blot detection of IFITM1 protein expression level (see results). Figure 4 ),

[0063] Cell viability assay

[0064] After the knockout of ifitm1 gene was confirmed by Western blot, the cell morphology and cell proliferation rate were detected (the results are shown in Figure 5 ). The cells to be tested were inoculated into a 96-well plate at a rate of 100 μL of culture medium per well, and cultured until the cells were fully attached to the wall. 10 μL of CCK-8 solution was added to each well and incubated for 2 h. The OD was measured using an ELISA reader. 450 nm The experiment was repeated three times.

[0065] Extraction and determination of HBV mRNA

[0066] HepG2.215 and ifitm1 KO-1, 2 and 3 HepG2.215 cells with good growth status were plated into 12-well plates and transfected when the cell density reached 100%. Empty vector pcDNA3.1-Flag was transfected into HepG2.215 cells, and empty vector pcDNA3.1-Flag and IFITM1 were transfected into ifitm1KO HepG2.215 cells respectively. After transfection, cells were collected and RNA-easy isolation reagent was used to detect the expression of IFITM1.

[0067] (R701-02-AA, Vazyme, China) kit was used to extract RNA, and reverse transcription was performed using a reverse transcription kit. The system was 5 μl RNA template, DEPC water was made up to 13 μl, Enzyme Mix 3 μl, and 5×Reaction buffer 4 μl; the reaction conditions were: 25°C 5min random primers were paired with RNA templates, genomic DNA was removed, 55°C 15min, reverse transcription reaction and rapid inactivation of dsDNA, 85°C 5min, inactivation of reverse transcriptase. After completion, the obtained template was frozen in a -20°C refrigerator for later use. After that, the fluorescence quantitative qRT-PCR experiment was performed using a 2×HQ SYBR qPCR kit (ZF503-2, ZomanBio, China). The detailed operation steps were carried out according to the instructions, and the primers used are shown in Table 2.

[0068] Extraction and determination of HBV rcDNA and cccDNA

[0069] HepG2.215 and ifitm1 KO-1, 2 and 3 HepG2.215 cells with good growth status were plated into 12-well plates and transfected when the cell density reached 100%. Empty vector pcDNA3.1-Flag was transfected into HepG2.215 cells, and empty vector pcDNA3.1-Flag and IFITM1 were transfected into ifitm1KO HepG2.215 cells, respectively. After transfection, the cells were collected, DNA was extracted using a genome purification kit, and then single-stranded DNA was removed with mung bean nuclease at 30°C for 30min to obtain covalently closed circular DNA (covalently closed circular DNA, cccDNA). Subsequently, the primers used for fluorescence quantitative PCR (qRT-PCR) experiments were performed with relaxed circular double-stranded DNA (rcDNA) primers and cccDNA primers, respectively, as shown in Table 2.

[0070]

[0071] Table 2: qRT-PCR detection primers

[0072] Western blot

[0073] HepG2.215 and ifitm1 KO HepG2.215 cells with good growth status were plated into 12-well plates. After the cell density reached 100%, transfection was performed. Fresh medium was replaced after 24 hours. The plates were washed twice with PBS before collecting adherent cells the next day. The harvested cells were lysed with 1 mL of cell lysis buffer (P0013; Beyotime; China), supplemented with 1 mM protease inhibitor (P001; NCMBiotech; China) and 10 mM dithiothreitol (DTT), and lysed on ice for 30 min. Cell lysis was performed by centrifugation at 12000 rpm for 30 min at 4 °C, and the supernatant was collected as a protein sample. The protein concentration was measured by bicinchoninic acid (BCA) protein detection kit. The quantified protein was mixed with 5× SDS-PAGE loading buffer (WB2001; NCMBiotech; China), boiled and subjected to SDS-PAGE. The proteins in the gel were transferred to a 0.45 mm polyvinylidene fluoride (PVDF) membrane, detected with specific antibodies, and visualized by chemiluminescence.

[0074] The HepG2.215 cell line with ifitm1 gene knockout constructed by the method can be used in the preparation of anti-HBV virus target drugs. The method of the present invention is simple, and a method for targeted knockout of human ifitm1 gene based on CRISPR-Cas9 is constructed to obtain a cell model with human ifitm1 gene knockout, which is used for the study of natural immunity-related diseases of host anti-HBV virus, etc., with huge social and economic benefits.

[0075] 3. Results

[0076] Construction of the recombinant vector ifitm1-sgRNA-LentiCRISPRV2-PURO targeting the ifitm1 gene

[0077] ifitm1 is 1233 bp in length and consists of two exons. After searching with DNA Star software, Figure 3 As shown in Figure 2, two sgRNAs were designed on exon 1: Target1 and 2, and one sgRNA was designed on exon 2: Target3. Figure 2 The sgRNA primers were designed as shown in Table 1. The BsmBI endonuclease site was introduced into the synthesized sgRNA oligonucleotide single strand, so that it could be annealed to form a double strand with sticky ends, and then ligated with the LentiCRISPRV2 vector ( Figure 1) to construct the recombinant plasmid ifitm1-sgRNA-LentiCRISPRV2, and verify whether it is correct by sequencing ( Figure 4 ). The recombinant plasmid ifitm1-sgRNA-LentiCRISPRV2 can transcribe the sgRNA targeting the target gene, and the plasmid can also express the Cas protein, so the plasmid can simultaneously complete the functions of targeting and cutting.

[0078] The puromycin resistance gene in the LentiCRISPRV2-PURO vector backbone can be used to screen positive clones. Verification of protein level of knockout effect of recombinant plasmid

[0079] After all cells in the negative control group died after selection with puromycin, the culture medium was changed to DMEM complete medium. After a certain number of cells were passaged, they were labeled as IFITM1 KO-1, IFITM1 KO-2 and IFITM1KO-3, and the remaining surviving cells were collected for cell lysis to detect the protein expression of IFITM1. Figure 5 As shown, the results of Western blot detection showed that compared with HepG2.215 cells, there was no IFITM1 expression in the cells after transfection of ifitm1-sgRNA1-LentiCRISPRV2, ifitm1-sgRNA2-LentiCRISPRV2 and ifitm1-sgRNA3-LentiCRISPRV2, respectively, indicating that the ifitm1 gene was successfully knocked out.

[0080] Morphology and viability of ifitm1 KO HepG2.215 cells

[0081] After successful knockout of IFITM1 protein was identified by Western blot, the cell morphology and viability of the resulting IFITM1 knockout cell line were identified. Figure 6 As shown, the cell morphology and cell proliferation rate of IFITM1 KO-1, 2 and 3 cells are the same as those of HepG2.215 cells, indicating that the knockout of IFITM1 does not affect the normal growth and proliferation of cells and can be expanded for experiments.

[0082] IFITM1 inhibits HBV infection

[0083] The above experimental results show that ifitm1 KO HepG2.215 cell line has no ifitm1 expression. Based on this, the present invention uses qRT-PCR to detect the differences in HBV covalently closed circular DNA (cccDNA) and relaxed circular double-stranded DNA (rcDNA) and mRNA transcription levels in cells compared with wild-type HepG2.215. The results are as follows Figure 7 As shown, the results showed that in ifitm1 KO HepG2.215 cells, the levels of HBV cccDNA, rcDNA and mRNA were significantly higher than those in HepG2.215 cells, and after replenishing IFITM1 in ifitm1KO HepG2.215 cells, the HBV level was restored, indicating that knocking out IFITM1 led to increased HBV infection.

[0084] It can be clearly seen from the above that the present invention obtains a HepG2.215 cell line with a knockout of the human ifitm1 gene based on CRISPR-Cas9 editing technology. Its principle and core key technology are to scientifically and rationally construct sgRNA targeting the human ifitm1 gene, and then clone the sgRNA into the LentiCRISPRV2-PURO plasmid with the Cas9 gene, and transfect the plasmid into HepG2.215 cells, and use the CRISPR-Cas9 system to achieve gene silencing, and finally obtain a positive cell line by drug screening, thereby obtaining a human ifitm1 knockout HepG2.215 cell line. After IFITM1 was knocked out, HBV in HepG2.215 cells increased significantly, which was used for research on natural immune-related diseases of the host against HBV virus, and also provided new ideas for the development of antiviral target drugs, which has great application research value and huge social and economic benefits.

[0085] The above is only one of the specific implementations of the present invention, but the technical features of the present invention are not limited thereto. Any modification, equivalent replacement or improvement made within the technical concept of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a HepG2.215 cell line with stable knockout of ifitm1 gene, characterized in that: The following steps are involved: (1) Download the human ifitm1 full gene sequence (GeneID: 8519) from the Genebank database, identify its CDS region (exons), and perform GG(N) on the CDS region using DNA Star software. 18 NGG or CC(N) 18 Search for the NCC motif, use the found motif as the positive strand of the sgRNA, add CACCG to the 5' end of the positive strand, add AAAC to the 5' end of the negative strand, and add C to the 3' end to synthesize a single-stranded sgRNA; (2) The double-stranded LentiCRISPRV2 vector was digested with BsmBI endonuclease. The digestion system was: 2 μg of template plasmid, 1 μL of Fast Digest BsmBI, 1 μL of Fast AP, 2 μL of 10× Fast Digest Buffer, supplemented with ddH2O to 20 μL system, mixed and incubated at 37°C for 30 min, and then subjected to 1% agarose gel electrophoresis to recover the linearized vector after digestion; (3) Add 3 μL of forward primer and 3 μL of reverse primer to 20 μL system with ddH2O, mix well and place in PCR instrument, and perform the procedure at 95°C for 10 min, 90°C for 3 min, 85°C for 3 min, 80°C for 3 min, with a gradient of 5°C, 25°C for 3 min, and 4°C hold. After the procedure is completed, take out the sample and dilute the annealed oligonucleotide into sterile water at a ratio of 1:

200. If the clone is to be constructed on the same day, it can be temporarily stored at 4°C, otherwise it can be stored at -20°C. (4) Insert the sgRNA prepared in step (3) into the multiple cloning site of the LentiCRISPRV2 expression plasmid vector to obtain a recombinant vector that simultaneously expresses the Cas9 protein gene and the targeting sgRNA sequence; that is, 100-200 ng of the linearized pLentiCRISPRv2 vector is connected at 16°C for 6 hours, and the connection system is: 10 μL of the linearized vector (gel recovery product), 2 μL of the inserted fragment (annealed oligo), 1 μL of T4 Ligase, 2 μL of 10×T4 Ligase Buffer and 5 μL of sterile double distilled water were mixed evenly, placed on a 16°C PCR instrument for 1 hour or placed in a 4°C refrigerator overnight for 12-15 hours; the ligation product was obtained, and then the ligation product was slowly transferred to the competent DH5α, and the positive clones were screened on an ampicillin-resistant plate and a single clone was picked, inoculated with liquid ampicillin-resistant LB medium, and the plasmid was extracted using a plasmid extraction kit, and the extracted plasmid was sequenced to verify whether it was the correct recombinant plasmid. If the sequencing results were 100% consistent, it proved that the recombinant CRISPR-Cas9-ifitm1-sgRNA plasmid was successfully constructed; (5) Prepare HepG2.215 cells in 6-well plates. When the cell density grows to 85%-95%, transfect each well with 4ug of CRISPR-Cas9-ifitm1-sgRNA1, CRISPR-Cas9-ifitm1-sgRNA2, and CRISPR-Cas9-ifitm1-sgRNA3 plasmids respectively. Set the untransfected cells as negative control. After 6h of transfection, replace with 10% fetal bovine serum medium. After 24h, discard the original medium and replace with 10% medium containing 4μg / mL puromycin for screening. After that, replace with fresh 10% growth medium containing 2μg / mL puromycin every 48h. After all the cells in the negative control group die, replace with drug-free 10% fetal bovine serum medium, collect the remaining surviving cells for cell lysis, verify the knockout effect, and finally mark them as IFITM1 KO-1, IFITM1 KO-2, and IFITM1 KO-3.

2. The method according to claim 1, characterized in that: Said The nucleotide sequence 5′→3′ of ifitm1-sgRNA1 is 5′-TGATCACGGTGGACCTTGGA-3′; The nucleotide sequence 5′→3′ of ifitm1-sgRNA2 is 5′-CTTCTTGAACTGGTGCTGTC-3′; The nucleotide sequence 5′→3′ of ifitm1-sgRNA3 is 5′-GGATGAATCCAATGGTCATG-3′.

3. The method according to claim 1, characterized in that: The function of the protein encoded by the ifitm1 gene in the host cell is lost by gene targeting technology; the double-stranded sgRNA obtained in the step (3) is gel-recovered and purified using an oligonucleotide recovery kit, and the purified double-stranded sgRNA is recovered and subjected to the next step.

4. The method according to claim 1, characterized in that: The sequencing identification method in step (4) is to perform agarose gel electrophoresis on the extracted plasmid after bacterial solution PCR, and send the bacterial solution corresponding to the positive band to be sequenced. If the sequencing results are 100% consistent, it is confirmed. CRISPR-Cas9-ifitm1-sgRNA1, 2, and 3 were successfully constructed.

5. The use of a method for constructing a HepG2.215 cell line with ifitm1 gene knockout based on CRISPR-Cas9 editing technology according to any one of claims 1 to 4, characterized in that: This method is used to study diseases related to the host's natural immunity against hepatitis B virus.

6. The use according to claim 5, characterized in that: The method is further applied to the host's natural immune defense system against hepatitis B virus.

7. The use according to claim 6, characterized in that: This method is further applied to the development of anti-HBV target drugs.

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