Monoclonal LMH cell line with TLR15 gene knockout and preparation method thereof

Abstract

The present application belongs to the biological field, and discloses a method for preparing a monoclonal LMH cell line with a knockout TLR15 gene, comprising the following steps: Step 1: preparing a lentivirus connected with a TLR15 guide RNA; Step 2: using a lentivirus to infect LMH cells, and screening LMH cells successfully infected with the lentivirus; Step 3: separating the LMH cells obtained in step 2 into single cells and adding them to a single cell culture medium for culture, identifying the cells obtained after culture, and obtaining a monoclonal LMH cell line with a knockout TLR15 gene. The present invention transfects LMH cells by a lentiviral transfection method, separates the transfected LMH cells into single cells, and then uses a special single cell culture medium for culture, so as to obtain a monoclonal LMH cell line with a knockout TLR15 gene, which effectively overcomes the problem that the traditional method cannot successfully prepare a monoclonal LMH cell line with a knockout TLR15 gene. At the same time, the present invention also provides a monoclonal LMH cell line with a knockout TLR15 gene.

Description

Technical Field

[0001] The invention relates to the biological field, and in particular to a TLR15 gene knocked-out monoclonal LMH cell line and a preparation method thereof. Background Art

[0002] Monoclonal usually refers to a homologous cell population induced or separated from a single mother cell or cell line. These cell populations have the same genetic and functional characteristics. Monoclonal technology is widely used in many biological researches, especially in gene editing, antibody research and development, cell line development, cell biology, oncology, stem cell research and other fields.

[0003] After knocking out the gene of the cell, a single cell is isolated and cultured to obtain a monoclonal cell with high consistency and purity. For LMH (chicken liver cancer cells), it is very difficult to prepare its monoclonal cell. After experiments, we found that the monoclonal propagation and expansion culture of LMH cells cannot be achieved using DMED / F12 culture medium containing 10% FBS and double antibodies. This means that even if we successfully knock out the target gene, the preparation of the monoclonal LMH cell line will not be achieved because the expansion culture technology of a single cell cannot achieve the expected results.

[0004] From the search of the prior art, the following documents mentioned the monoclonal preparation of LMH cells:

[0005] Publication No. CN118638783A, the subject is a LMH cell line with 14-3-3ε gene knocked out, its construction method and application, which discloses knocking out the 14-3-3ε gene of LMH cells by knockout plasmid; and expanding the culture of monoclonal cells by cell subcloning technology;

[0006] The specific cell subcloning technology disclosed is: after counting the cells, they are plated into a 96-well plate (ensuring that each well contains a single cell), and then placed in a cell culture incubator at 37°C and 5% CO2 for culture; on the 5th day of culture, half of the cell culture medium in each well is replaced; on the 10th day after culture, the cell plate is placed under a microscope for observation, and the cells in the cell wells with only single cell clumps are screened for digestion and expanded culture.

[0007] Since they did not disclose the culture medium used in the subcloning process, we were unable to repeat their experiments. However, during the verification of the present invention, we used DMED / F12 culture medium containing 10% FBS and dual antibodies for culture and found that it was completely impossible to achieve expansion and passaging.

[0008] The problem that this project needs to solve is: How to successfully develop a monoclonal LMH cell line with TLR15 gene knockout that can be stably propagated and expanded. Summary of the invention

[0009] The purpose of the present invention is to provide a method for preparing a monoclonal LMH cell line with a TLR15 gene knocked out. The present invention infects LMH cells by a lentiviral infection method, separates the infected LMH cells into single cells, and then cultured them in a special single cell culture medium to obtain a monoclonal LMH cell line with a TLR15 gene knocked out, thereby effectively overcoming the difficulty that the traditional method cannot successfully prepare a monoclonal LMH cell line with a TLR15 gene knocked out.

[0010] Meanwhile, the present invention also provides a monoclonal LMH cell line with TLR15 gene knocked out.

[0011] To achieve the above object, the present application discloses a method for preparing a monoclonal LMH cell line with TLR15 gene knockout, comprising the following steps:

[0012] Step 1: Prepare lentivirus linked to TLR15 guide RNA;

[0013] Step 2: Infect LMH cells with lentivirus, and screen LMH cells successfully infected with lentivirus;

[0014] Step 3: separating the LMH cells obtained in step 2 into single cells and adding them to a single cell culture medium for culture, identifying the cells obtained after culture, and obtaining a monoclonal LMH cell line with TLR15 gene knockout;

[0015] The single cell culture medium comprises a DMEM / F12 culture medium containing 18-22wt% FBS and a cell supernatant; the single cell culture medium also contains transferrin with a final concentration of 8-12 μg / mL, a growth factor with a final concentration of 20-30 ng / mL, and a bispecific antibody; the preparation method of the cell supernatant comprises: culturing chicken liver cancer cells with a DMEM / F12 culture medium containing 8-12wt% FBS, and collecting the supernatant after culturing for a period of time to obtain the cell supernatant;

[0016] The sequence of the TLR15 guide RNA is shown in SEQ ID NO. 1. The sequence of the TLR15 guide RNA is ATAGGATGAGGATCCTTAT.

[0017] TLR15 reference sequence: FJ915258 (provided by NCBI website)

[0018] The complete sequence of TLR15 gene is as follows (SEQ ID NO.2):

[0019]

[0020] The complete sequence of TLR15 CDs is shown below (SEQ ID NO.3):

[0021]

[0022] The core innovations of the present invention are:

[0023] 1. The TLR15 gene of LMH cells was successfully knocked out using lentivirus containing TLR15 guide RNA;

[0024] 2. Single cell subculture is achieved through single cell culture medium;

[0025] Among them, the role of the cell supernatant is to provide unknown nutrients to individual cells or signal transduction substances that stimulate cell proliferation; the role of growth factors is to promote cell proliferation; when the culture medium supernatant and the culture medium with growth factors, FBS, and transferrin are mixed, the successful culture of monoclonal cells of chicken liver cancer cell lines is achieved, and the survival rate of the traditional method for the culture of monoclonal cells of chicken liver cancer cell lines is increased from 0% to 70%.

[0026] In the above method for preparing a monoclonal LMH cell line with TLR15 gene knockout, step 1 is specifically as follows:

[0027] Step 11: Ligate the TLR15 guide RNA into the lentiCRISPR v2 plasmid to obtain the master plasmid;

[0028] Step 12: The main plasmid, psPAX2 plasmid and pMD2.G are mixed and transfected into the 293t cell line, and the culture supernatant is collected after cultivation; the culture supernatant contains the lentivirus; and the lentivirus is obtained by filtration.

[0029] In the above-mentioned method for preparing the monoclonal LMH cell line with TLR15 gene knockout, the mass ratio of the main plasmid, psPAX2 plasmid and pMD2.G is 4:3:1; and the culture medium used for transfection is opti-mem culture medium.

[0030] In the above method for preparing a monoclonal LMH cell line with TLR15 gene knockout, step 2 is specifically as follows:

[0031] The LMH cells after trypsin digestion were centrifuged, and the LMH cells separated by centrifugation were resuspended with a lentivirus solution containing lentivirus, and the resuspended solution was added to the well plate, and serum-free culture medium was added to the well plate, and the plate was placed in a cell culture incubator with a 5% CO2 atmosphere for 48 hours, and then the LMH cells were selected with puromycin, and the surviving cells were LMH cells infected with lentivirus.

[0032] In the above-mentioned method for preparing a monoclonal LMH cell line with TLR15 gene knockout, step 3 specifically comprises: digesting the LMH cells infected with lentivirus into single cells without clumping; adding a single cell culture medium to the wells of the well plate, and adding a LMH cell infected with lentivirus to each well; the single cell culture medium also contains puromycin; and culturing the well plate in a cell culture incubator containing 5% CO2 for 12 to 16 days.

[0033] In the above method for preparing the monoclonal LMH cell line with TLR15 gene knocked out, the growth factor is one or more combinations of epidermal growth factor, hepatocyte growth factor, transforming growth factor, and fibroblast growth factor.

[0034] In the above-mentioned method for preparing a monoclonal LMH cell line with TLR15 gene knockout, the volume ratio of the DMEM / F12 culture medium containing 18-22 wt % FBS to the cell supernatant is 1:1.

[0035] In the above-mentioned method for preparing the monoclonal LMH cell line with TLR15 gene knockout, the dual antibodies are penicillin and streptomycin.

[0036] In the above-mentioned method for preparing the monoclonal LMH cell line with TLR15 gene knockout, the preparation method of the cell supernatant is: chicken liver cancer cells are cultured in DMEM / F12 medium containing 8-12wt% FBS, and the supernatant is collected after culturing for 48 hours in a cell culture incubator containing 5% CO2 at 37°C to obtain the cell supernatant.

[0037] Finally, the present invention also discloses a monoclonal LMH cell line with TLR15 gene knocked out, which is prepared by any of the methods described above.

[0038] The beneficial effects of this application are:

[0039] The present invention transfects LMH cells by lentiviral transfection, separates the transfected LMH cells into single cells and then cultures them in a special single cell culture medium, thereby obtaining a monoclonal LMH cell line with TLR15 gene knocked out, effectively overcoming the difficulty that traditional methods cannot successfully prepare a monoclonal LMH cell line with TLR15 gene knocked out. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a comparative chart of TLR15 protein expression in TLR15 wild-type cell line, TLR15-KO-1, TLR15-KO-2, and TLR15-KO-3;

[0041] Figure 2 This is the result of western blot verification of TLR15-KO-1;

[0042] Figure 3 This is a statistical table of virus propagation results for TLR15 wild-type cell line and TLR15-KO-1. DETAILED DESCRIPTION

[0043] The present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. In the description of the present invention, it should be noted that, if no specific conditions are specified in the embodiments, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0044] Part I Reagents

[0045] LMH cell line: ATCC, catalog number: CRL-2117;

[0046] DMEM / F12 culture medium: Gibco, DMEM / F12 (1:1) culture medium catalog number: C11330500BT; Opti-MEM culture medium: Gibco, catalog number: 31985070;

[0047] Fetal bovine serum: VivaCell BIOSCIENCES, Fetal Bovine Serum, Catalog No.: C04001-500; Double antibody: Biosharp, Penicillin-Streptomycin Solution (100×), Catalog No.: BL505A;

[0048] Transferrin: Sigma, catalog number: T8158-100MG;

[0049] Epidermal growth factor: Sigma, catalog number: E9644-5MG;

[0050] Hepatocyte growth factor: SinoBiological Company, catalog number: 10463-HNAS;

[0051] Transforming growth factor: SinoBiological Company, catalog number: 10804-H08H-B;

[0052] Fibroblast growth factor: SinoBiological Company, catalog number: GMP-10014-HNAE;

[0053] Trypsin: Gibco, 0.25% Trysin-EDTA (1×), catalog number: 25200-072;

[0054] lentiCRISPR v2 master plasmid: addgene: #52961;

[0055] psPAX2 plasmid: addgene: #12260;

[0056] pMD2.G: addgene: #12259;

[0057] 293t cell line: ATCC: CRL-3216;

[0058] Puromycin: BBI:A610593-0025;

[0059] TLR15 antibody: NOVUS: NBP2-27191SS;

[0060] GAPDH antibody: Sigma: G9545.

[0061] Part II Preparation of TLR15 knockout LMH monoclonal cells

[0062] Example 1

[0063] Step 1: According to the instructions of the lentiCRISPR v2 master plasmid, the TLR15 guide RNA sequence (SEQ ID NO.1) was connected to the lentiCRISPR v2 master plasmid to obtain the master plasmid;

[0064] Step 2: According to the mass ratio of 4:3:1, the main plasmid, psPAX2, and pMD2.G, a total of 24 μg, were transfected into the 293T cell line. The culture medium was Opti-MEM medium. After 6 hours of transfection, the DMEM / F12 medium containing 2% FBS was changed. After another 72 hours, the supernatant was collected. The supernatant was placed in a 50ml centrifuge tube and centrifuged at 4°C, 5000rpm / min for 10 minutes. The supernatant was filtered with a 0.45μm filter and packaged to obtain the lentiviral liquid, which was stored at -80°C.

[0065] Step 3: Digest the prepared LMH cell line with trypsin, measure the cell concentration with a cell counter, and select approximately 2*10 6 The cells were centrifuged at 250 g for 5 min, the supernatant was discarded, the cells were resuspended with 1 ml of lentivirus liquid, added to a 6-well plate, the 6-well plate was supplemented with 1 ml of DMEM / F12 serum-free medium, and cultured in a cell culture incubator containing 5% CO2 for 48 h.

[0066] Step 4: The cells obtained in step 3 were digested with trypsin, transferred to a T25 culture flask with DMEM / F12 medium containing 10% FBS (containing 10 μg puro (puromycin)), and cultured in a cell culture incubator containing 5% CO2 for 72 hours. LMH cells that were not infected with lentivirus all died due to the action of puro.

[0067] Step 5: The puro-resistant LMH cell line remaining in step 4 was digested with trypsin, and the trypsin was neutralized with DMEM / F12 medium containing 10% FBS, and centrifuged at 250 g for 5 minutes, and the supernatant was discarded.

[0068] Step 6: Resuspend the LMH cell line in single cell culture medium and filter with a 300-mesh filter to separate the cells into single cells without clumping.

[0069] Step 7: 200 μL of single cell culture medium (1 μg / ml final concentration of puro) was added to each well of the 96-well plate in advance, and LMH cells were sorted into the 96-well culture plate by flow cytometry at a setting of 1 cell per well. The cells were cultured in a cell culture incubator containing 5% CO2 for 15 days.

[0070] The single cell survival rate was observed, and the cell lines with successful single cell propagation were selected for expansion culture. The culture medium used for expansion culture was DMEM / F12 culture medium containing 10% FBS.

[0071] Step 8: Randomly select three monoclonal cell lines for expansion and culture, extract cell DNA and perform gene sequencing.

[0072] The sequencing results are as follows Figure 1 Compared with the TLR15 wild-type cell line (TLR15-WT), the genes of the other three TLR15 knockout cell lines (TLR15-KO-1, TLR15-KO-2, and TLR15-KO-3) were changed to varying degrees.

[0073] (Note: ATG is the start codon of the TLR15 protein coding region. Three bases encode one amino acid. If the addition or deletion of bases is not a multiple of 3, the subsequent amino acids are incorrectly encoded, i.e., the TLR15 protein is not encoded).

[0074] A cell line (TLR15-KO-1) was randomly selected for protein validation. Since TLR15 can be activated by proteinase K (de Zoete et al. 2011), we performed subsequent protein validation ( Figure 2 ), the results showed that the TLR15 gene was successfully knocked out in the TLR15-KO cell line.

[0075] The single cell culture medium used above can be seen as follows:

[0076] The LMH cell line was plated at a concentration of 90% in a T75 culture flask, and the T75 culture flask had a DMEM / F12 medium; the DMEM / F12 medium contained 10 wt % fetal bovine serum, 100 U / mL penicillin and 0.1 mg / mL streptomycin;

[0077] The culture conditions were: culture in a cell culture incubator at 37°C with 5% CO2;

[0078] After 48 h of culture, the cell supernatant was collected into a sterile 50 ml centrifuge tube and stored at 4°C, or stored at -20°C for a long term.

[0079] The single-cell culture medium is composed of DMEM / F12 culture medium containing 20wt% FBS and the cell supernatant in the first part in a volume ratio of 1:1; the single-cell culture medium contains transferrin with a final concentration of 10 μg / mL, hepatocyte growth factor with a final concentration of 25 ng / mL, and dual antibodies with a final concentration of 100 U / mL penicillin and 0.1 mg / mL streptomycin.

[0080] Example 2

[0081] The TLR15 knockout LMH cells obtained in step 5 of Example 1 were subjected to steps 6 and 7, and the survival rate was observed.

[0082] In this example, the single cell culture medium used is substantially the same as that used in Example 1, except that:

[0083] The concentration of transferrin was 8 μg / mL, and the concentration of hepatocyte growth factor was 30 ng / mL; the DMEM / F12 culture medium contained 18 wt % of FBS.

[0084] Example 3

[0085] The TLR15 knockout LMH cells obtained in step 5 of Example 1 were subjected to steps 6 and 7, and the survival rate was observed.

[0086] In this example, the single cell culture medium used is substantially the same as that used in Example 1, except that:

[0087] The concentration of transferrin was 12 μg / mL, and the concentration of hepatocyte growth factor was 20 ng / mL; the DMEM / F12 culture medium contained 22 wt % of FBS.

[0088] Example 4

[0089] The TLR15 knockout LMH cells obtained in step 5 of Example 1 were subjected to steps 6 and 7, and the survival rate was observed.

[0090] In this example, the single cell culture medium used is substantially the same as that used in Example 1, except that:

[0091] The growth factor is epidermal growth factor.

[0092] Example 5

[0093] The TLR15 knockout LMH cells obtained in step 5 of Example 1 were subjected to steps 6 and 7, and the survival rate was observed.

[0094] In this example, the single cell culture medium used is substantially the same as that used in Example 1, except that:

[0095] The growth factor is fibroblast growth factor.

[0096] Example 6

[0097] The TLR15 knockout LMH cells obtained in step 5 of Example 1 were subjected to steps 6 and 7, and the survival rate was observed.

[0098] In this example, the single cell culture medium used is substantially the same as that used in Example 1, except that:

[0099] The growth factor is composed of hepatocyte growth factor and epidermal growth factor in a mass ratio of 1:1.

[0100] Example 7

[0101] The TLR15 knockout LMH cells obtained in step 5 of Example 1 were subjected to steps 6 and 7, and the survival rate was observed.

[0102] In this example, the single cell culture medium used is substantially the same as that used in Example 1, except that:

[0103] The growth factor is composed of hepatocyte growth factor and fibroblast growth factor in a mass ratio of 1:1.

[0104] Example 8

[0105] The TLR15 knockout LMH cells obtained in step 5 of Example 1 were subjected to steps 6 and 7, and the survival rate was observed.

[0106] In this example, the single cell culture medium used is substantially the same as that used in Example 1, except that:

[0107] The growth factor is composed of epidermal growth factor and fibroblast growth factor in a mass ratio of 1:1.

[0108] Comparative Example 1

[0109] The TLR15 knockout LMH cells obtained in step 5 of Example 1 were subjected to steps 6 and 7, and the survival rate was observed.

[0110] In this comparative example, a traditional culture medium was used to replace the single cell culture medium. The traditional culture medium was DMED / F12 culture medium to which 10% FBS, 100 U / mL penicillin and 0.1 mg / mL streptomycin were added.

[0111] The survival rates of the culture results of Examples 1 to 8 and Comparative Example 1 were statistically analyzed, and the statistical results are shown in Table 1 below;

[0112] Table 1 Survival rate of TLR15 knockout LMH monoclonal cells

[0113]

[0114]

[0115] Result analysis:

[0116] 1. It can be seen from Examples 1 to 8 that the culture medium composed of DMEM / F12 culture medium and cell supernatant is the basis for the successful culture of single cells;

[0117] 2. It can be seen from Examples 1 to 3 that using hepatocyte growth factor and controlling the concentrations of transferrin, hepatocyte growth factor, and FBS is an important means to improve the survival rate;

[0118] 3. It can be seen from Examples 1, 4 and 5 that there is no substantial difference in the survival rate between hepatocyte growth factor and epidermal growth factor and fibroblast growth factor;

[0119] 4. It can be seen from Examples 1, 4, 5, 6 to 8 that, while using hepatocyte growth factor, compounding with other growth factors is another effective means to improve the survival rate.

[0120] Based on the above analysis, it can be seen that the important factors for improving single cell survival are: the selection of basal culture medium, the dosage of each functional component and the selection of appropriate growth factor combination.

[0121] 4. As an important breakthrough of the present invention, the present invention overcomes the difficulty that the traditional method cannot successfully culture TLR15 knockout LMH monoclonal cells.

[0122] Part III Effects of TLR15-KO-1 on viral replication

[0123] The successfully expanded TLR15-KO-1 cells were used to culture FAdV4 (avian adenovirus type 4), and the culture method was as follows:

[0124] The TLR15-KO-1LMH cell line and the wild-type LMH cell line were plated at a density of 95% in a T25 cell culture flask (the culture medium was DMEM / F12 medium containing 10% FBS), and cultured in a 37°C cell culture incubator containing 5% CO2 for 12 hours. After 12 hours, the cell culture medium was discarded, and the cells were washed 3 times with PBS. The TLR15-KO-1LMH cell line and the wild-type LMH cell line were infected with FAdV4 at a multiplicity of infection MOI = 1, and were placed in a 37°C cell culture incubator containing 5% CO2 for adsorption for 1.5 hours. After 1.5 hours, the adsorption liquid was discarded, the cells were washed 3 times with PBS, and the cell maintenance medium containing 2% FBS was added for culture. The cell supernatant was aspirated 48 hours after infection and the DNA of the cell supernatant was extracted. The number of viral nucleic acid copies of FAdV4 in the cell supernatant of the TLR15-KO-1LMH cell line and the wild-type LMH cell line was analyzed by qPCR detection;

[0125] The primer information used for qPCR detection is as follows: Primer F: 5′-ATGGCGCAGATGGCTAAGG-3 (SEQ ID NO. 4);

[0126] R: 5'-AGCGCCTGGGTCAAACCGA-3' (SEQ ID NO. 5).

[0127] Specific culture results can be found Figure 3 ;

[0128] pass Figure 3 It can be seen that in the supernatant, the copy number of the LMH cell line without gene knockout during virus culture was about 1 order of magnitude lower than that of TLR15-KO-1 cells.

[0129] This indicates that the cell line obtained by the present invention can be used as high-quality research material in virus culture and propagation, vaccine development, antiviral mechanism research, etc.

[0130] In summary, the unique innovations of this case are:

[0131] 1. The TLR15 gene of LMH cells was successfully knocked out using a lentivirus containing TLR15 guide RNA, and it was verified that knocking out the gene and performing monoclonal culture could achieve virus culture and expansion;

[0132] 2. Single cell subculture is achieved through single cell culture medium;

[0133] A mixture of basal culture medium supplemented with growth factors and cell supernatant was used as a single cell culture medium. The cell supernatant provided nutrients or signal transduction substances that stimulated cell proliferation to single cells, and growth factors promoted cell proliferation, so that the non-viable TLR15 knockout LMH cells could replicate efficiently, providing basic conditions for the subsequent accurate verification of gene functions and virus culture. At the same time, through the screening of growth factors, hepatocyte growth factor that is particularly suitable for TLR15 knockout LMH cells was obtained, which plays a pivotal role in improving the survival rate of single cells.

Claims

1. A method for preparing a monoclonal LMH cell line with TLR15 gene knockout, characterized in that: The steps include: Step 1: Prepare lentivirus linked to TLR15 guide RNA; Step 2: Infect LMH cells with lentivirus, and screen LMH cells successfully infected with lentivirus; Step 3: separating the LMH cells obtained in step 2 into single cells and adding them to a single cell culture medium for culture, identifying the cells obtained after culture, and obtaining a monoclonal LMH cell line with TLR15 gene knockout; The single cell culture medium comprises a DMEM / F12 culture medium containing 18-22wt% FBS and a cell supernatant; the single cell culture medium also contains transferrin with a final concentration of 8-12 µg / mL, a growth factor with a final concentration of 20-30ng / mL, and a bispecific antibody; the preparation method of the cell supernatant is: chicken liver cancer cells are cultured in a DMEM / F12 culture medium containing 8-12wt% FBS, and the supernatant is collected after a period of culture to obtain the cell supernatant; The sequence of the TLR15 guide RNA is shown in SEQ ID NO.1; The growth factor is one or more combinations of epidermal growth factor, hepatocyte growth factor and fibroblast growth factor.

2. The method for preparing a monoclonal LMH cell line with TLR15 gene knockout according to claim 1, characterized in that: The step 1 is specifically as follows: Step 11: Ligate the TLR15 guide RNA into the lentiCRISPR v2 plasmid to obtain the master plasmid; Step 12: The main plasmid, psPAX2 plasmid and pMD2.G are mixed and transfected into the 293t cell line, and the culture supernatant is collected after cultivation; the culture supernatant contains the lentivirus; and the lentivirus is obtained by filtration.

3. The method for preparing a monoclonal LMH cell line with TLR15 gene knockout according to claim 2, characterized in that: The mass ratio of the main plasmid, psPAX2 plasmid and pMD2.G is 4:3:1; the culture medium used for transfection is Opti-MEM culture medium.

4. The method for preparing a monoclonal LMH cell line with TLR15 gene knockout according to claim 2, characterized in that: The step 2 is specifically as follows: The LMH cells after trypsin digestion were centrifuged, and the LMH cells separated by centrifugation were resuspended with a lentivirus solution containing lentivirus, and the resuspended solution was added to the well plate, and the well plate was supplemented with serum-free culture medium, and cultured in a cell culture incubator with a CO2 atmosphere of 5% for 48 hours, and then the LMH cells were selected with puromycin, and the surviving cells were LMH cells infected with lentivirus.

5. The method for preparing a monoclonal LMH cell line with TLR15 gene knockout according to claim 4, characterized in that: The step 3 specifically comprises: digesting the LMH cells infected with the lentivirus into single cells without clumping; adding a single cell culture medium into the wells of the well plate, and adding a LMH cell infected with the lentivirus into each well; the single cell culture medium also contains puromycin; and placing the well plate in a cell culture incubator containing 5% CO2 and culturing for 12 to 16 days.

6. The method for preparing a monoclonal LMH cell line with TLR15 gene knockout according to claim 1, characterized in that: The volume ratio of the DMEM / F12 culture medium containing 18-22wt% FBS to the cell supernatant is 1:

1.

7. The method for preparing a monoclonal LMH cell line with TLR15 gene knockout according to claim 1, characterized in that: The dual resistance is penicillin and streptomycin.

8. The method for preparing a monoclonal LMH cell line with TLR15 gene knockout according to claim 1, characterized in that: The preparation method of the cell supernatant is as follows: chicken liver cancer cells are cultured in a DMEM / F12 medium containing 8-12wt% FBS, and the supernatant is collected after culturing for 48 hours in a cell culture incubator containing 5% CO2 at 37°C to obtain the cell supernatant.

9. A monoclonal LMH cell line with TLR15 gene knockout, characterized in that: The method is prepared by any one of claims 1 to 8.

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