CN117487761A - Construction method and application of Cre gene expression pig bone marrow macrophage line - Google Patents
Construction method and application of Cre gene expression pig bone marrow macrophage line Download PDFInfo
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Abstract
The invention relates to the field of biotechnology, in particular to a construction method of a pig bone marrow macrophage line expressing Cre genes and application thereof; the method comprises a method for introducing SV40LT genes into electroporated primary pig bone marrow macrophages to immortalize the same, a method for continuously constructing a pig bone marrow macrophage line which stably expresses Cre genes on the cells, and a method for removing screening marker genes in gene deletion recombinant viruses by using the cell line. The method adopted by the invention is to introduce a transposon system containing SV40LT genes into primary pig bone marrow macrophages in an electroporation mode, and screen out high-quality immortalized pig bone marrow macrophages. The Cre gene is continuously electroporated and introduced on the immortalized pig bone marrow macrophage to ensure that the Cre gene is stably expressed, and the Cre gene can be applied to excision of the screening marker gene in the process of constructing the gene deletion vaccine.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a construction method of a pig bone marrow macrophage line expressing Cre genes and application thereof.
Background
The gene deletion vaccine makes it possible to distinguish natural infection wild toxin from vaccine immunity clinically, and is one effective tool for eliminating infectious diseases. In the development or production of vaccines, it is generally necessary to infect susceptible cells by gene-deleted viruses.
Susceptible cells: macrophages are susceptible cells to many infectious diseases of porcine origin, such as African Swine Fever Virus (ASFV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine Epidemic Diarrhea Virus (PEDV), porcine pseudorabies virus (PRV), and the like. However, the preparation of primary macrophages is time consuming and expensive, and the batches are unstable and may be contaminated, so that large-scale vaccine production with primary cells is impractical and also challenging from an ethical point of view. At the same time, one of the important obstacles limiting the development of safe and efficient vaccines is the lack of suitable stable passaged cell lines for virus isolation and propagation. To address this problem, global scientists continue to strive to establish immortalized porcine macrophage lines for the study of viral biological properties such as viral replication cycle, host immunomodulation and pathogenesis to design more efficient diagnostic systems, antiviral drugs and candidate vaccines.
The large T antigen of SV40 virus (SV 40 LT) is a common immortalized cell gene. There are six pig macrophage lines reported at home and abroad that are immortalized by SV40LT genes: IPAM constructed by Canadian vaccine and infectious disease research organization (VIDO) is a continuous cell line of pig alveolar macrophages, a vector pSV3-neo carrying SV40LT genes is chemically transfected by primary Pig Alveolar Macrophages (PAM), and three monoclonal cell strains 3D4/2, 3D4/21 and 3D4/31 are obtained through screening by a selection medium containing geneticin. IPAM expresses only low levels of CD14 and SW3 compared to primary cell PAM, CD163 and SLAII are barely detectable, and thus IPAM is not a mature macrophage (Weingartl HM et al Continuous porcine cell lines developed from alveolar macrophages: partial characterization andvirus subsspool availability. Journal virology methods.2002Jul;104 (2): 203-16). (II) IPKM constructed by national agricultural and food research organization (NARO) is a pig kidney-derived macrophage, and cell lines were obtained by infecting a recombinant lentivirus carrying SV40LT gene and porcine telomerase reverse transcriptase (pTERT) on primary pig kidney-derived macrophage (PKM) and screening them with a selection medium containing geneticin. IPKM has a similar morphology to primary kidney macrophage PKM, expressing macrophage-specific surface markers Iba1, KT022 and CD172a (Takenouchi T et al Immortalization and characterization of porcine macrophages that had been transduced with lentiviral vectors encoding the SV, large T antigen and porcine telomerase reverse transdatase. Front in terinary science.2017Aug 21; 4:132). (III) NARO in 2022 published IPIM constructed is a pig intestinal macrophage, immortalized cell method and IPKM, through recombinant lentivirus transfection mode to express SV40LT and pTERT. IPIM expresses macrophage specific surface molecules Iba-1, CD172a, CD204, CD203a and CD16, and part of the 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 Infection. Front in everinary science.2022Jul18; 9:91977). (IV) domestic patent application publication No. CN 105793416A published "immortalized pig alveolar macrophages" is obtained by transfecting transposon vectors pPB-CAG-SV40 TAg and pPB-CMV-hyPBase carrying SV40LT into primary Pig Alveolar Macrophages (PAM) to immortalize them, and flow assay shows that macrophage surface molecules CD163 and sialoadhesin P210 can be expressed, and the cell line has been cultured for 8 months (50-60 generations) by the time of patent publication. In an immortalized pig macrophage strain, a construction method and application thereof, disclosed in patent application publication No. CN 113528453A, a cell line is obtained by infecting recombinant lentivirus carrying SV40LT gene and human telomerase reverse transcriptase (hTERT) gene by primary pig bone marrow-derived macrophage (BMDM), a used lentivirus vector GLV2-CMV-MCS-PGK-puro expresses puromycin resistance gene, and the cell line is proved to express CD14 through flow detection. The cell line in the patent publication No. CN 114292873A immortalized pig bone marrow macrophages, the construction method and the application thereof is obtained by transferring a plasmid pIED-Neo-SV40TAg carrying an SV40LT gene in primary pig bone marrow macrophages (BMDM) and screening the plasmid pIED-Neo-SV40TAg by a selection medium containing geneticin.
Therefore, although SV40LT can immortalize pig macrophages according to the current literature data, the current data do not inquire about complete experimental data to prove whether the pig macrophages immortalized by SV40LT are fully mature or have tumorigenicity. In addition, the above 6 cell lines were all screened using drug resistance genes, and it was not known whether these drug resistance genes affected downstream applications.
The method for constructing the gene deletion virus comprises the following steps: currently, the mainstream techniques for constructing gene-deleted viruses include three types: traditional marker-assisted site-directed mutagenesis, viral gene deletion and CRISPR/Cas 9-mediated gene editing techniques based on constructed viral vectors (e.g., bacterial artificial chromosome technology, recombinant cosmid systems, 8-plasmid virus rescue systems of influenza virus, replication defective adenovirus vectors, etc.). The construction process of the virus vector is time-consuming and labor-consuming, the experimental operation difficulty is high, and the mature background technology and long-time verification are required. CRISPR/Cas9 technology has been very popular in recent years, and the principle of its recognition site is that a single guide RNA (sgRNA) recruits and binds endonuclease Cas9 to a target site located upstream of the pre-spacer adjacent motif (Protospacer adjacentmotif, PAM). Thus, target selection is limited by the sequence characteristics of PAM and there is a risk of off-target, possibly causing unnecessary mutations. Thus, conventional marker-assisted site-directed mutagenesis techniques, while having the disadvantage of low efficiency, are not limited in site sequence selection and are highly conserved. The construction of gene-deleted recombinant viruses in eukaryotic cells usually employs a combination of selectable markers in combination with homologous recombination, and the conventional process can be divided into three steps: the first step, a transfer vector and a viral genome are transfected in a virus-susceptible cell (or the virus is infected after transfection), homologous arm sequences on the transfer vector are recombined with homologous sequences on the viral genome, and a screening marker gene (such as a fluorescent gene or/and a drug resistance gene) on the transfer vector is introduced into the viral genome; secondly, separating and purifying recombinant viruses from the mixture of wild viruses and recombinant viruses by utilizing the characteristics of screening marker genes; and thirdly, removing the screening marker genes in the recombinant viruses. The method for early removing the screening marker gene can reuse the recombinant plasmid which only has two homologous arm sequences but does not contain the screening marker gene to carry out homologous recombination, and the step of replacing the screening marker gene is equivalent to the two steps before repeating, but the round of virus purification has no assistance of the screening marker gene, and the virus purification difficulty and the workload are extremely large. Therefore, in practical use, people prefer to use site-specific recombination systems, the most commonly used systems are Cre/LoxP and FLP/FRT, wherein Cre and FLP are specific recombinases, belong to the family of recombinase lambda integrases, the reaction types, target sites and recombination mechanisms are very similar, loxP and FRT are specific sites, and have similar structures. Thus, in designing the transfer vector, specific sites are flanked by identical orientations on both sides of the selectable marker gene. Then, the screening marker gene can be precisely and efficiently removed by expressing the specific recombinase in the virus-susceptible cells and transfecting the recombinant virus genome (or infecting the recombinant virus). However, the only factor that determines the efficiency of viral purification is the proportion of cells that express the specific recombinase. If conventional transfection methods are used, the conventional transfection methods are limited by cell characteristics, especially macrophages are recognized as cells which are difficult to transfect, so that 100% expression efficiency cannot be achieved by conventional transfection, and a virus purification step in the process of removing the selection marker gene cannot be avoided. If a macrophage cell line expressing specific recombinase exists, the difficulty of removing the screening marker gene can be easily solved, and the virus purification step of removing the screening marker gene is omitted.
From the above, it was found that constructing a gene-deleted vaccine of macrophage virus involves gene-deleted recombinant virus and easy cells. At present, the problem of lack of mature high-quality susceptible cells still exists, and a screening marker gene is often added in the process of constructing a gene deletion vaccine to assist in screening positive strains, but the problem of high workload of reversely screening negative strains because the screening marker cannot be carried in vaccine approval requirements is urgently solved. However, in the development of porcine viral vaccines of swine macrophages, it is difficult to remove the selectable marker gene due to transfection difficulties.
Disclosure of Invention
In order to overcome the difficulty that screening marker genes cannot be removed efficiently in the process of constructing a macrophage virus gene deletion vaccine at present, the invention provides a method for constructing immortalized pig macrophages by using SV40LT genes, which can obtain mature immortalized pig bone marrow macrophages which have no tumorigenicity, normal chromosome karyotype and no drug resistance genes, and a Cre gene is further introduced into the cells to obtain a stably expressed cell line, thereby providing convenience conditions for removing the screening marker genes in the process of constructing gene deletion recombinant viruses.
In order to achieve the above object, the present invention adopts the following technical scheme:
an immortalized pig bone marrow macrophage, characterized in that the immortalized pig bone marrow macrophage is
Immortalized porcine bone marrow macrophages IPBM14w3 was deposited at the chinese collection of typical cultures at 2023, 6, 7, accession number: the preservation number of the Chinese Wuhan university is CCTCC NO: C2023157;
or immortalized pig bone marrow macrophage IPBM14w3-Cre was deposited at the China center for type culture Collection, with a deposit address: the preservation number of the Chinese Wuhan university is CCTCC NO: C2023178.
A method of constructing said immortalized porcine bone marrow macrophages comprising the steps of:
(1) Taking femur and tibia of a pig, separating bone marrow cells, and performing in vitro induction culture to obtain macrophages, and then performing digestion and collection;
(2) Introducing a transposon vector containing an SV40LT gene and a transposase vector into primary porcine bone marrow macrophages by electroporation;
the transposon vector of the SV40LT gene comprises a nucleotide sequence shown in the CDS region of the "large T anti" gene in GenBank accession NC-001669.1;
(3) And screening the monoclonal cell strain of the surviving cells by a limiting dilution method, and carrying out continuous subculture to obtain the immortalized pig bone marrow cells IPBM14 w3.
(4) The Cre gene is introduced into the pig bone marrow macrophage IPBM14w3-Cre.
The method is characterized in that the pig bone marrow macrophage IPBM14w3-Cre in the step (4) is prepared by the following steps:
introducing a transposon vector containing Cre gene and a transposase vector into an immortalized pig bone marrow macrophage IPBM14w3 in an electroporation manner; the transposon vector of the Cre gene comprises a nucleotide sequence shown in a CDS region of the Cre gene in GenBank accession NC_ 005856.1; after electroporation, positive clone cell strains are screened by G418 selective medium, and the pig bone marrow macrophage IPBM14w3-Cre which stably expresses Cre is obtained. The invention also provides an application method of the pig bone marrow cell line which is constructed by the method and stably expresses the Cre gene in removing the screening marker red fluorescent gene in the recombinant virus deleted in the pig pseudorabies virus gE gene.
Application of immortalized pig bone marrow macrophages in preparing vaccine
Pig bone marrow macrophages IPBM14w3 was deposited with the chinese collection of typical cultures at 2023, 6, 7, accession number: the preservation number of the Chinese Wuhan university is CCTCC NO: C2023157.
Pig bone marrow macrophages IPBM14w3-Cre were deposited with the chinese collection of typical cultures at 2023, 6 and 20, accession number: the preservation number of the Chinese Wuhan university is CCTCC NO: C2023178.
The beneficial effects are that:
the pig bone marrow macrophages are difficult to spontaneously immortalize, common cell immortalization strategies require introducing immortalization genes such as SV40LT and the like into primary cells by means of lentiviral vectors, and simultaneously, screening marker genes are introduced for screening convenience, but whether the lentiviral vectors, exogenous immortalization genes and screening marker genes can be used for production becomes a current concern.
The invention provides a high-quality immortalized pig bone marrow macrophage, which introduces a transposon system carrying SV40LT genes into primary pig bone marrow macrophages by electroporation. The strategy not only avoids the integration of irrelevant sequences on the vector into the genome of the cell, but also has high efficiency, no assistance of drug resistance genes and small side effect on the cell. The inventor finds through a large number of screening that the cell chromosome karyotype provided by the invention is normal, has no tumorigenicity and no drug resistance gene, and highly expresses 6 macrophage specific surface molecules (CD 14, CD16, CD163, CD169, CD172a and CD203 a), and after passage for 70 generations, the IPBM14w3 can still keep similar cell morphology as primary pig bone marrow macrophages, and is a mature pig bone marrow macrophage line. Meanwhile, the porcine bone marrow macrophage line which is constructed on the cell line and can stably express Cre can conveniently and efficiently remove the screening tag in the gene deletion recombinant virus.
Specifically:
the invention provides a method for constructing immortalized pig bone marrow macrophages, which is characterized in that normal pig bone marrow cells are separated from pig bones and induced to differentiate into macrophages, and immortalization is carried out by adopting a transposon system containing SV40LT genes and an electroporation mode, so as to obtain the immortalized pig bone marrow macrophages. The immortalized pig bone marrow macrophage obtained by the method has the remarkable advantages of normal chromosome karyotype, no tumorigenicity, no drug resistance gene, high expression of macrophage specific cell surface molecules and the like. Meanwhile, the method for constructing the porcine bone marrow macrophage line for stably expressing the Cre gene can be used for removing the screening marker of the deletion recombinant virus, and as shown in the embodiment of the invention, the screening marker-red fluorescent gene in the recombinant porcine pseudorabies virus with the gE gene deletion can be effectively removed by utilizing the porcine bone marrow cell line for expressing the Cre gene, and the cell line can also be used for removing the screening marker genes of other cell-adaptive virus gene deletion recombinant viruses.
The specific analysis is as follows: first, SV40LT is used as a common immortalization gene, and in the research progress of immortalized pig macrophages, the obtained cell lines can not normally express specific macrophage specific surface molecules, and whether the cell lines have normal chromosome karyotype and tumorigenicity are not reported by experimental data. Second, macrophages are recognized as difficult cells to transfect as compared to other mammalian cells. On the one hand, because primary macrophages are almost non-proliferating cells in vitro, the possibility of exogenous nucleic acids reaching the nucleus during the rupture of the mitotic nuclear membrane is reduced, and because of the almost non-proliferating nature, primary macrophages are difficult to culture in vitro for a long period of time and therefore difficult to spontaneously immortalize; on the other hand, since macrophages are innate immune cells, they naturally have a mechanism to recognize foreign nucleic acids and initiate the immune response to clear "foreign bodies". The common virus vector transfection such as slow virus and retrovirus vector can integrate virus genes such as large terminal repeated sequence (LTR) into cell genome at the same time, and the virus transfection needs to carry out complex processes such as virus packaging and infection, thus having higher requirements on experimental equipment and biological safety level. Another conventional transfection method, chemical transfection, introduces exogenous nucleic acid and simultaneously causes macrophage activation and adverse reactions. Therefore, electroporation is simple and efficient compared with other transfection methods, has no biological and chemical side effects, but is not perfect, and if only a common vector is electroporated, the efficiency of integrating the target gene into the genome of the cell is extremely low, and the loss phenomenon occurs after passage for several times. Third, transposons are natural DNA transfer tools, and can effectively integrate exogenous genes into the genome and stably express them for a long period of time, similar to integrating viruses. Not only is it efficient, but also it is only integrated into the gene of interest, avoiding integration into other unrelated sequences on the vector, but transposon transfer is not perfect, e.g. transposons cause genomic instability. Thus, the first and second substrates are bonded together,
as shown in the embodiment of the invention, the immortalized pig bone marrow macrophages constructed by the method are high-quality pig macrophages. The cells are in the shape of a fried egg, have clear cell outline and complete cells, and have typical morphological characteristics similar to those of primary pig bone marrow macrophages. The cell line is stable in passage and has been passed over 70 times. The cell strain does not form tumor on nude mice, and 63 rd generation cells still retain a similar staining karyotype as primary pig cells. By flow-through assays, the cell line can express high levels of specific surface molecules of mature macrophages: CD14, CD16, CD163, CD169, CD172a and CD203a.
The Cre/LoxP recombinase system has complete functions, and Cre protein in the cell line successfully removes a screening marker-red fluorescent protein gene in gE deletion recombinant toxin. Therefore, the cell line IPBM14w3-Cre-1 stably expressing Cre gene can successfully remove the screening marker gene only by single infection and deletion of recombinant toxin.
Drawings
FIG. 1 is a transposon vector map of SV40LT (A) and Cre (B)
FIG. 2 is a cell morphology of the primary porcine bone marrow cells isolated and cultured and immortalized porcine bone marrow macrophages of 10, 20, 30, 40, 50, 60, 70 generations of example 1;
FIG. 3 is a flow chart of detection of macrophage specific cell surface molecules expressed by immortalized porcine bone marrow macrophages in example 1;
FIG. 4 is a graph of the results of the nodulation assay of immortalized porcine bone marrow macrophages in nude mice tested in example 1 (A) and their cytokaryotype (B);
FIG. 5 is a graph showing the identification result of Cre-expressing immortalized porcine bone marrow macrophages WB of 3 strains selected in example 2;
FIG. 6 is a graph showing the effect of removing the selection marker from the recombinant virus deleted from the porcine pseudorabies virus gE gene by using the porcine bone marrow macrophages expressing Cre gene in example 3, wherein A is a fluorescence micrograph and B is a nucleic acid electrophoresis result;
FIG. 7 is a comparison of the efficiency of transient transfection and stable expression of Cre protein in comparative example 3 in removal of gene deletion recombinant virus selection markers.
Detailed Description
The following examples are presented to better illustrate the technical solution of the present invention, but are not intended to limit the scope of the present invention.
EXAMPLE 1 construction of immortalized porcine bone marrow macrophages
1.1 isolated culture of Primary porcine bone marrow macrophages
1) Taking femur and tibia of a pig, and transferring the femur and tibia to a biosafety cabinet under low temperature and aseptic conditions;
2) After meat is removed, 75% alcohol is sprayed on the surface of the bone for disinfection, and the exposed cross section is sawed in the middle;
3) Flushing bone marrow with a syringe filled with marrow flushing liquid, and filtering to obtain marrow cell suspension;
4) Transferring the suspension into a centrifuge tube, centrifuging at 1500rpm and 4 ℃ for 5 minutes, and discarding the supernatant;
5) Resuspension cells with 3-5 times volume of erythrocyte lysate, standing for 2 min, stopping reaction with 20mL of cell washing liquid, centrifuging at 1500rpm and 4 ℃ for 5 min, and discarding supernatant;
6) Resuspension with cell washing liquid, centrifuging at 1500rpm and 4 deg.C for 5 min, discarding supernatant, and repeating washing for three times to obtain precipitate as pig bone marrow cells;
7) Cells were resuspended in growth medium (RPMI-1640 medium+10% FBS+10ng/mLpGM-CSF+1% diabody), transferred to 10cm dishes, 37℃and 5% CO 2 Half liquid exchange every three days, and standing culture for 7 days to obtain pig bone marrow macrophages.
As shown in FIG. 2, the upper left corner shows pig bone marrow macrophages (BMDM) induced by GM-CSF to differentiate pig bone marrow cells in the shape of "omelette", with clear cell contours and adherent growth. It can be seen that the isolated and cultured pig bone marrow macrophages of the present invention are normal.
1.2 electroporation of porcine bone marrow macrophages
1) Taking primary pig bone marrow macrophages in the step 1.1, digesting and collecting, re-suspending with electroporation liquid with proper volume, counting cells, and sub-packaging into centrifuge tubes, each tube 10 6 A cell;
2) Plasmids pTEG-SV40LT and pSB16 were added to each cell, wherein the pTEG-SV40LT vector map is shown in FIG. 1;
3) After the cells and the plasmid are evenly mixed, the mixture is transferred into a 2mm electric shock cup;
4) Parameters are set on the electrotransport meter: an exponential decay pulse is selected, the capacitance is 940 muF, the voltage is 150V-300V, and the time is 1 ms-10 ms;
5) Immediately transferring cells into a 12-hole plate containing a growth culture medium after electric pulse, and lightly blowing and uniformly mixing;
6)37℃，5％CO 2 and (5) standing and culturing for 3 days.
1.3 screening of immortalized porcine bone marrow macrophages
1) Taking the electroporation transfected pig bone marrow macrophages obtained in the step 1.2, diluting the pig bone marrow macrophages into single cells by a limiting dilution method, and inoculating the single cells into a 96-well plate;
2) Standing at 37 ℃ with 5% CO2, changing liquid every five days, and transferring into a 24-pore plate for continuous culture after the cells grow into single pores after digestion;
3) After the bottom of the 24 pore plates is full, transferring the 24 pore plates into the 6 pore plates; transferring the cell into a 6cm cell dish after the 6 pore plate is full; after 6cm cell dish is full, transferring into 10cm cell dish for subculture;
4) Gradually expanding culture, and screening monoclonal cell strains with uniform cell morphology and stable growth speed. Finally, the optimal cell line was selected and designated IPBM14 w3.
As shown in FIG. 2, comparing the cell morphology of primary porcine bone marrow macrophages (BMDM) with that of different generations of immortalized porcine bone marrow macrophages IPBM14w3, the first panel in the upper left hand corner is the cell morphology of isolated bone marrow cells after 7 days of induction in vitro with porcine GM-CSF, followed by the immortalized porcine bone marrow macrophages of generations 10, 20, 30, 40, 50, 60 and 70, in that order, it is seen that IPBM14w3 still maintains a cell morphology similar to that of primary porcine bone marrow macrophages. RNA of IPBM14w3 cells of different generations is extracted, and RT-PCR detection shows that SV40LT is expressed.
Flow assay of macrophage specific cell surface molecules for immortalized porcine bone marrow cells IPBM14w 3: CD14, CD16, CD163, CD172a, CD203a and CD169. The primary antibodies were selected from BIO-RAD company (CD 14 cat# MCA1218GA, CD16 cat# MCA1971GA, CD163 cat# MCA2311GA, CD172a cat# MCA2312GA, CD203a cat# MCA1973GA and CD169 cat# MCA2316 GA), and FITC-labeled secondary antibodies were from Invitrogen company (cat# A11001). As shown in FIG. 3, the cells of IPBM14w3 of the bone marrow cells of the pig all expressed CD14, CD16, CD163, CD172a, CD203a and CD169, indicating that IPBM14w3 is a mature macrophage.
Immortalized porcine bone marrow cells IPBM14w3 were inoculated subcutaneously into 4-week-old balb/c nude mice, 10 per mouse 7 The cells were observed for two months, as shown in fig. 4A, no tumor was formed on the surface of the nude mice after the inoculation of IPBM14w3 cells, and no abnormal situation was observed after dissection, while the positive control group inoculated with mouse myeloma cells SP2/0 cells formed obvious tumor nodules 13 days after inoculation; the negative control group was inoculated with primary chick embryo fibroblast CEF without tumor formation. Thus, IPBM14w3 does not have tumorigenicity.
Immortalized pig bone marrow cells IPBM14w3 63 rd generation cells are taken, and a general chromosome sheeting technology is adopted, and the clearer picture containing metaphase chromosomes is obtained through colchicine pretreatment, fixation, dyeing, sheeting, microscopic examination, photographing and other processes, as shown in figure 4B. Selecting pictures with high cell dispersivity and clear chromosome number, and counting 50 chromosomes containing clear mitotic metaphase cells. By analyzing the number of chromosomes, the number of chromosomes of the IPBM14w3 cell is found to be 2n.apprxeq.38, which is similar to that of normal pig somatic cells. Thus, IPBM14w3 has the chromosomal karyotype of normal cells.
EXAMPLE 2 construction of porcine bone marrow macrophage System stably expressing Cre protein
1) Taking the immortalized pig bone marrow macrophage IPBM14w3 obtained in the step 1.3 of the example 1, carrying out electroporation in the same way as in the step 1.2 of the example 1, and carrying out electroporation of pTEG-Cre-IRES2-NeoR and pSB16 in each tube of cells, wherein the map of pTEG-Cre-IRES2-NeoR vector is shown in figure 1, transferring cells into a 12-well plate containing a growth medium immediately after electric pulse, and gently blowing and mixing;
2)37℃，5％CO 2 culturing for 24 hours, adding a screening culture medium containing G418 (100 ug/mL), and continuing culturing;
3) Observing the survival condition of the cells every day, and changing the screening culture medium every 3 days;
4) Maintaining culture until obtaining cell holes with good survival condition, carrying out limited dilution on the cell of the survival hole in the same way as the step 1.3 of the example 1 to obtain cell clone, and gradually expanding culture;
5) And (3) detecting and identifying the condition of expressing the Cre gene by each cloned cell by RT-PCR to obtain the pig macrophage line for stably expressing the Cre gene.
Results: obtaining 3 pig bone marrow cell strains which stably express Cre and are respectively named as IPBM14w3-Cre-1, IPBM14w3-Cre-2 and IPBM14w3-Cre-3; as shown in FIG. 5, through Western blot detection, the 3 cells can express the exogenous protein of about 38kDa, which indicates that the 3 cells can effectively express Cre protein.
Note that: the IPBM14w3-Cre-1 is pig bone marrow macrophage IPBM14w3-Cre, and the preservation number is CCTCC NO: C2023178.
Example 3 removal of screening markers in pseudorabies virus Gene deletion recombinant Virus Using Cre protein expression
3.1 construction of porcine pseudorabies Virus gE Gene deletion recombinant Virus
The pig pseudorabies virus gE gene deletion recombinant strain JSY7ΔgE-LoxP-RFP used in the patent is a JSY7 strain (JSY 7 genome sequence GeneBank accession number is MT 150583.1) deletion part gE coding gene (124465 ～ 125483 th bit of nucleotide sequence of sequence number MT 150583.1), and carries a screening marker gene, namely red fluorescent gene TurboRFP, and two ends of the strain are respectively provided with a DNA sequence of a homodromous LoxP site: ataac ttcgtataatgtatgctatactagagtttat. In the invention, a gE gene deletion recombinant virus is constructed by adopting a conventional method for constructing a virus gene deletion recombinant virus, for example, a method disclosed in a patent document CN114657151A, CN113249341A or CN 104830810A.
3.2 removal of the selectable marker Gene from the Gene deletion recombinant toxin
1) Inoculating cells: the Cre gene-expressing porcine bone marrow macrophage IPBM14w3-Cre-1 obtained in example 2 was inoculated into 6-well plates, 10 per well 6 Individual cells, 37 ℃,5% co 2 Culturing overnight;
2) Inoculating virus: inoculating the porcine pseudorabies virus gE gene deletion recombinant strain JSY7ΔgE-LoxP-RFP constructed in the step 3.1 into a 6-well plate, wherein the infection amount is MOI=0.1;
3) Collecting virus liquid: observing red fluorescence and cytopathic conditions in cell holes after virus inoculation every day until more than 80% of cytopathic conditions in the holes are obvious and even die and fall off, collecting virus cultures, dividing the virus cultures into two parts, repeatedly freezing and thawing one part for 3 times, centrifuging at 10000rpm for 5 minutes, collecting supernatant, and preserving at-80 ℃ for later use; another fraction was collected to extract genome.
4) Inoculating cells: same as step 1) IPBM14w3-Cre-1 cells were seeded in 6-well plates, 10 per well 6 Individual cells, 37 ℃,5% co 2 Culturing overnight;
5) Inoculating virus: inoculating 100 microliters/well of the virus solution collected in step 3) into 6-well plate cells; .
6) Fluorescence was observed: red fluorescence and lesions of three days of inoculated virus cell wells were observed continuously.
As a result of the experiment, see FIG. 6, the "P1" in FIG. 6A is that in step 3), the IPBM14w3-Cre-1 cells were infected with gE gene deleted recombinant virus JSY7ΔgE-LoxP-RFP for the first time, a small amount of red fluorescent cells were still seen after three days, the first-generation virus solution was collected, and the IPBM14w3-Cre-1 cells were again infected in the same manner, as shown in "P2" in FIG. 6A, and red fluorescent expressing cells were not seen. Meanwhile, as shown in fig. 6A, the parent cell IPBM14w3 infected with wild virus JSY7 did not express fluorescent protein, while repeated infection with jsy7Δge-LoxP-RFP still showed red fluorescence. Primers (a front primer is 5'-CCGGGAAGATAGCCATGGTG-3' and a rear primer is 5'-CGTCACCGTCGTAGTAGTCCTCG-3') are designed on two sides of the gE gene, PCR amplification is carried out, the result of nucleic acid electrophoresis is shown in figure 6B, and a genome sample of a parent cell IPBM14w3 infected with wild virus JSY7 can be amplified into 1834bp strips, which are in accordance with the expectations; genomic samples of parent cells IPBM14w3 infected with JSY7ΔgE-LoxP-RFP can be amplified to have a 3339bp band, which is in line with expectations; the genomic sample of the IPBM14w3-Cre-1 cell line infected with JSY7ΔgE-LoxP-RFP for the first time only expands 1074bp bands, which accords with the expectations, and shows that the Cre/LoxP recombinase system has complete functions, and Cre protein in the cell line successfully removes the screening marker-red fluorescent protein gene in gE deletion recombinant toxin. Therefore, the cell line IPBM14w3-Cre-1 stably expressing Cre gene can successfully remove the screening marker gene only by single infection and deletion of recombinant toxin. Comparative example 1 efficiency of removal of deletion recombinant virus selection markers by comparison of transient transfection and stable expression of Cre protein lines
In order to compare the efficiency of screening marker genes in the removal of gene deletion recombinant viruses of the IPBM14w3-Cre-1 cell line provided by the invention with the conventional transient transfection method for expressing Cre. According to the electroporation method described in example 1.2, the eukaryotic expression vector pcDNA3-Cre of Cre was electroporated with IPBM14w3 of the parent cell line, and after 24 hours, the gE gene deletion recombinant strain JSY7ΔgE-LoxP-RFP was inoculated, and after 48 hours, the cell genome was extracted, and the detection was carried out according to the PCR amplification method mentioned in example 3, and as a result of nucleic acid electrophoresis, as shown in FIG. 7, only one 1074bp band could be amplified from a sample of JSY7ΔgE-LoxP-RFP infected with IPBM14w3-Cre-1 cells; while the IPBM14w3 electroporated pcDNA3-Cre and infected with JSY7ΔgE-LoxP-RFP still amplified two bands of 3339bp and 1074bp. Therefore, the IPBM14w3-Cre-1 cell line not only can remove the deletion recombinant virus screening marker with high efficiency, but also is simple to operate, and avoids the step of repeated virus purification.
In conclusion, the immortalized pig bone marrow macrophage chromosome karyotype is normal, has no tumorigenicity and no drug resistance gene, expresses macrophage specific surface molecules at high level, and is a mature pig macrophage line. The cell line with stable expression, such as pig bone marrow macrophage line expressing Cre gene, can be used to eliminate the screening mark in gene deletion recombinant virus effectively, and is convenient for constructing gene deletion vaccine.
While the foregoing is directed to the preferred embodiments of the present invention, it should be noted that modifications and equivalents may be made to the embodiments described herein. Modifications and substitutions within the spirit and principles of the present invention should be construed as being included within the scope of the present invention.
Claims (4)
1. An immortalized pig bone marrow macrophage, characterized in that the immortalized pig bone marrow macrophage is
Pig bone marrow macrophages IPBM14w3 was deposited with the chinese collection of typical cultures at 2023, 6, 7, accession number: the preservation number of the Chinese Wuhan university is CCTCC NO: C2023157;
or pig bone marrow macrophage IPBM14w3-Cre was deposited with China center for type culture Collection, with a deposit address: the preservation number of the Chinese Wuhan university is CCTCC NO: C2023178.
2. A method of constructing the immortalized porcine bone marrow macrophage of claim 1, comprising the steps of:
(1) Taking femur and tibia of a pig, separating bone marrow cells, and performing in vitro induction culture to obtain macrophages, and then performing digestion and collection;
(2) Introducing a transposon vector containing an SV40LT gene and a transposase vector into primary porcine bone marrow macrophages by electroporation;
the transposon vector of the SV40LT gene comprises a nucleotide sequence shown in the CDS region of the "large T anti" gene in GenBank accession NC-001669.1;
(3) Screening monoclonal cell strains of the surviving cells by a limiting dilution method, and continuously carrying out subculture to obtain immortalized pig bone marrow cells IPBM14 w3;
(4) The Cre gene is introduced into the pig bone marrow macrophage IPBM14w3-Cre.
3. The method of claim 2, wherein step (4) porcine bone marrow macrophage IPBM14w3-Cre is prepared by:
introducing a transposon vector containing Cre gene and a transposase vector into an immortalized pig bone marrow macrophage IPBM14w3 in an electroporation manner; the transposon vector of the Cre gene comprises a nucleotide sequence shown in a CDS region of the Cre gene in GenBank accession NC_ 005856.1; after electroporation, positive clone cell strains are screened by G418 selective medium, and the pig bone marrow macrophage IPBM14w3-Cre which stably expresses Cre is obtained.
4. Use of an immortalized porcine bone marrow macrophage according to claim 1 for isolated culture of an adapted virus or for the preparation of a vaccine.
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