CN114292873A - Immortalized pig bone marrow macrophage, construction method and application thereof - Google Patents

Abstract

The application discloses immortalized pig bone marrow macrophages, a construction method and application thereof. The construction method comprises the following steps: transfecting pig bone marrow macrophages with a first recombinant plasmid containing a nucleotide sequence shown in SEQ ID NO.1, culturing the transfected cells, and separating cell monoclonals. Furthermore, the immortalized porcine bone marrow macrophages are transformed by transfecting second recombinant plasmids and third recombinant plasmids containing nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO. 3. The construction and modification methods of the immortalized pig bone marrow macrophage line provided by the application are simple and easy to operate, and the obtained immortalized pig bone marrow macrophage line has the remarkable characteristic of continuous passage and high sensitivity to viruses after modification.

Description

Immortalized pig bone marrow macrophage, construction method and application thereof

Technical Field

The application specifically relates to immortalized pig bone marrow macrophages, a construction method and application thereof, and belongs to the technical field of biology.

Background

Macrophages are one of the important immune defense cells of the body, are widely distributed throughout the body, are present in the lymphoid organs, liver, lung, gastrointestinal tract, central nervous system, bone and skin, are important components of the host's innate immunity and specific immunity, and are also key regulatory and effector cells in the immune response (Verdeguer F and Aouadi m. In nonspecific immunity, macrophages can kill and eliminate pathogens and foreign body-mediated inflammatory reactions through phagocytosis and participate in innate immune response; and, in specific immunity, processes and presents antigens as antigen presenting cells and initiates adaptive immune responses, while secreting cytokines to exert immunomodulatory effects (MurrayP J. immune regulation by monocytes [ J ]. Semin Immunol, 2018, 35: 12-18). Macrophages in different differentiation states have different susceptibility to viruses, and viruses can prevent phagocytosis of macrophages, inhibit antigen presentation capacity of macrophages, reduce and regulate proinflammatory cytokines and influence acquired immune response of hosts by infecting the macrophages. Therefore, the study of macrophages is important for the future study of viruses.

Macrophage polarization is a process stimulated by microenvironment signals to acquire different phenotypic and functional characteristics, and is generally obtained by culturing bone marrow mononuclear cells in vitro with GM-CSF, and generally includes two pathways, namely a classical activation pathway and a selective activation pathway. The classically activated M1 type macrophage is stimulated by IFN-alpha and Lipopolysaccharide (LPS), and is characterized by secreting Th1 related cytokines IL-12, IL-6, IL-1 and TNF-alpha, participating in inflammatory reaction of organisms and presenting stronger antigen presenting and phagocytosis capacity. Selectively activated macrophages of type M2 are characterized by secretion of Th 2-related factors IL-10, among others, which reduce local inflammatory responses and promote tissue repair (Garcia-Nicolas O, Baumann, VielleNJ, et al, Virus and genetic-associated infection of tissue-repair by porous produced and respiratory syndrome viruses [ J ]. Virus Res, 2014, 179: 204-11). Depending on the stimulating factor, M2-type macrophages can be further differentiated into M2a (induced by IL-4 and IL-13), M2b (induced by immune complexes and TLR or IL-1R agonists), and M2c macrophages (induced by IL-10).

Macrophages are the main cells targeted by a plurality of porcine viruses, and the macrophages derived from bone marrow are widely distributed in the body and have strong plasticity, and have the advantages of high yield, easy transfection, long growth cycle and the like, so the macrophages have higher potential research significance. At present, life science research usually relates to two types of cells, one type is primary cells, the other type is immortalized cells or cell lines, and the existing pig bone marrow macrophages have the problems of strict requirements on growth conditions, limited survival time and yield, difficulty in passage and the like during in vitro culture, so that an immortalized pig bone marrow macrophage line needs to be established by adopting an immortalization technology, but related reports are rarely seen so far.

Disclosure of Invention

The application mainly aims to provide immortalized pig bone marrow macrophages, a construction method and application thereof, so as to overcome the defects in the prior art.

In order to achieve the above purpose, the present application adopts a technical solution comprising:

one aspect of the present application provides a method for constructing immortalized porcine bone marrow macrophages, comprising: transfecting pig bone marrow macrophages with a first recombinant plasmid comprising the nucleotide sequence shown in SEQ ID No.1 or an increased or truncated sequence thereof, culturing the transfected cells, and isolating cell monoclonals.

In some embodiments, the first recombinant plasmid is constructed by a method comprising:

BglII and BCLI loci are added into the nucleotide sequence of SV40TAg through PCR to obtain a recombinant SV40TAg gene;

the recombinant SV40TAg gene was cloned into the pIED-Neo plasmid using BglII and BCLI sites, thereby generating the first recombinant plasmid, defined as pIED-Neo-SV40TAg plasmid.

In some embodiments, the method for constructing immortalized porcine bone marrow macrophages specifically comprises:

selecting leg bones of healthy piglets of 15 days old, flushing marrow cavities by using a complete culture medium to obtain bone marrow cells,

inducing the bone marrow cells to differentiate into porcine bone marrow macrophages by GM-CSF induction culture solution.

Further, the complete medium contains 10V/V% of fetal bovine serum.

Further, the GM-CSF induction medium contained 1 wt% primocin antibiotic, 10 wt% FBS and 100ng/mLGM-CSF, and the balance was 1640 medium.

In some embodiments, the method for constructing immortalized porcine bone marrow macrophages further comprises: and (2) resuspending the porcine bone marrow macrophages in a complete culture medium, culturing for 50-80 min at 35-38 ℃, and transferring the non-adherent cells after culture into a new complete culture medium for culture, thereby realizing the amplification of the porcine bone marrow macrophages.

In some embodiments, the method for constructing immortalized porcine bone marrow macrophages specifically comprises the following steps:

(1) selecting healthy piglets of 15 days old, taking leg bones, and flushing marrow cavities by using a complete culture medium to obtain marrow cells;

(2) dividing the bone marrow cells into multi-well plates (e.g., 6-well plate, 24-well plate), culturing with GM-CSF induction medium and inducing differentiation into porcine bone marrow macrophages;

(3) the culture supernatant in the multi-well plate was replaced and transfected with pIED-Neo-SV40TAg plasmid;

(4) and (3) carrying out static culture on the transfected pig bone marrow macrophages, forming a cell community by partial cells, and separating cell monoclonals to obtain the immortalized pig bone marrow macrophage system.

Further, in the step (4), the inoculation amount of the porcine bone marrow macrophages is 0.5 x 10⁵～2×10⁵Per well.

Further, in the step (4), the addition amount of the complete medium is 0.8 to 2 ml/well.

Further, the step (4) further comprises: treating the transfected pig bone marrow macrophage line with puromycin of 0.5-5 mug/ml for 1-4 days, screening out positive cell clones, and selecting a monoclonal cell strain to obtain the immortalized pig bone marrow macrophage line.

In another aspect of the present application, there is provided an immortalized porcine bone marrow macrophage constructed by any of the methods described herein.

Another aspect of the present application provides a method of remodeling immortalized porcine bone marrow macrophages, comprising: transfecting the immortalized pig bone marrow macrophage by a second recombinant plasmid containing a nucleotide sequence shown in SEQ ID NO.2 and/or a third recombinant plasmid containing a nucleotide sequence shown in SEQ ID NO.3, and screening out a positive cell strain in the immortalized pig bone marrow macrophage to obtain the virus-sensitive immortalized pig bone marrow macrophage.

In some embodiments, the method for modifying immortalized porcine bone marrow macrophages specifically comprises:

(1) transfecting the immortalized pig bone marrow macrophage by the second recombinant plasmid, treating the transfected immortalized pig bone marrow macrophage line by 400-1200 mu g/ml neomycin for one week, screening out positive cell clones in the immortalized pig bone marrow macrophage line, and selecting a monoclonal cell strain to obtain an immortalized pig bone marrow macrophage line successfully transfected with CD163-CD169-CD 51;

(2) transfecting the immortalized pig bone marrow macrophage line transfected with CD163-CD169-CD51 successfully by using the third recombinant plasmid, treating the immortalized pig bone marrow macrophage line transfected again by using puromycin of 0.5-5 mu g/ml for one week, screening positive cell clones in the immortalized pig bone marrow macrophage line, and selecting a monoclonal cell strain to obtain the immortalized pig bone marrow macrophage line transfected with Ibal-KT022-Cd172a successfully, namely the immortalized pig bone marrow macrophage sensitive to virus.

In some embodiments, the second recombinant plasmid is a pIED-Neo-CD163-CD169-CD151 plasmid.

Further, the construction method of the second recombinant plasmid comprises the following steps: the nucleotide sequence shown in SEQ ID NO.2 was cloned into the first recombinant plasmid by PCR to obtain the second recombinant plasmid, defined as pIED-Neo-CD163-CD169-CD151 plasmid.

In some embodiments, the third recombinant plasmid is the pIED-Ibal-KT022-Cd172a plasmid.

Further, the method for constructing the third recombinant plasmid comprises the following steps: the nucleotide sequence shown in SEQ ID NO.3 was cloned into the first recombinant plasmid by PCR to obtain the third recombinant plasmid, which was defined as pIED-Ibal-KT022-Cd172a plasmid.

In some embodiments, the method of remodeling immortalized porcine bone marrow macrophages further comprises: and (3) carrying out suspension domestication on the virus-sensitive immortalized pig bone marrow macrophages by using a serum-free suspension culture medium.

Preferably, the serum-free medium contains inorganic salts, vitamins, amino acids, sugars, trace elements and the like.

Preferably, the serum-free medium contains xanthan gum, interleukin-2, epidermal growth factor, insulin and the like

Preferably, the serum-free medium contains yeast hydrolysate, soybean hydrolysate, or the like.

Further, the serum-free medium comprises inorganic salt, vitamins, amino acid, sugar, trace elements, xanthan gum, interleukin-2, epidermal growth factor, insulin, yeast hydrolysate and soybean hydrolysate.

Yet another aspect of the present application provides a serum-free medium comprising inorganic salts, vitamins, amino acids, sugars, trace elements, xanthan gum, interleukin-2, epidermal growth factor, insulin, yeast hydrolysate, soy hydrolysate.

Yet another aspect of the present application provides a kit comprising the first recombinant plasmid, the second recombinant plasmid, the third recombinant plasmid, the immortalized porcine bone marrow macrophage or the virus-sensitive immortalized porcine bone marrow macrophage.

Compared with the prior art, the technical scheme provided by the embodiment of the application has at least the following advantages:

(1) the provided method for constructing the immortalized pig bone marrow macrophage line is to separate normal pig bone marrow macrophages from pig bone marrow, transfect the normal pig bone marrow macrophages with pIED-Neo-SV40TAg plasmid to obtain the immortalized pig bone marrow macrophage line which grows in an extension state, has clear cell outline and complete cells, has typical morphological characteristics similar to primary pig bone marrow macrophages, and has obvious characteristics of continuous passage, for example, after the immortalized pig bone marrow macrophage line is passed for 10 generations, the cell morphology and characteristics have no obvious difference with the 1 st generation cells;

(2) the method for modifying immortalized pig bone marrow macrophage system is characterized in that a cell strain is transfected by coding genes of CD163, CD169, CD51, Ibal, KT022 and Cd172a, so that the cell strain contains 6 receptors such as CD163, CD169, CD51, Ibal, KT022 and Cd172a, and the sensitivity of cells to viruses is remarkably improved by increasing cell surface characteristic receptors.

Drawings

FIG. 1 is a schematic diagram showing the structure of pIED-Neo-SV40TAg plasmid in example 1 of the present application.

FIG. 2 shows the results of gel electrophoresis in example 2 of the present application.

FIG. 3 is a schematic structural diagram of pIED-Neo-CD163-CD169-CD151 plasmid in example 3 of the present application.

FIG. 4 shows the results of gel electrophoresis in example 3 of the present application.

FIG. 5 is a schematic structural diagram of pIED-Ibal-KT022-Cd172a plasmid in example 4 of the present application.

FIG. 6 shows the results of gel electrophoresis in example 4 of the present application.

FIGS. 7 a-7 b are the respective profiles observed for the immortalized porcine bone marrow macrophage cell line engineered in example 6 of the present application.

Detailed Description

The present application is further illustrated by way of example below. The reagents and starting materials used in the following examples are commercially available, and the test methods in which specific conditions are not specified are generally carried out under conventional conditions or conditions recommended by the respective manufacturers. Further, unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ conventional techniques in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques are well described in the literature.

EXAMPLE 1 isolation culture of porcine bone marrow macrophages

Selecting thighbones and shinbones of healthy piglets of 15 days old, removing surface tissues, and carefully scratching periosteum during operation so as not to be polluted during later flushing. The treated bone was soaked in 75% alcohol for 5min, then transferred to a clean bench and washed with sterile PBS, both ends of the bone were cut off by means of a bone forceps, and a puncture needle was inserted into both ends for washing. The bone marrow was washed by pipetting 1640 medium containing 10 wt% serum and 1 wt% antibiotic with a 20mL syringe, washing both ends of the bone once into 24-well-plates (24 well plates), centrifuging at 550 Xg for 6min in a low temperature horizontal centrifuge at 4 ℃ and discarding the supernatant. After washing twice with 1640 medium containing 10 wt% serum and 1 wt% antibiotic, centrifuging at 4 deg.C and 550 Xg for 6min, discarding the supernatant, lysing with 5mL of erythrocyte lysate for 5min, mixing the lysate with the lysate by inversion at two minute intervals, centrifuging at 4 deg.C and 550 Xg for 6min, washing twice with medium, discarding the supernatant, and resuspending the centrifuged cells in 1640 medium containing 1 wt% primocin antibiotic, 10 wt% FBS and 100ng/mL GM-CSF to obtain a single cell suspension. Resuspend the cells at 2X 10⁷The density of the bottom surface is 100mm²The culture medium was changed after 3 days, and after 6 to 7 days, cell morphology was observed under an inverted microscope to obtain porcine bone marrow macrophages (BMDM).

Example 2 construction of pIED-Neo-SV40TAg plasmid

To construct pIED-Neo-SV40TAg, SV40TAg gene containing BglII and BCLI sites was synthesized in Nanjing Kingsler Biotech Co., Ltd and cloned into pIED-Neo vector to obtain pIED-Neo-SV40TAg plasmid (see FIG. 1). The size of the target gene is verified by carrying out gel electrophoresis after the plasmid is digested, as shown in figure 2, target bands appear at positions of 8668bp and 333bp, and the construction success of the pIED-Neo-SV40TAg plasmid is verified by sequencing.

Example 3 construction of pIED-Neo-CD163-CD169-CD151 plasmid

The codon-optimized CD163-CD169-CD151 gene (SEQ ID NO: 2) was synthesized by Nanjing Kingsler Biotech Co., Ltd and cloned into pIED-Neo-SV40TAg vector to obtain pIED-Neo-CD163-CD169-CD151 plasmid (see FIG. 3).

Specifically, the pIED-Neo-SV40TAg vector was used as a template, and CD163-CD169-CD151-F (SEQ ID NO: 4) and CD163-CD169-CD151-R (SEQ ID NO: 5) were used as upstream and downstream primers for PCR amplification, and the amplification system is shown in Table 1.

TABLE 1 amplification System for the CD163-CD169-CD151 Gene

The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 45 seconds, annealing at 54 ℃ for 45 seconds, extension at 72 ℃ for 1 minute, 35 cycles; extension at 72 ℃ for 10 min.

The size of the target gene is verified by carrying out gel electrophoresis on the PCR product, and as shown in figure 4, a target band appears at the position of 10505bp, so that the successful amplification of the target gene is proved.

Example 4 construction of pIED-Ibal-KT022-Cd172a plasmid

Codon-optimized Ibal-KT022-Cd172a gene (SEQ ID NO: 2) was synthesized in Nanjing Kingsler Biotech Ltd and cloned into pIED-Neo-SV40TAg vector to obtain pIED-Ibal-KT022-Cd172a plasmid (FIG. 5).

Specifically, pIED-Neo-SV40TAg vector is used as a template, Ibal-KT022-Cd172a-F, Ibal-KT022-Cd172a-R is used as an upstream primer and a downstream primer for PCR amplification (the gene sequence of Ibal-KT022-Cd172a-F, Ibal-KT022-Cd172a-R is shown as SEQ ID NO: 6 and 7), and the amplification system is shown in Table 2.

TABLE 2 Ibal-KT022-Cd172a Gene amplification System

The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 45 seconds, annealing at 54 ℃ for 45 seconds, extension at 72 ℃ for 1 minute, 35 cycles; extension at 72 ℃ for 10 min.

The size of the target gene is verified by carrying out gel electrophoresis on the PCR product, and as shown in FIG. 6, a target band appears at the 4601bp position, thus proving that the target gene is successfully amplified.

Example 5 transfection of porcine bone marrow macrophages

Porcine bone marrow macrophages harvested in example 1 were transfected separately. Specifically, porcine bone marrow macrophages can be seeded in 6-well plates with approximately 1X 10 cells per well⁵On the next day, after the cells adhered to the wall, the medium was changed, 1mL of complete medium (containing 10V/V% fetal bovine serum) was added, transfection was performed with pIED-Neo-SV40TAg plasmid, the cells were continued to be cultured after mixing, the state of the cells was observed after 12 hours, and the cells were changed to fresh medium, and when the cells had grown to the bottom of the plate, they were passaged to a T25 flask. After transfection, cells are amplified for 3-4 generations and then screened, partial cells form a cell community, cell monoclone is separated to obtain an immortalized pig bone marrow macrophage system, and the screened cells are amplified.

EXAMPLE 6 reconstruction of immortalized porcine bone marrow macrophages

The transfected porcine bone marrow macrophages obtained in example 5 were transfected with the pIED-Neo-CD163-CD169-CD151 plasmid containing the nucleotide sequence shown in SEQ ID NO.2, and positive cell lines were selected, and monoclonal cell lines were selected. Specifically, 400-1200 mug/ml neomycin is adopted to treat the transfected immortalized pig bone marrow macrophage cell line during screening positive clones, and the positive cell clone formed after one week of treatment is the immortalized pig bone marrow macrophage cell line successfully transfected with CD163-CD169-CD 51.

After an immortalized pig bone marrow macrophage line which is transfected with CD163-CD169-CD51 successfully is obtained, pIED-Ibal-KT022-Cd172a plasmid is used for transfecting a cell strain, positive cell strains are screened, and a monoclonal cell strain is selected, wherein the pIED-pac-Ibal-KT022-Cd172a plasmid contains a nucleotide sequence shown in SEQ ID NO. 3. Specifically, when positive clones are screened, the immortalized pig bone marrow macrophage line after re-transfection is treated by 0.5-5 mug/ml puromycin, and the positive cell clone formed after one week of treatment is the immortalized pig bone marrow macrophage line successfully transfected with Ibal-KT022-Cd172a (fig. 7 a-7 b).

EXAMPLE 8 culture of immortalized porcine bone marrow macrophages

After the successful immortalized pig bone marrow macrophage line transfected with Ibal-KT022-Cd172a is obtained, a serum-free suspension culture medium is developed for the immortalized pig bone marrow macrophage line, and cells are suspended and domesticated. Specifically, the serum-free culture medium contains inorganic salt, vitamins, amino acids, sugar, trace elements, xanthan gum, interleukin-2, epidermal growth factor, insulin, yeast hydrolysate and soybean hydrolysate.

It should be understood that the above-mentioned embodiments are not intended to limit the embodiments of the present application, and any other changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of the present application should be construed as being equivalent to the above-mentioned embodiments and shall be included in the protection scope of the present application.

Sequence listing

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<120> immortalized pig bone marrow macrophage, construction method and application thereof

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ggctctgcac aaccactaca cgcagaagag cctctccctg tctccgggta aatgactcga 5280

gaatcgatgc ctgcaggtgc ccggccacac cggtgcccct ctccctcccc cccccctaac 5340

gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc 5400

accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg 5460

agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg 5520

aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc 5580

aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa 5640

gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa 5700

agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta 5760

ccccattgta tgggatctga tctggggcct cggtacacat gctttacatg tgtttagtcg 5820

aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca 5880

cgatgataag cttgccacaa ccccgggaga tgaggatcta attccatggt tcgaccattg 5940

aactgcatcg tcgccgtgtc ccaaaatatg gggattggca agaacggaga cctaccctgg 6000

cctccgctca ggaacgagtt caagtacttc caaagaatga ccacaacctc ttcagtggaa 6060

ggtaaacaga atctggtgat tatgggtagg aaaacctggt tctccattcc tgagaagaat 6120

cgacctttaa aggacagaat taatatagtt ctcagtagag aactcaaaga accaccacga 6180

ggagctcatt ttcttgccaa aagtttggat gatgccttaa gacttattga acaaccggaa 6240

ttggcaagta aagtagacat ggtttggata gtcggaggca gttctgttta ccaggaagcc 6300

atgaatcaac caggccacct cagactcttt gtgacaagga tcatgcagga atttgaaagt 6360

gacacgtttt tcccagaaat tgatttgggg aaatataaac ttctcccaga atacccaggc 6420

gtcctctctg aggtccagga ggaaaaaggc atcaagtata agtttgaagt ctacgagaag 6480

aaagactaag tcgagctttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag 6540

atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacc 6600

ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact 6660

tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 6720

aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 6780

atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc 6840

ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 6900

gtaaagcctg gcgcggctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 6960

ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc gatcgcgcgg 7020

ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 7080

cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 7140

cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 7200

accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 7260

acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 7320

cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 7380

acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 7440

atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 7500

agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 7560

acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 7620

gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 7680

gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 7740

gcaaacaaac caccgctggt agcggttttt ttgtttgcaa gcagcagatt acgcgcagaa 7800

aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 7860

aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 7920

ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 7980

acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 8040

ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 8100

gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 8160

taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 8220

tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 8280

gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 8340

cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 8400

aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 8460

cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 8520

tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 8580

gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 8640

tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 8700

gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 8760

ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 8820

cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 8880

agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 8940

gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctgaccccgt aattgattac 9000

t 9001

<210> 2

<211> 16415

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

gacggatcgg gagatctgat cagtattttc tccttacgca tctgtgcggt atttcacacc 60

gcatacgcgg atctgcgcag caccatggcc tgaaataacc tctgaaagag gaacttggtt 120

aggtaccttc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa 180

gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac 240

caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 300

ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 360

ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc 420

cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt 480

ttgcaaaaag cttgattctt ctgacacaac agtctcgaac ttaaggctag agccaccatg 540

attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 600

tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 660

caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag 720

gacgaggcag cgcggctatc gtggctggct acgacgggcg ttccttgcgc agctgtgctc 780

gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 840

ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 900

cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 960

gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 1020

catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcacgcat gcccgacggc 1080

gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 1140

cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 1200

gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 1260

gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 1320

gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc 1380

catcacgatg gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg 1440

tgtgaacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 1500

tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 1560

gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 1620

cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 1680

tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 1740

aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 1800

acatctacgt attagtcatc gctattactc gagtccttag ggagcgatcc agcacgagga 1860

gaggccggga gggggcggga cggggcgggg cctctgggag agtgggttgc ggggaggctg 1920

gcttttggca ggaagtaacg catttgctgg actcgagtga tgcggttttg gcagtacatc 1980

aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 2040

aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 2100

gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 2160

ctctggcaag cgtttaaact taagcttggt accgagctcg gatccgccac catggacaaa 2220

ctcagaatgg tgctacatga aaactctgga tctgcagact ttagaagatg ttctgcccat 2280

ttaagttcct tcacttttgc tgtagtcgct gttctcagtg cctgcttggt cactagttct 2340

cttggaggaa aagacaagga gctgaggcta acgggtggtg aaaacaagtg ctctggaaga 2400

gtggaggtga aagtgcagga ggagtgggga actgtgtgta ataatggctg ggacatggat 2460

gtggtctctg ttgtttgtag gcagctggga tgtccaactg ctatcaaagc cactggatgg 2520

gctaatttta gtgcaggttc tggacgcatt tggatggatc atgtttcttg tcgagggaat 2580

gagtcagctc tctgggactg caaacatgat ggatggggaa agcataactg tactcaccaa 2640

caggatgctg gagtaacctg ctcagatgga tctgatttag agatgaggct ggtgaatgga 2700

ggaaaccggt gcttaggaag aatagaagtc aaatttcaag gacggtgggg aacagtgtgt 2760

gatgataact tcaacataaa tcatgcttct gtggtttgta aacaacttga atgtggaagt 2820

gctgtcagtt tctctggttc agctaatttt ggagaaggtt ctggaccaat ctggtttgat 2880

gatcttgtat gcaatggaaa tgagtcagct ctctggaact gcaaacatga aggatgggga 2940

aagcacaatt gcgatcatgc tgaggatgct ggagtgattt gcttaaatgg agcagacctg 3000

aaactgagag tggtagatgg agtcactgaa tgttcaggaa gattggaagt gaaattccaa 3060

ggagaatggg gaacaatctg tgatgatggc tgggatagtg atgatgccgc tgtggcatgt 3120

aagcaactgg gatgtccaac tgctgtcact gccattggtc gagttaacgc cagtgaggga 3180

actggacaca tttggcttga cagtgtttct tgccatggac acgagtctgc tctctggcag 3240

tgtagacacc atgaatgggg aaagcattat tgcaatcata atgaagatgc tggtgtgaca 3300

tgttctgatg gatcagatct ggaactgaga cttaaaggtg gaggcagcca ctgtgctggg 3360

acagtggagg tggaaattca gaaactggta ggaaaagtgt gtgatagaag ctggggactg 3420

aaagaagctg atgtggtttg caggcagctg ggatgtggat ctgcactcaa aacatcatat 3480

caagtttatt ccaaaaccaa ggcaacaaac acatggctgt ttgtaagcag ctgtaatgga 3540

aatgaaactt ctctttggga ctgcaagaat tggcagtggg gtggacttag ttgtgatcac 3600

tatgacgaag ccaaaattac ctgctcagcc cacaggaaac ccaggctggt tggaggggac 3660

attccctgct ctggtcgtgt tgaagtacaa catggagaca cgtggggcac cgtctgtgat 3720

tctgacttct ctctggaggc ggccagcgtg ctgtgcaggg aactacagtg cggcactgtg 3780

gtttccctcc tggggggagc tcactttgga gaaggaagtg gacagatctg ggctgaagaa 3840

ttccagtgtg aggggcacga gtcccacctt tcactctgcc cagtagcacc ccgccctgac 3900

gggacatgta gccacagcag ggacgtcggc gtagtctgct caagatacac acaaatccgc 3960

ttggtgaatg gcaagacccc atgtgaagga agagtggagc tcaacattct tgggtcctgg 4020

gggtccctct gcaactctca ctgggacatg gaagatgccc atgttttatg ccagcagctt 4080

aaatgtggag ttgccctttc tatcccgaga ggagcacctt ttgggaaagg aagtgagcag 4140

gtctggaggc acatgtttca ctgcactggg actgagaagc acatgggaga ttgttccgtc 4200

actgctctgg gcgcatcact ctgttcttca gggcaagtgg cctctgtaat ctgctcaggg 4260

aaccagagtc agacactatc cccgtgcaat tcatcatcct cggacccatc aagctctatt 4320

atttcagaag aaaatggtgt tgcctgcata gggagtggtc aacttcgcct ggtcgatgga 4380

ggtggtcgtt gtgctgggag agtagaggtc tatcatgagg gctcctgggg caccatctgt 4440

gatgacagct gggacctgaa tgatgcccat gtggtgtgca aacagctgag ctgtggatgg 4500

gccattaatg ccactggttc tgctcatttt ggggaaggaa cagggcccat ttggctggat 4560

gagataaact gtaatggaaa agaatctcat atttggcaat gccactcaca tggttggggg 4620

cggcacaatt gcaggcataa ggaggatgca ggagtcatct gctcagagtt catgtctctg 4680

agactgatca gtgaaaacag cagagagacc tgtgcagggc gcctggaagt tttttacaac 4740

ggagcttggg gcagcgttgg caggaatagc atgtctccag ccacagtggg ggtggtatgc 4800

aggcagctgg gctgtgcaga cagaggggac atcagccctg catcttcaga caagacagtg 4860

tccaggcaca tgtgggtgga caatgttcag tgtcctaaag gacctgacac actatggcag 4920

tgcccatcat ctccatggaa gaagagactg gccagcccct cagaggagac atggatcaca 4980

tgtgccaaca aaataagact tcaagaagga aacactaatt gttctggacg tgtggagatc 5040

tggtacggag gttcctgggg cactgtgtgt gacgactcct gggaccttga agatgctcag 5100

gtggtgtgcc gacagctggg ctgtggctca gctttggagg caggaaaaga ggccgcattt 5160

ggccagggga ctgggcccat atggctcaat gaagtgaagt gcaaggggaa tgaaacctcc 5220

ttgtgggatt gtcctgccag atcctggggc cacagtgact gtggacacaa ggaggatgct 5280

gctgtgacgt gctcagaaat tgcaaagagc cgagaatccc tacatgccac aggtcgctca 5340

tcttttgttg cacttgcaat ctttggggtc attctgttgg cctgtctcat cgcattcctc 5400

atttggactc agaagcgaag acagaggcag cggctctcag ttttctcagg aggagagaat 5460

tctgtccatc aaattcaata ccgggagatg aattcttgcc tgaaagcaga tgaaacggat 5520

atgctaaatc cctcaggaga ccactctgaa gtacaatgag cggccgcccc ctctccctcc 5580

ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat 5640

atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct 5700

gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg 5760

ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta 5820

gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag 5880

ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg 5940

atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat 6000

gcccagaagg taccccattg tatgggatct gatctggggc ctcggtacac atgctttaca 6060

tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc 6120

ctttgaaaaa cacgatgata atatggccac cgcgggccac catggacttc ctgctcctgc 6180

tcctcctcct ggcttcatct gctctagcag gcctggcctc gtggacggtt tccagccccg 6240

agaccgtgca gggcatcaag ggctcctgcc tcatcatccc ctgcaccttc ggcttcccgg 6300

ccaacgtgga ggtgccccat ggcatcacag ccatctggta ctatgactac tcaggcaagc 6360

gcctggtagt gagccactcc aggaacccaa aggtggtgga gaaccacttc caaggccggg 6420

ccctgctgtt ggggcaggtt gaacagagga cgtgcagcct gctgctgaag gacctgcagc 6480

cccaggactc gggctcctat aacttccgct ttgagatcag cgagggcaac cgctggtcag 6540

atgtcaaagg cacagttgtc accgtgacag aggtgcccag cgtgcccacc attgccttgc 6600

cagccaagct gcatgagggc atggaggtgg acttcaactg ctccactccc tatgtgtgcc 6660

cgacggagcc ggtcaaccta cagtggcaag gccaggaccc cacccgctcc gtcacctccc 6720

acctccagaa gcttgagccc tcgggcacca gccacatgga gaccctgcac atggccctgt 6780

cctggcagga ccatggccgg attctgagct gccaggtctc agcagccgaa cgcaggatgc 6840

agaaggagat tcacctccaa gtgcagtatg cccccaaggg tgtggagatc cttttcagcc 6900

actccggacg gaacgtcctt ccaggtgatc tggtcaccct cagctgccag gtgaatagca 6960

gcaaccctca ggtcagttcc gtgcagtggg tcaaggatgg gacgaagctc aaagaccaga 7020

aacgtgtact gcagttgcgc cgggcagcct gggctgatgc tggcgtctac acctgccaag 7080

ccgggaatgc cgtgggctct tcagtctcac ccccggtcag cctccacgtc ttcatggctg 7140

aggtccaggt aagccctgtg ggctccatcc tggagaacca gacggtgacg ctggcctgca 7200

atacacctaa ggaagcgccc agcgagctgc gctacagctg gtacaagaac cacgccctgc 7260

tggagggctc tcacagccgc accctccggc tgcactcagt taccagggcg gattcgggct 7320

tctacttctg cgaggtgcag aacgcccggg gcagagagcg ctctccccct gtcagcgtgg 7380

tggtcagcca cccacccctc accccggacc taactgcctt cctggagaca caggcggggc 7440

tggtgggcat cctccaatgc tctgtggtca gcgagccccc agctactctg gtgttgtcac 7500

acgggggcct catcttggcc tctacctccg gggagggtga ccacagccca cgcttcagtg 7560

tcgcctctgc ccccaactcc ctgcgcctgg agattcaaga cctggggcca acagacagtg 7620

gggaatacat gtgctcagcc agcagttctc ttgggaatgc gtcctccacc ctggacttcc 7680

atgccaatgc agcccgcctc ctcatcagcc cagcagcaga ggtggtggaa gggcaggcgg 7740

tgacactgag ctgcaggagc agcctgagcc tgatgcctga cacccgtttt tcctggtacc 7800

tgaacggggc cctgattctc gaggggccca gcagcagcct cctgctccca gcagcctcca 7860

gcacagatgc cggctcatac cactgccggg cccagaacag ccacagcacc agcgggccct 7920

cctcacctgc tgttctcacc gtgctctacg ccccacgcca gcccgtgttc actgcccagc 7980

tggaccctga tactgcagga gctggggccg gacgccaagg cctcctcttg tgccgtgtgg 8040

acagcgaccc cccagcccag ctgcagctgc tccacagggg ccgtgttgtg gcctcttctc 8100

tgtcatgggg gggcggctgc tgcacctgcg gaggctgttt ccaccgcatg aaggtcacca 8160

aagcacccaa cctactgcgt gtagagatcc gagacccggt gctggaggat gagggtgtgt 8220

acctgtgcga ggccagcagc gccctgggca acgcctccgc ctctgcaacc ttggatgccc 8280

aggccactgt cctggtcatc acaccgtcac acacgctgca ggaaggcatt gaagccaacc 8340

tgacttgcaa cgtgagccgt gaagccagcg gccctgccaa cttctcctgg ttccgagatg 8400

gggcgctatg ggcccagggc cctctggaca ccgtgacgct gctacctgtg gccagaactg 8460

atgctgccct ctatgcttgc cgcatcgtca ccgaggctgg tgctggcctc tccacccctg 8520

tggccctgaa tgtgctctat ccccccgatc ctccaaagtt gtcagccctc ctggacgtgg 8580

accagggcca cacggctgtg ttcgtctgta ctgtggacag tcgccctctt gcccagttgg 8640

ccctgttccg tggggaacac ctcctggccg ccagctcggc actccggctc ccccctcgtg 8700

gccgcctcca ggccaaagcc tcggccaact ccttgcagct agaggtccga gacttgagcc 8760

ttggggactc tggcagctac cactgtgagg ccaccaacat ccttggatca gccaacactt 8820

ctcttacctt ccaggtccga ggagcctggg tccgggtgtc accgtcgcct gagctccagg 8880

agggccaggc tgtggtcctg agctgccagg tacccatagg ggtcctggag gggacctcat 8940

atcgttggta tcgggatggc cagcccctcc aggagtccac ttcggccacg ctccgttttg 9000

cagccataac tctgagccag gctggagcct accattgcca agcccaagct ccaggctcag 9060

ccaccacgga cctggctgcc cctgtcagcc tccacgtgac ctacgcacct cgccaggcca 9120

cactcaccac cctgatggac tcaggcctcg ggcgactggg cctccttctg tgccgtgtga 9180

acagtgaccc tcctgcccag ctccgactgc tccatgggag ccgcctcgtg gcctctactc 9240

tacaaggtgt ggaggagctt gcaggcagct ctccccgcct acaggtggcc acagccccca 9300

acacgctgcg cctggagatc cacaacgcag tgctggagga tgaaggcgtc tacacctgcg 9360

aggccaccaa caccctgggt cagaccttgg cctccgccgc cttcgatgcc caggctatga 9420

gagtgcaggt gtggcccaat gccaccgtgc aagaggggca gctggtgaac ctgacctgcc 9480

ttgtatggac cacgcacctg gcccagctca cctacacgtg gtaccgagac cagcagcagc 9540

tcccaggtgc tgcccactcc atcctcctgc ccaatgtcac tgtcacagat gccgcctcct 9600

accgctgtgg catattgatc cctggccagg cactccgcct ctccagacct gtcgccctgg 9660

atgtcctcta cgcaccccgc agactgcgcc tgacccatct cttggagagc cgtggtgggc 9720

agctggccgt ggtgctgtgc actgtggaca gtcgcccagc tgcccagctg accctcagcc 9780

atgctggccg cctcctggcc tcctcaaccg cagcctctgt ccccaacacc ctgcgcctgg 9840

agctgtggga gccccggccc agtgatgagg gtctctacag ctgctcggcc cgcagtcctc 9900

tgggccaggc caacacatcc ctggagctgc ggctagaggg cgtgcaggtg gcactggctc 9960

catcggccac tgtgccggag ggggcccctg tcacagtgac ctgtgaagac cctgctgccc 10020

gcccacccac tctctatgtc tggtaccaca acagccgttg gctgcaggag gggtcggctg 10080

cctccctctc gtttccagcg gctacacggg ctcacgcggg cgcctatacc tgccaggtcc 10140

aggatgccca gggcacacgc atctcccagc ccgcagcact gcacatcctc tatgcccctc 10200

gggatgctgt cctttcctcc ttctgggact caagggccag ccctatggcc gtggtacagt 10260

gcactgtgga cagcgagcca cctgccgaga tgaccctgtc ccatgatggc aaggtgctgg 10320

ccaccagcca tggggtccac ggcttagcag tggggacagg ccatgtccag gtggcccgca 10380

acgccctgca gctgcgggtg cagaatgtgc cctcacgtga caaggacacc tacgtctgca 10440

tggaccgcaa ctccttgggc tcagtcagca ccatggggca gctgcagcca gaaggtgtgc 10500

acgtggtagc tgagccaggg ctggatgtgc ctgaaggcac agcgctgaac ctgagctgtc 10560

gcctccctag tggccctggg cacataggca actccacctt tgcttggttc cggaacggtc 10620

ggcagctaca cacagagtct gtgcccaccc ttaccttcac ccatgtggcc cgcgcccaag 10680

ctggcttgta ccactgccag gctgagctcc ccgccggggc tgccacctct gctccagtct 10740

tgctccgggt gctctaccct cccaagacgc ccaccatgac tgtttttgtg gagcccgagg 10800

gtggcatcca gggcattctg gactgccgag tggacagtga gcccctagcc agcctgaccc 10860

tccacctggg cagtcggctg gtggcctcca gccagcctca ggctgcccct gccaagccgc 10920

acatccgcgt ctcagccagt cccaatgcct tgcgagtgga catggaggag ctgaagccca 10980

gtgaccaggg ggagtatgtg tgctcggcct ccaatgccct gggctctgcc tctgctgcca 11040

cctacttcgg aaccagagcc ctgcatcgcc tgcatctgtt ccagcacctt ctctggttcc 11100

tggggctgct ggcgagcctc ctcttcctac tgttgggcct gggggtctgg tacgcctgga 11160

gacggggaaa tttttacaag ctgagaatgg gcgaatattc agtagagatg gtatctcgga 11220

aggaaaccac gcagatgtcc actgaccagg aagaagttac tggaatcggt gatgatgcgg 11280

gctctgtgaa ccaggcggca tttgatcctg cccacctctg tgaaaacaca cagtctgtga 11340

aaagcacagt ctgatctaga cccctctccc tccccccccc ctaacgttac tggccgaagc 11400

cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct 11460

tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt 11520

ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct 11580

ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc 11640

ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag 11700

gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc 11760

tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga 11820

tctgatctgg ggcctcggta cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt 11880

ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggc 11940

cagctagcgc caccatgggc gaattcggcg agaagggcgc gccatgcggc accatctgcc 12000

tcaagtacct gcttttcacc ttcaactgct gcttctggct ggctggcctg gccgtcatgg 12060

cagtgggcat ctggacgctg gccctcaaga gcgactacat cagcctcctg gcctcgggca 12120

cctacctggc cacagcctac atcctagtgg tggcgggcat tgttgtcatg gtgaccggtg 12180

ttctgggctg ctgtgccacc ttcaaggagc ggaggaacct gctgcggctg tacttcatcc 12240

tgctgctgct catctttctg ctggagatca tcgccggagt cctggcctac gtctactacc 12300

agcagctgag tgcagagctc aaggcgaacc tgaaggacac tctgagcaag cgctaccgcc 12360

agccgggcca cgagggtgtg accagcgcca cggataagct gcagcaggag ttccactgct 12420

gtggcagcaa caactcccag gactggcggg acagtgagtg gattctctcg ggtgaggcag 12480

gcgaccgtgt ggttcctgac agctgctgca agacggtggt ggcgggctgc gggcggcggg 12540

accacgcctc caacatctac aaagtggagg gcggctgcat caccaggctg gagaccttca 12600

tccgggagca cctgaggatc atcggggctg tgggcatcgg catcgcctgt gtgcaggtgt 12660

tcggcatgat cttcacgtgc tgcctgtaca agagcctgaa gctggagcac tactgaatcg 12720

atgcctgcag gtgcccggcc acaccggtgc ccctctccct cccccccccc taacgttact 12780

ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata 12840

ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt 12900

cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa 12960

gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag 13020

cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca 13080

cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc 13140

aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat 13200

tgtatgggat ctgatctggg gcctcggtac acatgcttta catgtgttta gtcgaggtta 13260

aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgatga 13320

taagcttgcc acaaccccgg gagatgagga tctaattcca tggttcgacc attgaactgc 13380

atcgtcgccg tgtcccaaaa tatggggatt ggcaagaacg gagacctacc ctggcctccg 13440

ctcaggaacg agttcaagta cttccaaaga atgaccacaa cctcttcagt ggaaggtaaa 13500

cagaatctgg tgattatggg taggaaaacc tggttctcca ttcctgagaa gaatcgacct 13560

ttaaaggaca gaattaatat agttctcagt agagaactca aagaaccacc acgaggagct 13620

cattttcttg ccaaaagttt ggatgatgcc ttaagactta ttgaacaacc ggaattggca 13680

agtaaagtag acatggtttg gatagtcgga ggcagttctg tttaccagga agccatgaat 13740

caaccaggcc acctcagact ctttgtgaca aggatcatgc aggaatttga aagtgacacg 13800

tttttcccag aaattgattt ggggaaatat aaacttctcc cagaataccc aggcgtcctc 13860

tctgaggtcc aggaggaaaa aggcatcaag tataagtttg aagtctacga gaagaaagac 13920

taagtcgagc tttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg 13980

attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gaccccggct 14040

ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta 14100

ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 14160

ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 14220

gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt 14280

gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 14340

cctggcgcgg ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 14400

tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggcgatcgc gcggggagag 14460

gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 14520

ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 14580

caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 14640

aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 14700

atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 14760

cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 14820

ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 14880

gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 14940

accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 15000

cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 15060

cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 15120

gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 15180

aaaccaccgc tggtagcggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 15240

gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 15300

cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 15360

attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 15420

accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 15480

ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 15540

gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 15600

agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 15660

ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 15720

ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 15780

gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 15840

ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 15900

tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 15960

tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 16020

cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 16080

tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 16140

gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 16200

tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 16260

ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 16320

attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 16380

cgcgcacatt tccccgaaaa gtgccacctg acgtc 16415

<210> 3

<211> 6812

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

gacggatcgg gagatctgat cagtattttc tccttacgca tctgtgcggt atttcacacc 60

gcatacgcgg atctgcgcag caccatggcc tgaaataacc tctgaaagag gaacttggtt 120

aggtaccttc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa 180

gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac 240

caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 300

ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 360

ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc 420

cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt 480

ttgcaaaaag cttgattctt ctgacacaac agtctcgaac ttaaggctag agccaccatg 540

attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 600

tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 660

caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag 720

gacgaggcag cgcggctatc gtggctggct acgacgggcg ttccttgcgc agctgtgctc 780

gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 840

ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 900

cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 960

gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 1020

catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcacgcat gcccgacggc 1080

gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 1140

cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 1200

gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 1260

gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 1320

gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc 1380

catcacgatg gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg 1440

tgtgaacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 1500

tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 1560

gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 1620

cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 1680

tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 1740

aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 1800

acatctacgt attagtcatc gctattactc gagtccttag ggagcgatcc agcacgagga 1860

gaggccggga gggggcggga cggggcgggg cctctgggag agtgggttgc ggggaggctg 1920

gcttttggca ggaagtaacg catttgctgg actcgagtga tgcggttttg gcagtacatc 1980

aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 2040

aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 2100

gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 2160

ctctggcaag cgtttaaact taagcttggt accgagctcg gatccgccac cgccaccatg 2220

gaacagtggc atagctttcc tgatcaaccg gaggacactg acagttgtat ggaatctgtc 2280

aagttcgatg ctcgctcaat gacagctttg cttcctccaa atcctaaaaa tggcccaacg 2340

cttcaagaga aaatgaagtc tttcaaagct gcactgattg ccctttatct ccttgtgttt 2400

gtcgtgctgg tgcctatcat tggagtactg gcagctcatc tcctgaaatg ggaaatgaag 2460

aattgtgcag ttggctcaat tagtgcagga aaaggaaatg acagtgaaaa tgacatgaga 2520

tttcgtgacg tcgttacaga acatatgagc aacatggaga agagaattca gtatctttca 2580

gataatgaag ccaatctcgt agaatccgag catttccaaa atttcactgc aatgactgat 2640

caaagattta atgatgtcct ttcccagcta aataacttgc tttccttcat tcaggaacat 2700

ggaaatgtaa taaatgaaat ctctaagtca ttaataagtc tgaacaccac attgcttgat 2760

ttgcagctca atgtcgaaac actgaaaggc aaagtccaag agaatacatt taaacaacaa 2820

gaggagatga gtaaattaga ggagcgtgtg tacaatgtgt cagcagaaat tatgtctctg 2880

aacgagaaac aagcgcattt ggaacaggaa ataaaaggag aagtgaaact gttgaataac 2940

gtcacgaatg atcttaggct gaaggactgg gagcattctc agacactgaa aaatatcact 3000

ttagttcaag gtcctcctgg acctccaggt gaaaaaggag atagaggtcc tgctggagaa 3060

gttggtccac gcggcattcc aggtccacta ggtcctccag gtcttaaagg tgatcgggga 3120

tcaattggct ttcctggaag tcgaggattc ccaggaccaa tggggaagac cgggaggcca 3180

ggaaatcctg gacaaaaagg ccagaaggga gaaaaaggga gtggaagcat gctaaaacca 3240

tctaaaacag ttcgtctggt tggtggtagt ggccctcacg aaggcagagt ggaaattttc 3300

catgatggcc agtggggtac agtttgtgat gaccgctggg aattgcgtgg tggactggtc 3360

atctgcagga gtttgggata ccagggtgtt aaaaatgtgc ataaacaagc ttactttgga 3420

gcaggtactg gtccaatatg gctgaatgat gtgttttgtt ttgggagaga atcatctatt 3480

gaagaatgta atattagaca gtggggtgtg agaacctgtt cacatgctga agatgctgga 3540

gtcacctgca ctacataagc ctgcaggtgc ccggccacac cggtgcccct ctccctcccc 3600

cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat 3660

gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt 3720

cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt 3780

gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc 3840

gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc 3900

acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat 3960

agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc 4020

ccagaaggta ccccattgta tgggatctga tctggggcct cggtacacat gctttacatg 4080

tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct 4140

ttgaaaaaca cgatgataag cttgccacaa ccccgggaga tgaggatcta attccgccac 4200

catgagccag accctggatc tgcagggcgg caaagcgttt ggcctgctga aagcgcagca 4260

ggaagaacgc ctgaacgaaa ttaacaaaca gtttctggat gatccgaaat atagcagcga 4320

tgaagatctg agcagcaaac tggaagcgtt taaacagaaa tatatggaat ttgatctgaa 4380

cggcaacggc gatattgata ttatgagcct gaaacgcatg ctggaaaaac tgggcgtgcc 4440

gaaaacccat ctggaactga aaaaactgat taaagaagtg agcagcggca gcggcgaaac 4500

ctttagctat agcgattttc tgaaaatgat gctgggcaaa cgcagcgcga ttctgaaaat 4560

gattctgatg tatgaagaaa aagcgcgcga acaggaaaaa ccgaccggcc cgccggcgaa 4620

aaaagcgatt agcgaactgc cgtaagcctg caggtgcccg gccacaccgg tgcccctctc 4680

cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg 4740

tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg 4800

gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag 4860

gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt 4920

ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc 4980

aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga 5040

gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga 5100

aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tacacatgct 5160

ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg 5220

ttttcctttg aaaaacacga tgataagctt gccacaaccc cgggagatga ggatctaatt 5280

ccgccaccat ggagcccgcc cgcccggccc ccggccgcct cgggccgctg ctctgcctgc 5340

tgctcgcctt gttgagcgcc tggacaggag cggcaggtga ggaggggctg caggtgcttc 5400

agcctgagag gtccgtgtct gtcgcagctg gagagacggc cactctgccc tgcactgcga 5460

cctctctgat ccccattggg cccttcaagt ggttcaaggg gacaggacca gcccgggagc 5520

taatctacga tttcaaagga gatcctcggg gccactcccc ccgagtaaca aatgcttcag 5580

atgccacaag gagagacaac aaggactttt ccatccgcat ccggaacatc accccagcag 5640

acgccgggac ctactactgt gtgaagttcc ggagagagag ccctgcggac gtggagttta 5700

ggtctgggcc aggcactcag ctcactgtga gtgccaagcc ctctcggccc gtggtctcag 5760

gccctgcggc gagggccaca cctgagcaga cagtgcgctt cacctgcaaa tcccacggct 5820

tctcccccag aaacatctcc ctgaaatggt tcaaaaatgg caatgagctc ccagcctccc 5880

agaccagcgt ggagcccgag ggaaacgttt cctacagcat ctccagcacc accgaggtgc 5940

tgctggcccc gggggatgtg cactcccagg tcatctgcga ggtggcccac gtcaccctgc 6000

aagggggccc tcctctccgt ggaactgcca acttgtctga gaccatccga gttccgccca 6060

ccctggaggt tacccaacag cccccgacgg ggagccaggt gaatgtcacc tgcctggtga 6120

agaagttcta cccccagcgc ctacagctga cctggctgga gaacagaaac gtgtcccgga 6180

cagaaacggc ctcgaccctc atggagaaca aggatgggac cttcaaccgg accagctggc 6240

tcctggtgaa ctcatctgcc cacagggagg ctgtgctgct cacctgtcag gtggagcatg 6300

atgggcagcc ggcggtcacc aaaaactata ccctggaggt tgtcactcac cagaaggagc 6360

agggtgcaga tgcagtcctt gacaactccg acaactggaa aagcatcttt atcacggtgg 6420

gcgtggtgtg tgccttgctg gtggccctgc tcgtcgctgc tctctacctc ctccgaatca 6480

gacagaagaa agccaaaggc tccacgtctt ctacaaggtt gcatgagccc gagaagaacg 6540

ccagagaaat aacccagatc caggacaaca atgacattac ctatgcagac ctgaacctgc 6600

ccaaggggaa gaaacccgcc cccaaggccg aagaacccaa cgaccacacg gagtatgcca 6660

gcattcaggc ccgcccacac cccgggtcgg aggccaacct tacttatgct gacctggata 6720

tggtgcacct caaccggggc cccaagcatc cagcccccaa gcctgagcca tcctgctcgg 6780

agtatgccag cgtccaggtc cggaggaagt ga 6812

<210> 4

<211> 39

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

ataggatcca tggacaaact cagaatggtg ctacatgaa 39

<210> 5

<211> 36

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

atagaattct cagtagtgct ccagcttcag gctctt 36

<210> 6

<211> 36

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

ataggatccg ccaccatgga acagtggcat agcttt 36

<210> 7

<211> 33

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

ataggatcct cacttcctcc ggacctggac gct 33

Claims (10)

1. A construction method of immortalized pig bone marrow macrophages is characterized by comprising the following steps: transfecting pig bone marrow macrophages with a first recombinant plasmid containing a nucleotide sequence shown in SEQ ID NO.1, culturing the transfected cells, and separating cell monoclonals.

2. The method for constructing immortalized porcine bone marrow macrophages according to claim 1, wherein the first recombinant plasmid is constructed by the method comprising:

BglII and BCLI loci are added into the nucleotide sequence of SV40TAg through PCR to obtain a recombinant SV40TAg gene;

the recombinant SV40TAg gene was cloned into the pIED-Neo plasmid using BglII and BCLI sites, thereby generating the first recombinant plasmid, defined as pIED-Neo-SV40TAg plasmid.

3. The method for constructing immortalized porcine bone marrow macrophages according to claim 1, comprising:

selecting leg bones of healthy piglets of 15 days old, flushing marrow cavities by using a complete culture medium to obtain bone marrow cells,

inducing the bone marrow cells to differentiate into porcine bone marrow macrophages by GM-CSF induction culture solution;

wherein the complete medium contains 10V/V% fetal bovine serum;

the GM-CSF induction medium contains 1 wt% primocin antibiotic, 10 wt% FBS and 100ng/mLGM-CSF, and the balance is 1640 medium.

4. The method for constructing immortalized porcine bone marrow macrophages according to claim 3, further comprising: and (2) resuspending the porcine bone marrow macrophages in a complete culture medium, culturing for 50-80 min at 35-38 ℃, and transferring the non-adherent cells after culture into a new complete culture medium for culture, thereby realizing the amplification of the porcine bone marrow macrophages.

5. An immortalized porcine bone marrow macrophage cell constructed by the method of any one of claims 1 to 4.

6. A method for modifying immortalized porcine bone marrow macrophages, comprising: transfecting the immortalized pig bone marrow macrophage cell of claim 5 with a second recombinant plasmid comprising the nucleotide sequence shown in SEQ ID No.2 and/or a third recombinant plasmid comprising the nucleotide sequence shown in SEQ ID No.3, and screening positive cell strains to obtain the virus-sensitive immortalized pig bone marrow macrophage cell.

7. The method for remodeling immortalized porcine bone marrow macrophages according to claim 6, comprising:

(1) transfecting the immortalized pig bone marrow macrophage by the second recombinant plasmid, treating the transfected immortalized pig bone marrow macrophage line by 400-1200 mu g/ml neomycin for one week, screening out positive cell clones in the immortalized pig bone marrow macrophage line, and selecting a monoclonal cell strain to obtain an immortalized pig bone marrow macrophage line successfully transfected with CD163-CD169-CD 51;

(2) transfecting the immortalized pig bone marrow macrophage line transfected with CD163-CD169-CD51 successfully by using the third recombinant plasmid, treating the immortalized pig bone marrow macrophage line transfected again by using puromycin of 0.5-5 mu g/ml for one week, screening positive cell clones in the immortalized pig bone marrow macrophage line, and selecting a monoclonal cell strain to obtain the immortalized pig bone marrow macrophage line transfected with Ibal-KT022-Cd172a successfully, namely the immortalized pig bone marrow macrophage sensitive to virus.

8. The method for modifying immortalized porcine bone marrow macrophages according to claim 6 or 7, wherein the second recombinant plasmid is constructed by the method comprising: the nucleotide sequence shown in SEQ ID NO.2 was cloned into the first recombinant plasmid by PCR to obtain the second recombinant plasmid, defined as pIED-Neo-CD163-CD169-CD151 plasmid.

9. The method of remodeling immortalized porcine bone marrow macrophages according to claim 6 or 7, wherein: the construction method of the third recombinant plasmid comprises the following steps: the nucleotide sequence shown in SEQ ID NO.3 was cloned into the first recombinant plasmid by PCR to obtain the third recombinant plasmid, which was defined as pIED-Ibal-KT022-Cd172a plasmid.

10. The method of remodeling immortalized porcine bone marrow macrophage according to claim 6 or 7, further comprising: carrying out suspension domestication on the virus-sensitive immortalized pig bone marrow macrophages by a serum-free suspension culture medium;

wherein the serum-free culture medium contains inorganic salt, vitamins, amino acids, sugar, microelements, xanthan gum, interleukin-2, epidermal growth factor, insulin, yeast hydrolysate and soybean hydrolysate.

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