PK-15 cell line capable of stably expressing porcine Mx2 protein as well as preparation and application of PK-15 cell line

Abstract

The invention provides a PK-15 cell line capable of stably expressing porcine Mx2 protein as well as preparation and application of the PK-15 cell line. The PK-15 cell line capable of stably expressing porcine Mx2 gene is constructed through a lentiviral vector system. A specific primer is designed according to the nucleotide sequence of the porcine Mx2 gene in a GeneBank database, recombinant lentivirus plasmids are constructed after target genes are amplified, HEK293T cell packaging lentivirus is transfected by utilizing a three-plasmid system, PK-15 cells are infected by the obtained recombinant lentivirus, and the PK-15 cell line for stably expressing the porcine Mx2 gene is obtained through puromycin screening. Experimental results show that the PK-15 cell line PK-15-poMx2 capable of stably expressing the porcine Mx2 protein is successfully constructed by the method disclosed by the invention. Compared with normal PK-15 cells, the expression quantity of Mx2 in the porcine Mx2 stable expression cell line is remarkably increased, and the porcine Mx2 stable expression cell line has an inhibiting effect on replication of PRV (Porcine Reproductive Virus). Therefore, the successful construction of the cell line for stably expressing the porcine Mx2 protein provides an important tool for deeply exploring the functional characteristics and the antiviral action mechanism of the porcine Mx2 protein.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a PK-15 cell line stably expressing porcine Mx2 protein, and its preparation and application. Background Art

[0002] Myxovirus resistance protein (Mx) is an interferon (IFN)-induced dynamin-like GTPase present in all vertebrates and has broad-spectrum antiviral activity against a variety of viruses. Most mammals encode two similar Mx proteins, namely type I myxovirus resistance protein Mx1 and type II myxovirus resistance protein Mx2, which are also known as MxA and MxB in humans. Mx1 and Mx2 proteins have similar structures, each containing an amino-terminal domain (NTD), a GTPase domain (G domain), and a carboxyl-terminal stalk domain, and all domains are connected by a trimeric bundle signal element (BSE). Mx1 protein has broad inhibitory activity against a variety of RNA and DNA viruses, such as influenza A virus (IAV) and hepatitis B virus (HBV). Mx2 protein was initially considered an interferon-stimulated gene (ISG) significantly upregulated by type I interferon (IFNα and IFNβ) and has no antiviral activity against influenza A virus (IAV) or vesicular stomatitis virus (VSV). Therefore, for a long time, it was thought that Mx2 did not have antiviral activity, although it was strongly induced by IFN. Until 2013, three different research groups found that human Mx2 could effectively inhibit HIV-1 infection in vitro. Subsequent extensive studies further confirmed that Mx2 could inhibit primate immunodeficiency viruses but not other retroviruses such as murine leukemia virus (MLV). With the continuous in-depth research, it was found that Mx2 protein could also restrict the replication of herpesviruses, hepatitis B virus (HBV), and hepatitis C virus (HCV), etc., and the mechanisms of Mx2's inhibitory effects on different virus families are different. For example, the amino-terminal domain of Mx2 is important for restricting the replication of herpesviruses and HIV-1. For HIV-1, the tri-arginine motif at the amino terminus of Mx2 restricts HIV-1 replication by binding to the HIV-1 capsid, while for human herpesviruses, the first 25 amino acid residues (nuclear localization signal) at the amino terminus of Mx2 play an important role. In contrast, the deletion of the nuclear localization signal (the first 25 amino acid residues) at the amino terminus of Mx2 does not affect its antiviral activity against HBV. The GTPase activity of Mx2 protein, including GTP binding and hydrolysis activities, seems to be dispensable for the anti-HIV-1 activity of Mx2. However, the anti-HBV activity of Mx2 protein requires GTP binding but not GTP hydrolysis activity. In contrast, both GTP binding and hydrolysis activities of Mx2 protein are essential for anti-herpesvirus activity. In addition, Mx2 restricts HCV replication by interfering with the interaction between NS5A and CypA through interaction with the NS5A protein of HCV. This indicates that Mx2 exerts its antiviral effects through different, and possibly cell type-specific, mechanisms.

[0003] Mx proteins are highly conserved in vertebrates. It has been reported that Mx1 and Mx2 have also been detected in pigs. The molecular weight of the protein encoded by porcine Mx2 is 82 kDa. The nucleotide and amino acid sequence homologies between porcine Mx2 and human MxB are 76.5% and 69.2% respectively. Studies have shown that porcine Mx2 also has antiviral activity. For example, porcine Mx2 can inhibit the replication of influenza virus, porcine reproductive and respiratory syndrome virus (PRRSV), and vesicular stomatitis virus (VSV), but the specific mechanism is not yet clear. In addition, porcine Mx2 also has anti-classical swine fever virus (CSFV) activity. Porcine Mx2 can co-precipitate with the NS5B protein of CSFV, but there is no co-localization. It is speculated that porcine Mx2 exerts its antiviral activity against CSFV through indirect interaction with the CSFV NS5B protein. Porcine Mx2 can also inhibit the replication of pseudorabies virus (PRV) by inhibiting the synthesis of early genes of PRV, and the oligomerization and GTPase activity of porcine Mx2 are necessary for its anti-PRV activity. Therefore, porcine Mx2 plays an important role in the antiviral defense of pigs.

[0004] In summary, Mx2 protein is an important protein that plays an antiviral role in the innate immune response and plays an important role in the antiviral defense mechanism of pigs. Targeting porcine Mx2 protein, through drug design and screening, to develop new antiviral drugs that can enhance the antiviral activity of porcine Mx2 protein or mimic its function, can provide new ways for the treatment of viral diseases such as swine influenza, classical swine fever, and porcine reproductive and respiratory syndrome. To deeply study the structure and function of porcine Mx2 protein and its antiviral mechanism, the present invention constructed a PK-15 cell line PK-15-poMx2 that stably expresses porcine Mx2 protein, detected the viability of the cell line using the CCK-8 method, analyzed the expression of porcine Mx2 in the PK-15-poMx2 cell line by Western blot method, and preliminarily evaluated the PK-15-poMx2 cell line using the model virus PRV. The successful construction of the PK-15 cell line (PK-15-poMx2) that stably expresses porcine Mx2 provides a powerful tool for in-depth study of the structure and function of porcine Mx2 protein and its antiviral mechanism. Summary of the Invention

[0005] The object of the present invention is a PK-15 cell line that stably expresses porcine Mx2 protein and its preparation and application.

[0006] The present invention provides a PK-15 cell line that stably expresses porcine Mx2 protein. The cell line integrates the porcine Mx2 gene into the PK-15 cell genome through a lentiviral vector system to achieve stable expression of porcine Mx2 protein.

[0007] The lentiviral vector system includes the pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro vector, the auxiliary packaging plasmid pMD2.G, and psPAX2. The nucleotide sequence GeneBank accession number of the porcine Mx2 gene is AB258432.1. The cell line was obtained by puromycin screening.

[0008] The present invention provides the application of the PK-15 cell line stably expressing porcine Mx2 protein in screening antiviral drugs or preparing a cell model for antiviral infection. The cell line is also used to study the antiviral mechanism of porcine Mx2 protein. The cell line is used to evaluate the inhibitory effect of porcine Mx2 protein on pseudorabies virus (PRV). The present invention provides a method for constructing a PK-15 cell line stably expressing porcine Mx2 protein, which includes the following steps: (1) Construct the recombinant lentiviral plasmid pLV-GFP-poMx2; (2) Obtain recombinant lentivirus through the lentiviral packaging system; (3) Infect PK-15 cells with the recombinant lentivirus, and obtain the PK-15 cell line stably expressing porcine Mx2 through puromycin screening.

[0009] In step (1), the recombinant lentiviral plasmid pLV-GFP-poMx2 is obtained by ligating the lentiviral expression vector pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro with the porcine Mx2 gene, transforming the ligation product into JM109 competent cells, picking monoclonal colonies for culture, and then extracting the plasmid for sequencing verification.

[0010] In step (2), the lentiviral packaging system uses HEK293T cells for packaging. Transfect HEK293T cells with plasmids, and co-transfect HEK293T cells with the recombinant plasmid pLV-GFP-Mx2, the packaging plasmid psPAX2, and pMD2.G. The mass ratio of the three is pLV-GFP-Mx2:pSPAX2:pMD2.G = 4:3:1.

[0011] Beneficial effects: 1. Successfully constructed a PK-15 cell line stably expressing porcine Mx2. Western blot results show that the expression level of porcine Mx2 protein is significantly increased.

[0012] 2. Experimental data show that the mRNA level, protein expression level, and virus titer of PRV in PK-15-poMx2 cells are significantly lower than those in PK-15-WT cells (P ≤ 0.05), verifying the inhibitory effect of porcine Mx2 on PRV.

[0013] 3. The PK-15-poMx2 cell line of the present invention can be used for screening antiviral drugs, studying the antiviral mechanism of porcine Mx2, and preparing a cell model for antiviral infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Restriction enzyme digestion identification result of recombinant plasmid pLV-GFP-poMx2, M: 15000 bp DNA Marker.

[0015] Figure 2 : Fluorescence expression of HEK293T cells during lentivirus packaging, A: 24 h after transfection of lentivirus recombinant plasmid and auxiliary plasmid, observed under fluorescence microscope (10-fold magnification); B: 48 h after transfection of lentivirus recombinant plasmid and auxiliary plasmid, observed under fluorescence microscope (10-fold magnification).

[0016] Figure 3 : Construction and identification of PK-15-poMx2 cell line, A: Fluorescence expression after lentivirus infecting PK-15 cells for 24 h; B: Fluorescence expression in PK-15 cells after screening for one week; C: Detection of protein level of porcine Mx2 in PK-15-poMx2 cell line.

[0017] Figure 4 : CCK-8 cell viability detection result.

[0018] Figure 5 : Influence of PK-15-poMx2 cell line on PRV replication, A: Detection of mRNA level of PRV replication; B: Detection of protein level of PRV replication; C: Detection of progeny virus titer of PRV (*: P ≤ 0.05; **: P ≤ 0.01). DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention is further described below by specific embodiments. The plasmids, cell strains and main reagents used in the present invention are as follows: The vector plasmid pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro, auxiliary packaging plasmids pMD2.G and psPAX2, HEK293T cells, PK-15 cells, vero cells, and PRV-Bartha-K61 strain are all preserved in the laboratory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; restriction enzymes ( Xba Ⅰ and EcoRⅠ) Purchased from NEB; the high-fidelity enzyme PrimeSTAR HS DNA Polymerase and DNA Ligation Kit Ver.2.1 were both purchased from Takara Biotechnology (Beijing) Co., Ltd.; puromycin hydrochloride was purchased from Thermo Fisher Scientific; Cell Counting Kit-8 (CCK-8 kit) was purchased from Beyotime; GAPDH antibody was purchased from Proteintech; Mx2 antibody was purchased from abcam; UL34 antibody was stored in the laboratory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; HRP-labeled goat anti-mouse IgG and HRP-labeled goat anti-rabbit IgG were both purchased from CW Biotech Co., Ltd.

[0020] The analysis method is as follows: Statistical analysis was performed using GraphPad Prism 9.5.1 software. Independent samples t-test was used for significance analysis. ns. P>0.05 indicates no significant difference between data, *. P ≤ 0.05 indicates a significant difference between data, **. P ≤ 0.01 indicates a highly significant difference between data. ***. P ≤ 0.001 indicates an extremely high statistical significance of the difference between data. ****. P ≤ 0.0001.

[0021] Example 1 Construction of a PK-15 cell line stably expressing porcine Mx2 protein 1. Construction of recombinant lentiviral plasmid Specific primers were designed according to the nucleotide sequence of porcine Mx2 gene (accession number: AB258432.1) in the GeneBank database (see Table 1), and EcoR I and Xba I restriction enzyme cutting sites were introduced at the 5' and 3' ends of the primers respectively to amplify the target gene fragment. After digestion and purification of the lentiviral expression vector pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro and the target fragment, they were ligated using DNA Ligation Kit Ver.2.1; the ligation product was transformed into JM109 competent cells, and after picking monoclonal cultures, plasmids were extracted for sequencing verification, and finally the recombinant plasmid pLV-GFP-Mx2 was obtained.

[0022] To verify the correctness of the recombinant plasmid pLV-GFP-poMx2, use EcoR I and XbaThe double digestion experiment was carried out with restriction endonuclease I. The results of agarose gel electrophoresis showed that an obvious DNA band appeared at about 8000 bp, corresponding to the cut vector fragment; another band was about 2100 bp, which was consistent with the size of the target gene fragment. The experimental results indicated that the recombinant lentiviral plasmid pLV-GFP-poMx2 was successfully constructed ( Figure 1 ).

[0023] 2. Establishment of lentivirus packaging and stable porcine Mx2-expressing cell line HEK293T cells were inoculated into 6-well plates at a density of 1×10 6 cells / mL, 2 mL per well. When the cell density reached about 80%, plasmid co-transfection was carried out. A total of 2.5 μg of plasmid mixture was added to each well, and the specific ratio was pLV-GFP-Mx2:pSPAX2:pMD2.G = 4:3:1. After transfection, the cells were placed in an incubator at 37°C and 5% CO 2 . The fluorescence expression was observed under an inverted fluorescence microscope at 24 h and 48 h after transfection ( Figure 2 ), and the cell supernatant was collected 48 h after transfection. The cell debris was removed by centrifugation at 1200 r / min for 10 min, and the virus solution was aliquoted and stored at -80°C.

[0024] When the density of PK-15 cells reached 60% - 70%, the above-packaged virus solution was used for infection. After 24 h of lentivirus infection of PK-15 cells, a weak fluorescence signal was observed under an inverted fluorescence microscope ( Figure 3 A), indicating that the lentivirus successfully infected PK-15 cells. The culture was continued until 48 h after infection, and then replaced with DMEM complete medium containing 10% fetal bovine serum and continued to be cultured. After 2 - 3 d, the fluorescence expression was observed under an inverted fluorescence microscope. After the fluorescence expression was stable, the complete medium containing puromycin (2 μg / mL) was replaced for screening, and the untransfected PK-15 cells were used as the control group. The complete medium containing puromycin (2 μg / mL) was replaced every day for the first two days, and then changed to be replaced every 2 d with the complete medium containing puromycin (2 μg / mL) for continued screening. After continuous screening for one week, a strong fluorescence signal was observed under an inverted fluorescence microscope ( Figure 3 B), indicating that porcine Mx2 was stably expressed in PK-15 cells. The cell line was continuously screened with the complete medium containing puromycin (2 μg / mL) until all the control group cells died. The screened cells were continuously passaged for expansion culture and used for subsequent verification.

[0025] 3. Identification of stable porcine Mx2-expressing cell line The negative control PK-15-WT cells and the porcine Mx2 stable transfected cells PK-15-poMx2 were simultaneously inoculated into a 12-well plate. After the cells grew to confluence, they were washed three times with PBS, 200 μL of RIPA lysis buffer was added, and after ultrasonic disruption, 4× protein loading buffer was added. The proteins were denatured by heating at 98 °C for 10 min. 10 μL of the sample was added to each well of the protein electrophoresis gel, and electrophoresis was carried out at a constant voltage of 80 V for 30 min, at a constant voltage of 120 V for 1 h, and transferred at a constant current of 200 mA for 2 h. Blocking was carried out with 5% non-fat milk powder at room temperature for 2 h. Anti-Mx2 antibody (1:1000) was used as the primary antibody and incubated overnight at 4 °C, and Goat Anti-Rabbit IgG (1:5000) was used as the secondary antibody and incubated at room temperature for 2 h, and developed with an ECL chemiluminescence kit. The results showed that the porcine Mx2 protein level was significantly increased in the stable transfected cell line ( Figure 3 C).

[0026] Example 2 Detection of the activity of the cell line stably expressing porcine Mx2 The CCK-8 detection method was used to determine the cell activity. PK-15-WT cells and PK-15-poMx2 cells were respectively inoculated into 96-well cell culture plates. 10 μL of CCK-8 solution was added to the wells at different time points (0, 12, 24, 36, 48 h). Three replicate groups were set at each time point, and the plates were incubated in an incubator at 37 °C and 5% CO2 for 1 h. The OD values of each well were detected with a microplate reader at a wavelength of 450 nm, and a curve was plotted. The results showed that the cell viabilities of PK-15-WT cells and PK-15-poMx2 cells were basically the same ( Figure 4 ), indicating that overexpression of porcine Mx2 did not affect the viability of PK-15 cells.

[0027] Example 3 Evaluation of the antiviral effect of the PK-15 poMx2 stable transfected cell line against PRV To verify the effect of stable expression of porcine Mx2 on PRV replication, PK-15-WT cells and PK-15-poMx2 cells were respectively infected with PRV, and samples were collected 12 h later. RT-qPCR and Western blot were used to detect the replication of PRV. The specific operations were as follows: RT-qPCR method: PK-15-WT cells and PK-15-poMx2 cells were respectively inoculated into 6-well plates. After the cells grew to confluence, they were infected with PRV. Cell samples were collected 12 h after infection, and total cellular RNA was extracted and reverse transcribed into cDNA. Using the cDNA as a template, RT-qPCR detection was carried out. The porcine GAPDH gene was used as an internal reference gene, and the relative mRNA level of the PRV-gB gene was calculated. The primers used are shown in Table 1.

[0028] Western blot method: PK-15-WT cells and PK-15-poMx2 cells were respectively seeded in 6-well plates. After the cells grew to confluence, they were infected with PRV. Cell samples were collected 12 h post-infection, and total proteins were extracted after lysis. After 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred onto NC membranes. After blocking with 5% non-fat milk powder at room temperature for 2 h, primary antibodies (Mx2 antibody, UL34 antibody) were added and incubated overnight at 4 °C. After washing 3 times with PBST, secondary antibodies (HRP-labeled goat anti-mouse IgG, HRP-labeled goat anti-rabbit IgG) were added and incubated for 2 h, and then developed with an ECL chemiluminescence kit.

[0029] The results showed that, compared with PK-15-WT cells, the replication of PRV in the PK-15-poMx2 cell line was significantly inhibited, and its mRNA level and protein expression level were both significantly decreased ( Figure 5 Figures 5A and 5B).

[0030] In addition, to verify the effect of stable expression of porcine Mx2 on the PRV titer, PK-15-WT cells and PK-15-poMx2 cells were infected with PRV for 12 h, and then the virus fluids were harvested for TCID 50 detection. The operation was as follows: PK-15-WT cells and PK-15-poMx2 cells were respectively seeded in 6-well plates. After the cells grew to confluence, they were infected with PRV. When 80% of the cytopathic effect occurred, the 6-well plates were placed at -40 °C and frozen and thawed 3 times, and then centrifuged at 1000 r / min for 5 min to remove cell debris. The obtained samples were serially diluted from 10 -1 to 10 -10 with serum-free DMEM medium, and then respectively inoculated into 96-well plates with monolayer Vero cells. Each dilution was replicated 16 times, and 100 μL of virus fluid was added to each well. The plates were placed in an incubator at 37 °C and 5% CO 2 2, and the cytopathic effect was observed 72 h later. The TCID 50 of the amplified virus was calculated according to the Reed Muench method.

[0031] The results showed that the virus titer of PRV in PK-15 cells stably expressing porcine Mx2 was significantly lower than that of the control cells (P ≤ 0.05) ( Figure 5C) It shows that the stable overexpression of porcine Mx2 in vitro significantly inhibits the proliferation of PRV. In summary, the technology of constructing a cell line stably expressing the target gene using the lentiviral packaging system has been widely applied in life science research. Lentiviral vectors can integrate foreign genes into the host cell genome to achieve stable gene expression. Lentiviral vectors can generally accommodate foreign gene fragments of about 8 kb, which can meet the needs of introducing multiple genes or larger functional gene clusters. However, some small viral vectors such as adeno-associated virus vectors have limited ability to carry gene fragments and are difficult to meet such complex requirements. Therefore, in this study, the lentiviral packaging technology was used to construct a PK-15 cell line stably expressing porcine Mx2, and the protein expression level of poMx2 in the constructed cell line was analyzed by Western blot. The effects of the cell line on PRV replication were evaluated using real-time fluorescence quantitative PCR, Western blot, and TCID 50 The results showed that the protein expression level of porcine Mx2 in the constructed cell line was significantly increased, indicating that the PK-15 cell line stably expressing porcine Mx2 was successfully constructed. Compared with normal PK-15 cells, the replication of PRV in the PK-15-poMx2 stable transfected cell line was significantly inhibited, and its mRNA level, protein expression level, and progeny virus titer were all significantly decreased, indicating that porcine Mx2 protein has anti-PRV activity. This result not only verifies the key role of porcine Mx2 in antiviral infection but also shows that the porcine Mx2 stable transfected cell line can be used as an effective model for studying the regulation of host innate immune responses by porcine viruses. The successfully constructed PK-15 cell line stably expressing porcine Mx2 in this invention provides a powerful tool for studying the protein structure and function of porcine Mx2 and its antiviral mechanism, and also provides a new idea for the prevention and control of porcine viral diseases.

Claims

1. A PK-15 cell line stably expressing porcine Mx2 protein, characterized in that: The cell line integrates the porcine Mx2 gene into the PK-15 cell genome through a lentiviral vector system to achieve stable expression of the porcine Mx2 protein.

2. The PK-15 cell line according to claim 1, characterized in that The lentiviral vector system includes pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro vector, auxiliary packaging plasmid pMD2.G and psPAX2.

3. The PK-15 cell line according to claim 1, characterized in that The nucleotide sequence GeneBank accession number of the pig Mx2 gene is AB258432.

1.

4. The PK-15 cell line according to claim 1, characterized in that The cell line was obtained by puromycin selection.

5. Use of the PK-15 cell line stably expressing porcine Mx2 protein according to claim 1 in screening antiviral drugs or preparing a cell model for antiviral infection.

6. A method for constructing a PK-15 cell line stably expressing porcine Mx2 protein, characterized in that: The following steps are involved: (1) Construction of recombinant lentiviral plasmid pLV-GFP-poMx2; (2) Obtaining recombinant lentivirus through lentiviral packaging system; (3) The recombinant lentivirus was used to infect PK-15 cells, and a PK-15 cell line stably expressing porcine Mx2 was obtained by puromycin selection.

7. The method according to claim 6, characterized in that In step (1), the recombinant lentiviral plasmid pLV-GFP-poMx2 is obtained by connecting the lentiviral expression vector pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro with the porcine Mx2 gene, transforming the ligation product into JM109 competent cells, picking a single clone for culture, and extracting the plasmid for sequencing verification.

8. The method according to claim 6, characterized in that In step (2), the lentiviral packaging system is packaged using HEK293T cells, and the plasmid is used to transfect HEK293T cells. The recombinant plasmid pLV-GFP-Mx2, the packaging plasmid psPAX2 and pMD2.G are co-transfected into HEK293T cells, and the mass ratio of the three is pLV-GFP-Mx2:pSPAX2:pMD2.G = 4:3:1.

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