A human embryonic kidney cell line stably expressing ruminant SLAM protein and its construction method and application

Abstract

The present application relates to the technical field of genetic engineering, and specifically discloses a 293T cell line stably expressing ruminant SLAM protein, a construction method and application thereof, which comprises introducing an HA tag at the 3' end of a ruminant SLAM gene to obtain a SLAM-HA target gene, connecting the SLAM-HA target gene to a lentivirus vector CD513B-Cherry, transforming DH5a competent cells, extracting a positive plasmid to obtain a lentivirus vector plasmid; mixing the lentivirus vector plasmid with an auxiliary plasmid, and respectively transfecting 293T cells to obtain packaged SLAM lentivirus; and transducing the SLAM lentivirus into human renal epithelial cells to obtain a human renal epithelial cell line stably expressing ruminant SLAM protein. The present application successfully constructs a cell line stably expressing ruminant SLAM protein, which can obviously mediate the infection and proliferation of PPRV, and has potential for preparing a vaccine.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a human embryonic kidney cell line that stably expresses SLAM protein in ruminants, its construction method, and its applications. Background Technology

[0002] Peste des petits ruminants (PPR) is a highly contagious viral disease in small ruminants caused by the peste des petits ruminants virus (PPRV). It poses a significant threat to the health of economically important animals such as goats and sheep, as well as wild ruminants like blue sheep and white-tailed deer. The Food and Agriculture Organization of the United Nations and the World Organisation for Animal Health have designated PPR as a disease to be eradicated globally. Epidemiological research and vaccine development are the two most crucial aspects of controlling and eradicating PPR. Both virus isolation in epidemiological studies and strain propagation in vaccine development require the use of cell lines.

[0003] The receptor for peste des petits ruminants (PPRV) is SLAM, and SLAM in its natural host, ruminants, effectively mediates PPRV infection and proliferation. However, constructing cell lines that stably express SLAM from multiple ruminants is a technical challenge, and currently known methods are insufficient to construct cell lines that stably express SLAM proteins and efficiently mediate PPRV infection and proliferation. Therefore, there is an urgent need to develop a method for constructing cell lines that stably express ruminant SLAM proteins and efficiently mediate PPRV infection and proliferation. Summary of the Invention

[0004] To develop a method for constructing a cell line that stably expresses ruminant SLAM protein and efficiently mediates PPRV infection and proliferation, this invention provides a 293T cell line that stably expresses ruminant SLAM protein, its construction method, and its applications. This invention successfully constructed a cell line that stably expresses ruminant SLAM protein, which can significantly mediate PPRV infection and proliferation, and has the potential for use in vaccine preparation.

[0005] This invention provides a method for constructing a 293T cell line that stably expresses SLAM protein in ruminants, comprising the following steps:

[0006] Download the SLAM gene of the desired ruminant from the whole genome database of ruminants, introduce the HA tag into the 3' end of the SLAM gene to obtain the SLAM-HA target gene, then link the SLAM-HA target gene into the CD513B-Cherry lentiviral vector, transform DH5α competent cells, and extract positive plasmids to obtain lentiviral vector plasmids.

[0007] The CD513B-Cherry lentiviral vector construction process is as follows: the GFP fluorescent reporter gene of the CD513B plasmid is replaced with the Cherry fluorescent reporter gene, and an HTLV promoter is added in front of the Cherry gene to obtain the CD513B-Cherry lentiviral vector.

[0008] The obtained lentiviral vector plasmid was mixed with helper plasmids pMDLg / pRRE, pRSV-Rev, and pMD2.G and co-transfected into 293T cells to obtain packaged SLAM lentivirus.

[0009] After transducing SLAM lentivirus into human renal epithelial cells, a human renal epithelial cell line stably expressing ruminant SLAM protein was obtained.

[0010] Furthermore, the construction process of the lentiviral vector plasmid is as follows: using the lentiviral vector CD513B-Cherry as a template, fragment 1 is amplified using the upstream primer shown in SEQ ID NO.13 and the downstream primer shown in SEQ ID NO.14; fragment 3 is amplified using the upstream primer shown in SEQ ID NO.15 and the downstream primer shown in SEQ ID NO.16.

[0011] Then, using fusion PCR technology, fragment 1, the SLAM-HA target gene, and fragment 3 were fused, so that the SLAM-HA target gene was linked to the lentiviral vector CD513B-Cherry, transformed into DH5α competent cells, and positive plasmids were extracted to obtain the lentiviral vector plasmid.

[0012] Furthermore, the ruminant is any one of the following: Javan mouse deer, giraffe, musk deer, white-tailed deer, American antelope, and goat.

[0013] Furthermore, the SLAM gene sequence of the Javan mouse deer is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.7;

[0014] The SLAM gene sequence of the giraffe is shown in SEQ ID NO.2, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.8;

[0015] The SLAM gene sequence of the musk deer is shown in SEQ ID NO.3, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.9;

[0016] The SLAM gene sequence of the white-tailed deer is shown in SEQ ID NO.4, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.10.

[0017] The SLAM gene sequence of the American antelope is shown in SEQ ID NO.5, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.11;

[0018] The SLAM gene sequence of the goat is shown in SEQ ID NO.6, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.12.

[0019] Furthermore, when the ruminant is Javan mouse deer, the primer pairs for amplifying its SLAM-HA target gene are shown in SEQ ID NO.23-SEQ ID NO.24;

[0020] When the ruminant is a giraffe, the primer pairs for amplifying its SLAM-HA target gene are shown in SEQ ID NO.19-SEQ ID NO.20;

[0021] When the ruminant is the musk deer, the primer pairs for amplifying its SLAM-HA target gene are shown in SEQ ID NO.27-SEQ ID NO.28;

[0022] When the ruminant is the white-tailed deer, the primer pairs for amplifying its SLAM-HA target gene are shown in SEQ ID NO.21-SEQ ID NO.22;

[0023] When the ruminant is the American antelope, the primer pairs for amplifying its SLAM-HA target gene are shown in SEQ ID NO.25-SEQ ID NO.26;

[0024] When the ruminant is a goat, the primer pairs for amplifying its SLAM-HA target gene are shown in SEQ ID NO.17-SEQ ID NO.18.

[0025] Further, the SLAM lentivirus packaging process is as follows: the obtained lentiviral vector plasmid is mixed with helper plasmids pMDLg / pRRE, pRSV-Rev, and pMD2.G in a ratio of 3:1:1:1 to obtain a plasmid mixture. The plasmid mixture is then mixed with the transfection reagent TurboFact and incubated. The cell culture medium is then discarded, and the cells are washed with serum-free Opti-MEM medium. 1 mL of 2% FBSDMEM is then added. The incubated plasmid mixture is transfected and packaged, the culture medium is collected, centrifuged, and the supernatant is collected to obtain the packaged SLAM lentivirus.

[0026] Furthermore, the ratio of the plasmid mixture to the transfection reagent TurboFact is 3ug:6μL.

[0027] Furthermore, the centrifugation conditions were: 4℃ for 10 min.

[0028] This invention provides a SLAM lentivirus, which is packaged from the SLAM lentivirus described above.

[0029] The present invention also provides the application of the SLAM lentivirus described above in the preparation of PPR diagnostic reagents and / or vaccines.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1. The method provided in this invention constructs cell lines stably expressing SLAM proteins from Javan chevrotain, giraffe, musk deer, white-tailed deer, antelope, and goat, demonstrating the efficiency of SLAM-mediated PPRV infection and proliferation in Javan chevrotain, giraffe, musk deer, white-tailed deer, antelope, and goat. Compared to cells not expressing SLAM, HEK-293T cells expressing SLAM from these six ruminants significantly mediate PPRV infection and proliferation. Firstly, the lesioning time of PPRV in 293T-Slam cell lines is significantly longer than that in 293T-Cherry cells; secondly, the viral titer in 293T-Slam cell lines is approximately 100-fold higher than that in 293T-Cherry cells.

[0032] 2. The ruminant Slam gene was stably integrated into the 293T cell genome chromosome using a lentivirus system, resulting in good genetic stability and resistance to loss. The constructed 293T-Slam cell line grows rapidly and is easy to culture, meeting both small-scale needs for scientific research and large-scale needs for vaccine production. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 The lentiviral vector plasmid map obtained in Example 1 of this invention;

[0035] In the figure, A is the map of the lentiviral vector plasmid CD513B-Cherry-Goat Slam-HA obtained in Example 1;

[0036] B is a map of the lentiviral vector plasmid CD513B-Cherry-Giraffe Slam-HA obtained in Example 1;

[0037] C is a map of the lentiviral vector plasmid CD513B-Cherry-WTD Slam-HA obtained in Example 1;

[0038] D is a map of the lentiviral vector plasmid CD513B-Cherry-JMD Slam-HA obtained in Example 1;

[0039] E is a map of the lentiviral vector plasmid CD513B-Cherry-CFMD Slam-HA obtained in Example 1;

[0040] F is a map of the lentiviral vector plasmid CD513B-Cherry-ProngHorn Slam-HA obtained in Example 1.

[0041] Figure 2 This is a gel image of bacterial culture PCR identification of the lentiviral vector plasmid obtained in Example 1 of the present invention. + represents the positive control, i.e., the gel-recovered fragments of the SLAM gene of each species, and - represents the negative control, i.e., water.

[0042] In the figure, A represents the single-clone colony PCR identification results of lentiviral vector plasmids CD513B-Cherry-Goat Slam-HA and CD513B-Cherry-Giraffe Slam-HA. M1 represents marker D2000, lanes 1-5 represent five replicates of lentiviral vector plasmid CD513B-Cherry-Goat Slam-HA as a sample, and lanes 6-12 represent seven replicates of lentiviral vector plasmid CD513B-Cherry-Giraffe Slam-HA as a sample.

[0043] B represents the single-clone colony PCR identification result of the lentiviral vector plasmid CD513B-Cherry-WTD Slam-HA, where M1 represents marker D2000, and lanes 1-10 represent replicates of the lentiviral vector plasmid CD513B-Cherry-WTD Slam-HA.

[0044] C represents the single-clone colony PCR identification result of the lentiviral vector plasmid CD513B-Cherry-JMD Slam-HA, where lanes 1-13 represent replicates of the lentiviral vector plasmid CD513B-Cherry-JMD Slam-HA, and M2 represents marker D2000 plus.

[0045] D represents the single-clone colony PCR identification results of lentiviral vector plasmids CD513B-Cherry-ProngHorn Slam-HA and CD513B-Cherry-CFMDSlam-HA. Lanes 1-5 represent replicates of lentiviral vector plasmid CD513B-Cherry-ProngHorn Slam-HA, M2 represents marker D2000 plus, and lanes 6-8 represent replicates of lentiviral vector plasmid CD513B-Cherry-CFMDSlam-HA.

[0046] Figure 3 This is a fluorescence image of Cherry expression after co-transfection of 293T cells with the lentiviral vector plasmid and helper plasmid constructed in Example 1.

[0047] Figure 4 The positive clone after subcloning of the SLAM cell line obtained in Example 1 of this invention.

[0048] Figure 5 The expression of SLAM in the SLAM cell line constructed in Example 1 was identified by Western Blot.

[0049] Figure 6 The expression of SLAM in the SLAM cell line obtained in Example 1 was identified by IFA.

[0050] Figure 7 The cytopathic fluorescence images of different 293T-SLAM cell lines obtained in Example 1 after being infected with rPPRV-GFP.

[0051] Figure 8 The proliferation of rPPRV-GFP on different 293T-SLAMs obtained in Example 1 was observed using a fluorescence microscope.

[0052] Figure 9 To evaluate the proliferation of rPPRV-GFP on different 293T-SLAMs obtained in Example 1 using TCID50;

[0053] In the figure, A represents the collection of supernatants from each cell line at 24h, 48h, 72h, and 96h to determine the rPPRV-GFP titer;

[0054] B represents the determination of rPPRV-GFP titers in each cell line after repeated freeze-thaw cycles 72 hours post-infection. Detailed Implementation

[0055] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0056] Example 1: Construction of 293T cells stably expressing SLAM from different families of ruminants.

[0057] I. Experimental Materials and Instruments

[0058] 1. Cells, plasmids, and antibodies

[0059] 293T cells were preserved in our laboratory. The plasmid CD513B-Cherry used to construct the lentiviral vector, as well as the helper plasmids pMDLg / pRRE, pRSV-Rev, and pMD2.G, were all preserved in our laboratory. HA, β-Tubulin, and Cherry protein-specific antibodies were purchased from Cell Signaling Technology (CST) and Wuhan Sanying Biotechnology Co., Ltd.

[0060] 2. Main reagents

[0061] Various restriction endonucleases and T4 ligases were purchased from NEB (Nexis Biosciences, Inc.); TurboFect™ Transfection Reagent was purchased from Thermo Fisher Scientific; Opti-MEM medium, fetal bovine serum, and DMEM medium were purchased from Gibco; PrimerSTAR MAX high-fidelity polymerase was purchased from TaKaRa Biosciences Co., Ltd.; and DNA gel extraction kit and plasmid mini-extraction kit were purchased from OMEGA.

[0062] 3. Main Instruments

[0063] Clean bench (Jinan Xinbeixi BIOBASE BBS-DDC); UV gel imaging system (Syngene, UK); protein electrophoresis system (Beijing Junyi Dongfang); inverted fluorescence microscope (OLYMP USDP80); autoclave (Shanghai Boxun); standard PCR instrument (Bori TC-XP).

[0064] II. Experimental Methods

[0065] 1. Obtaining target genes

[0066] Ruminants include animals from the families of cherubidium, giraffe, musk deer, deer, pronghorn, and bovidae. Representative animals from each family were selected: Javan mouse deer (JMD), giraffe, Chinese forest musk deer (CFMD), white-tailed deer (WTD), prawn (PH), and goat. The SLAM genes of these six species were downloaded from a ruminant genome database (as shown in Table 1), and the amino acid sequences of the SLAM proteins for the six species are shown in Table 2. Due to the lack of specific antibodies for the SLAM of these species, this invention introduces an HA tag sequence at the 3' end of the SLAM gene, denoted as the SLAM-HA gene, for easier subsequent identification. The sequence was sent to Beijing Qingke Biotechnology Co., Ltd. for synthesis.

[0067] Table 1. Nucleotide sequences of SLAM proteins from six species.

[0068]

[0069]

[0070]

[0071] Table 2. Amino acid sequences of SLAM proteins from six species.

[0072]

[0073]

[0074] 2. Construction of lentiviral vector plasmid CD513B-Cherry-SLAM HA

[0075] Primers were designed based on the six target genes and the vector nucleotide sequence. First, the three fragments required for fusion PCR were amplified, namely fragment 1, fragment 2 and fragment 3. Fragment 1 was denoted as F1, fragment 2 as F2 and fragment 3 as F3. Fragment 1 is the sequence between the SgrAI restriction site and T2A in the CD513B-Cherry vector, and fragment 3 is the fragment between the cherry gene and KpnI (T2A is located between the cherry gene and SLAM-HA in the vector). Fragment 2 is the SLAM-HA gene of ruminants, selected from any one of Goat-F2, Giraffe-F2, WTD-F2, JMD-F2, PH-F2, and CFMD-F2. Using fusion PCR technology and two restriction enzyme sites (SgrAI and KpnI), the fusion fragment (SLAM-HA) containing these sites was ligated into the lentiviral vector CD513B-Cherry. This was transformed into DH5α competent cells, and single colonies were picked for colony PCR identification. Plasmids were extracted from positive bacterial colonies and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The correctly sequenced lentiviral vector plasmids were named CD513B-Cherry-JMD Slam-HA, CD513B-Cherry-Giraffe Slam-HA, CD513B-Cherry-CFMD Slam-HA, CD513B-Cherry-WTD Slam-HA, CD513B-Cherry-ProngHorn Slam-HA, and CD513B-Cherry-Goat, respectively. Slam-HA. In these vectors, SLAM is linked to the Cherry gene via T2A; theoretically, expressing Cherry also expresses SLAM, facilitating subsequent screening of positive cells. The vector maps of the six constructed lentiviral vectors are shown below. Figure 1 As shown. The specific construction steps are as follows:

[0076] (1) Primer design

[0077] SLAM sequences from six ruminants from different families—Java Mouse Deer (JMD), Giraffe, Chinese Forest Musk Deer (CFMD), White Tail Deer (WTD), Prong Horn (PH), and Goat—were analyzed. Specific primers were designed based on the CD513B-Cherry restriction site of the lentiviral vector. The upstream primer for fragment 1 introduced the SgrAI restriction site, fragment 2 contained the SLAM-HA gene of the six ruminants, and the downstream primer for fragment 3 introduced the KpnI restriction site. The target gene was cloned by fusion PCR. The primers were synthesized at Xi'an Qingke Biotechnology Co., Ltd. Primer information is shown in Table 3.

[0078] Table 3 Primer sequences

[0079]

[0080] (2) Amplification and fusion of fragment 1, fragment 2 and fragment 3

[0081] Using CD513B-Cherry as a template, fragment 1 was amplified using the upstream primer shown in SEQ ID NO. 13 and the downstream primer shown in SEQ ID NO. 14. Using the SLAM-HA gene of each ruminant as a template, fragment 2 of different ruminants was amplified using the primers shown in SEQ ID NO. 17-SEQ ID NO. 28 in Table 3, representing the SLAM-HA genes of different ruminants. For the Javan mouse deer, the primer pairs for amplifying its SLAM-HA gene are shown in SEQ ID NO. 23-SEQ ID NO. 24; for the giraffe, the primer pairs are shown in SEQ ID NO. 19-SEQ ID NO. 20; for the musk deer, the primer pairs are shown in SEQ ID NO. 27-SEQ ID NO. 28; and for the white-tailed deer, the primer pairs are shown in SEQ ID NO. 21-SEQ ID NO. 28. As shown in NO.22; when the ruminant is the American antelope, the primer pairs for amplifying its SLAM-HA target gene are shown in SEQ ID NO.25-SEQ ID NO.26; when the ruminant is the goat, the primer pairs for amplifying its SLAM-HA target gene are shown in SEQ ID NO.17-SEQ ID NO.18.

[0082] Fragment 3 was obtained by amplification using CD513B-Cherry as a template and the upstream primer shown in SEQ ID NO.15 and the downstream primer shown in SEQ ID NO.16.

[0083] Using fragments 1, 2, and 3 simultaneously as templates, the fusion fragments were amplified using fusion PCR technology with the upstream primer shown in SEQ ID NO. 13 and the downstream primer shown in SEQ ID NO. 16. The amplification system is shown in Table 4, and the amplification program is shown in Table 5.

[0084] Table 4 PCR amplification system

[0085] Components volume PrimerSTAR MAX DNA Polymerase (2×) 20μL Upstream primer F (10 μM) 1μL Downstream primer R (10 μM) 1μL template 1μL ddH2O 17μL Total volume 40μL

[0086] Table 5 PCR Procedure

[0087]

[0088] 3. PCR identification of lentiviral vector plasmids

[0089] The results are as follows Figure 2 As shown, the target gene SLAM-HA was successfully ligated into the lentiviral vector plasmid CD513B-Cherry. Lentiviral vector plasmids CD513B-Cherry-JMD Slam-HA, CD513B-Cherry-Giraffe Slam-HA, CD513B-Cherry-CFMD Slam-HA, CD513B-Cherry-WTD Slam-HA, CD513B-Cherry-ProngHorn Slam-HA, and CD513B-Cherry-Goat Slam-HA were obtained. The vector maps of the six lentiviral vector plasmids are shown below. Figure 1 As shown.

[0090] 4. Packaging of lentiviral vector plasmids

[0091] The constructed lentiviral vector plasmids expressing SLAM of the above six species, CD513B-Cherry-JMD Slam-HA, CD513B-Cherry-Giraffe Slam-HA, CD513B-Cherry-CFMD Slam-HA, CD513B-Cherry-WTDSlam-HA, CD513B-Cherry-ProngHorn Slam-HA, and CD513B-Cherry-Goat Slam-HA, were mixed with helper plasmids pMDLg / pRRE, pRSV-Rev, and pMD2.G in a ratio of 3:1:1:1, and then transfected into 293T cells.

[0092] The specific procedure is as follows: 293T cells were evenly seeded in 24-well cell plates, and transfected when the density reached 80%. Recombinant plasmids CD513B-Cherry-JMD Slam-HA, CD513B-Cherry-Giraffe Slam-HA, CD513B-Cherry-CFMD Slam-HA, CD513B-Cherry-WTD Slam-HA, CD513B-Cherry-ProngHorn Slam-HA, and CD513B-Cherry-Goat Slam-HA were mixed with helper plasmids pMDLg / pRRE, pRSV-Rev, and pMD2.G in a 3:1:1:1 ratio to obtain a 3 μg plasmid mixture. This mixture was then mixed with 6 μL of TurboFact transfection reagent and incubated for 15 min. The cell culture medium was discarded, and the cells were washed twice with 300 μL of serum-free Opti-MEM medium, followed by 1 mL of 2% FBSDMEM. The incubated plasmid mixture was gently and evenly added to the cell wells and placed in a cell culture incubator. 16 hours after transfection, lentivirus packaging solution was added and cultured for 72 hours. 1 mL of cell supernatant was collected, centrifuged at 4°C for 10 minutes, and the supernatant was collected to obtain six packaged SLAM lentiviruses.

[0093] Six lentiviral vectors expressing SLAM were co-transfected with helper plasmids into 293T cells. Cherry expression was observed under a fluorescence microscope 72 hours later. Results are as follows: Figure 3 As shown, 293T cells successfully expressed the Cherry protein of lentivirus, indicating that lentivirus was packaged.

[0094] 5. Lentiviral transduction and screening of positive cells

[0095] Healthy 293T cells were evenly seeded into 6-well cell culture plates. After 12 hours, when confluence reached approximately 60%, the supernatant was discarded. The six SLAM lentiviruses prepared in the previous steps were mixed with DMEM medium containing 10% fetal bovine serum at a ratio of 1 mL:1 mL. Polybrene (hexammonium bromide) was added at a ratio of 1:5000, and the mixture was gently added to the 6-well cell culture plates. After 72 hours of transduction, the medium was replaced with a 10-fold selection concentration (30 μg / mL) and changed every 3 days. After 6 days, the medium was replaced with a normal selection concentration (3 μg / mL) complete medium and changed every 3 days. After 9 days, the cells in the infected group were confirmed to be confluent based on Cherry expression under a fluorescence microscope, while all cells in the control group had died. The cells were digested, counted, and diluted to a concentration of 10 cells / mL. The cells were then seeded into 96-well cell culture plates, with 100 μL of cell dilution added to each well to ensure that each well contained only one cell. 96-well plates were cultured in a 37°C, 5% CO2 incubator, with culture medium replenished every 3 days. After approximately 12 days, the cells were observed under a microscope. Cells from wells expressing Cherry protein and exhibiting only a single cell cluster were selected, digested with trypsin, and seeded into 24-well plates for expansion culture. Once the cells reached confluence, they were passaged, and Western blotting and indirect immunofluorescence were performed to identify SLAM protein expression (control group: 293T-Cherry). One positive clone from each 293T-SLAM cell line was selected for passage culture. The 293T cells expressing these six SLAM species were named 293T-Goat SLAM, 293T-Giraffe SLAM, 293T-WTDSLAM, 293T-JMD SLAM, 293T-PH SLAM, and 293T-CFMD SLAM, respectively, and the cells were cryopreserved.

[0096] 6. Determine the minimum killing concentration of puromycin against 293T cells.

[0097] 293T cells were seeded in 24-well plates and cultured to 80% confluence with DMEM medium containing 10% fetal bovine serum. Then, the cells were cultured in DMEM medium containing different concentrations of puromycin and 2% fetal bovine serum. The different puromycin concentrations were defined as: 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, and 10 μg / mL, with three replicates for each concentration. The medium was changed daily for four consecutive days. The lowest puromycin concentration in the medium where all cells died after 4 days was determined to be the optimal puromycin selection concentration. The results showed that all cells died on day 4 in a medium with a concentration of 3 μg / mL. Therefore, the minimum lethal puromycin concentration for 293T cells was determined to be 3 μg / mL, which was also the selection concentration for positive cells.

[0098] 7. Specific steps for Western blotting:

[0099] (1) Protein sample collection: Six 293T-SLAM cells expressing different ruminant SLAMs were seeded in 12-well plates. When the cell density reached more than 90%, cell proteins were collected. To prevent protein degradation, protease inhibitors were added to the cell lysis buffer. The liquid in the wells containing the cells to be collected was discarded. The cells were washed twice with PBS, and 80 μL of lysis buffer containing PMSF was added. After all cells were lysed into a fluid state, they were collected into EP tubes and lysed on ice for 10 min. Then, 20 μL of 1× protein loading buffer was added, and the tubes were boiled in a metal bath at 100°C for 10 min.

[0100] (2) Electrophoresis: Prepare a 10% protein separating gel. Use a pipette to load 10 μL of sample into each well, and add an appropriate amount of protein marker. Perform electrophoresis on the stacking gel and separating gel using voltages of 80V and 120V respectively. Stop electrophoresis when the bromophenol blue reaches near the bottom of the gel.

[0101] (3) Transfer: The PVDF membrane must be activated with methanol for 1 minute before use. The protein glue is applied to the activated PVDF membrane. The membrane is transferred in the direction of negative electrode-protein glue-PVDF membrane-positive electrode. After the clamp is fixed, it is placed in the transfer tank and pre-cooled transfer solution is added. The transfer tank is placed in an ice bath, and a constant current of 100mA is set. The transfer time is 2h.

[0102] (4) Sealing: After the transfer is completed, the PVDF membrane is placed in the sealing solution for sealing. The box containing the sealing solution is placed on the decolorizing shaker and shaken slowly. After sealing for 2 hours, the sealing solution is washed away with TBST.

[0103] (5) Primary antibody incubation: Dilute the primary antibody to an appropriate working concentration with TBST. Place the PVDF membrane with transferred protein into the corresponding primary antibody (the antibodies used include HA, Cherry, and β-Tubulin specific antibodies) and incubate overnight at 4°C. After incubation, recover the primary antibody. Add TBST and wash on a shaker at 80 rpm for 6–8 min. Discard the washing buffer, add TBST again and wash for 8 min. Repeat the washing 5 times.

[0104] (6) Secondary antibody incubation: Select the corresponding secondary antibody based on the primary antibody, dilute the secondary antibody to an appropriate working concentration with TBST, place the membrane in the secondary antibody, and incubate on a shaker at room temperature for 1 hour. After incubation, recover the secondary antibody. Add TBST, shake and wash for 8 minutes at 80 rpm on a shaker, discard the washing buffer, add TBST again and wash for 8 minutes, repeating the washing 5 times.

[0105] (7) Protein detection: Prepare ECL luminescent solution for color development and expose it in the imaging system.

[0106] Specific steps of indirect immunofluorescence assay (IFA):

[0107] When the density of 293T-SLAM cells in the overnight cultured 96-well plates reached 80%, IFA was performed. A rabbit-derived monoclonal antibody against HA protein was selected to detect SLAM expression. The secondary antibody was goat anti-rabbit IgG labeled with the 488 fluorescent tag. The specific steps are as follows:

[0108] (1) Fixation: Discard the culture medium, wash twice with PBS, add 50 μL of 4% paraformaldehyde to the well to cover the cells, and do not allow the cells to dry during the process. After fixing for about 30 minutes, discard the paraformaldehyde and wash 3 times with PBS.

[0109] (2) Permeabilization: Discard the PBS, add 50 μL of 0.2% Triton X-100 solution to treat the cells for 5 min, discard the liquid, and wash the cells with PBS;

[0110] (3) Blocking: Discard PBS, add 50 μL of pre-prepared 1% BSA and place in a 37℃ incubator for 30 min;

[0111] (4) Incubation of primary antibody: Dilute the primary antibody to working concentration using 1% BSA according to the monoclonal antibody instructions, discard the BSA from the previous step, add 40 μL of primary antibody, and incubate overnight at 4°C.

[0112] (5) Washing: Wash the cells 5 times with PBS and gently shake the cell plate for 5 minutes each time to remove unbound primary antibody;

[0113] (6) Incubation of secondary antibody: Dilute the fluorescent secondary antibody to working concentration with 1% BSA, add 50 μL to the cell wells, wrap the cell plate with aluminum foil to protect it from light, and incubate at 37°C for 1 h.

[0114] (7) Washing: Discard the secondary antibody solution, wash the cells with PBS and gently shake the cell plate 5 times for 5 minutes each time to wash away the unbound secondary antibody;

[0115] (8) Staining cell nuclei: Dilute Hoechst 33342 solution at a ratio of 1:500 to obtain the working concentration, add 50 μL to the cell wells, and incubate in the dark for 15 min;

[0116] (9) Washing: Discard the Hoechst 33342 solution, wash the cells three times with PBS and gently shake the cell plate for 2 min each time, and finally add 50 μL of PBS to the wells;

[0117] (10) Observe the luminescence under an inverted fluorescence microscope and compare it with the control group to determine the gene expression.

[0118] Example 2: Application of the constructed 293T cells that stably express SLAM in ruminants.

[0119] I. Experimental Materials

[0120] (1) 293T cells that stably express SLAM of different families of ruminants were prepared in Example 1, namely 293T-SLAM cell lines: 293T-Goat SLAM, 293T-Giraffe SLAM, 293T-WTD SLAM, 293T-JMD SLAM, 293T-PHSLAM, and 293T-CFMD SLAM.

[0121] (2) The construction process of the 293T-Cherry cell line is as follows: The lentiviral vector plasmid CD513B-Cherry is mixed with helper plasmids pMDLg / pRRE, pRSV-Rev, and pMD2.G in a ratio of 3:1:1:1 to obtain a 3ug plasmid mixture. The plasmid mixture is then mixed with 6μL of transfection reagent TurboFact. After incubation, the cell culture medium is discarded, and 300μL of serum-free Opti-MEM medium is added to wash twice. Then, 1mL of 2% FBS DMEM is added. The incubated plasmid mixture is gently and evenly added to the cell wells. 16h after transfection, lentiviral packaging solution is added, and the cell culture is carried out for 72h. 1mL of cell supernatant is collected, centrifuged at 4℃ for 10min, and the supernatant is collected to obtain the packaged Cherry lentivirus. Healthy 293T cells were evenly seeded into 6-well cell culture plates. After 12 hours, when confluence reached approximately 60%, the supernatant was discarded. The prepared Cherry lentivirus was mixed 1 mL:1 mL with DMEM medium containing 10% fetal bovine serum, and Polybrene (hexammonium bromide) was added at a 1:5000 ratio. After gentle mixing, the mixture was added to the 6-well cell culture plates. 72 hours after transduction, the medium was replaced with a 10-fold selection concentration (30 μg / mL) and changed every 3 days. After 6 days, the medium was replaced with a normal selection concentration (3 μg / mL) complete medium and changed every 3 days. After 9 days, the cells were confirmed to be confluent based on Cherry expression under a fluorescence microscope, while all cells in the control group had died. The cells were digested, counted, and diluted to a concentration of 10 cells / mL. The cells were then seeded into 96-well cell culture plates, with 100 μL of cell dilution added to each well to ensure only one cell per well. The 96-well plates were cultured in a 37°C, 5% CO2 cell culture incubator, with culture medium replenished every 3 days. After about 12 days, the cells were observed under a microscope. Cells expressing Cherry protein and forming only one cell cluster were selected from the wells. After being digested with trypsin, the cells were seeded into 24-well plates for expansion culture. After the cells reached confluence, they were passaged to obtain the 293T-Cherry cell line.

[0122] (3) rPPRV-GFP is a recombinant virus rescued using the PPRV reverse genetics system established in previous laboratory studies. The specific construction method is as follows:

[0123] First, RNA was extracted from the PPRV vaccine strain Nigeria / 75 / 1 according to the Trizol RNAiso Plus reagent instructions. Then, cDNA was obtained according to the StarScript II First-strand cDNA Synthesis Mix reverse PCR kit instructions. Using the cDNA as a template, PCR was performed to ligate the obtained whole-genome fragment into the PCMV-add vector (this pCMV-add vector is obtained by replacing the T7 promoter with the CMV promoter and introducing the HDVribozyme sequence from the pBluescript SK plasmid), thus obtaining the recombinant plasmid pT7-PPRV containing the whole genome of PPRV Nigeria / 75 / 1. Based on this plasmid, a green fluorescent protein (GFP) gene was inserted between the P and M genes to construct the full-length PPRV plasmid pT7-PPRV-GFP with the chimeric GFP gene. Using plasmid pT7-PPRV as a template, the gene sequences of PPRV Nigeria / 75 / 1 nucleoprotein (NP), phosphoprotein (P), and large polymerase protein (L) were amplified, and the amplified fragments were cloned into the pCI-neo eukaryotic expression plasmid to obtain the helper plasmid PPRV-NPL. The three plasmids pT7-PPRV-GFP, PPRV-NPL, and pCAGGS-T7 (obtained by cloning the T7RANA polymerase sequence into the pCAGGS eukaryotic vector) were co-transfected into BHK21 cells at a mass ratio of 2:2:1. Cells were observed under a fluorescence microscope until they expressed a large amount of GFP and produced cytopathic effects, thus obtaining the rPPRV-GFP strain.

[0124] II. Experimental Methods

[0125] 1. Observe the cytopathic effects produced by rPPRV-GFP infection of the six 293T-SLAM cells obtained in Example 1.

[0126] Six strains of 293T-SLAM cells obtained in Example 1 were seeded into 24-well plates. When the cell density reached 90%, the culture medium was discarded, and the cells were washed twice with pure DMEM medium. rPPRV-GFP was diluted with an infection index of 2 MOI and added to the cell wells. The plates were incubated at 37°C, with the plates shaken every 30 minutes. After 2 hours, the virus solution was discarded, and the medium was replaced with DMEM containing 2% fetal bovine serum. Forty-eight hours post-infection, viral GFP expression and the size of syncytia produced by the cells were observed under a microscope. 293T-Cherry cells were used as a control group.

[0127] The results are as follows Figure 7As shown, after infection, microscopic observation revealed that 293T-Cherry cells could not form syncytia, but SLAM-expressing cells fused together to form distinct syncytia.

[0128] 2. Proliferation of rPPRV-GFP in six 293T-SLAM cell lines

[0129] Six types of SLAM-293T cells obtained in Example 1 were seeded with 1 mol rPPRV-GFP and cultured in 1 mL of DMEM medium containing 2% fetal bovine serum. Every 24 h after infection, 100 μL of supernatant was collected to measure TCID50 (half-tissue culture infection dose), and 100 μL of medium was added to the cell wells until 96 h. Viral GFP expression was observed under a fluorescence microscope at each time point. The specific steps for measuring TCID50 were as follows: DogSLAM-Vero cells constructed in the laboratory were seeded at 1 × 10⁻⁶ cells... 4 Virus supernatants were inoculated into 96-well plates at a density of 100 cells / well and incubated overnight. The collected viral supernatant was serially diluted 10-fold with pure MEM in sterile 1.5 mL centrifuge tubes, starting from 10... -1 Up to 10 -10 The cells were seeded in 96-well plates, with 100 μL seeded in 3 wells for each concentration gradient. A blank control group was set up with only pure MEM seeded. After culturing in an incubator at 37°C for 4 days, the expression of viral GFP was observed under an inverted fluorescence microscope. The TCID50 of rPPRV-GFP was calculated according to the Reed-Muench method and the growth curve was plotted. 293T-Cherry cells were used as a control group.

[0130] The results are as follows Figure 8 As shown, although 293T-Cherry cells exhibited green fluorescence after infection, and this fluorescence increased after 72 hours, the fluorescence intensity was significantly lower than that of the six SLAM cell lines, and no obvious syncytia were produced. Figure 9 As shown, the viral titer in 293T-Cherry cells after PPRV infection was only 1×10⁻⁶. 3.75 TCID50 / mL; however, in cell lines expressing SLAM, the viral titer reached 1×10⁻⁶. 5 TCID 50 / mL or higher, with 293T-Cherry-JMD titers reaching as high as 1×10⁻⁶. 5.75 The TCID50 / mL viral titer was 100 times that of 293T-Cherry cells.

[0131] In summary, this invention provides the amino acid sequences of SLAM proteins from Javan chevrotain, giraffe, musk deer, white-tailed deer, antelope, and goat. Stable cell lines expressing SLAM proteins from these six ruminants were constructed. The efficiency of SLAM proteins from these animals in mediating PPRV infection and proliferation was elucidated. The advantages are that, compared to cells without SLAM expression, HEK-293T cells expressing SLAM proteins from these six ruminants significantly mediate PPRV infection and proliferation. Firstly, the lesioning time of PPRV in 293T-Slam cell lines is significantly longer than that in 293T-Cherry cells. Secondly, the viral titer in 293T-Slam cell lines is approximately 100-fold higher than that in 293T-Cherry cells. Furthermore, the ruminant Slam gene was stably integrated into the 293T cell genome chromosome using a lentivirus system, resulting in good genetic stability and resistance to loss. In addition, the constructed 293T-Slam cell line grows rapidly and is easy to culture, meeting both small-scale needs for scientific research and large-scale needs for vaccine production.

[0132] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0133] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for constructing a 293T cell line stably expressing ruminant SLAM protein, characterized in that, Includes the following steps: Download the SLAM gene of the desired ruminant from the whole genome database of ruminants, introduce the HA tag into the 3' end of the SLAM gene to obtain the SLAM-HA target gene, then link the SLAM-HA target gene into the CD513B-Cherry lentiviral vector, transform DH5α competent cells, and extract positive plasmids to obtain lentiviral vector plasmids. The CD513B-Cherry lentiviral vector construction process is as follows: the GFP fluorescent reporter gene of the CD513B plasmid is replaced with the Cherry fluorescent reporter gene, and an HTLV promoter is added in front of the Cherry gene to obtain the CD513B-Cherry lentiviral vector. The obtained lentiviral vector plasmid was mixed with helper plasmids pMDLg / pRRE, pRSV-Rev, and pMD2.G and co-transfected into 293T cells to obtain packaged SLAM lentivirus. After transducing SLAM lentivirus into human renal epithelial cells, a human renal epithelial cell line stably expressing ruminant SLAM protein was obtained; The ruminants are any one of the following: Javan mouse deer, giraffe, musk deer, white-tailed deer, American antelope, and goat; The SLAM gene sequence of the Javan mouse deer is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.7; The SLAM gene sequence of the giraffe is shown in SEQ ID NO.2, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.8; The SLAM gene sequence of the musk deer is shown in SEQ ID NO.3, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.9; The SLAM gene sequence of the white-tailed deer is shown in SEQ ID NO.4, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

10. The SLAM gene sequence of the American antelope is shown in SEQ ID NO.5, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

11. The SLAM gene sequence of the goat is shown in SEQ ID NO.6, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

12.

2. The method for constructing a 293T cell line stably expressing ruminant SLAM protein according to claim 1, characterized in that, The lentiviral vector plasmid was constructed as follows: using the lentiviral vector CD513B-Cherry as a template, fragment 1 was amplified using the upstream primer shown in SEQ ID NO.13 and the downstream primer shown in SEQ ID NO.14; fragment 3 was amplified using the upstream primer shown in SEQ ID NO.15 and the downstream primer shown in SEQ ID NO.

16. Then, using fusion PCR technology, fragment 1, the SLAM-HA target gene, and fragment 3 were fused, so that the SLAM-HA target gene was linked to the lentiviral vector CD513B-Cherry, transformed into DH5α competent cells, and positive plasmids were extracted to obtain the lentiviral vector plasmid.

3. The method for constructing a 293T cell line stably expressing ruminant SLAM protein according to claim 1, characterized in that, When the ruminant is Javan mouse deer, the primer pairs for amplifying its SLAM-HA target gene are shown in SEQ ID NO.23-SEQ ID NO.24; When the ruminant is a giraffe, the primer pairs for amplifying its SLAM-HA target gene are shown in SEQ ID NO.19-SEQ ID NO.20; When the ruminant is the musk deer, the primer pairs for amplifying its SLAM-HA target gene are shown in SEQ ID NO.27-SEQ ID NO.28; When the ruminant is the white-tailed deer, the primer pairs for amplifying its SLAM-HA target gene are shown in SEQ ID NO.21-SEQ ID NO.22; When the ruminant is the American antelope, the primer pairs for amplifying its SLAM-HA target gene are shown in SEQ ID NO.25-SEQ ID NO.26; When the ruminant is a goat, the primer pairs for amplifying its SLAM-HA target gene are shown in SEQ ID NO.17-SEQ ID NO.

18.

4. The method for constructing a 293T cell line stably expressing ruminant SLAM protein according to claim 1, characterized in that, The SLAM lentivirus packaging process is as follows: The obtained lentiviral vector plasmid is mixed with helper plasmids pMDLg / pRRE, pRSV-Rev, and pMD2.G in a ratio of 3:1:1:1 to obtain a plasmid mixture. The plasmid mixture is then mixed with the transfection reagent TurboFact and incubated. The cell culture medium is then discarded, and the cells are washed with serum-free Opti-MEM medium. 1 mL of 2% FBS DMEM is then added. The incubated plasmid mixture is transfected and packaged. The culture medium is collected, centrifuged, and the supernatant is collected to obtain the packaged SLAM lentivirus.

5. The method for constructing a 293T cell line stably expressing ruminant SLAM protein according to claim 4, characterized in that, The ratio of the plasmid mixture to the transfection reagent TurboFact was 3 μg: 6 μL.

6. The method for constructing a 293T cell line stably expressing ruminant SLAM protein according to claim 4, characterized in that, Centrifugation conditions: 4 ℃ for 10 min.

7. A SLAM lentivirus, characterized in that, Obtained by the method described in claim 4.

8. The use of the SLAM lentivirus of claim 7 in the preparation of PPR diagnostic reagents and / or vaccines.

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