Co-expression and antiviral activity analysis method of CH25H-IFITM3-ISG15

Abstract

The invention discloses a co-expression and antiviral activity analysis method of CH25H-IFITM3-ISG15. The method comprises the following steps: selecting HEK-293T, HEK-293, BHK-21, a VSV-GFP virus, an H3N2 subtype influenza virus and a pCK carrier, and preserving the selected HEK-293T, HEK-293, BHK-21, the VSV-GFP virus, the H3N2 subtype influenza virus and the pCK carrier; a recombinant plasmid pCK-CII is obtained; the method comprises the following steps: carrying out DNA (deoxyribonucleic acid) sequencing analysis, cell transfection and collection, Western blot analysis on pCK-CII expression, and pCK-CII anti-VSV-GFP and H3N2 subtype influenza virus activity experiments. According to the invention, the in-vitro anti-VSV-GFP and H3N2 subtype influenza virus activity on different source cells is preliminarily verified, a reference is provided for further research on the antiviral activity of a host limiting factor on other viruses, and a new idea is provided for research and development of broad-spectrum antiviral drugs.

Description

Technical Field

[0001] The present invention relates to the field of gene expression and antiviral analysis, and in particular to a method for co-expression of CH25H-IFITM3-ISG15 and analysis of its antiviral activity. Background Art

[0002] Host restriction factors (HRFs) are a class of antiviral proteins that can be induced to be highly expressed by interferons in cells. These proteins can play an inhibitory role at various stages of the viral life cycle. According to current antiviral research on HRFs, cholesterol 25-hydroxylase (CH25H), interferon-induced transmembrane protein 3 (IFITM3), and interferon-stimulated gene 15 (ISG15) are the three proteins with good antiviral effects and the most extensive research. Therefore, the present invention selects these three host restriction factors for antiviral activity research.

[0003] Cholesterol 25-hydroxylase (CH25H) has a molecular weight of 32 kDa. It is an interferon-induced enzyme that restricts virus-cell membrane fusion during virus entry and widely inhibits vesicular stomatitis virus (VSV), murine gammaherpesvirus 68 (MHV-68), SARS virus (SARS-CoV), etc. IFITM3 is one of the five members of the human interferon-induced transmembrane protein family, with a molecular weight of 17 kDa. The antiviral properties of IFITM3 were discovered by researchers using small-interfering RNA (SiRNA) technology when developing intrinsic inhibitors for influenza A virus (IAV), West Nile virus (WNV) and dengue virus (DENV) infections. Mechanistic studies of IFITM3 have shown that it does not rely on specific recognition of viral components to restrict viral entry. Instead, it restricts viral entry by changing the mechanical properties of the cell membrane to be unfavorable for the membrane fusion reaction between the virus and the target cell. IFITM3's restriction of virus-cell fusion occurs after hemifusion and before the formation of an expanded fusion pore for viral proteins and nucleic acids to enter the host cell cytoplasm. Interferon-stimulated gene 15 (ISG15), with a molecular weight of 17 kDa, has been shown to be strongly induced by type I interferons. Studies have shown that in addition to being strongly induced by type I interferons, ISG15 is also induced by viral and bacterial infections, lipopolysaccharide (LPS), retinoic acid or certain genotoxic stressors, indicating that the expression of ISG15 represents the host's response to pathogenic stimuli. The antiviral activity of ISG15 is mainly through the destruction of the viral replication machinery by conjugating with viral nucleoproteins, destroying its ability to oligomerize and form viral nucleoproteins, thereby inhibiting viral RNA synthesis.

[0004] The present invention cloned the human CH25H-IFITM3-ISG15 (CII) gene sequence, and the three genes were connected by a cleavage peptide. The 3' end of each gene was designed with an HA tag, the 5' of the entire fragment was designed with a kozak sequence, and the 3' end was equipped with a His tag. In this study, the recombinant fragment was connected to the pCK vector for expression, and the inhibition of the activity of H3N2 subtype influenza virus (IAV) and recombinant vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP) in cells from different sources such as HEK-293 and BHK-21 was analyzed to explore the antiviral effect of the co-expression of the three, and to establish a foundation for the research and development of antiviral nucleic acid drugs for gene therapy. Summary of the invention

[0005] In view of this, the present invention provides a method for co-expression of CH25H-IFITM3-ISG15 and analysis of its antiviral activity.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for co-expression of CH25H-IFITM3-ISG15 and analysis of antiviral activity comprises the following steps:

[0008] Step 1: Select HEK-293T, HEK-293, BHK-21, VSV-GFP virus, H3N2 subtype influenza virus, and pCK vector for storage;

[0009] Step 2: Referring to the CDS sequences of the three genes CH25H, IFITM3, and ISG15, HAtag tags were added to the C-termini of the three genes, and the three genes were connected in series; the Kozak sequence was inserted into the N segment of the connected recombinant fragment to enhance the translation efficiency, and the His tag was inserted into the C segment; finally, the recombinant fragment CII gene sequence was codon optimized, synthesized and connected to the pVAX vector to obtain the recombinant plasmid pCK-CII;

[0010] Step 3: Using pVAX-CII as a template, amplify the CII gene by PCR technology, separate and recover the amplified fragment after agarose gel electrophoresis, and seamlessly clone it into the linearized pCK vector. Obtain the recombinant plasmid pCK-CII by conventional methods such as plasmid transformation, single colony screening, plasmid extraction, and double enzyme digestion identification, and perform DNA sequencing analysis;

[0011] Step 4: Perform cell transfection and collect cells;

[0012] Step 5: Perform Western blot analysis of pCK-CII expression;

[0013] Step 6: Conduct pCK-CII anti-VSV-GFP virus activity experiment;

[0014] Step 7: Conduct an activity experiment of pCK-CII against H3N2 subtype influenza virus.

[0015] Preferably, in step 2, the method for connecting the three genes in series is: adding a HAtag tag to the C-terminus of the three genes, and connecting them to the IFITM3 gene using P2A behind the HA tag of CH25H, and similarly connecting them to the ISG15 gene using T2A behind the HA tag of IFITM3, so that the three genes are connected in series.

[0016] Preferably, in step 3, the concentration of agarose gel electrophoresis is 1%.

[0017] Preferably, in step 3, the CII gene PCR amplification primers are as shown in Table 1:

[0018] Table 1:

[0019]

[0020] Preferably, in step 4, the method for cell transfection is: HEK 293T cells are inoculated in a 6-well plate and cultured in a 5% CO2 incubator overnight; before transfection, the original culture medium is discarded and replaced with Opti-MEM, and the recombinant plasmid pCK-CII and its control vector pCK are mixed with Lip3000 transfection reagent in a ratio of 1:1, allowed to stand at room temperature for 20 minutes, and evenly added to the 6-well plate. After 6 hours, it is replaced with 10% + 1% DMEM and cultured at 37°C for 48 hours.

[0021] Preferably, in step 5, the method for Western blot analysis of pCK-CII expression is: the collected cell samples are prepared into protein samples for SDS-PAGE electrophoresis, and then transferred to the NC membrane by rapid wet transfer. After the transfer, it is blocked with 5% skim milk at room temperature for 2 hours, and incubated with Anti-HA, Anti-IFITM3, and Anti-ISG15 as primary antibodies at room temperature for 2 hours, and goat anti-rabbit IgG as secondary antibodies at room temperature for 0.5 hours. Between each step, the membrane needs to be washed 3 times with TBST, each time for 10 minutes, and finally reacted with chemiluminescent liquid ECL, and exposed to light for development.

[0022] Preferably, in step 6, the pCK-CII anti-VSV-GFP virus activity experiment is divided into immunofluorescence method and flow cytometry method.

[0023] Preferably, the immunofluorescence method is as follows: BHK-21 cells are inoculated into a 12-well plate (1×105 cells / well), and after the cells grow to 70% to 80%, pCK-CII (2 μg) is transfected into the cells, and the cells are incubated at 37°C and 5% CO2 for 24 hours, and VSV-GFP (0.01 MOI) is inoculated into the 12-well plate. After incubation for 1 hour, the virus solution is discarded, and the cells are washed twice with PBS, and 2 ml of DMEM containing 2% serum is added to continue culturing. After 12 hours, the supernatant is discarded and the cells are washed with PBS, and the fluorescence is observed using a fluorescence microscope.

[0024] Preferably, the flow cytometry method is: after taking pictures and recording under a fluorescence microscope, the cells are digested with trypsin, blown evenly, centrifuged at 3000 rpm for 5 min, the supernatant is discarded, the cells are resuspended with 1 ml of PBS, and the number of cells expressing green fluorescent protein (GFP) is calculated by flow cytometry.

[0025] Preferably, in step 7, the method for conducting the pCK-CII anti-H3N2 subtype influenza virus activity experiment is: HEK293 cells are inoculated in a 6-well plate, and after the cells grow to 70% to 80%, pCK-CII is transfected into the cells, and after incubation at 37°C and 5% CO2 for 24 hours, H3N2 influenza virus is inoculated into the 6-well plate, and the supernatant is discarded after 12 hours, and the cells are collected to prepare protein samples for WB analysis.

[0026] Compared with the prior art, the present invention has achieved the following technical effects:

[0027] The present invention constructs a recombinant pCK-CII plasmid that expresses three antiviral proteins, CH25H, IFITM3, and ISG15, in vitro, and preliminarily verifies the in vitro anti-VSV-GFP and H3N2 subtype influenza virus activities on different source cells, providing a reference for further research on the antiviral activity of host restriction factors against other viruses, and providing a new idea for the research and development of broad-spectrum antiviral drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A schematic diagram of a recombinant plasmid pCK-CII for co-expression of CH25H-IFITM3-ISG15 and an antiviral activity analysis method of the present invention;

[0029] Figure 2 This is a diagram showing the result of target fragment amplification and double enzyme digestion identification of a method for co-expression and antiviral activity analysis of CH25H-IFITM3-ISG15 of the present invention;

[0030] A. Amplification of the target fragment of CII gene (M: DL5000 DNA Marker; 1. pVAX-CII; 2-4. PCR amplification results; 5. Negative control); B Double enzyme digestion identification (M: DL5000 DNA Marker; 1. pCK-CII digested by HindⅢ and X BaⅠ);

[0031] Figure 3 This is an expression verification diagram of a method for co-expression and antiviral activity analysis of CH25H-IFITM3-ISG15 of the present invention after HEK 293T cells were transfected with pCK-CII;

[0032] A. HA tag antibody was used to detect the expression of pCK-CII protein (M: 26616 protein marker; 1. pCK-CII; 2. pCK; 3 blank controls); B. IFITM3 specific antibody was used to detect the expression of pCK-CII protein (M: 26616 protein marker; 1. pCK-CII; 2. pCK; 3 blank controls); C. ISG15 specific antibody was used to detect the expression of pCK-CII protein (M: 26616 protein marker; 1. pCK-CII; 2. pCK; 3 blank controls);

[0033] Figure 4 This is a diagram of an immunofluorescence analysis of a method for co-expression of CH25H-IFITM3-ISG15 and analysis of antiviral activity of pCK-CII on BHK-21 cells (100×);

[0034] Figure 5 This is a flow cytometry analysis of the anti-VSV-GFP viral activity of pCK-CII on BHK-21 cells in a method for co-expression and antiviral activity analysis of CH25H-IFITM3-ISG15 of the present invention;

[0035] Figure 6 This is a diagram showing a Western blot analysis of the anti-H3N2 subtype influenza virus activity of pCK-CII on HEK 293 cells in accordance with a method for co-expression and antiviral activity analysis of CH25H-IFITM3-ISG15 of the present invention. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in 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.

[0037] A method for co-expression of CH25H-IFITM3-ISG15 and analysis of antiviral activity comprises the following steps:

[0038] Step 1: Select HEK-293T, HEK-293, BHK-21, VSV-GFP virus, H3N2 subtype influenza virus, and pCK vector for storage;

[0039] Step 2: Referring to the CDS sequences of the three genes CH25H, IFITM3 and ISG15, HAtag tags were added to the C-termini of the three genes, and P2A was used to connect the CH25H HA tag to the IFITM3 gene, and T2A was used to connect the ISG15 gene to the IFITM3 HA tag, so that the three genes were connected in series; the Kozak sequence was inserted into the N segment of the series-connected recombinant fragment to enhance the translation efficiency, and the His tag was inserted into the C segment; finally, the recombinant fragment CII gene sequence was codon-optimized, synthesized and connected to the pVAX vector to obtain the recombinant plasmid pCK-CII;

[0040] Step 3: Using pVAX-CII as a template, amplify the CII gene by PCR technology. The amplified fragment is separated and recovered after 1% agarose gel electrophoresis, seamlessly cloned into the linearized pCK vector, and the recombinant plasmid pCK-CII is obtained by conventional methods such as plasmid transformation, single colony screening, plasmid extraction and double enzyme digestion identification, and DNA sequencing analysis is performed; wherein, the CII gene PCR amplification primers are shown in Table 1:

[0041] Table 1:

[0042]

[0043] Step 4: HEK293T cells were seeded in a 6-well plate and cultured overnight in a 5% CO2 incubator; before transfection, the original culture medium was discarded and replaced with Opti-MEM, and the recombinant plasmid pCK-CII and its control vector pCK were mixed with Lip3000 transfection reagent at a ratio of 1:1, allowed to stand at room temperature for 20 minutes, and evenly added to the 6-well plate. After 6 hours, it was replaced with 10% + 1% DMEM and cultured at 37°C for 48 hours;

[0044] Step 5: Prepare the collected cell samples into protein samples for SDS-PAGE electrophoresis, and then transfer to the NC membrane by rapid wet transfer. After the transfer, block with 5% skim milk at room temperature for 2 hours, incubate with Anti-HA, Anti-IFITM3, and Anti-ISG15 as primary antibodies at room temperature for 2 hours, and incubate with goat anti-rabbit IgG as secondary antibodies at room temperature for 0.5 hours. Wash the membrane 3 times with TBST between each step, each time for 10 minutes, and finally react with chemiluminescent solution ECL, and expose and develop;

[0045] Step 6: Immunofluorescence: BHK-21 cells were inoculated into 12-well plates (1×105 cells / well). After the cells grew to 70% to 80%, pCK-CII (2 μg) was transfected into the cells and incubated at 37°C, 5% CO2 for 24 h. VSV-GFP (0.01 MOI) was inoculated into the 12-well plates. After incubation for 1 h, the virus solution was discarded, and the cells were washed twice with PBS. 2 ml of DMEM containing 2% serum was added for continued culture. After 12 h, the supernatant was discarded and the cells were washed with PBS. The fluorescence was observed using a fluorescence microscope.

[0046] Flow cytometry method: After taking photos and recording under a fluorescence microscope, the cells were digested with trypsin, blown evenly, centrifuged at 3000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1 ml of PBS. The number of cells expressing green fluorescent protein (GFP) was counted by flow cytometry;

[0047] Step 7: HEK 293 cells were seeded in 6-well plates. After the cells grew to 70% to 80%, pCK-CII was transfected into the cells. After incubation at 37°C and 5% CO2 for 24 hours, H3N2 influenza virus was inoculated into the 6-well plates. After 12 hours, the supernatant was discarded and the cells were collected to prepare protein samples for WB analysis.

[0048] Embodiment 1:

[0049] Material:

[0050] HEK-293T, HEK-293, BHK-21, VSV-GFP virus, H3N2 subtype influenza virus, and pCK vector were stored in this laboratory; Bioflux gel recovery kit was purchased from Hangzhou Biori Technology Co., Ltd.; DNA Marker was purchased from Bao Bioengineering Co., Ltd. (Dalian); Transl-T1 competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; Lip3000 transfection reagent was purchased from Thermo Fisher Scientific; penicillin-streptomycin solution was purchased from Gibco; plasmid DNA mini-extraction kit was purchased from Axygen; plasmid DNA large-scale extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd.; Anti-HA and Anti-IFITM3 antibodies were purchased from Beijing Biopsies Biotechnology Co., Ltd.; Anti-ISG15 antibody was purchased from Cell Signaling Technology (CST); influenza virus H3N2 antibody (NP) was purchased from Beijing Sino Biological Technology Co., Ltd.; HRP-goat anti-rabbit IgG was purchased from Beyotime Biotechnology Co., Ltd.

[0051] Gene design and synthesis:

[0052] Referring to the CDS sequences of three genes CH25H (GeneBank accession number: NM003956.4), IFITM3 (GeneBank accession number: NM021034.3), and ISG15 (GeneBank accession number: NM005101.4) published by GeneBank, HAtag tags were added to the C-termini of the three genes, and P2A was used to connect the CH25H HA tag with the IFITM3 gene. Similarly, T2A was used to connect the ISG15 gene behind the HA tag of IFITM3, so that the three genes were connected in series; the Kozak sequence was inserted into the N segment of the series-connected recombinant fragment to enhance the translation efficiency, and the His tag was inserted into the C segment. Finally, the recombinant fragment CII gene sequence was codon-optimized, synthesized by Nanjing GenScript Biotechnology Co., Ltd., and connected to the pVAX vector. Since the expression efficiency of pCK is better than that of pVAX vector, this study cloned the recombinant fragment between the HindIII and XbaI restriction sites in the pCK vector to obtain the recombinant plasmid pCK-CII.

[0053] Construction and identification of recombinant expression plasmid:

[0054] Using pVAX-CII as a template, the CII gene was amplified by PCR technology. The primers and fluorescent probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The specific information is shown in Table 1. The amplified fragments were separated and recovered after 1% agarose gel electrophoresis, seamlessly cloned into the linearized pCK vector, and the recombinant plasmid pCK-CII was obtained by conventional methods such as plasmid transformation, single colony screening, plasmid extraction, and double enzyme digestion identification, and DNA sequencing analysis was performed. Subsequent experiments were carried out after the sequencing was correct.

[0055] Table 1 Primers for PCR amplification of CII gene

[0056]

[0057] Cell transfection:

[0058] HEK 293T cells were seeded in 6-well plates and cultured overnight in a 5% CO2 incubator. Before transfection, the original culture medium was discarded and replaced with Opti-MEM. The recombinant plasmid pCK-CII and its control vector pCK were mixed with Lip3000 transfection reagent at a ratio of 1:1, allowed to stand at room temperature for 20 minutes, and evenly added to the 6-well plate. After 6 hours, it was replaced with 10% + 1% DMEM, cultured at 37°C for 48 hours, and the cells were collected.

[0059] Western blot analysis of pCK-CII expression:

[0060] The collected cell samples were prepared into protein samples for SDS-PAGE electrophoresis, and then transferred to the NC membrane by rapid wet transfer. After the transfer, the membrane was blocked with 5% skim milk at room temperature for 2 hours, incubated with Anti-HA, Anti-IFITM3, and Anti-ISG15 as primary antibodies at room temperature for 2 hours, and goat anti-rabbit IgG as secondary antibodies at room temperature for 0.5 hours. The membrane was washed 3 times with TBST between each step, each time for 10 minutes, and finally reacted with chemiluminescent solution ECL and exposed to light for development.

[0061] pCK-CII anti-VSV-GFP virus activity experiment:

[0062] Immunofluorescence:

[0063] BHK-21 cells were inoculated into 12-well plates (1×105 cells / well). After the cells grew to 70% to 80%, pCK-CII (2 μg) was transfected into the cells and incubated at 37°C, 5% CO2 for 24 h. VSV-GFP (0.01 MOI) was inoculated into the 12-well plates. After incubation for 1 h, the virus solution was discarded, and the cells were washed twice with PBS. 2 ml of DMEM containing 2% serum was added for continued culture. After 12 h, the supernatant was discarded and the cells were washed with PBS. Fluorescence was observed using a fluorescence microscope.

[0064] Flow cytometry method:

[0065] After taking photos under a fluorescence microscope, the cells were digested with trypsin, blown evenly, centrifuged at 3000 rpm for 5 min, the supernatant was discarded, the cells were resuspended in 1 ml of PBS, and the number of cells expressing green fluorescent protein (GFP) was counted by flow cytometry.

[0066] pCK-CII anti-H3N2 subtype influenza virus activity experiment:

[0067] HEK 293 cells were seeded in 6-well plates (1×106 cells / well). After the cells grew to 70% to 80%, pCK-CII (4 μg) was transfected into the cells. After incubation at 37°C, 5% CO2 for 24 h, H3N2 (0.01 MOI) influenza virus was inoculated into the 6-well plates. After 12 h, the supernatant was discarded and the cells were collected to prepare protein samples for WB analysis.

[0068] result:

[0069] Construction and identification of recombinant plasmid pCK-CII gene:

[0070] The schematic diagram of the recombinant plasmid pCK-CII is as follows Figure 1 As shown, after synthesis, it was first cloned into the vector pVAX, and then the CII gene was amplified by PCR, as shown Figure 2 , as shown in A, it was connected to the eukaryotic expression plasmid pCK to obtain the recombinant expression plasmid pCK-CII. The double enzyme digestion results showed two specific bands, such as Figure 2 , as shown in B, and then the target fragment was sequenced, and the sequencing results were completely consistent with the reference sequence, indicating that the construction was successful.

[0071] Expression identification of pCK-CII:

[0072] The recombinant plasmid pCK-CII was transfected into 293T cells for 48 hours, and the transient expression of the recombinant protein CII was analyzed by Western blot. The expression of CII protein was detected by HA tag antibody, IFITM3 specific antibody, and ISG15 specific antibody. The molecular weight of CH25H was 32kDa, the molecular weight of IFITM3 was 14kDa, and the molecular weight of ISG15 was 17kDa. Figure 3 As shown, CH25H can detect a specific band at about 35 kDa using HA antibody, while a specific band between 15-25 kDa is detected using specific antibodies IFITM3 and ISG15, indicating that transient expression of CII can be cleaved into three free monomeric proteins.

[0073] Immunofluorescence analysis of CII anti-VSV-GFP viral activity on BHK-21 cells:

[0074] The fluorescence observation results showed that the fluorescence intensity of the infection model group (NC) and the transient empty plasmid group (Vector) was comparable at 12 h after infection, but both were significantly higher than the CII group. Figure 4 As shown, it was confirmed that CII had an inhibitory effect on VSV-GFP infection in BHK cells.

[0075] Flow cytometry analysis of the anti-VSV-GFP viral activity of pCK-CII on BHK-21 cells:

[0076] The results of flow cytometry analysis showed that the fluorescence rates of the infection model group were 80.92%, 74.50%, and 79.29%, respectively; the fluorescence rates of the empty vector group were 65.24%, 56.81%, and 56.12%, respectively; and the fluorescence rates of the transient CII plasmid group were 33.27%, 43.30%, and 27.20%, respectively. The flow cytometry data were statistically analyzed using GraphPad software. The results are shown in Figure 5 As shown, the ratio of fluorescent cells in the CII group was significantly lower than that in the Vector group (p<0.01) and the MOD group (p<0.001), indicating that transient transfection of pCK-CII plasmid can inhibit the infection of VSVG in BHK-21 cells.

[0077] pCK-CII anti-H3N2 influenza virus experiment on 293 cells:

[0078] The virus was inoculated 24 hours after transfection, and samples were collected for WB experiment 12 hours after inoculation. The gray value of the WB experiment results was analyzed using Image J software. The results showed that Figure 6 As shown, the expression level of influenza virus NP protein after transfection with CII plasmid was 0.52 times that of the empty vector group and 0.41 times that of the infection model group, indicating that CII can inhibit the replication of H3N2 influenza virus in 293 cells.

[0079] The present invention connects three host restriction factors, CH25H, IFITM3 and ISG15, in series, and uses a single plasmid to express three target proteins to verify their antiviral potential in the early stage of viral infection. In cell experiments, we verified the anti-VSV-GFP activity of the pCK-CII plasmid on BHK-21 cells and the anti-H3N2 subtype influenza virus activity on HEK 293 cells. In BHK-21 cells, the fluorescence intensity of the CII overexpression group was significantly lower than that of the Vector group and the NC group. Through flow cytometry data analysis, the number of cells expressing green fluorescence in the CII group was less than that in the Vector group and the NC group. Statistical analysis showed that the difference was very significant (P<0.01), which indicated that pCK-CII could effectively restrict the entry of VSV-GFP virus into cells at 12h after VSV-GFP infection. Similarly, in terms of anti-H3N2 subtype influenza virus activity, Western blot analysis of the expression abundance of influenza virus NP protein showed that the CII group could significantly inhibit the expression of H3N2 subtype influenza virus NP protein, which indicated that pCK-CII could effectively inhibit the invasion of H3N2 subtype influenza virus into cells and inhibit its replication at 12h after H3N2 subtype influenza virus infection.

[0080] The present invention constructs a recombinant pCK-CII plasmid that expresses three antiviral proteins, CH25H, IFITM3, and ISG15, in vitro, and preliminarily verifies the in vitro anti-VSV-GFP and H3N2 subtype influenza virus activities on different source cells, providing a reference for further research on the antiviral activity of host restriction factors against other viruses, and providing a new idea for the research and development of broad-spectrum antiviral drugs.

[0081] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for co-expression of CH25H-IFITM3-ISG15 and analysis of antiviral activity, characterized in that: The following steps are involved: Step 1: Select HEK-293T, HEK-293, BHK-21, VSV-GFP virus, H3N2 subtype influenza virus, and pCK vector for storage; Step 2: Referring to the CDS sequences of the three genes CH25H, IFITM3, and ISG15, HAtag tags were added to the C-termini of the three genes, and the three genes were connected in series; the Kozak sequence was inserted into the N segment of the connected recombinant fragment to enhance the translation efficiency, and the His tag was inserted into the C segment; finally, the recombinant fragment CII gene sequence was codon optimized, synthesized and connected to the pVAX vector to obtain the recombinant plasmid pCK-CII; Step 3: Using pVAX-CII as a template, amplify the CII gene by PCR technology, separate and recover the amplified fragment after agarose gel electrophoresis, and seamlessly clone it into the linearized pCK vector. Obtain the recombinant plasmid pCK-CII by conventional methods such as plasmid transformation, single colony screening, plasmid extraction, and double enzyme digestion identification, and perform DNA sequencing analysis; Step 4: Perform cell transfection and collect cells; Step 5: Perform Western blot analysis of pCK-CII expression; Step 6: Conduct pCK-CII anti-VSV-GFP virus activity experiment; Step 7: Conduct an activity experiment of pCK-CII against H3N2 subtype influenza virus.

2. The method for co-expression and antiviral activity analysis of CH25H-IFITM3-ISG15 according to claim 1, characterized in that: In step 2, the method for connecting the three genes in series is: adding HAtag labels to the C-termini of the three genes, and connecting them to the IFITM3 gene using P2A behind the HA tag of CH25H, and similarly connecting them to the ISG15 gene using T2A behind the HA tag of IFITM3, so that the three genes are connected in series.

3. The method for co-expression and antiviral activity analysis of CH25H-IFITM3-ISG15 according to claim 1, characterized in that: In step 3, the concentration of agarose gel electrophoresis is 1%.

4. The method for co-expression and antiviral activity analysis of CH25H-IFITM3-ISG15 according to claim 1, characterized in that: In step 3, the CII gene PCR amplification primers are shown in Table 1: Table 1:

5. The method for co-expression and antiviral activity analysis of CH25H-IFITM3-ISG15 according to claim 1, characterized in that: In step 4, the cell transfection method is as follows: HEK 293T cells are inoculated in a 6-well plate and cultured in a 5% CO2 incubator overnight; before transfection, the original culture medium is discarded and replaced with Opti-MEM, and the recombinant plasmid pCK-CII and its control vector pCK are mixed with Lip3000 transfection reagent in a ratio of 1:1, allowed to stand at room temperature for 20 minutes, and evenly added to the 6-well plate. After 6 hours, it is replaced with 10% + 1% DMEM and cultured at 37°C for 48 hours.

6. The method for co-expression and antiviral activity analysis of CH25H-IFITM3-ISG15 according to claim 1, characterized in that: In the step 5, the method for Western blot analysis of pCK-CII expression is as follows: the collected cell samples are prepared into protein samples for SDS-PAGE electrophoresis, and then transferred to the NC membrane by rapid wet transfer. After the transfer, it is blocked with 5% skim milk at room temperature for 2 hours, and incubated with Anti-HA, Anti-IFITM3, and Anti-ISG15 as primary antibodies at room temperature for 2 hours, and goat anti-rabbit IgG as secondary antibodies at room temperature for 0.5 hours. Between each step, the membrane needs to be washed 3 times with TBST, each time for 10 minutes, and finally reacted with chemiluminescent liquid ECL, and exposed to light for development.

7. The method for co-expression and antiviral activity analysis of CH25H-IFITM3-ISG15 according to claim 1, characterized in that: In step 6, the pCK-CII anti-VSV-GFP virus activity experiment is divided into immunofluorescence method and flow cytometry method.

8. The method for co-expression and antiviral activity analysis of CH25H-IFITM3-ISG15 according to claim 7, characterized in that: The immunofluorescence method is as follows: BHK-21 cells are inoculated into a 12-well plate (1×105 cells / well), and after the cells grow to 70% to 80%, pCK-CII (2 μg) is transfected into the cells, and the cells are incubated at 37° C. and 5% CO2 for 24 hours. VSV-GFP (0.01 MOI) is inoculated into the 12-well plate, and the virus solution is discarded after incubation for 1 hour, and the cells are washed twice with PBS, and 2 ml of DMEM containing 2% serum is added for continued culture. After 12 hours, the supernatant is discarded, the cells are washed with PBS, and the fluorescence is observed using a fluorescence microscope.

9. The method for co-expression and antiviral activity analysis of CH25H-IFITM3-ISG15 according to claim 7, characterized in that: The flow cytometry method is as follows: after taking pictures and recording under a fluorescence microscope, the cells are digested with trypsin, blown evenly, centrifuged at 3000 rpm for 5 min, the supernatant is discarded, the cells are resuspended with 1 ml of PBS, and the number of cells expressing green fluorescent protein (GFP) is calculated by flow cytometry.

10. The method for co-expression and antiviral activity analysis of CH25H-IFITM3-ISG15 according to claim 1, characterized in that: In step 7, the method for conducting the pCK-CII anti-H3N2 subtype influenza virus activity experiment is as follows: HEK293 cells are inoculated in a 6-well plate, and after the cells grow to 70% to 80%, pCK-CII is transfected into the cells, and after incubation at 37° C. and 5% CO2 for 24 hours, H3N2 influenza virus is inoculated into the 6-well plate, and after 12 hours, the supernatant is discarded, and the cells are collected to prepare protein samples for WB analysis.

Images