Porcine bone marrow macrophage, construction method therefor, and use thereof

Abstract

The present invention relates to the technical field of biology, in particular to a porcine bone marrow macrophage, a construction method therefor, and the use thereof. The method comprises a method of performing electroporation on a primary porcine bone marrow macrophage to introduce pTERT, pCDK4(R24C) and pCCND1 genes into same so as to immortalize the porcine bone marrow macrophage; a method of continuously constructing, on the basis of the cell, a porcine bone marrow macrophage cell line stably expressing the Cre gene; and a method of removing a screening marker gene on a gene-deleted recombinant virus strain by means of using the cell line. The methods comprise: by means of electroporation, separately introducing into the primary porcine bone marrow macrophage transposon vectors and transposase vectors which respectively contain pTERT, pCDK4(R24C) and pCCND1, and screening out an immortalized porcine bone marrow macrophage; and continuing to perform electroporation on the immortalized porcine bone marrow macrophage to introduce the Cre gene so as to enable same to stably express a Cre protein. The present invention can be used for removing screening marker genes during the process of constructing gene-deleted recombinant virus strains.

Description

Pig bone marrow macrophage and its construction method and application Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a porcine bone marrow macrophage and a construction method and application thereof. Background Art

[0002] Since the 1980s, the emergence of gene-deleted vaccines for pseudorabies in pigs has ushered in a new era in the prevention and control of animal infectious diseases. Using genetically engineered live virus vaccines that lack virulence-determining genes (TK, gE, and / or gI), combined with appropriate differential diagnostic techniques, these vaccines enable clinical differentiation between naturally infected animals and vaccine-immunized animals, making them an effective tool for eradicating infectious diseases. Decades of clinical application have fully demonstrated the advantages of gene-deleted vaccines: strong immunogenicity and protection; excellent safety profile with no relapse into virulence; and, when combined with differential diagnostics, eradication effectiveness. Vaccine development and production typically requires infection of susceptible cells with the gene-deleted virus.

[0003] Macrophages are one of the most susceptible cells to African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), and pseudorabies virus (PRV). Large-scale vaccine production using primary cells is not feasible because preparing primary macrophages is time-consuming and expensive, batches are unstable, and contamination is a possibility. Furthermore, there is a lack of suitable stable cell lines for the isolation and propagation of macrophage-tropic viruses. To address this challenge, scientists worldwide have been working to establish immortalized porcine macrophage cell lines.

[0004] There are currently five immortalized porcine macrophage cell lines reported in foreign literature: WSL, IPAM, IPKM, IPIM, and ZMAC-4. (I) WSL, constructed by the Friedrich-Löffler Federal Institute for Animal Health (FLI) in Germany, is a naturally immortalized wild boar embryonic porcine lung cell line. This cell line expresses SLA II and SW3 at high levels, but hardly expresses CD163 and CD169, indicating that this cell line is an immature macrophage precursor (Sánchez EG et al., Phenotyping and susceptibility of established porcine cell lines to African Swine Fever Virus infection and viral production. Scientific reports. 2017 Sep 4; 7(1): 10369). (2) The IPAM, an immortalized cell line of porcine alveolar macrophages, was constructed by the Vaccine and Infectious Diseases Organization (VIDO) of Canada. Primary porcine alveolar macrophages (PAMs) were transfected with the vector pSV3-neo carrying the SV40 large T antigen (SV40LT) gene. Three monoclonal cell lines, 3D4 / 2, 3D4 / 21, and 3D4 / 31, were obtained by screening in a culture medium containing geneticin G418. Compared with primary PAM cells, IPAMs only express low levels of CD14 and SW3, and almost no expression of CD163 and SLAII. Therefore, IPAMs are not mature macrophages (Weingartl HM et al., Continuous porcine cell lines developed from alveolar macrophages: partial characterization and virus susceptibility. Journal of virological methods. 2002 Jul; 104(2): 203-16). (3) The IPKM constructed by the National Agriculture and Food Research Organization (NARO) of Japan is a porcine kidney-derived macrophage. The cell line was obtained by infecting primary porcine kidney-derived macrophages (PKM) with a recombinant lentivirus carrying the SV40LT gene and porcine telomerase reverse transcriptase (pTERT), and then screening with a culture medium containing geneticin G418.IPKM has a similar morphology to primary renal macrophages (PKM) and expresses macrophage-specific surface molecules Iba1, KT022, and CD172a (Takenouchi T et al. Immortalization and characterization of porcine macrophages that had been transduced with lentiviral vectors encoding the SV40 large T antigen and porcine telomerase reverse transcriptase. Frontiers in veterinary science. 2017; 4:132.). (IV) The IPIM constructed by NARO in 2022 is a porcine intestinal macrophage. The immortalization method is similar to that of IPKM, and SV40LT and pTERT are expressed by lentiviral transfection. IPIM expresses macrophage-specific surface molecules Iba-1, CD172a, CD204, CD203a, and CD16, and some cells express CD163, CD169, and MHC-II (Takenouchi T et al., Isolation and immortalization of macrophages derived from fetal porcine small intestine and their susceptibility to porcine viral pathogen infections. Frontiers in veterinary science. 2022 Jul 18;9:919077). (V) ZMAC-4, selected and cultivated by Aptimmune Biologics, Inc. and the University of Illinois, is derived from porcine embryonic lung lavage fluid. This cell line is an untransformed macrophage cultured in a "static suspension culture" format. M-CSF is added to the culture medium for maintenance. After 34 days, the cell number can be expanded more than 100-fold. As of the time of publication, the cell number has been passaged more than 75 times in the 8 months since publication, with no signs of decreased proliferation. A batch of ZMAC-4 cells frozen on March 26, 2008, was used to create a master cell bank, which has been approved by the U.S. Department of Agriculture's Center for Veterinary Biologics for use in commercial vaccine production.This cell line expresses several surface markers of primary PAMs, such as CD14, CD163, CD172, and the E26 family transcription factor PU.1, but does not express CD203a, indicating that they are not as mature as primary cell PAMs (Portugal R et al., A porcine macrophage cell line that supports high levels of replication of OURT88 / 3, an attenuated strain of African swine fever virus. Emerging microbes & infections. 2020 Dec; 9(1): 1245-1253).

[0005] Currently, there are four immortalized porcine macrophages published in China: (1) "Immortalized Porcine Alveolar Macrophages" published with patent application publication number CN 105793416A is an immortalized cell line that was transfected with the transposon vectors pPB-CAG-SV40 T Ag and pPB-CMV-hyPBase carrying the SV40LT gene into primary porcine alveolar macrophages (PAMs). Flow cytometry analysis revealed the expression of macrophage surface markers CD163 and sialoadhesin P210. As of the time of patent publication, the cell line had been cultured for 8 months (50 to 60 generations). (2) Patent application publication number CN 113528453A, "An Immortalized Porcine Macrophage Cell Line, Its Construction Method, and Application," describes a cell line obtained by infecting primary porcine bone marrow macrophages (BMDM) with a lentivirus carrying the SV40LT gene and the human telomerase reverse transcriptase (hTERT) gene. The lentiviral vector GLV2-CMV-MCS-PGK-puro expresses a puromycin resistance gene, and flow cytometry analysis shows that this cell line can express CD14. (3) Patent application publication number CN 114292873 A, "Immortalized Porcine Bone Marrow Macrophages, Its Construction Method, and Application," describes a cell line obtained by transfecting primary porcine bone marrow macrophages (BMDM) with the plasmid pIED-Neo-SV40TAg carrying the SV40LT gene and screening in a culture medium containing puromycin. (4) Patent application publication number CN 113151171 A, “Porcine alveolar macrophage healthy cell line and construction method and application”, is a porcine alveolar macrophage cell line obtained by screening porcine alveolar macrophages (PAM) using a special cell culture medium combined with natural domestication.

[0006] Therefore, according to the currently available literature, there is still no mature porcine macrophage cell line in the world that has the advantages of being free of exogenous viral oncogenes, exogenous viral vector genes, and drug resistance genes, and having stable passage and simple operation. There are literature reports that the use of non-viral human genes (hTERT, hCyclin D1, and hCDK4 mutants) can establish a normal diploid OSE cell line from primary human ovarian surface epithelial cells (OSE) without chromosomal instability (Sasaki R et al., Oncogenic transformation of human ovarian surface epithelial cells with defined cellular oncogenes. Carcinogenesis. 2009 Mar; 30(3): 423-31.). The effectiveness of this three-gene combination in immortalizing cells has also been demonstrated in other cell types, including human myogenic cells (Shiomi, K et al., 2011), bovine and porcine fibroblasts (Donai et al., 2014), monkey fibroblasts and fetal bovine colon epithelial cells (Kuroda K et al., 2015), as well as cells from several other endangered species. However, it remains unknown whether the corresponding porcine three-gene combination (pTERT, pCyclin D1, and pCDK4 mutant) can immortalize porcine macrophages, and even more so whether these immortalized porcine macrophages retain macrophage properties.

[0007] Summary of the Invention

[0008] The present invention provides a method for constructing immortalized porcine macrophages, which can obtain immortalized porcine bone marrow macrophages without viral oncogenes, viral vector genes and drug resistance genes. The Cre gene is further introduced into the cells to obtain a stably expressed cell line, providing convenient conditions for removing the selection marker gene in the process of constructing gene deletion toxins.

[0009] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0010] An immortalized porcine bone marrow macrophage, characterized in that the immortalized porcine bone marrow macrophage cell line is:

[0011] Immortalized porcine bone marrow macrophages IPBM 27w7 were deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China on June 20, 2023, with the deposit number CCTCC NO: C2023177;

[0012] Or immortalized porcine bone marrow macrophages IPBM27w7-Cre, deposited on June 20, 2023, in China Center for Type Culture Collection, Wuhan University, Wuhan, China, CCTCC NO: C2023176.

[0013] A method for constructing the immortalized porcine bone marrow macrophages comprises the following steps:

[0014] (1) Obtain the femur and tibia of the pig, isolate the bone marrow cells, induce and culture them into macrophages in vitro, and then digest and collect them;

[0015] (2) The transposon vector and transposase vector containing the pTERT, pCDK4(R24C), and pCCND1 genes were introduced into primary porcine bone marrow macrophages by electroporation;

[0016] The pTERT transposon vector includes the nucleotide sequence shown in the CDS region of the "TERT" gene in GenBank accession number NM_001244300.2;

[0017] The pCDK4(R24C) transposon vector comprises the nucleotide sequence of the CDS region of the "CDK4" gene as in GenBank accession number NM_001123097.1, and in addition, the amino acid at position 24 is mutated from arginine (R) to cysteine ​​(C);

[0018] The pCCND1 transposon vector comprises the nucleotide sequence of the CDS region of the "CCND1" gene in GenBank accession number XM_021082686.1;

[0019] (3) The surviving cells were subjected to limiting dilution method to screen monoclonal cell lines, and serial subculture was performed to obtain immortalized porcine bone marrow macrophages IPBM 27w7;

[0020] (4) Introducing the Cre gene into the immortalized porcine bone marrow macrophages IPBM 27w7 obtained in step (3) to obtain immortalized porcine bone marrow macrophages IPBM27w7-Cre.

[0021] In step (4), a transposon vector and a transposase vector containing the Cre gene are introduced into immortalized porcine bone marrow macrophages IPBM 27w7 by electroporation; the transposon vector of the Cre gene includes the nucleotide sequence shown in the CDS region of the "Cre" gene in GenBank accession number NC_005856.1; after electroporation, positive clone cell lines are screened using G418 selective culture medium to obtain immortalized porcine bone marrow macrophages IPBM27w7-Cre that stably express Cre.

[0022] The present invention also provides a method for using the porcine bone marrow cell line stably expressing the Cre gene constructed by the method described in the above technical solution in removing the screening marker red fluorescent gene in the recombinant virus with the gE gene deleted of the porcine pseudorabies virus.

[0023] The invention relates to the use of the immortalized pig bone marrow macrophages in preparing vaccines.

[0024] Immortalized porcine bone marrow macrophages IPBM 27w7 were deposited in China Center for Type Culture Collection on June 20, 2023, at Wuhan University, Wuhan, China, with the deposit number CCTCC NO: C2023177.

[0025] Immortalized porcine bone marrow macrophages IPBM27w7-Cre were deposited in the China Center for Type Culture Collection on June 20, 2023, at Wuhan University, Wuhan, China, CCTCC NO: C2023176. Beneficial effects:

[0026] 1. The present invention provides a new type of immortalized porcine bone marrow macrophages

[0027] Primary cells isolated from living tissue typically have only a limited number of passages in vitro, largely determined by the length of telomeres at the ends of chromosomes. Due to the semiconservative nature of DNA replication, telomeres continuously shorten as cells replicate, eventually becoming too short for the cell to divide normally, leading to cell death. Telomerase, a complex involved in telomere synthesis, plays a particularly significant role in maintaining telomere length, thereby enhancing cell division capacity. Therefore, overexpression of telomerase reverse transcriptase (TERT) is a commonly used strategy for cell immortalization. In actual research, overexpression of TERT alone is insufficient for immortalizing all cell types, such as primary porcine bone marrow macrophages. Oncogene introduction is another common immortalization method. In addition to the commonly expressed viral oncogenes (such as SV40LT and HPV E6E7) or intracellular proto-oncogenes (such as HRAS), several tumor suppressor genes, such as p53 and pRb, also contribute to cell immortalization. Blocking the pRb pathway (p16-CDK4 / cyclin D1-pRb) has been reported to accelerate cell progression. The cell cycle protein Cyclin D1 binds to the corresponding cyclin-dependent kinase CDK4 to form a specific CDK4-CyclinD1 complex that regulates the cell cycle or transcription. The CDK4 (R24C) mutant binds to CyclinD1 and is unable to activate pRb, potentially bypassing the senescence phase (M1) and crisis phase (M2) of cell immortalization. However, because porcine bone marrow macrophages differ from ordinary primary cells, introducing these individual genes alone will not achieve immortalization or will have poor results. Furthermore, some of the immortalized biochemical porcine macrophages disclosed in the prior art lack specific surface antigens, which is different from primary porcine macrophages and limits their application.

[0028] The present invention provides a novel method for constructing immortalized porcine bone marrow macrophages, namely, introducing a transposon vector and a transposase vector of the pTERT, pCDK4 (R24C) and pCCND1 genes into primary porcine bone marrow macrophages by electroporation, wherein the three-gene immortalizing gene combination of pTERT, pCDK4 (R24C) and pCCND1 has not been reported in the literature as immortalized porcine bone marrow macrophages. At the same time, the present invention found that after using pTERT or hTERT alone to introduce primary porcine bone marrow macrophages, immortalization could not be achieved (less than 6 generations); while the combination of pTERT, pCDK4 (R24C) and pCCND1 introduced into bone marrow macrophages can achieve immortalization, with an immortalization effect equivalent to that of the oncogene SV40LT, and the three-gene combination is a non-viral oncogene, avoiding the introduction of exogenous viral oncogenes; the present invention constructed a total of 3 porcine bone marrow macrophage cell lines stably expressing Cre, named IPBM 27w7-Cre-1, IPBM27w7-Cre-2 and IPBM27w7-Cre-3; among them, the porcine bone marrow macrophage IPBM 27w7 highly expressed CD14, CD16, CD163, CD172a, CD203a and CD169, and after 30 stable passages, it still maintained a cell morphology similar to that of primary porcine bone marrow macrophages, with clear cell contours and stable cell passage. In summary, the porcine bone marrow macrophage line IPBM 27w7 is a high-quality porcine macrophage with significant advantages: it is free of viral oncogenes, viral vector genes, and selection tags, and its morphology is similar to that of primary porcine bone marrow macrophages, with clear cell outlines, stable cell passage, and simple operation. Flow cytometry analysis demonstrated that this cell line expresses high levels of macrophage-specific surface molecules: CD14, CD16, CD163, CD169, CD172a, and CD203a, indicating that it is a mature macrophage. Furthermore, this cell line has been deposited (CCTCC NO: C2023177).

[0029] 2. No foreign genes or screening tags are special requirements for ideal vaccines

[0030] The introduction of exogenous genes during vaccine production is unavoidable, and these exogenous genes can pose risks. For example, immortalization typically requires the introduction of viral oncogenes (such as the SV40LT gene) via lentiviral vectors. Furthermore, screening tags are often incorporated to facilitate screening. However, these exogenous viral vectors, oncogenes, and screening tags all introduce new risks and isolation difficulties. Guidelines issued by organizations such as the World Health Organization (WHO), the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the Chinese Pharmacopoeia, as well as pharmacopoeias, clearly define safety testing standards for production cells. A comprehensive understanding of production cells is crucial, including cell characteristics, stability, purity, the presence of exogenous and endogenous factors, and tumorigenicity. Testing for tumorigenicity and exogenous factors is a mandatory safety test requirement in all organizations' regulations. Therefore, when selecting potential production cells, there is a tendency to choose options with lower risk, aiming to minimize or avoid the introduction of viral or oncogene genes into the vaccine to mitigate risk.

[0031] Methods for constructing gene-deleted viruses: Currently, the mainstream technologies for constructing gene-deleted viruses include three categories: traditional marker-assisted site-directed mutagenesis, viral gene deletion based on constructed viral vectors (for example, bacterial artificial chromosome technology, recombinant cosmid system, influenza virus 8-plasmid virus rescue system and replication-defective adenovirus vector), and CRISPR / Cas9-mediated gene editing technology. The construction process of viral vectors is time-consuming and labor-intensive, and the experimental operation is difficult, requiring mature background technology and long-term verification. CRISPR / Cas9 technology has been very popular in recent years, but its target selection is limited by the sequence characteristics of the protospacer adjacent motif (PAM), and there is a risk of off-target, which may cause unnecessary mutations. Therefore, although traditional marker-assisted site-directed mutagenesis has the disadvantage of low efficiency, it is not limited in site sequence selection and is highly conservative. The construction of gene-deficient recombinant viruses in eukaryotic cells usually uses a combination of selection markers and homologous recombination. The conventional process can be divided into three steps: first, the transfer vector and the viral genome are transfected in cells susceptible to the virus (or the virus is infected after transfection), so that the homologous arm sequences on the transfer vector and the homologous sequences on the virus are recombined, and the selection marker gene on the transfer vector (such as a fluorescent gene and / or a drug resistance gene) is introduced into the viral genome; second, the characteristics of the selection marker gene are used to separate and purify the recombinant virus from the mixture of wild-type virus and recombinant virus; third, the selection marker gene is removed from the recombinant virus. The early method of removing the selection marker gene can reuse the recombinant plasmid with only the homologous arm sequences on both sides but without the selection marker gene for homologous recombination to replace the selection marker gene. This step is equivalent to repeating the first two steps, but there will be no selection marker gene to assist in the purification of the virus this time, making the virus purification extremely difficult and labor-intensive. Currently, the most widely used site-specific recombination systems are the Cre / LoxP and FLP / FRT systems. Cre and FLP are both specific recombinases belonging to the λ integrase family of recombinases. They catalyze similar reactions, target sites, and recombination mechanisms. LoxP and FRT are specific sites and share similar structures. Therefore, when designing transfer vectors, specific sites are placed in the same orientation on either side of the selectable marker gene. Then, by expressing the specific recombinase in virus-susceptible cells and transfecting the recombinant viral genome (or infecting the recombinant virus), the selectable marker gene can be precisely removed. However, the sole factor determining the efficiency of virus purification is the proportion of cells expressing the specific recombinase. Conventional transfection methods, however, are limited by cellular characteristics, particularly macrophages, which are generally considered difficult to transfect. Therefore, achieving 100% expression efficiency is unavoidable, and the virus purification step required to remove the selectable marker gene is unavoidable. However, the availability of a macrophage cell line expressing the specific recombinase could easily address the challenge of removing the selectable marker gene, eliminating the need for virus purification.

[0032] As can be seen from the above, constructing a gene-deleted vaccine for a macrophage-tropic virus involves both the gene-deleted virus and susceptible cells. However, there is still a lack of high-quality, easily manipulated susceptible cell lines. Furthermore, during the construction of gene-deleted vaccines, selection marker genes are often added to aid in the selection of positive strains. However, vaccine approval requirements prohibit the inclusion of such selection markers, and the labor-intensive reverse screening of negative strains remains a pressing issue. While the Cre / LoxP recombinase system is commonly used to remove a single selection marker gene, in the development of macrophage-tropic porcine viral vaccines, this is difficult due to transfection challenges.

[0033] The porcine bone marrow macrophage cell line stably expressing the Cre gene, constructed using the methods described herein, can be used to remove the selection marker of gene-deleted recombinant viruses. For example, as shown in the examples herein, the Cre-expressing porcine bone marrow macrophage cell line described herein can effectively remove the selection marker—the red fluorescent gene—from a recombinant virus containing the gE gene of pseudorabies virus. The cell line can also be used to remove selection marker genes from other gene-deleted recombinant viruses adapted to this cell line. Therefore, the Cre-expressing porcine bone marrow macrophage cell line provided by the present invention can efficiently and simply remove selection marker genes during the construction of gene-deleted vaccines.

[0034] 3. Conventional transfection methods—chemical transfection—can cause macrophage activation and adverse reactions when introducing exogenous nucleic acids. Compared to other transfection methods, electroporation is simpler and more efficient, without biological or chemical side effects. This invention utilizes electroporation for transfection. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a map of the transposon vectors pTERT (A), pCDK4 (R24C) (B), pCCND1 (C), and Cre (D);

[0036] FIG2 is a morphological diagram of primary porcine bone marrow cells (A) and induced differentiated porcine bone marrow macrophages (B) isolated in Example 1;

[0037] FIG3 is a cell morphology diagram of the immortalized porcine bone marrow macrophages at passages 10 (A), 20 (B), and 30 (C) obtained by screening in Example 1;

[0038] FIG4 is a flow cytometry graph showing the detection of macrophage-specific cell surface molecules expressed by immortalized porcine bone marrow macrophages in Example 1;

[0039] FIG5 is a diagram showing the WB identification results of three immortalized porcine bone marrow macrophages expressing Cre screened in Example 2;

[0040] FIG6 is a diagram showing the effect of using pig bone marrow macrophages expressing the Cre gene to remove the selection marker in the recombinant virus with gE gene deletion of pseudorabies virus in Example 3;

[0041] FIG7 is a comparison of the transfection efficiency of three common transfection methods on porcine macrophages in Comparative Example 2;

[0042] FIG8 is a comparison of the efficiency of removing the gene-deleted recombinant virus selection marker by transient transfection and stable expression of Cre protein in Comparative Example 3. DETAILED DESCRIPTION

[0043] The following examples are provided to better illustrate the technical solutions of the present invention, but are not intended to limit the scope of protection of the present invention.

[0044] Example 1 Construction of Immortalized Porcine Bone Marrow Macrophages

[0045] 1.1 Isolation and culture of primary porcine bone marrow macrophages

[0046] 1) Remove the femur and tibia of the pig and transfer them to a biosafety cabinet under low temperature and sterile conditions;

[0047] 2) After removing the meat, spray the bone surface with 75% alcohol to disinfect it, and saw it in the center to expose the cross section;

[0048] 3) Flush out the bone marrow with a syringe filled with bone marrow flushing fluid and filter to obtain a bone marrow cell suspension;

[0049] 4) Transfer the suspension into a centrifuge tube, centrifuge at 1500 rpm, 4°C for 5 minutes, and discard the supernatant;

[0050] 5) Resuspend the cells in 3-5 volumes of red blood cell lysis buffer, let stand for 2 minutes, terminate the reaction with 20 mL of cell wash buffer, centrifuge at 1500 rpm, 4°C for 5 minutes, and discard the supernatant;

[0051] 6) Resuspend in cell washing solution, centrifuge at 1500 rpm, 4°C for 5 minutes, discard the supernatant, and repeat washing three times to obtain the precipitate of porcine bone marrow cells;

[0052] 7) Resuspend the cells in growth medium (RPMI-1640 medium + 10% FBS + 10 ng / mL pGM-CSF + 1% double antibody), transfer to a 10 cm culture dish, and incubate at 37°C, 5% CO2, changing half the medium every three days. Incubate for 7 days to obtain porcine bone marrow macrophages.

[0053] As shown in Figure 2, the left image shows the morphology of freshly isolated porcine bone marrow cells (BM), and the right image shows the morphology of porcine bone marrow macrophages (BMDM) cultured after GM-CSF induction. Freshly isolated porcine bone marrow cells are spherical, suspended, and not adherent to the wall. Porcine bone marrow macrophages that have undergone induced differentiation have a "fried egg" shape, with clear cell outlines and adherent growth. This shows that the porcine bone marrow macrophages isolated and cultured in the present invention are normal.

[0054] 1.2 Electroporation of porcine bone marrow macrophages

[0055] 1) Take the primary porcine bone marrow macrophages from step 1.1, digest and collect them, resuspend them in appropriate electroporation solution, count the cells, and dispense them into centrifuge tubes, 10 per tube. 6 cells;

[0056] 2) Add plasmids pTEG-pTERT, pTEG-pCDK4(R24C), pTEG-pCCND1, and pSB16 to each tube of cells. The vector maps of pTEG-pTERT, pTEG-pCDK4(R24C), and pTEG-pCCND1 are shown in Figure 1;

[0057] 3) After the cells and plasmid are mixed, transfer them into a 2 mm electroporation cuvette;

[0058] 4) Set the parameters on the electroporator: select exponential decay pulse, capacitance of 940 μF, voltage between 150 V and 300 V, and duration of 1 ms to 10 ms;

[0059] 5) Immediately after the electric pulse, transfer the cells to a 12-well plate containing growth medium and gently pipette to mix.

[0060] 6) Incubate at 37°C, 5% CO2 for 3 days.

[0061] 1.3 Screening of immortalized porcine bone marrow macrophages

[0062] 1) Take the electroporated porcine bone marrow macrophages obtained in step 1.2, dilute them into single cells by limiting dilution method and inoculate them into 96-well plates;

[0063] 2) Incubate at 37°C, 5% CO2, changing the medium every five days. Once the cells have filled a single well, transfer them to a 24-well plate after digestion and continue culturing.

[0064] 3) After the bottom of the 24-well plate is fully grown, transfer the cells to a 6-well plate; after the 6-well plate is fully grown, transfer the cells to a 6-cm cell dish; after the 6-cm cell dish is fully grown, transfer the cells to a 10-cm cell dish for subculture;

[0065] 4) Gradually expand the culture to screen for monoclonal cell lines with uniform morphology and stable growth. Ultimately, the optimal cell line was identified and named IPBM 27w7, with a CCTCC accession number of C2023177.

[0066] Figure 3 shows the cell morphology of immortalized porcine bone marrow macrophages (IPBM) 27w7 at passages 10, 20, and 30. It can be seen that IPBM 27w7 cells maintain a similar morphology to primary porcine bone marrow macrophages (Figure 2, right), with clear cell outlines and stable cell passages.

[0067] Immortalized porcine bone marrow IPBM 27w7 cells were used to detect macrophage-specific cell surface molecules: CD14, CD16, CD163, CD172a, CD203a, and CD169. Primary antibodies were obtained from BIO-RAD (CD14 Catalog No. MCA1218GA, CD16 Catalog No. MCA1971GA, CD163 Catalog No. MCA2311GA, CD172a Catalog No. MCA2312GA, CD203a Catalog No. MCA1973GA, and CD169 Catalog No. MCA2316GA). FITC-labeled secondary antibodies were obtained from Invitrogen (Catalog No. A11001). The flow cytometry results are shown in Figure 4 . The porcine bone marrow macrophages IPBM 27w7 highly expressed CD14, CD16, CD163, CD172a, CD203a, and CD169, indicating that IPBM 27w7 is a mature macrophage.

[0068] Example 2 Construction of a porcine bone marrow macrophage cell line stably expressing Cre protein

[0069] 1) Immortalized porcine bone marrow macrophages (IPBM) 27w7 cells obtained in step 1.3 were electroporated using the same procedure as in step 1.2. Each tube of cells was electroporated with pTEG-Cre-IRES2-NeoR and pSB16 (the map of the pTEG-Cre-IRES2-NeoR vector is shown in Figure 1). Immediately after electroporation, the cells were transferred to a 12-well plate containing growth medium and gently pipetted to mix.

[0070] 2) Incubate at 37°C, 5% CO2 for 24 hours, add screening medium containing G418 (100 μg / mL), and continue incubation;

[0071] 3) Observe cell survival every day and replace the screening medium every 3 days;

[0072] 4) Maintain the culture until cells with good survival are obtained. As in step 1.3, perform limiting dilution of cells in the surviving wells to obtain cell clones and gradually expand the culture.

[0073] 5) RT-PCR detection was used to identify the expression of the Cre gene in each cloned cell, and a porcine macrophage cell line stably expressing the Cre gene was obtained.

[0074] Results: Three porcine bone marrow cell lines stably expressing Cre were obtained, named IPBM27w7-Cre-1, IPBM27w7-Cre-2, and IPBM27w7-Cre-3. As shown in Figure 5, Western blot analysis showed that all three cell lines could express exogenous proteins of approximately 38 kDa, indicating that all three cell lines could effectively express Cre protein.

[0075] Note: IPBM27w7-Cre-1 in the examples refers to immortalized porcine bone marrow macrophages IPBM27w7-Cre, CCTCC NO: C2023176.

[0076] Example 3 Removal of the selection marker in the pseudorabies virus gene-deleted recombinant virus by expressing Cre protein

[0077] 3.1 Construction of recombinant viruses with gE gene deletion of porcine pseudorabies virus

[0078] The porcine pseudorabies virus gE gene-deficient recombinant virus JSY7ΔgE-LoxP-RFP used in this patent is a JSY7 strain (JSY7 genome sequence GeneBank accession number MT150583.1) with a partial deletion of the gE coding gene (nucleotide sequences 124465 to 125483 of sequence number MT150583.1). It also carries the selection marker gene, the red fluorescent gene TurboRFP, and has a DNA sequence of LoxP sites in the same orientation at both ends: ATAACTTCGTATAATGTATGCTATACGAAGTTAT. The gE gene-deficient recombinant virus is constructed using conventional methods for constructing viral gene-deficient recombinant viruses, such as those disclosed in patent documents CN114657151A, CN113249341A, or CN104830810A.

[0079] 3.2 Removal of the selection marker gene from the gene-deleted recombinant virus

[0080] 1) Cell inoculation: The Cre gene-expressing pig bone marrow macrophages IPBM27w7-Cre-1 obtained in Example 2 were inoculated into 6-well plates, with 10 cells per well. 6 cells, cultured overnight at 37°C in 5% CO2;

[0081] 2) Virus inoculation: The porcine pseudorabies virus gE gene-deficient recombinant virus JSY7ΔgE-LoxP-RFP constructed in step 3.1 was inoculated into a 6-well plate at an infection dose of 0.1;

[0082] 3) Virus culture collection: Observe the red fluorescence and cytopathic effects of the cell wells after virus inoculation daily until more than 80% of the cells in the wells show obvious cytopathic effects or even die and fall off. Collect the virus culture and divide it into two aliquots. One aliquot is frozen and thawed three times, centrifuged at 10,000 rpm for 5 minutes, and the supernatant is collected and stored at -80°C until further use. The other aliquot is used to collect cells and extract the genome.

[0083] 4) Seeding cells: Same as step 1) Seed IPBM27w7-Cre-1 cells in 6-well plates, 10 cells per well. 6 cells, cultured overnight at 37°C in 5% CO2;

[0084] 5) Virus inoculation: The virus solution collected in step 3) was inoculated into cells in a 6-well plate at 100 μl / well.

[0085] 6) Fluorescence observation: Observe the red fluorescence and pathological changes in the virus-inoculated cell wells for three consecutive days.

[0086] The experimental results are shown in Figure 6. "P1" in Figure 6A represents the first infection of IPBM27w7-Cre-1 cells with the gE gene-deficient recombinant virus JSY7ΔgE-LoxP-RFP in step 3). Three days later, a small number of red fluorescent cells were still visible. The first-generation virus liquid was collected and reinfected with IPBM27w7-Cre-1 using the same method. As shown in "P2" in Figure 6A, no cells expressing red fluorescence were observed. Furthermore, as shown in Figure 6A, the parental IPBM27w7 cells infected with the wild-type virus JSY7 did not express fluorescent protein, but red fluorescence was still observed after repeated infection with JSY7ΔgE-LoxP-RFP. Primers were designed on both sides of the gE gene (forward primer: 5'-CCGGGAAGATAGCCATGGTG-3'; back primer: 5'-CGTCACCGTCGTAGTAGTCCTCG-3') and PCR amplification was performed. The results of nucleic acid electrophoresis are shown in Figure 6B. The genomic sample of the parental cell IPBM 27w7 infected with the wild-type virus JSY7 amplified a band of 1834 bp, which was consistent with expectations; the genomic sample of the parental cell IPBM 27w7 infected with JSY7△gE-LoxP-RFP amplified a band of 3339 bp, which was consistent with expectations; the genomic sample of the IPBM14w3-Cre-1 cell line infected with JSY7△gE-LoxP-RFP for the first time only amplified a band of 1074 bp, which was consistent with expectations, indicating that the Cre / LoxP recombinase system is fully functional and the Cre protein in the cell line successfully removes the selection marker in the gE-deficient recombinant virus - the red fluorescent protein gene. Therefore, the cell line IPBM27w7-Cre-1, which stably expresses the Cre gene, only needs a single infection with the deletion recombinant virus to successfully remove the selection marker gene.

[0087] Comparative Example 1 Immortalization Effects of Different Immortalization Gene Combinations on Primary Porcine Bone Marrow Macrophages

[0088] In order to select a suitable immortalization gene to immortalize primary porcine bone marrow macrophages, the electroporation method of porcine bone marrow macrophages in 1.2 of Example 1 was used. After electroporation under the same conditions, the primary porcine macrophages were routinely passaged, and the number of passages was often less than 3. After a large number of repeated experiments, it was concluded that surviving cells that were passaged more than 6 times after the introduction of the immortalization gene may have the potential for partial immortalization. Primary porcine bone marrow macrophages are difficult to spontaneously immortalize. After a large number of experiments, it was found that the porcine three-gene combination (TERT+CDK4(R24C)+CyclinD) not only has an immortalization effect equivalent to the oncogene SV40LT, but also has the significant advantage of being a non-viral oncogene.

[0089] Table 1 Passaging of primary porcine bone marrow macrophages after electroporation with different immortalizing gene combinations

[0090] Comparative Example 2 Comparison of the transfection efficiency of three common transfection methods on porcine macrophages

[0091] Common transfection methods for introducing exogenous nucleic acids into cells include chemical transfection, viral transfection, and electroporation. The fluorescent plasmid pEGFP-C1 was transfected into primary porcine bone marrow macrophages (BMDM) and the immortalized porcine bone marrow cell line (IPBM 27w7) to compare transfection efficiency. Commercially available conventional chemical transfection reagents, lipofectamine 2000 (Invitrogen), Mirus TransIT-X2 (Mirus), and jetPEI macrophag (PolyPlus), were used, and pEGFP-C1 was transfected into BMDM according to the instructions. After 24 hours, as shown in Figure 7A, cells expressing green fluorescent protein were barely visible under the FITC channel of a fluorescence microscope. Using the same method, IPBM27w7 cells were transfected with pEGFP-C1, and sporadic cells expressing green fluorescent protein were observed (see Figure 7B). Under the bright field of the microscope, the cell state of BMDM and IPBM deteriorated after chemical transfection, indicating that the chemical transfection reagent is toxic to porcine bone marrow macrophages, which is particularly evident on BMDM (see Figures 7A and 7B). BMDM was inoculated with a dose of 30MOI using a lentivirus expressing green fluorescence. After 5 days, as shown in Figure 7C, although lentiviral infection had little effect on the cell state, not only was the operation process cumbersome, but only a small amount of cells expressed green fluorescent protein. Electroporation plasmid pEGFP-C1 was performed on BMDM and IPBM 27w7 according to the operating method of 1.2 in Example 1. As shown in Figure 7D, the electroporation method had a significantly high level of transfection efficiency on both BMDM and IPBM 27w7, and had little effect on the cell state.

[0092] Comparative Example 3 Comparison of the efficiency of removing gene-deleted recombinant virus selection markers by transient transfection and stable expression of Cre protein

[0093] To compare the efficiency of selecting marker genes in the gene-deleted recombinant virus removed by conventional transient transfection and the IPBM27w7-Cre-1 cell line provided by the present invention, the parental cell line IPBM 27w7 was electroporated with the Cre eukaryotic expression vector pcDNA3-Cre according to the electroporation method described in 1.2 of Example 1. 24 hours later, the gE gene-deleted recombinant strain JSY7ΔgE-LoxP-RFP was inoculated. 48 hours later, the cell genome was extracted and tested using the PCR amplification method described in Example 3. The results of nucleic acid electrophoresis are shown in Figure 8. The sample from the IPBM27w7-Cre-1 cell line infected with JSY7ΔgE-LoxP-RFP amplified only a single band of 1074 bp; however, the sample from the IPBM 27w7 cell line electroporated with pcDNA3-Cre and then infected with JSY7ΔgE-LoxP-RFP still amplified two bands of 3339 bp and 1074 bp. Therefore, the IPBM27w7-Cre-1 cell line can not only efficiently remove the gene-deleted recombinant virus selection marker, but also is easy to operate, avoiding the repeated virus purification steps.

[0094] In summary, the method provided by the present invention constructs immortalized porcine bone marrow macrophages that are free of viral oncogenes, viral vector genes, and selection markers, exhibit stable cell passages, and express high levels of macrophage-specific surface molecules, resulting in a mature porcine macrophage cell line. This cell line can also be used to construct stable expression cell lines, such as a porcine bone marrow macrophage cell line expressing the Cre gene, effectively removing selection markers from gene-deletion recombinant viruses, facilitating the construction of gene-deletion vaccines.

[0095] The above are preferred embodiments of the present invention. It should be noted that those skilled in the art may modify or substitute equivalents for the technical solutions described in the aforementioned embodiments. Any modifications and substitutions made within the spirit and principles of the present invention shall be included in and considered within the scope of protection of the present invention.

Claims

1. An immortalized porcine bone marrow macrophage, characterized in that: The immortalized porcine bone marrow macrophage cell line is: Immortalized porcine bone marrow macrophages IPBM 27w7 were deposited in China Center for Type Culture Collection on June 20, 2023, with the deposit address: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C2023177; Or immortalized porcine bone marrow macrophages IPBM27w7-Cre, deposited in China Center for Type Culture Collection on June 20, 2023, deposit address: Wuhan University, Wuhan, China, CCTCC NO: C2023176.

2. A method for constructing the immortalized porcine bone marrow macrophages according to claim 1, comprising the following steps: (1) Obtaining the femur and tibia of a pig, isolating bone marrow cells, inducing and culturing them into macrophages in vitro, and then digesting and collecting them; (2) introducing the transposon vector and transposase vector containing pTERT, pCDK4(R24C) and pCCND1 genes into primary porcine bone marrow macrophages by electroporation; The pTERT transposon vector includes the nucleotide sequence shown in the CDS region of the "TERT" gene in GenBank accession number NM_001244300.2; The pCDK4(R24C) transposon vector includes the nucleotide sequence of the CDS region of the "CDK4" gene in GenBank accession number NM_001123097.1, and in addition, the 24th amino acid is mutated from arginine (R) to cysteine ​​(C); The pCCND1 transposon vector sub-vector includes the nucleotide sequence of the CDS region of the "CCND1" gene in GenBank accession number XM_021082686.1; (3) The surviving cells were subjected to limiting dilution method to select monoclonal cell lines, and then serially subcultured to obtain immortalized porcine bone marrow macrophages IPBM 27w7; (4) Introducing the Cre gene into the immortalized porcine bone marrow macrophage IPBM 27w7 obtained in step (3) to obtain immortalized porcine bone marrow macrophage IPBM27w7-Cre.

3. The method according to claim 2, characterized in that In step (4), a transposon vector and a transposase vector containing the Cre gene are introduced into immortalized porcine bone marrow macrophages IPBM 27w7 by electroporation; the transposon vector of the Cre gene includes the nucleotide sequence shown in the CDS region of the "Cre" gene in GenBank accession number NC_005856.1; after electroporation, positive clone cell lines are screened by G418 selective culture medium to obtain immortalized porcine bone marrow macrophages IPBM27w7-Cre that stably express Cre.

4. Use of the immortalized porcine bone marrow macrophages according to claim 1 in isolating and culturing viruses and preparing vaccines.

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