Immortalized alpaca kidney cell line as well as construction method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an immortalized alpaca kidney cell line and a construction method and application thereof. The immortalized alpaca kidney cell line disclosed by the invention is named as immortalized alpaca kidney cells (Alpaca kidney cells) BNalpaca 1.0, and the preservation number of the immortalized alpaca kidney cell line is CGMCC (China General Microbiological Culture Collection Center) No.46058. The construction method of the immortalized alpaca kidney cell line comprises the following steps: extracting and culturing alpaca primary kidney cells; infecting alpaca primary kidney cells by using SV40 large T antigen immortalized lentivirus; carrying out resistance screening on the immortalized alpaca kidney cells; carrying out alpaca kidney cell immortalized cell detection and identification; and obtaining the immortalized alpaca kidney cell line. The immortalized alpaca kidney cell constructed by the invention has the advantages of simple culture, repeated passage, good homogeneity, high expression quantity of constructed exogenous genes and the like, and provides a more powerful tool cell for research on alpaca immunity; the immortalized alpaca kidney cell line provides a new tool for virus infection mechanism, drug in-vitro function detection and antibody drug development and screening.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an immortalized alpaca kidney cell line and a construction method and application thereof. Background Art

[0002] Primary cells reflect the pathological and physiological state of a certain type of cells in the body more realistically than ordinary cell lines, and are widely used in research fields such as embryonic development, tissue maintenance and remodeling, and tumorigenesis mechanisms. However, primary cells are often only able to grow stably for a few specific generations, and continuous propagation cannot be guaranteed, which is not conducive to in vitro cell experiments. Immortalized cells can enable cells to acquire the ability to continue to grow and proliferate.

[0003] Cell immortalization refers to the process in which cells cultured in vitro escape from the proliferation and aging crisis due to their own changes or the influence of external conditions, thereby acquiring unlimited proliferation ability. However, the probability of spontaneous cell immortalization is very low. The construction of immortalized cell lines mainly adopts exogenous induction methods, including chemicals, radiation mutagenesis, viral infection, telomerase activation, etc. At present, there are very few reports on the immortalization of alpaca kidney cells.

[0004] Currently, the commonly used immunization methods for alpacas are protein immunization and peptide immunization, and there are no special alpaca tool cells to assist in completing cellular immunity.

[0005] Alpaca kidney cells can also be used for virus research. By observing the growth and replication of viruses in alpaca kidney cells, we can understand the characteristics of viruses, including the virus infection cycle and proliferation rate, which is helpful for studying the mechanism of viral infection. In the process of drug development, alpaca kidney cells can be used as an in vitro model to test the toxicity of drugs. Newly developed drugs may have an impact on kidney cells. By applying drugs to alpaca kidney cells and observing changes in cell morphology, activity, metabolism, etc., the safety and effectiveness of drug development can be improved.

[0006] Therefore, if an immortalized alpaca kidney cell line can be obtained, it will promote the research and application of alpaca kidney cells in cellular immunity, viral infection mechanisms, and drug development processes. Summary of the invention

[0007] The purpose of the present invention is to fill the gap in the prior art and provide an immortalized alpaca kidney cell line and a construction method and application thereof.

[0008] The inventive concept of the present invention is as follows: Simian vacuolating virus 40 (SV40) is used to infect primary alpaca kidney cells, so that they are separated from the normal aging and death process and acquire the ability to continuously divide, and finally immortalized alpaca kidney cells are constructed with the advantages of simple culture, multiple passages, and good uniformity.

[0009] The technical solution of the present invention to solve the technical problem is as follows:

[0010] In a first aspect of the present invention, an immortalized alpaca kidney cell line is provided, the name of which is immortalized alpaca kidney cells (Alpaca kidney cells) BNalpaca 1.0, and the deposit number thereof is: CGMCC No.46058.

[0011] In the second aspect of the present invention, a method for constructing the immortalized alpaca kidney cell line as described in the first aspect is provided. The construction method comprises the following steps:

[0012] S1: Extraction and culture of primary alpaca kidney cells;

[0013] S2: Infection of primary alpaca kidney cells with SV40 immortalized lentivirus;

[0014] S3: Resistance screening of immortalized alpaca kidney cells;

[0015] S4: Detection and identification of immortalized alpaca kidney cells;

[0016] S5: Obtain immortalized alpaca kidney cell line.

[0017] Preferably, the step S1, extraction and cultivation of alpaca kidney primary cells, comprises the following steps:

[0018] 1) Take fresh and healthy alpaca kidney, cut off part of the alpaca kidney, and cut it into small pieces;

[0019] 2) Place the small pieces in a cell culture dish containing EDTA solution and incubate at room temperature;

[0020] 3) digesting the tissue pieces in step 2) with trypsin overnight;

[0021] 4) Cut the kidney pieces into smaller pieces and digest them with trypsin;

[0022] 5) Make a suspension, filter, centrifuge, and discard the supernatant;

[0023] 6) Add culture medium to prepare single cell suspension for cell culture;

[0024] 7) Replace the culture medium, and then replace the medium every 3 days to obtain primary cells.

[0025] Preferably, in step S2, the virus used for infection is obtained by the following method:

[0026] 1) Plasmid construction: The large T antigen gene of SV40 virus was constructed into the lentiviral vector pLVX-IRES-G418, and finally the plasmid was extracted;

[0027] 2) Plate 293T cells in logarithmic growth phase before transfection;

[0028] 3) On the day of transfection, dilute the lentiviral vector plasmid, auxiliary plasmid psPAX2 plasmid and PMD2G plasmid in Opti-MEM medium at a mass ratio of 2:1:1, mix gently, add polyethyleneimine PEI transfection reagent to the diluted plasmid, mix gently, and let stand at room temperature;

[0029] 4) Take out the HEK-293T cells, add the above system dropwise into the culture plate, shake gently, and place in an incubator for culture;

[0030] 5) Take the supernatant of the lentiviral particles after transfection, centrifuge, filter, and collect the viral supernatant.

[0031] Preferably, in step S2, the SV40 large T antigen immortalized lentivirus infection comprises the following steps:

[0032] 1) Inoculating primary alpaca kidney cells and infecting them with viruses;

[0033] 2) removing the original culture medium, adding a complete culture medium containing an infection enhancer, and then adding an appropriate amount of lentivirus SV40, i.e., large T antigen, for incubation; the infection enhancer is F108, and the complete culture medium is RPMI 1640 culture medium. In order to enhance viral infection, the cells are centrifuged, and the cells are placed in an incubator after centrifugation;

[0034] 3) After infection, remove the virus-containing solution, add complete culture medium, and continue culturing.

[0035] Preferably, the step S3, resistance screening of immortalized alpaca kidney cells, comprises the following steps: after the cells are expanded, G418 antibiotic is added for resistance screening, and the cells are frozen when the cells are in the logarithmic growth phase and the cell viability reaches more than 95%.

[0036] More preferably, the step S1, extraction and cultivation of alpaca kidney primary cells, comprises the following steps:

[0037] 1) Take fresh and healthy alpaca kidney, cut part of the alpaca kidney, and cut it into small pieces of 0.5-1 square centimeter;

[0038] 2) Place the small pieces in a cell culture dish containing EDTA solution and incubate at room temperature for 5 minutes;

[0039] 3) digesting the tissue pieces in step 2) with trypsin at 4°C overnight;

[0040] 4) After the kidney pieces were cut into smaller pieces, they were digested with trypsin at 37°C for 60 minutes;

[0041] 5) Make a suspension, pass through a 70-mesh cell strainer, centrifuge at 300 g for 5 minutes, and discard the supernatant;

[0042] 6) Add RPMI 1640 medium containing 20% ​​FBS and 1% P / S to prepare a single cell suspension, inoculate it into a cell culture dish, and place it in a 37°C, 5% CO2 constant temperature cell culture incubator;

[0043] 7) After 72 hours, the culture medium was replaced, and then every 3 days to obtain primary cells.

[0044] More preferably, in step S2, the virus used for infection is obtained by the following method:

[0045] 1) Plasmid construction: The large T antigen gene of SV40 virus was constructed into the lentiviral vector pLVX-IRES-G418, and finally the plasmid was extracted;

[0046] 2) 24 h before transfection, 293T cells in logarithmic growth phase were plated in 6-well plates;

[0047] 3) On the day of transfection, dilute the lentiviral vector plasmid, auxiliary plasmid psPAX2 plasmid and PMD2G plasmid in Opti-MEM medium at a mass ratio of 2:1:1, mix gently, add 3 times the mass of polyethyleneimine (PEI) transfection reagent to the diluted plasmid, mix gently, and let stand at room temperature for 15 minutes;

[0048] 4) Take out the HEK-293T cells, add the above system dropwise into the culture plate, shake gently, and place in an incubator for culture;

[0049] 5) Take the supernatant of the lentiviral particles 72 hours after transfection, centrifuge at 1500 rpm for 5 minutes, and then filter the virus supernatant with a 0.45 μM filter to collect the virus supernatant.

[0050] More preferably, in step S2, the SV40 immortalized lentivirus infection comprises the following steps:

[0051] 1) Alpaca kidney primary cells were inoculated at a density of 2.5E5 cell / mL and infected with viruses 24 hours later.

[0052] 2) Remove the original culture medium, add a complete culture medium containing an infection enhancer, and then add an appropriate amount of lentivirus SV40 (large T antigen) for incubation. The infection enhancer is F108, and the complete culture medium is RPMI 1640 culture medium containing 20% ​​FBS and 1% P / S. The content of the infection enhancer in the complete culture medium is 1%. In order to enhance viral infection, place the cells in a horizontal centrifuge at 400g and centrifuge at 32 degrees for 60 minutes. After centrifugation, place the cells in a 37-degree CO2 incubator.

[0053] 3) 6 hours after infection, remove the virus-containing solution, add complete culture medium, and continue culturing for 48 hours.

[0054] More preferably, the step S3, resistance screening of immortalized alpaca kidney cells, comprises the following steps: after the cells are expanded, G418 antibiotic is added for resistance screening, and when the cells are in the logarithmic growth phase and the cell viability reaches more than 95%, the cells are cultured at a rate of 1×10 6 Cells / tubes are cryopreserved.

[0055] In the third aspect of the present invention, there is provided a use of the immortalized alpaca kidney cell line as described in the first aspect in constructing a stable cell line of alpaca kidney cells overexpressing human CD19.

[0056] The method for constructing a stable cell line overexpressing human CD19 comprises the following steps:

[0057] S1: Recovery and culture of immortalized alpaca kidney cells;

[0058] S2: Based on the human CD19 sequence information, the pLVX-IRES-Human CD19-Puro lentiviral plasmid was expressed;

[0059] S3: Use the humanCD19 lentivirus obtained in step S2 to infect immortalized alpaca kidney cells;

[0060] S4: Verify the expression level of the alpaca kidney cell-human CD19 stable cell line obtained in S3.

[0061] The present invention also provides a stable cell line of alpaca kidney cells overexpressing humanCD19, which is prepared by the preparation method described in the third aspect.

[0062] In a fourth aspect of the present invention, there is provided a use of the immortalized alpaca kidney cell line as described in the first aspect in constructing a stable cell line of alpaca kidney cells overexpressing alpaca CD40LG.

[0063] The method for constructing a stable cell line overexpressing alpaca CD40LG comprises the following steps:

[0064] S1: Recovery and culture of immortalized alpaca kidney cells;

[0065] S2: Based on the sequence information of alpaca CD40LG, construct the pLVX-IRES-hygromycin-alpaca CD40LG lentiviral plasmid;

[0066] S3: Use the alpaca CD40LG lentivirus obtained in step S2 to infect immortalized alpaca kidney cells;

[0067] S4: Verify the expression level of the stable cell line of alpaca kidney cells-alpaca CD40LG obtained in S3.

[0068] The present invention also provides a stable cell line of alpaca kidney cells overexpressing alpacaCD40LG, which is prepared by the preparation method described in the fourth aspect.

[0069] In the fifth aspect of the present invention, there is provided the use of the immortalized alpaca kidney cell line as described in the first aspect as a tool cell for studying the mechanism of viral infection.

[0070] In the sixth aspect of the present invention, there is provided the use of the immortalized alpaca kidney cell line as described in the first aspect as a tool cell for in vitro drug function detection.

[0071] In the seventh aspect of the present invention, there is provided the use of the immortalized alpaca kidney cell line as described in the first aspect as a tool cell for antibody drug development and screening.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] 1) From Example 1 Figure 3 It can be seen that the immortalized alpaca kidney cells constructed in the present invention have the advantages of simple culture, multiple passages, good uniformity, and high expression of exogenous genes. Figure 4 It can be seen that the immortalized alpaca kidney cells established proliferate rapidly, with a doubling time of 40 hours, and the proliferation rate of cells of different densities remains consistent. Therefore, the immortalized alpaca kidney cell line of the present invention will provide a more powerful tool cell for the study of alpaca immunity, and is expected to enhance the domestic alpaca cell immunity research platform.

[0074] 2) The immortalized alpaca kidney cell line constructed by the present invention provides a new tool for viral infection mechanism, in vitro drug function detection, antibody drug development and screening, and has broad application prospects.

[0075] The deposit information of the microorganisms involved in the present invention is as follows:

[0076] Depository: China General Microbiology Center (CGMCC);

[0077] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China;

[0078] Classification and naming: Immortalized alpaca kidney cells (Alpaca kidney cells) BNalpaca 1.0;

[0079] Deposit number: CGMCC No.46058;

[0080] Date of preservation: October 24, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 This is a picture of the cell morphology of alpaca kidney primary cells under a microscope.

[0082] Figure 2 This is a diagram of the cell morphology of immortalized alpaca kidney cells.

[0083] Figure 3 This is the result of RT-qPCR test on immortalized alpaca kidney cells; Figure 3 a: SV40 mRNA expression; Figure 3 b: Dissolution curve of SV40 gene in BNalpaca 1.0 cells; Figure 3 c: Dissolution curve of SV40 gene in BNalpaca 1.0 cells, with a single dissolution peak.

[0084] Figure 4 This is the growth curve of immortalized alpaca kidney cells.

[0085] Figure 5 Establish a process for stable cell lines.

[0086] Figure 6 This is the flow cytometry validation result of BNalpaca 1.0-human CD19 stable cell line.

[0087] Figure 7 This is the flow cytometry validation result of BNalpaca 1.0-lpacaCD40LG stable cell line. DETAILED DESCRIPTION

[0088] In order to make the above-mentioned objects, features and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. However, the present invention is not limited to the given examples. The methods used are conventional methods unless otherwise specified.

[0089] Example 1

[0090] An immortalized alpaca kidney cell line and a construction method thereof, comprising the following process:

[0091] S1: Extraction and culture of primary alpaca kidney cells

[0092] 1. Take fresh and healthy alpaca kidneys. In a clean bench, use sterilized surgical scissors to cut part of the alpaca kidneys and cut them into small pieces of 0.5-1 square centimeter.

[0093] 2. Place the cut pieces in a cell culture dish containing 10 mL of 0.02% ethylenediaminetetraacetic acid (EDTA) solution and incubate at room temperature for 5 minutes.

[0094] 3. Add the tissue pieces from step 2 to a culture dish containing 10 mL of 0.25% trypsin and digest overnight at 4°C.

[0095] 4. Separation: Take out the kidney pieces, cut them into smaller pieces, and add them to a culture dish containing 10 mL of 0.25% trypsin for digestion at 37°C for 60 minutes.

[0096] 5. Use a 1 mL pipette to blow gently and repeatedly to make a suspension. Pass the suspension through a 70-mesh cell strainer and centrifuge at 300 g for 5 minutes. Discard the supernatant.

[0097] 6. Add RPMI 1640 + 20% FBS + 1% P / S culture medium to prepare a single cell suspension, inoculate it into a 10 cm cell culture dish, and place it in a 37°C, 5% CO2 constant temperature cell culture incubator.

[0098] 7. Replace the culture medium after 72 hours, and then replace it every 3 days.

[0099] Obtain primary cells and observe cell morphology under a microscope. Figure 1 As shown in Figure 2, the cells are epithelial-like and grow firmly attached to the wall.

[0100] S2: SV40 (large T antigen) immortalized lentiviral infection

[0101] 1. Lentivirus packaging:

[0102] 1.1. Plasmid construction: The SV40 large T antigen gene was constructed into the lentiviral vector pLVX-IRES-G418 through primer synthesis, ligation, enzyme digestion, and transformation, and finally the plasmid was extracted. The amino acid sequence of the large T antigen is shown in SEQ NO: 5.

[0103] 1.2. 24 h before transfection, plate 293T cells in logarithmic growth phase into 6-well plates.

[0104] 1.3. On the day of transfection, dilute the lentiviral vector plasmid, auxiliary plasmid psPAX2 plasmid and PMD2G plasmid in Opti-MEM medium at a ratio of 2:1:1, mix gently, add 3 times the mass of polyethyleneimine (PEI) transfection reagent to the diluted plasmid, mix gently, and let stand at room temperature for 15 minutes.

[0105] 1.4. Take out the HEK-293T cells, add the above system dropwise into the culture plate, shake gently, and place in an incubator for culture.

[0106] 1.5. 72h after transfection, the supernatant of lentiviral particles: centrifuge at 1500rpm for 5 minutes, and then filter the supernatant with a 0.45μM filter. Collect the supernatant.

[0107] 2. Viral infection

[0108] 2.1.2.5×10 5 Alpaca kidney primary cells were inoculated into 6-well plates at a density of 10 cells / mL and virus infection was performed 24 hours later.

[0109] 2.2. Remove the original culture medium in the 6-well plate, add 2 mL of complete culture medium containing infection enhancer, and then add an appropriate amount of lentivirus SV40 (large T antigen) for incubation. The enhancer is F108, which is a synthetic surfactant that can be purchased from Sigma, catalog number 07579-250G-F, which can increase the permeability of the cell membrane and enhance the transduction rate of the lentivirus. The complete culture medium is RPMI 1640+20% FBS+1% P / S culture medium. The content of infection enhancer in the complete culture medium is 1%. In order to enhance viral infection, centrifuge horizontally at 400g and centrifuge at 32 degrees for 60 minutes. After centrifugation, place the cells in a 37-degree CO2 incubator.

[0110] 2.3. 6 hours after infection, remove the virus-containing solution, add complete culture medium, and continue culturing for 48 hours.

[0111] 2.4. Observe the cell status, expand the cells into T25 cell culture flasks, add G418 antibiotics for resistance screening. When the cells are in the logarithmic growth phase and the cell viability reaches more than 95%, freeze the cells at 1E6 cells / tube and keep some cells for detection and identification.

[0112] S3: Immortalized Alpaca Kidney Cells Assay

[0113] 3.1 Observe the cytological morphology under a microscope. The morphology of immortalized alpaca kidney cells is as follows: Figure 2 As shown, the cytological morphology is spindle-shaped, which is consistent with that of primary cells.

[0114] 3.2 The primary alpaca kidney cells (Control) not infected with SV40 virus and the kidney cells (BNalpaca 1.0) of the 15th passage after infection with SV40 virus were respectively taken, RNA of each cell was extracted, and the expression level of SV40 gene in each cell was detected by real-time quantitative polymerase chain reaction (RT-qPCR). Figure 3 shown. Figure 3a shows that the kidney primary cells infected with SV40 lentivirus can be serially passaged, and the 15th generation cells (BNalpaca 1.0) were used to detect the expression of SV40 large T antigen. The results showed that compared with the control primary cells, the expression of SV40 large T antigen in BNalpaca 1.0 was significantly increased; Figure 3 b and c show that the dissolution curve of the SV40 gene in the cell is good and the dissolution peak is single.

[0115] The forward primer sequence of the SV40 primers used for RT-qPCR is shown in SEQ ID NO: 1;

[0116] The reverse primer sequence of the SV40 primer used for RT-qPCR is shown in SEQ ID NO: 2;

[0117] The forward primer sequence of the internal reference GAPDH primer is shown in SEQ ID NO: 3;

[0118] The reverse primer sequence of the internal reference GAPDH primer is shown in SEQ ID NO:4.

[0119] 3.3 Growth curve of immortalized alpaca kidney cells

[0120] BNalpaca 1.0 cells were inoculated into 96-well plates at cell densities of 2500 cells / well, 5000 cells / well, 7500 cells / well, and 10000 cells / well, respectively, and each well plate was set up for 3 replicates. The cells were cultured in a cell culture incubator at 37°C and 5% CO2. The cells were photographed and recorded every 4 hours using Sartorius' Incucyte S3 (live cell imaging analysis system) for 4 consecutive days. The cell growth curve obtained by instrument data analysis is shown in the figure below. Figure 4 As shown, the results showed that the established BNalpaca 1.0 cells proliferated rapidly, with a doubling time of 40 hours, and the proliferation rate of cells at different densities remained consistent. The immortalized cells obtained were named immortalized alpaca kidney cells (Alpaca kidney cells) BNalpaca1.0, and the cells were deposited in the China General Microbiological Culture Collection Center (CGMCC), Chinese Institute of Microbiology, with the deposit number: CGMCC No.46058.

[0121] Example 2 Application of immortalized alpaca kidney cells to construct a stable strain overexpressing human CD19

[0122] 1. Based on the immortalized alpaca kidney cell line, a stable alpaca kidney cell strain of human CD19 was constructed. The construction process and specific construction method were the same as those in S2 of Example 1. The difference was that the target plasmid was replaced with the pLVX-IRES-Puro plasmid to construct the pLVX-IRES-Human CD19-Puro lentiviral plasmid, and the infected cells were replaced with the immortalized alpaca kidney cells (Alpaca kidney cells) BNalpaca 1.0 that had been transformed with SV40 in Example 1. The construction process was as follows Figure 5 The pLVX-IRES-Human CD19-Puro lentiviral plasmid was constructed based on the human CD19 sequence (UniProt No.: P15391-1).

[0123] 2. After the construction is completed, collect the cultured alpaca kidney-human CD19 overexpressing cells.

[0124] 3. After digestion with trypsin, wash twice with PBS.

[0125] 4. Resuspend the cells with MACS buffer (PBS + 2% FBS + 2mM EDTA), count and plate. Add human CD19 detection antibody and incubate at 4°C for 1 hour.

[0126] 5. Wash twice with MACS buffer, then add fluorescent secondary antibody and incubate at 4°C for 30 min.

[0127] 6. Wash twice with MACS buffer and then test the fluorescence signal on a flow cytometer. Figure 6 As shown, the results showed that the expression level of human CD19 was significantly increased in BNalpaca1.0-human CD19 overexpressing cells.

[0128] Example 3 Application of immortalized alpaca kidney cells to construct stable strains overexpressing alpacaCD40LG

[0129] 1. Based on the immortalized alpaca kidney cell line, an alpaca CD40LG stable alpaca kidney cell strain was constructed. The construction process and specific construction method were the same as those in S2 of Example 1, except that the target plasmid was replaced with pLVX-IRES-hygromycin to construct the pLVX-IRES-hygromycin-alpaca CD40LG lentiviral plasmid, and the infected cells were replaced with the immortalized alpaca kidney cells (Alpaca kidney cells) BNalpaca 1.0 that had been transformed with SV40 in Example 1. The construction process was as follows: Figure 5The pLVX-IRES-hygromycin-alpaca CD40LG lentiviral plasmid was constructed based on the alpacaCD40LG sequence (UniProt No. A0A6I9IH35).

[0130] 2. After the construction is completed, collect the cultured alpaca kidney-alpaca CD40LG overexpressing cells.

[0131] 3. After digestion with trypsin, wash twice with PBS.

[0132] 4. Resuspend the cells with MACS buffer (PBS + 2% FBS + 2mM EDTA), count and plate, add 100nM alpacaCD40 hFc protein for detection, incubate at 4°C for 1h.

[0133] 5. Wash twice with MACS buffer, then add anti-hFC fluorescent secondary antibody and incubate at 4°C for 30 min.

[0134] 6. Wash twice with MACS buffer and then test the fluorescence signal on a flow cytometer. Figure 7 As shown, the results showed that the expression level of alpaca CD40LG was significantly increased in BNalpaca1.0-alpaca CD40LG overexpressing cells.

[0135] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An immortalized alpaca kidney cell line, characterized in that: The name is immortalized alpaca kidney cells (Alpaca kidney cells) BNalpaca 1.0, and its deposit number is: CGMCC No.46058.

2. The method for constructing an immortalized alpaca kidney cell line according to claim 1, characterized in that: The construction method comprises the following steps: S1: Extraction and culture of primary alpaca kidney cells; S2: Infection of primary alpaca kidney cells with lentivirus immortalized with SV40 large T antigen; S3: Resistance screening of immortalized alpaca kidney cells; S4: Detection and identification of immortalized alpaca kidney cells; S5: Obtain immortalized alpaca kidney cell line.

3. The method for constructing an immortalized alpaca kidney cell line according to claim 2, characterized in that: The step S1, extraction and cultivation of alpaca kidney primary cells, comprises the following steps: 1) Take fresh and healthy alpaca kidney, cut off part of the alpaca kidney, and cut it into small pieces; 2) Place the small pieces in a cell culture dish containing EDTA solution and incubate at room temperature; 3) digesting the tissue pieces in step 2) with trypsin overnight; 4) Cut the kidney pieces into smaller pieces and digest them with trypsin; 5) Make a suspension, filter, centrifuge, and discard the supernatant; 6) Add culture medium to prepare single cell suspension for cell culture; 7) Replace the culture medium, and then replace the medium every 3 days to obtain primary cells.

4. The method for constructing an immortalized alpaca kidney cell line according to claim 2, characterized in that: In step S2, the virus used for infection is obtained by the following method: 1) Plasmid construction: The large T antigen gene of SV40 virus was constructed into the lentiviral vector pLVX-IRES-G418, and finally the plasmid was extracted; 2) Plate 293T cells in logarithmic growth phase before transfection; 3) On the day of transfection, dilute the lentiviral vector plasmid, auxiliary plasmid psPAX2 plasmid and PMD2G plasmid in Opti-MEM medium at a mass ratio of 2:1:1, mix gently, add polyethyleneimine PEI transfection reagent to the diluted plasmid, mix gently, and let stand at room temperature; 4) Take out the HEK-293T cells, add the above system dropwise into the culture plate, shake gently, and place in an incubator for culture; 5) Take the supernatant of the lentiviral particles after transfection, centrifuge, filter, and collect the viral supernatant.

5. The method for constructing an immortalized alpaca kidney cell line according to claim 2, characterized in that: In step S2, the SV40 large T antigen immortalized lentivirus infection comprises the following steps: 1) Inoculating primary alpaca kidney cells and infecting them with viruses; 2) removing the original culture medium, adding a complete culture medium containing an infection enhancer, and then adding an appropriate amount of lentivirus SV40, i.e., large Tantigen, for incubation; the infection enhancer is F108, and the complete culture medium is RPMI 1640 culture medium. In order to enhance viral infection, the cells are centrifuged and placed in an incubator after centrifugation; 3) After infection, remove the virus-containing solution, add complete culture medium, and continue culturing.

6. The method for constructing an immortalized alpaca kidney cell line according to claim 2, characterized in that: The step S3, resistance screening of immortalized alpaca kidney cells, comprises the following steps: after the cells are expanded, G418 antibiotic is added for resistance screening, and the cells are frozen when the cells are in the logarithmic growth phase and the cell viability reaches more than 95%.

7. Use of the immortalized alpaca kidney cell line according to claim 1 in constructing a stable cell line of alpaca kidney cells overexpressing human CD19, characterized in that: The construction comprises the following steps: S1: Recovery and culture of immortalized alpaca kidney cells; S2: Based on the human CD19 sequence information, the pLVX-IRES-Human CD19-Puro lentiviral plasmid was expressed; S3: Use the humanCD19 lentivirus obtained in step S2 to infect immortalized alpaca kidney cells; S4: Verify the expression level of the alpaca kidney cell-human CD19 stable cell line obtained in S3.

8. Use of the immortalized alpaca kidney cell line according to claim 1 in constructing a stable cell line of alpaca kidney cells overexpressing alpaca CD40LG, characterized in that: The construction comprises the following steps: S1: Recovery and culture of immortalized alpaca kidney cells; S2: Based on the sequence information of alpaca CD40LG, construct the pLVX-IRES-hygromycin-alpaca CD40LG lentiviral plasmid; S3: Use the alpaca CD40LG lentivirus obtained in step S2 to infect immortalized alpaca kidney cells; S4: Verify the expression level of the stable cell line of alpaca kidney cells-alpaca CD40LG obtained in S3.

9. Use of the immortalized alpaca kidney cell line as claimed in claim 1 as a tool cell for studying the mechanism of viral infection.

10. Use of the immortalized alpaca kidney cell line as claimed in claim 1 as a tool cell for in vitro drug function detection.

11. Use of the immortalized alpaca kidney cell line as claimed in claim 1 as a tool cell for antibody drug development and screening.

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