CN114807226A

CN114807226A - Recombinant plasmid for expressing canine IL-12 and preparation method and application of cell strain for expressing canine IL-12 protein

Info

Publication number: CN114807226A
Application number: CN202210481190.8A
Authority: CN
Inventors: 郭霄峰; 陈桂娥; 马娇洁; 赖艳琴
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2022-05-05
Filing date: 2022-05-05
Publication date: 2022-07-29

Abstract

The invention provides a recombinant plasmid for expressing canine IL-12, a preparation method and application of a cell strain for expressing canine IL-12 protein, wherein the recombinant plasmid is an expression vector connected with a gene segment with a nucleotide sequence shown as SEQ ID NO. 7. The invention uses the gene segment containing IGK signal peptide and His label nucleic acid sequence to connect with expression vector to prepare recombinant plasmid, the recombinant plasmid is introduced into mammal cell line and screened to obtain cell line of high expression dog IL-12 protein, the protein expression of the cell line of high expression dog IL-12 protein is far higher than that of the recombinant plasmid prepared by IL-12 gene segment without IGK signal peptide, which is introduced into mammal cell line and screened to obtain cell line of high expression dog IL-12 protein; the invention provides a new method for obtaining the high-expression canine IL-12 protein and also provides a basis for further research and utilization of the canine IL-12 protein.

Description

Recombinant plasmid for expressing canine IL-12 and preparation method and application of cell strain for expressing canine IL-12 protein

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a recombinant plasmid for expressing canine IL-12, a preparation method of a cell strain for expressing canine IL-12 protein and application of the cell strain.

Background

Interleukin-12 (IL-12) is found in culture medium of EB virus transfected human B lymphoblast Cell line by Wistar in the United states, also known as cytotoxic-lymphocyte maturation Factor (CLMF), or Natural Killer Cell stimulating Factor (NKSF), and is a cytokine which is found to have the strongest inducing and regulating effect on immune cells in vivo and has the widest scope. IL-12 is a heterodimer formed by disulfide bonding of two subunits, p35 and p40, and only when the two subunits act together, a biologically active IL-12 can be obtained.

IL-12 in canine animal research is less, the current research mainly as immune enhancer in disease research. Jeffry Currera et al studied the electroporation-mediated combination of bleomycin, a chemotherapeutic drug, and IL-12 plasmid DNA, in dogs with tumors for multi-cycle electrochemotherapy gene therapy (ECGT), and the results showed that ECGT treatment was very effective in inducing tumor regression by anti-tumor immune response in all examined tissue types except sarcoma, and could rapidly eradicate or eliminate large squamous cell carcinoma (DOI: 10.1111/jcmm.12382). Nasim Akhtar et al constructed an endothelial cell model by IL12, studied IL12ADDIN to inhibit tumor growth of malignant endothelial cells in canine angiosarcoma, and showed that IFN-gamma produced by IL-12 induction can inhibit proliferation of malignant mouse endothelial cells (doi: 10.1593/neo.03334); nina Milevoj et al used electrochemical therapy, and coded canine IL12 plasmid-transformed cell reduction surgery in combination to treat malignant melanoma in the oral cavity, and the results showed that electrochemical therapy and IL-12-GET combination therapy were effective in treating malignant melanoma in the oral cavity of dogs (DOI:10.1016/j. rvsc.2018.11.001). Studies on the immune effect of IL-12 on canine parvovirus VP2 DNA vaccine revealed that IL-12 can enhance the immune response of canine parvovirus VP2 DNA vaccine (Studies on the immune enhancement effect of Li Xiujin interleukin-12 on canine parvovirus VP2 DNA vaccine [ J ]. zootechnical and veterinary, 2010,42: 189.).

The prior art discloses the gene of canine IL-12, but there is no research on the expression of canine IL-12 protein by using the gene of canine IL-12, and how to obtain high-expression canine IL-12 protein by using the gene of canine IL-12 is not obvious.

Therefore, there is a need to find methods for high expression of canine IL-12 protein.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the existing preparation of the canine IL-12 protein and provide a recombinant plasmid for expressing the canine IL-12 and a preparation method and application of a cell strain for expressing the canine IL-12 protein.

The invention aims to provide a recombinant plasmid for expressing canine IL-12.

The present invention also aims to provide a method for preparing a cell line expressing canine IL-12 protein.

The invention also aims to provide the cell strain for expressing the canine IL-12 protein prepared by the preparation method, in particular to the cell strain for highly expressing the canine IL-12 protein.

The invention also aims to provide a preparation method of the canine IL-12 protein.

The invention also aims to provide the canine IL-12 protein prepared by the preparation method.

The invention also aims to provide the application of the canine IL-12 protein in promoting lymphocyte proliferation.

The invention also aims to provide the application of the canine IL-12 protein in preparing medicaments for treating canine diseases.

The above object of the present invention is achieved by the following technical means:

the invention firstly provides a recombinant plasmid for expressing canine IL-12, wherein the recombinant plasmid is an expression vector connected with a gene fragment with a nucleotide sequence shown as SEQ ID NO. 7 (enzyme-digested IGK-CaIL12 gene fragment).

Preferably, the expression vector is pcDNA3.1.

Preferably, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 9.

The gene segment in the recombinant plasmid of the invention contains the nucleic acid sequence of IGK signal peptide and the nucleic acid sequence of His label, which provides convenience for the secretion and purification of the expression protein after the recombinant plasmid is transfected.

The construction method of the recombinant plasmid comprises the steps of taking a canine IL-12 gene with a nucleotide sequence shown as SEQ ID NO.1 as a template, synthesizing the canine IL-12 gene by using a primer, carrying out enzyme digestion on the canine IL-12 gene and an expression vector by using restriction endonucleases NheI and NotI respectively, and connecting the enzyme-digested canine IL-12 gene and the expression vector by using T4 ligase to obtain the recombinant plasmid; the nucleotide sequence of the primer is shown as SEQ ID NO. 2 and SEQ ID NO. 3.

Preferably, the expression vector is pcDNA3.1.

The application of the recombinant plasmid in the preparation of the canine IL-12 protein is also within the protection scope of the invention.

A preparation method of a cell strain for expressing canine IL-12 protein comprises the steps of introducing the recombinant plasmid into a mammalian cell line, and performing pressurized screening on the obtained cells; and (3) screening and identifying the cells obtained by pressurized screening to obtain the cell strain expressing the canine IL-12 protein.

Preferably, the pressure screening is performed by using a culture medium containing the geneticin (G418) with the final concentration of 790-810 mug/mL.

Further preferably, the pressure screening is performed using a medium containing G418 at a final concentration of 800. mu.g/mL.

Preferably, the mammalian cell line is the CHO-K1 cell line.

The cell strain for expressing the canine IL-12 protein prepared by the preparation method is also within the protection scope of the invention.

Preferably, the cell line is a cell line highly expressing canine IL-12 protein.

The application of the cell strain in preparing the canine IL-12 protein is also within the protection scope of the invention.

A preparation method of canine IL-12 protein comprises the steps of culturing the cell strain until the cell confluency is 90-96%, taking cells, cracking, filtering to remove cell debris, and purifying the filtrate to obtain the canine IL-12 protein.

Preferably, the cell line is cultured until the cell confluence is 95%, and the cells are taken.

Preferably, the cell extraction is specifically: and (3) culturing the cell strain until the cell confluency is 90-96%, centrifuging, and taking a precipitate.

Preferably, the lysis is to lyse the cells by using a cell lysate containing 0.8 to 1.2 wt% of PMSF.

Further preferably, the lysis is performed by lysing the cells using a cell lysate containing 1 wt% PMSF.

Preferably, the filtration is performed with a sterile filter of 0.4 to 0.5 μm.

Further preferably, the filtration is with a 0.45 μm sterile filter.

Preferably, the purification is performed using a protein purifier.

Preferably, the purification is carried out by eluting the protein by a linear elution procedure using a protein purifier with solution A1 and solution B1 as mobile phases; the solution A1 was 19mL of 0.5MNaH ₂ PO ₄ ，81mL 0.5M Na ₂ HPO ₄ Adding water to 1L of NaCl and imidazole, wherein the concentration of NaCl is 0.5M, the concentration of imidazole is 8-12 mM, and the pH value is adjusted to 7.4; the solution B1 was 19mL of 0.5M NaH ₂ PO ₄ ，81mL 0.5M Na ₂ HPO ₄ NaCl and imidazole, adding water to 1L, wherein the concentration of the NaCl is 0.5M, the concentration of the imidazole is 490-510 mM, and the pH value is adjusted to 7.4.

Preferably, the solution A1 is 19mL of 0.5M NaH ₂ PO ₄ ，81mL 0.5M Na ₂ HPO ₄ Adding NaCl and imidazole into water to 1L to ensure that the concentration of the NaCl is 0.5M and the concentration of the imidazole is 10mM, and adjusting the pH value to 7.4; the solution B1 was 19mL of 0.5M NaH ₂ PO ₄ ，81mL 0.5M Na ₂ HPO ₄ NaCl, imidazole, 1L of water to make NaCl concentration 0.5M and imidazole concentration 500mM, regulatingThe pH was 7.4.

Further preferably, the mobile phase solution A1 is 47% and the solution B1 is 53%.

The canine IL-12 protein prepared by the preparation method of the canine IL-12 protein is also within the protection scope of the invention.

The use of the canine IL-12 protein in promoting lymphocyte proliferation is also within the scope of the present invention.

Preferably, the use of canine IL-12 protein concentration is 200 u g/mL.

The application of the canine IL-12 protein in preparing the medicaments for treating the canine diseases is also within the protection scope of the invention.

A therapeutic agent for canine diseases, comprising the canine IL-12 protein.

Compared with the prior art, the invention has the following beneficial effects:

the invention uses gene segment and expression vector containing IGK signal peptide nucleic acid sequence and His label nucleic acid sequence to prepare recombinant plasmid, the recombinant plasmid is introduced into mammal cell line and screened to obtain high expression dog IL-12 protein cell line, the protein expression quantity of the high expression dog IL-12 protein cell line is far higher than that of the recombinant plasmid prepared by IL-12 gene segment without IGK signal peptide, which is introduced into mammal cell line and screened to obtain high expression dog IL-12 protein cell line. The invention provides a new method for obtaining the high-expression canine IL-12 protein and also provides a basis for further research and utilization of the canine IL-12 protein.

Drawings

FIG. 1 shows the results of PCR amplification using primers IGK-CaIL12 and CaIL12 according to the present invention; wherein M is Marker, and 1-2 are amplification products of primer CaIL 12; 3 is a negative control amplification product of the primer CaIL12 without adding a template, 4-5 are amplification products of the primer IGK-CaIL12, and 6 is a negative control amplification product of the primer IGK-CaIL12 without adding a template.

FIG. 2 is a map of a recombinant plasmid of the present invention; wherein a is the map of plasmid pcDNA3.1, b is the map of CaIL12 recombinant plasmid, and c is the map of IGK-CaIL12 recombinant plasmid.

FIG. 3 shows the PCR identification result of the recombinant plasmid bacterial liquid of the present invention; wherein a is CaIL12 recombinant plasmid bacterial liquid, and b is IGK-CaIL12 recombinant plasmid bacterial liquid; a. in b, M1 is Maker DL2000, M2 is Maker DL5000, 1-9 are 9 single-colony bacterial liquids selected after transformation, and 10 is a negative control without a bacterial liquid template.

FIG. 4 shows the result of bacterial liquid double digestion identification of the present invention; wherein M1 is Maker DL10000, M2 is Maker DL2000, 1 is the correct bacterial liquid of PCR identification of recombinant plasmid of pcDNA3.1 and CaIL12, 2: PCR of the recombinant plasmids pcDNA3.1 and IGK-CaIL12 identified the correct bacterial solution.

FIG. 5 shows Western Blotting detection results of CHO-K1 cell line of the present invention; wherein M is protein Marker, S1 is supernatant of cell culture solution of pcDNA3.1 group, S2 is supernatant of cell culture solution of pcDNA3.1-CaIL12 group, S3 is supernatant of cell culture solution of pcDNA3.1-IGK-CaIL12 group, C1 is cell precipitation of pcDNA3.1 group, C2 is cell precipitation of pcDNA3.1-CaIL12 group, and C3 is cell precipitation of pcDNA3.1-IGK-CaIL12 group.

FIG. 6 shows the expression of canine IL-12 protein detected by Western Blot of a monoclonal cell strain of CaIL12 of strain 7 according to the present invention; a is the protein band of the canine IL-12 protein expressed by the cell strain monoclonal to CaIL12, and b is the gray value of the canine IL-12 protein expressed by the cell strain monoclonal to CaIL 12.

FIG. 7 shows the expression of canine IL-12 protein detected by Western Blot of a 6 strain IGK-CaIL12 monoclonal cell strain of the invention; a is the protein band of the canine IL-12 protein expressed by the cell strain monoclonal to IGK-CaIL12, and b is the gray value of the canine IL-12 protein expressed by the cell strain monoclonal to IGK-CaIL 12.

FIG. 8 shows the gray values of canine IL-12 protein expressed by the 6 strain CaIL12 monoclonal cell strain and the 6 strain IGK-CaIL12 monoclonal cell strain of the present invention.

FIG. 9 shows the transcription of the recombinant gene of IGK-CaIL12-4 cell line of the present invention.

FIG. 10 shows the results of an indirect immunofluorescence assay of the present invention; fluorescence represents the protein expression fluorescence map, DAPI represents the nuclear staining map, and large represents the superposition of the fluorescence map and the nuclear staining map.

FIG. 11 shows Western blotting detection results for His tag protein standard and purified canine IL-12 protein of the present invention; 1 is His tag protein standard with concentration of 32.25 μ g/mL, 2 is purified canine IL-12 protein.

FIG. 12 is a graph of the gray scale values for the His-tag protein standard and the purified canine IL-12 protein band at a concentration of 32.25. mu.g/mL in accordance with the present invention.

FIG. 13 shows the results of measurement of stimulation index of splenic lymphocytes under different concentrations of canine IL-12 protein of the present invention.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 amplification of the Canine IL-12 Gene

PCR amplification

Two pairs of specific primers IGK-CaIL12 and CaIL12 were designed based on the canine IL-12(CaIL12) gene whose nucleotide sequence is shown in SEQ ID NO:1, and the primer sequences are shown in Table 1, underlined for restriction sites.

TABLE 1 primer sequences

The DNA sequence of the CaIL12 gene shown in SEQ ID NO.1 is used as a template, and primers IGK-CaIL12 and CaIL12 are used for PCR amplification. The PCR reaction system (25. mu.L) was: 2 × Tap Plus Master Mix, 9.5 μ L; upstream primer (10. mu. mol/L), 0.5. mu.L; downstream primer (10. mu. mol/L), 0.5. mu.L; DNA template, 1. mu.L; ddH ₂ O, 13.5. mu.L. Reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing for 30s, extension at 72 ℃ for 2min, and running for 30 cycles; finally, extension is carried out for 10min at 72 ℃; the annealing temperature for amplification with primer CaIL12 was 74 ℃; the annealing temperature for amplification with primer IGK-CaIL12 was 85 ℃.

Mixing 1 wt% of agarose gel with 0.05% of Godview nucleic acid dye by volume fraction, pouring the mixture into a nucleic acid electrophoresis gel plate, and standing for 30min until the mixture is solidified; and (3) carrying out nucleic acid gel electrophoresis on the PCR amplification product for 110V30min, and observing the PCR amplification result by using an ultraviolet light gel irradiating instrument.

The amplified target band was cut with a clean blade and placed in a 1.5mL sterile centrifuge tube. And (3) cutting, recovering and purifying the PCR amplification product according to the instruction of the Omega Bio-Tek agarose gel recovery kit to obtain a purified PCR amplification product.

2. Results

The results of PCR amplification by using primers IGK-CaIL12 and CaIL12 are shown in FIG. 1, wherein M is Marker, and 1-2 are amplification products of CaIL12 primer; 3 is a negative control amplification product of the primer CaIL12 without adding a template, 4-5 are amplification products of the primer IGK-CaIL12, and 6 is a negative control amplification product of the primer IGK-CaIL12 without adding a template.

The results in FIG. 1 show that: the bands of about 1634bp appeared in lanes 1-2, which are consistent with the expected size, indicating that the gene CaIL12 was successfully amplified, and the bands of about 1694bp appeared in lanes 3-4, which are consistent with the expected size, indicating that the gene IGK-CaIL12 was successfully amplified.

EXAMPLE 2 construction of recombinant plasmid

1. Enzyme-cleaved ligation

The pcDNA3.1 plasmid, the amplification product CaIL12 gene purified in example 1 and the IGK-CaIL12 gene were double digested with restriction endonucleases NheI and NotI, respectively.

The digestion system (20. mu.L) was: 10 XFastdigest Buffer, 2. mu.L; NheI, 1 μ L; NotI, 1. mu.L; CaIL12 gene, 2. mu.L (IGK-CaIL12 gene, 3. mu.L); pcDNA3.1 plasmid, 2.5. mu.L; ddH ₂ O, make up 20 μ L. Enzyme digestion is carried out for 1h in a metal bath at 37 ℃, and the enzyme digestion products are recovered according to the specification of an agarose gel recovery kit of Omega Bio-Tek, thus obtaining a CaIL12 gene fragment (1579bp) with the nucleotide sequence shown as SEQ ID NO. 6, an IGK-CaIL12 gene fragment (1639bp) with the nucleotide sequence shown as SEQ ID NO. 7 and a pcDNA3.1 plasmid after enzyme digestion.

Connecting the CaIL12 gene fragment (SEQ ID NO: 6) and the enzyme-digested pcDNA3.1 plasmid by using T4 DNase, and performing metal bath at 16 ℃ overnight to obtain a CaIL12 recombinant plasmid; ligation of IGK-CaIL12 Gene Using T4 DNaseThe fragment (SEQ ID NO: 7) and the digested pcDNA3.1 plasmid were heated in a metal water bath at 16 ℃ overnight to obtain the IGK-CaIL12 recombinant plasmid. The ligation system (10. mu.L) was: CaIL12 gene, 2. mu.L (IGK-CaIL12 gene, 3. mu.L); pcDNA3.1 plasmid, 1 μ L; t4 DNA ligase, 1. mu.L; 10 XT 4 Buffer, 1 μ L; ddH ₂ O, make up to 10. mu.L.

The map of the recombinant plasmid is shown in FIG. 2, wherein a is the map of the plasmid pcDNA3.1, b is the map of the CaIL12 recombinant plasmid, and c is the map of the IGK-CaIL12 recombinant plasmid.

2. Identification of recombinant plasmids

(1) PCR identification of bacterial liquid

Respectively transforming the two constructed recombinant plasmids into Top10 competent cells, specifically, taking out the Top10 competence from-80 ℃, melting the competence on ice, respectively adding 10 mu L of each of the two recombinant plasmids into 100 mu LTop10 competent cells, slightly and elastically mixing the competent cells uniformly, standing the mixture for 30min in ice bath, thermally shocking the mixture for 90s in 42 ℃ in water bath, and quickly inserting the mixture into the ice to stand for 2 min; then adding the mixture into 900 mu LLB liquid culture medium, mixing uniformly, placing in a shaking table, shaking and culturing for 1h at the constant temperature of 37 ℃ and 200 r/min; centrifuging the culture solution at room temperature and 3000r/min for 3min, discarding supernatant, resuspending thallus, uniformly coating the resuspended thallus on LB solid medium plate containing ampicillin, and culturing at 37 deg.C in an inverted culture box for 16h when the thallus is completely absorbed. Single colonies were picked and inoculated into LB liquid medium for 14h, and PCR was performed using each single colony bacterial solution as a template under the two primer pairs and reaction conditions of example 1.

(2) PCR identification result of bacterial liquid

The PCR identification result of the recombinant plasmid bacterial liquid is shown in FIG. 3, wherein a is the recombinant plasmid bacterial liquid CaIL12, and b is the recombinant plasmid bacterial liquid IGK-CaIL 12; a. in b, M1 is Maker DL2000, M2 is Maker DL5000, 1-9 are 9 single-colony bacterial liquids selected after transformation, and 10 is a negative control without a bacterial liquid template.

In FIG. 3, a shows that the single colonies in

lanes

2, 5 and 9 among the 9 single colonies showed bands amplified by PCR, and the size was about 1634bp, which is consistent with the expected size; in FIG. 3, b shows that the single colonies in lanes 1-4 and 6-9 among the 9 single colonies showed bands after PCR amplification, and the size was about 1694bp, which is consistent with the expected size.

(3) Bacterial liquid double enzyme digestion identification

Plasmid extraction is carried out on bacterial liquid with correct PCR identification by referring to the specification of a plasmid extraction kit of magenta company, restriction endonucleases NheI and NotI are used for carrying out double enzyme digestion identification on the plasmid, the enzyme digestion product is placed in agarose gel for electrophoresis (110v, 30min), and the electrophoresis result is observed in an ultraviolet transilluminator.

(4) Bacterial liquid double enzyme digestion identification result

The double enzyme digestion identification result of the bacterial liquid is shown in fig. 4, wherein M1 is Maker DL10000, M2 is Maker DL2000, 1 is the correct bacterial liquid for the PCR identification of the recombinant plasmids of pcdna3.1 and CaIL12, 2: PCR of the recombinant plasmids pcDNA3.1 and IGK-CaIL12 identified the correct bacterial solution.

FIG. 4 shows that two bands appear at 1634bp and 5339bp of lane 1, indicating that the CaIL12 recombinant plasmid is successfully constructed; two bands appear in lane 2 at 1694bp and 5339bp, indicating successful construction of the IGK-CaIL12 recombinant plasmid.

The recombinant plasmids identified correctly by PCR and double enzyme digestion were sent to Biotechnology engineering (Shanghai) Ltd for sequencing, and the recombinant plasmids identified correctly for sequencing were named pcDNA3.1-CaIL12(SEQ ID NO:8) and pcDNA3.1-IGK-CaIL12(SEQ ID NO:9), respectively.

EXAMPLE 3 screening and identification of cell lines expressing canine IL-12 protein

1. Extraction of recombinant plasmid

Extraction of the endotoxin-depleted recombinant plasmid was carried out according to the instructions of the plasmid extraction kit from Qiagen.

Resuscitating a bacterial solution containing the recombinant plasmids pcDNA3.1-CaIL12, pcDNA3.1-IGK-CaIL12 and pcDNA3.1 no-load plasmids (negative control) successfully constructed in the embodiment 2, inoculating the bacterial solution into 5mL of LB liquid culture medium containing ampicillin in a 1% inoculation amount, and performing shake culture at 37 ℃ and 200r/min in a shaking table for 12 hours to obtain a seed solution; inoculating the seed liquid with the shake bacteria in 100mL LB liquid culture medium containing ampicillin at 1%, shaking and culturing at 37 deg.C and 200r/min for 14h, and extracting pcDNA3.1-CaIL12 recombinant plasmid, pcDNA3.1-IGK-CaIL12 recombinant plasmid and pcDNA3.1 plasmid with the kit.

Recovery and passage of CHO-K1 cells

Taking out frozen CHO-K1 cells from liquid nitrogen tank, rapidly placing in 37 deg.C constant temperature water bath, standing for 2min, centrifuging at room temperature of 800rpm for 5min, performing the next operation in biosafety cabinet, sucking out supernatant with pipetting gun, slowly adding 1mLRPMI-1640 culture medium along the wall, gently mixing, centrifuging at room temperature of 800rpm for 5min, sucking out supernatant with pipetting gun, adding 1mLRPMI-1640 nutrient medium (RPMI-1640 culture medium containing 10% fetal calf serum (v/v%) and 1% streptomycin (v/v%), gently blowing and beating resuspended cells and transferring to 60mm cell culture dish, adding 4 mMI-1640 nutrient medium and sufficiently blowing and mixing LRPMR, adding 5% CO ₂ Culturing in a constant-temperature cell culture box at 37 ℃.

Taking out the cells from the incubator, spraying 75% alcohol on the surface, placing the cells in a super clean bench, removing supernatant, washing the cells for 2 times by using sterilized PBS, adding 0.5mL of 0.25% pancreatin, digesting the cells for 1min at normal temperature, removing the pancreatin, adding an RPMI 1640 culture medium containing 10% fetal bovine serum (v/v%), performing air-assisted beating to obtain single cells, and performing cell passage by one third (1: 3).

Transfection of CHO-K1 cell and preliminary identification of protein expression of CHO-K1 cell line

(1) Transfection of CHO-K1 cells

And (3) paving the CHO-K1 cells which are passaged to the third generation and have good growth state on a 6-hole cell culture plate, and transfecting when the cells grow to reach 70-80% of confluence degree. Cells were washed 2 times with sterile PBS and then 1mL CTS was added per well ^TM Opti-MEM ^TM I Medium and Lipofectamine ^TM Transfection was performed with 3000 reagents (seimer feishell science, L3000001). The transfection solution is a mixed solution of solution A and solution B: preparing solution A and solution B, adding solution B into solution A, mixing, and standing at room temperature for 15 min;

solution A: MEM medium, 125. mu.L/well; lipofectamine ^TM 3000, 5.625. mu.L/well;

and B, liquid B: MEM medium, 125. mu.L/well; p3000 ^TM Reagent, 5. mu.L/well; pcDNA3.1 plasmid, 2. mu.L (pcDNA3.1-CaIL12 plasmid, 2.3 μ L/well; pcDNA3.1-IGK-CaIL12 plasmid, 1.8. mu.L/well).

The pcDNA3.1 plasmid is marked as pcDNA3.1 group; the pcDNA3.1-CaIL12 plasmid was designated as pcDNA3.1-CaIL 12; the pcDNA3.1-IGK-CaIL12 plasmid was designated pcDNA3.1-IGK-CaIL 12.

Adding the transfection solution into the cell culture well, gently shaking while adding to uniformly distribute the transfection solution, and placing in 5% CO ₂ Culturing for 6h in a constant-temperature cell culture box at 37 ℃; then, the cells were washed 2 times with an appropriate amount of PBS, replaced with RPMI-1640 medium containing 10% fetal bovine serum (v/v%), and placed in 5% CO ₂ Culturing in a constant-temperature cell culture box at 37 ℃ for 24 hours; then, the RPMI-1640 medium (pressure screening medium) containing 10% fetal bovine serum (v/v%) and geneticin (G418) at a final concentration of 800. mu.g/mL was replaced every 2 to 3 days, the pressure screening was stopped when the CHO-K1 normal cells in the negative cell well to which no plasmid was added were completely dead, and the protein expression of the cell lines of pcDNA3.1 group, pcDNA3.1-CaIL12 group and pcDNA3.1-IGK-CaIL12 group were tested.

(2) Initial identification of protein expression of CHO-K1 cell line

Separating cell culture solutions of cell strains of pcDNA3.1 group, pcDNA3.1-CaIL12 group and pcDNA3.1-IGK-CaIL12 group into supernatant and cells respectively; the separated cells are cracked by 1 wt% of PMSF cell lysate, the supernatant is obtained by centrifugation after cracking, 5 xSDS protein electrophoresis sample loading buffer (WB-0091, Guangzhou Dingguo biotechnology Co., Ltd.) is boiled for 10min at 100 ℃, protein samples are prepared, and the expression condition of the canine IL-12 protein in the CHO-K1 cell strain is detected by Western Blotting.

Specifically, the protein sample prepared in this example was subjected to electrophoresis with SDS-polyacrylamide gel prepared by the PAGE gel rapid preparation kit (10%) of Hayase biomedical science and technology, Inc., with 80v for the upper layer and 20min for the lower layer, and 110v for the lower layer and 60min for the lower layer. After electrophoresis, performing electrotransfer, setting constant current at 200mA, performing time 90min, after electrotransfer, washing PBST for 3 times, each time for 5min, sealing at room temperature for 2h by using PBST containing 5 wt% of skimmed milk powder, then washing for 3 times by using PBST, and then putting into a container containing 1: his-tag antibody (mouse monoclonal antibody, Annu (Beijing) Biotechnology limited, Cat No.10001-0-AP) diluted 10000, GAPDH as an internal reference, was incubated overnight at 4 deg.C, washed 3 times with PBST, and added with a solution containing 1: and incubating HRP-goat anti-mouse IgG secondary antibody diluted by 10000 for 1h at 37 ℃, washing for 3 times by using PBST, adding ECL chemiluminescence liquid under the condition of keeping out of the sun, and exposing in a chemiluminescence imaging analysis system and observing the result after 1-2 min.

(3) As a result, the

The Western Blotting detection result of the CHO-K1 cell strain is shown in FIG. 5; wherein M is protein Marker, S1 is supernatant of cell culture solution of pcDNA3.1 group, S2 is supernatant of cell culture solution of pcDNA3.1-CaIL12 group, S3 is supernatant of cell culture solution of pcDNA3.1-IGK-CaIL12 group, C1 is cell precipitation of pcDNA3.1 group, C2 is cell precipitation of pcDNA3.1-CaIL12 group, and C3 is cell precipitation of pcDNA3.1-IGK-CaIL12 group.

FIG. 5 shows that protein bands appeared at both C2 and C3, indicating that both cell lines of pcDNA3.1-CaIL12 and pcDNA3.1-IGK-CaIL12 successfully expressed canine IL-12 protein.

4. Screening, culturing and identification of monoclonal cells

(1) Screening and culturing of monoclonal cells

Cells of the CaIL 12-positive cell line and the IGK-CaIL 12-positive cell line which successfully express the canine IL-12 protein and are obtained by pressure screening are washed 2 times by PBS respectively and then digested by 0.25 wt% of pancreatin. After digestion, cells were counted, diluted to 1 cell/100. mu.L each with RPMI-1640 medium containing 10% fetal bovine serum (v/v%), 1% streptomycin (v/v%), and 800. mu.g/mLG 418 at the final concentration, and then the diluted cells were added to 96-well cell plates at 100. mu.L per well, respectively. Observing whether monoclonal cells appear in each hole of a 96-hole plate every day, selecting the monoclonal cells to continue culturing, transferring the cells from the 96-hole plate to a 48-hole plate, a 24-hole plate, a 12-hole plate and a 6-hole plate after the monoclonal cells grow to be aggregated into clusters, and gradually carrying out amplification culture to obtain a cell strain of the CaIL12 monoclonal and a cell strain of the IGK-CaIL12 monoclonal.

(2) Identification of monoclonal cell lines

The protein expression of the monoclonal cell line was examined by the initial identification method of protein expression of CHO-K1 cell line in 3(2) of this example.

Separating cell culture fluid of the cell strain monoclonal to CaIL12 and the cell strain monoclonal to IGK-CaIL12 into supernatant and cells respectively; the separated cells are cracked by 1 wt% of PMSF cell lysate, centrifuged to obtain supernatant, 5 xSDS protein electrophoresis loading buffer (WB-0091, Guangzhou Dingguo biotechnology Co., Ltd.) is added into the supernatant, the mixture is boiled at 100 ℃ for 10min to prepare a protein sample, and the expression condition of the canine IL-12 protein in the monoclonal cell strain is detected by Western Blotting. And scanning the gray value of the protein band detected by Western Blot by using Image J software, and calculating the protein content.

(3) Results

The protein bands of the canine IL-12 protein detected by Western Blot of 6 strains of CaIL12 monoclonal cell strains are shown in FIG. 6; FIG. 6 shows that 6 CaIL12 monoclonal cell lines all expressed canine IL-12 protein, and the protein bands were clear and unique;

the protein band of the canine IL-12 protein detected by Western Blot of 6 strain IGK-CaIL12 monoclonal cell strain is shown in FIG. 7; FIG. 7 shows that 6 IGK-CaIL12 monoclonal cell lines all expressed canine IL-12 protein, and the protein bands other than the protein band No. 2 were clear and unique and were purified proteins.

The gray scale values of canine IL-12 protein expressed by the 6 strain CaIL12 monoclonal cell strain and the 6 strain IGK-CaIL12 monoclonal cell strain are shown in FIG. 8; FIG. 8 shows that the grey values of canine IL-12 protein expressed by the cell lines from IGK-CaIL12 monoclonal are all significantly higher than those of canine IL-12 protein expressed by the cell lines from CaIL12 monoclonal, indicating that the content of canine IL-12 protein expressed by the cell lines from IGK-CaIL12 monoclonal is much higher than that of the cell lines from CaIL12 monoclonal, further indicating that the cell lines from IGK-CaIL12 monoclonal transfected and screened by the recombinant plasmid IGK-CaIL12 are superior to the cell lines from IGK-CaIL12 monoclonal transfected and screened by the recombinant plasmid CaIL 12.

FIG. 8 shows that the canine IL-12 protein expressed by the cell line monoclonal to No. 2 IGK-CaIL12 has the highest gray level, while FIG. 7 shows that the protein band expressed by the cell line 2 is not unique, the protein is not pure, and it cannot be judged whether the canine IL-12 protein content is the highest, the canine IL-12 protein expressed by the cell line monoclonal to No. 4 IGK-CaIL12 has the gray level second to No. 2, and the cell line monoclonal to No. 4 IGK-CaIL12(IGK-CaIL12-4) is used as the cell line with the highest expressed protein content.

Example 4 high expression canine IL-12 protein cell line qPCR identification

The IGK-CaIL12-4 cell line screened in example 3 was used as an assay target to test the transcription level of the gene highly expressing canine IL-12 protein.

The IGK-CaIL12-4 cell lines in example 3 were collected, total cellular RNA was extracted using HiPure Unvirosal RNA Mini Kit (Kyoto, McDNAs) from Kyoto, Meiji Biotech, Inc., and the transcript level of the recombinant gene was detected by qRT-PCR. CHO-K1 cells and CHO-K1 cells transfected with pcDNA3.1 plasmid (CHO-K1-No load) were used as controls.

The transcription condition of the recombinant gene of the IGK-CaIL12-4 cell strain is shown in FIG. 9, and FIG. 9 shows that the relative expression of the recombinant gene of the IGK-CaIL12-4 cell strain is significantly higher than that of a control, which indicates that the recombinant gene of the IGK-CaIL12-4 cell strain can be efficiently expressed.

Example 5 detection of immunogenicity of Canine IL-12 protein

1. Method of producing a composite material

The immunogenicity of the canine IL-12 protein was tested using an indirect immunofluorescence assay.

CHO-K1 cells, pcDNA3.1 plasmid-transfected CHO-K1 cells (CHO-K1-No load), IGK-CaIL12-4 cell line of example 3 (CHO-K1-IGK-CaIL12-4) were cultured on a culture plate in RPMI-1640 medium containing 10% fetal bovine serum (v/v%), 1% streptomycin (v/v%) and 800. mu.g/mLG 418, and the cells were washed with PBS 3 times by discarding the medium until the cell confluency became 90%; then fixing the cells for 1h by using methanol precooled at 4 ℃, removing the methanol, washing the cells for 3 times by using PBS (phosphate buffer solution), adding the prepared 1 v/v% triton, permeating the cells for 30min at room temperature, removing the triton, washing the cells for 3 times by using PBS, and respectively adding 1: 25 dilution of rabbit derived canine IL-12 polyclonal antibody (CaIL12p70 rabbit polyclonal antibody), adding blank rabbit serum to control group IGK-CaIL12-4 cell strain cells (rabbit negative control), adding control group CHO-K1 cells, pcDNA3.1 plasmid transfected CHO-K1 cells, and IGK-CaIL12-4 cell strain cells to 1: 100 dilution of mouse His-tag antibody, 4 degrees C overnight incubation; cells were washed 3 times with PBS, and rabbit derived canine IL-12 polyclonal antibody panel was raised using sterile PBS at 1: 100 dilutions of FITC-labeled goat anti-rabbit IgG were used as secondary antibody, and the murine His-tag antibody panel was incubated with sterile PBS in 1: 100 diluted FITC labeled goat anti-mouse IgG is used as a secondary antibody, and incubation is carried out for 1h at a constant temperature of 37 ℃ in the absence of light. Recovering the secondary antibodies respectively, washing the cells for 3 times by PBS, adding a proper amount of DAPI to stain the cell nucleus for 3min, abandoning the DAPI, washing the cells for 1 time by PBS, and observing the staining result by an inverted fluorescence microscope.

The results of the indirect immunofluorescence experiments are shown in FIG. 10, where fluorescence represents the fluorescence pattern for protein expression, DAPI represents the nuclear staining pattern, and Merged represents the superposition of the fluorescence pattern and the nuclear staining pattern. FIG. 10 shows that the IGK-CaIL12-4 cell line produces fluorescence after incubation and staining of either rabbit canine IL-12 polyclonal antibody or murine His-tag antibody, while the remaining control group produces no fluorescence, indicating that the canine IL-12 protein expressed by the IGK-CaIL12-4 cell line has good immunogenicity.

EXAMPLE 6 Canine IL-12 protein purification

The cell culture was continued using the cell line from the IGK-CaIL12-4 monoclonal of example 3, and when the confluency of the cells reached 95%, the cell culture solution was centrifuged, the centrifuged cell pellet was collected, the cells were lysed using a cell lysate containing 1 wt% PMSF, and then filtered through a 0.45 μm sterile filter to remove cell debris, and the filtrate (protein solution) was placed on ice for future use.

Protein purification was performed using a protein purifier from burle corporation. Opening the computer, the protein purifier and the automatic collecting pipe, double-clicking the computer ChromLab application program, and carrying out the following operations when the application program displays stand by:

s1. firstly, ddH for ultrasonic degassing ₂ Flushing the pump A and the pump B to remove the absolute ethyl alcohol; the column was replaced with 1mL of His-tagged protein, and 20mL of ddH degassed with ultrasound were passed from Pump A ₂ O, the speed is 1mL/min, and absolute ethyl alcohol in the His label protein purification column is removed;

s2, introducing 30mL of solution A1 subjected to ultrasonic degassing from a pump A at a speed of 1mL/min, balancing, and dissolvingLiquid a 1: 19mL of 0.5M NaH ₂ PO ₄ ，81mL 0.5M Na ₂ HPO ₄ Adding double distilled water into NaCl and imidazole, oscillating and mixing uniformly, fixing the volume to 1L, wherein the concentration of NaCl is 0.5M, the concentration of imidazole is 10mM, and adjusting the pH value to 7.4;

s3, introducing the prepared protein solution from the pump A at the speed of 1mL/min, and loading to combine the protein in the protein solution with the tag on the His tag protein purification column;

s4, repeating the step S2, and eluting redundant foreign proteins;

s5, introducing a solution A1 from a pump A, introducing a solution B1 from a pump B, eluting protein by using a linear elution program with the solution A1 and the solution B1 as mobile phases, wherein when the solution A1 is 47% and the solution B1 is 53%, an elution peak appears, protein elution is carried out under the elution condition, and meanwhile, eluent is collected; solution B1: 19mL of 0.5MNaH ₂ PO ₄ ，81mL 0.5M Na ₂ HPO ₄ Adding double distilled water into NaCl and imidazole, oscillating and mixing uniformly, then fixing the volume to 1L, wherein the NaCl concentration is 0.5M, the imidazole concentration is 500mM, and adjusting the pH value to 7.4.

S6, passing 30mL of solution B1 through the pump B at the speed of 1mL/min, and washing the bound protein on the column.

S7, simultaneously introducing 30mL of ddH subjected to ultrasonic degassing from a pump A and a pump B ₂ And O, washing the instrument, introducing ethanol with the volume fraction of 20% after washing to fill the whole system, and finally detaching the His-tagged protein purification column and storing at 4 ℃.

And (4) concentrating the eluent collected in the step (S5), and fully filtering the eluent by using an ultrafiltration tube with the cut-off degree of 30kD to obtain concentrated protein, namely the purified canine IL-12 protein. Will BenchMark ^TM The His-tag protein standard (LC 5606, seimer feishell science) was diluted with PBS to a concentration of 32.25 μ g/mL, Western blotting was performed with the concentrated protein (purified canine IL-12 protein), the gray scale values of the protein bands were scanned using Image J software, and the content of the concentrated protein was calculated from the gray scale values and the standard concentration.

His-tag protein standard with a concentration of 32.25. mu.g/mL, and purified canine IL-12 protein 2. Western blotting detection results of the His tag protein standard and the purified canine IL-12 protein are shown in FIG. 11; wherein 1 is His tag protein standard with the concentration of 32.25 μ g/mL, and 2 is purified canine IL-12 protein.

The grayscale values of the His-tag protein standard and the purified canine IL-12 protein band at a concentration of 32.25 μ g/mL are shown in fig. 12, and from the grayscale values of the His-tag protein standard and the purified canine IL-12 protein band in fig. 12 and the concentration of the His-tag protein standard, it can be deduced that the purified canine IL-12 protein concentration is 30.10 μ g/mL.

Example 7 lymphocyte proliferation assay

Splenic lymphocytes from 3 normal mice were taken and the proliferative response of the canine IL-12 protein purified in example 6 to lymphocytes was determined.

1. Preparation of lymphocytes

The whole operation is carried out on a sterilization operating table. The method comprises the following specific steps:

(1) killing the mice by a neck-breaking method, and standing in 75% alcohol for 5 min;

(2) taking out spleen of a mouse, placing the spleen in a small dish, washing the surface of the spleen by PBS after autoclaving, removing fat on the surface of the spleen, adding RPMI-1640 culture medium by using a sterilized grinder, grinding the spleen until the color becomes transparent, then filtering by using a 200-mesh cell screen, centrifuging the filtrate at 2000rpm/min for 10min, discarding supernatant, and keeping precipitate;

(3) adding 5mL of erythrocyte lysate into the precipitate, carefully blowing and uniformly mixing, standing at room temperature for 10min to completely lyse erythrocytes in the spleen, centrifuging at 2000r/min for 10min, and indicating that the erythrocytes are completely lysed when the cell precipitate is white;

(4) discarding the supernatant, adding 5mL of RPMI-1640 culture medium to wash the cell precipitate, and centrifuging at 2000r/min for 10 min;

(5) the supernatant was discarded, 2mL of RPMI-1640 medium containing 10% FBS (v/v%) was added, the obtained splenic lymphocytes were resuspended, and counted using a hemocytometer to prepare a 5X 10 cell ⁵ Each 100. mu.L of spleen lymphocyte suspension.

2. Lymphocyte proliferation assay

Prepared in the previous stepSpleen lymphocyte suspension was added to a 96-well cell plate at 100. mu.L per well, each mouse was subjected to 3 duplicate wells, and divided into 11 groups, and then 100. mu.L of concanavalin (Con A, positive control) at a final concentration of 10. mu.g/mL was added to each well of

group

1, 100. mu.L of RPMI-1640 medium containing 10% FBS (v/v%) was added to each well of group 2 (as a negative control), spleen lymphocyte suspension was not added to

group

3, 200. mu.LRMI-1640 complete medium (blank control) was added to each well of

group

3, and 100. mu.L was added to each well of the remaining 8 groups, and the canine IL-12 protein purified in example 6 (CaIL12 protein, drug addition/experimental group), 96-well cell plates were placed at 37 ℃ with 5% CO ₂ Culturing in an incubator for 48 h.

After the culture is finished, 20 mu L of CCK8 solution is added into each hole of a 96-hole cell plate, the mixture is gently shaken and uniformly mixed, and the mixture is placed at 37 ℃ and 5 percent CO ₂ Incubate in the incubator for 2h, and determine the OD value of each well of culture medium at 450nm by using a microplate reader. The Stimulation Index (SI) was calculated according to the following formula,

stimulation index-OD value of stimulated/unstimulated wells,

the stimulated wells are the wells into which the concanavalin and the CaIL12 protein were added, and the unstimulated wells are the wells for negative control cells.

3. Results

The results of measurement of stimulation index of splenic lymphocytes at different concentrations of canine IL-12 protein are shown in FIG. 13, and FIG. 12 shows that the stimulation index of canine IL-12 protein concentration ranging from 40. mu.g/mL to 2000. mu.g/mL decreases with increasing concentration, the stimulation index is the maximum and greater than 1 at a canine IL-12 protein concentration of 200. mu.g/mL, and the stimulation index has an effect of promoting splenic lymphocyte proliferation at a canine IL-12 protein concentration of 200. mu.g/mL.

As can be seen, the canine IL-12 protein had the effect of promoting the proliferation of splenic lymphocytes at a concentration of 200. mu.g/mL.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> southern China university of agriculture

<120> recombinant plasmid for expressing canine IL-12 and preparation method and application of cell strain for expressing canine IL-12 protein

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

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<213> Artificial Sequence (Artificial Sequence)

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atgaggagcc tccccacagc ctcaccgagc ccaggaatat tccagtgcct caaccactcc 60

caaaacctgc tgagagccgt cagcaacacg cttcagaagg ccagacaaac tctagattat 120

attccctgca cttccgaaga gattgatcat gaagatatca caaaggataa aaccagcaca 180

gtggaggcct gcttaccact ggaattaacc atgaatgaga gttgcctggc ttccagagag 240

atctctttga taactaacgg gagttgcctg gcctctggaa aggcctcttt tatgacggtc 300

ctgtgcctta gcagcatcta tgaggacttg aagatgtacc agatggaatt caaggccatg 360

aacgcaaagc ttttaatgga tcccaagagg cagatctttc tggatcaaaa catgttgaca 420

gctatcgatg agctgttaca ggccctgaat ttcaacagtg tgactgtgcc acagaaatcc 480

tcccttgaag agccggattt ttataaaact aaaatcaagc tctgcatact tcttcatgct 540

ttcagaattc gtgcggtgac catcgataga atgatgagtt atctgaattc ttccggatca 600

tctacaggat catctatatg ggaactggag aaagatgttt atgttgtaga gttggactgg 660

caccctgatg cccccggaga aatggtggtc ctcacctgcc atacccctga agaagatgac 720

atcacttgga cctcagcgca gagcagtgaa gtcctaggtt ctggtaaaac tctgaccatc 780

caagtcaaag aatttggaga tgctggccag tatacctgcc ataaaggagg caaggttctg 840

agccgctcac tcctgttgat tcacaaaaaa gaagatggaa tttggtccac tgatatctta 900

aaggaacaga aagaatccaa aaataagatc tttctgaaat gtgaggcaaa gaattattct 960

ggacgtttca catgctggtg gctgacggca atcagtactg atttgaaatt cagtgtcaaa 1020

agtagcagag gcttctctga cccccaaggg gtgacatgtg gagcagtgac actttcagca 1080

gagagggtca gagtggacaa cagggattat aagaagtaca cagtggagtg tcaggaaggc 1140

agtgcctgcc cctctgccga ggagagccta cccatcgagg tcgtggtgga tgctattcac 1200

aagctcaagt atgaaaacta caccagcagc ttcttcatca gagacatcat caaaccagac 1260

ccacccacaa acctgcagct gaagccattg aaaaattctc ggcacgtgga ggtcagctgg 1320

gaataccccg acacctggag caccccacat tcctacttct ccctgacatt ttgcgtacag 1380

gcccagggca agaacaatag agaaaagaaa gatagactct gcgtggacaa gacctcagcc 1440

aaggtcgtgt gccacaagga tgccaagatc cgcgtgcaag cccgagaccg ctactatagt 1500

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ctagctagcg ccaccatgga gacagacaca ctcctgctat gggtactgct gctctgggtt 60

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taaagcggcc gcctaatggt gatggtgatg atgactgcag gacacagatg 50

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<213> Artificial Sequence (Artificial Sequence)

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ctagctagcg ccaccatgag gagcctcccc acagc 35

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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ctagccacca tggagacaga cacactcctg ctatgggtac tgctgctctg ggttccaggt 60

tccactggtg acaggagcct ccccacagcc tcaccgagcc caggaatatt ccagtgcctc 120

aaccactccc aaaacctgct gagagccgtc agcaacacgc ttcagaaggc cagacaaact 180

ctagattata ttccctgcac ttccgaagag attgatcatg aagatatcac aaaggataaa 240

accagcacag tggaggcctg cttaccactg gaattaacca tgaatgagag ttgcctggct 300

tccagagaga tctctttgat aactaacggg agttgcctgg cctctggaaa ggcctctttt 360

atgacggtcc tgtgccttag cagcatctat gaggacttga agatgtacca gatggaattc 420

aaggccatga acgcaaagct tttaatggat cccaagaggc agatctttct ggatcaaaac 480

atgttgacag ctatcgatga gctgttacag gccctgaatt tcaacagtgt gactgtgcca 540

cagaaatcct cccttgaaga gccggatttt tataaaacta aaatcaagct ctgcatactt 600

cttcatgctt tcagaattcg tgcggtgacc atcgatagaa tgatgagtta tctgaattct 660

tccggatcat ctacaggatc atctatatgg gaactggaga aagatgttta tgttgtagag 720

ttggactggc accctgatgc ccccggagaa atggtggtcc tcacctgcca tacccctgaa 780

gaagatgaca tcacttggac ctcagcgcag agcagtgaag tcctaggttc tggtaaaact 840

ctgaccatcc aagtcaaaga atttggagat gctggccagt atacctgcca taaaggaggc 900

aaggttctga gccgctcact cctgttgatt cacaaaaaag aagatggaat ttggtccact 960

gatatcttaa aggaacagaa agaatccaaa aataagatct ttctgaaatg tgaggcaaag 1020

aattattctg gacgtttcac atgctggtgg ctgacggcaa tcagtactga tttgaaattc 1080

agtgtcaaaa gtagcagagg cttctctgac ccccaagggg tgacatgtgg agcagtgaca 1140

ctttcagcag agagggtcag agtggacaac agggattata agaagtacac agtggagtgt 1200

caggaaggca gtgcctgccc ctctgccgag gagagcctac ccatcgaggt cgtggtggat 1260

gctattcaca agctcaagta tgaaaactac accagcagct tcttcatcag agacatcatc 1320

aaaccagacc cacccacaaa cctgcagctg aagccattga aaaattctcg gcacgtggag 1380

gtcagctggg aataccccga cacctggagc accccacatt cctacttctc cctgacattt 1440

tgcgtacagg cccagggcaa gaacaataga gaaaagaaag atagactctg cgtggacaag 1500

acctcagcca aggtcgtgtg ccacaaggat gccaagatcc gcgtgcaagc ccgagaccgc 1560

tactatagtt catcctggag cgactgggca tctgtgtcct gccatcatca ccatcaccat 1620

taggcggccg 1630

<210> 7

<211> 1570

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctagccacca tgaggagcct ccccacagcc tcaccgagcc caggaatatt ccagtgcctc 60

aaccactccc aaaacctgct gagagccgtc agcaacacgc ttcagaaggc cagacaaact 120

ctagattata ttccctgcac ttccgaagag attgatcatg aagatatcac aaaggataaa 180

accagcacag tggaggcctg cttaccactg gaattaacca tgaatgagag ttgcctggct 240

tccagagaga tctctttgat aactaacggg agttgcctgg cctctggaaa ggcctctttt 300

atgacggtcc tgtgccttag cagcatctat gaggacttga agatgtacca gatggaattc 360

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cttcatgctt tcagaattcg tgcggtgacc atcgatagaa tgatgagtta tctgaattct 600

tccggatcat ctacaggatc atctatatgg gaactggaga aagatgttta tgttgtagag 660

ttggactggc accctgatgc ccccggagaa atggtggtcc tcacctgcca tacccctgaa 720

gaagatgaca tcacttggac ctcagcgcag agcagtgaag tcctaggttc tggtaaaact 780

ctgaccatcc aagtcaaaga atttggagat gctggccagt atacctgcca taaaggaggc 840

aaggttctga gccgctcact cctgttgatt cacaaaaaag aagatggaat ttggtccact 900

gatatcttaa aggaacagaa agaatccaaa aataagatct ttctgaaatg tgaggcaaag 960

aattattctg gacgtttcac atgctggtgg ctgacggcaa tcagtactga tttgaaattc 1020

agtgtcaaaa gtagcagagg cttctctgac ccccaagggg tgacatgtgg agcagtgaca 1080

ctttcagcag agagggtcag agtggacaac agggattata agaagtacac agtggagtgt 1140

caggaaggca gtgcctgccc ctctgccgag gagagcctac ccatcgaggt cgtggtggat 1200

gctattcaca agctcaagta tgaaaactac accagcagct tcttcatcag agacatcatc 1260

aaaccagacc cacccacaaa cctgcagctg aagccattga aaaattctcg gcacgtggag 1320

gtcagctggg aataccccga cacctggagc accccacatt cctacttctc cctgacattt 1380

tgcgtacagg cccagggcaa gaacaataga gaaaagaaag atagactctg cgtggacaag 1440

acctcagcca aggtcgtgtg ccacaaggat gccaagatcc gcgtgcaagc ccgagaccgc 1500

tactatagtt catcctggag cgactgggca tctgtgtcct gccatcatca ccatcaccat 1560

taggcggccg 1570

<210> 8

<211> 6911

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gccaccatga ggagcctccc cacagcctca ccgagcccag gaatattcca gtgcctcaac 960

cactcccaaa acctgctgag agccgtcagc aacacgcttc agaaggccag acaaactcta 1020

gattatattc cctgcacttc cgaagagatt gatcatgaag atatcacaaa ggataaaacc 1080

agcacagtgg aggcctgctt accactggaa ttaaccatga atgagagttg cctggcttcc 1140

agagagatct ctttgataac taacgggagt tgcctggcct ctggaaaggc ctcttttatg 1200

acggtcctgt gccttagcag catctatgag gacttgaaga tgtaccagat ggaattcaag 1260

gccatgaacg caaagctttt aatggatccc aagaggcaga tctttctgga tcaaaacatg 1320

ttgacagcta tcgatgagct gttacaggcc ctgaatttca acagtgtgac tgtgccacag 1380

aaatcctccc ttgaagagcc ggatttttat aaaactaaaa tcaagctctg catacttctt 1440

catgctttca gaattcgtgc ggtgaccatc gatagaatga tgagttatct gaattcttcc 1500

ggatcatcta caggatcatc tatatgggaa ctggagaaag atgtttatgt tgtagagttg 1560

gactggcacc ctgatgcccc cggagaaatg gtggtcctca cctgccatac ccctgaagaa 1620

gatgacatca cttggacctc agcgcagagc agtgaagtcc taggttctgg taaaactctg 1680

accatccaag tcaaagaatt tggagatgct ggccagtata cctgccataa aggaggcaag 1740

gttctgagcc gctcactcct gttgattcac aaaaaagaag atggaatttg gtccactgat 1800

atcttaaagg aacagaaaga atccaaaaat aagatctttc tgaaatgtga ggcaaagaat 1860

tattctggac gtttcacatg ctggtggctg acggcaatca gtactgattt gaaattcagt 1920

gtcaaaagta gcagaggctt ctctgacccc caaggggtga catgtggagc agtgacactt 1980

tcagcagaga gggtcagagt ggacaacagg gattataaga agtacacagt ggagtgtcag 2040

gaaggcagtg cctgcccctc tgccgaggag agcctaccca tcgaggtcgt ggtggatgct 2100

attcacaagc tcaagtatga aaactacacc agcagcttct tcatcagaga catcatcaaa 2160

ccagacccac ccacaaacct gcagctgaag ccattgaaaa attctcggca cgtggaggtc 2220

agctgggaat accccgacac ctggagcacc ccacattcct acttctccct gacattttgc 2280

gtacaggccc agggcaagaa caatagagaa aagaaagata gactctgcgt ggacaagacc 2340

tcagccaagg tcgtgtgcca caaggatgcc aagatccgcg tgcaagcccg agaccgctac 2400

tatagttcat cctggagcga ctgggcatct gtgtcctgcc atcatcacca tcaccattag 2460

gcggccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc 2520

tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 2580

cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg 2640

tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa 2700

tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg 2760

gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 2820

ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 2880

cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 2940

ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 3000

tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 3060

gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 3120

ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 3180

gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt 3240

ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 3300

gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat 3360

ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg 3420

cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc 3480

gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta 3540

ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca 3600

ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct 3660

tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc 3720

gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc 3780

ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc 3840

gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg 3900

ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc 3960

atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac 4020

caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat 4080

caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc 4140

aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg 4200

aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg 4260

gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc 4320

gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc 4380

gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg 4440

accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa 4500

ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc 4560

tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat 4620

aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 4680

gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga 4740

gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 4800

cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 4860

aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 4920

agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 4980

ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 5040

ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 5100

tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 5160

tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 5220

gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 5280

ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 5340

tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 5400

agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5460

atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5520

acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 5580

actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 5640

tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggttttt 5700

ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 5760

tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 5820

gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 5880

tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 5940

ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 6000

taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 6060

cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 6120

gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 6180

gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 6240

tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 6300

gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 6360

ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 6420

ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 6480

cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 6540

ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 6600

gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 6660

ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 6720

ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 6780

tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 6840

ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 6900

cacctgacgt c 6911

<210> 9

<211> 6971

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt tccaggttcc 960

actggtgaca ggagcctccc cacagcctca ccgagcccag gaatattcca gtgcctcaac 1020

cactcccaaa acctgctgag agccgtcagc aacacgcttc agaaggccag acaaactcta 1080

gattatattc cctgcacttc cgaagagatt gatcatgaag atatcacaaa ggataaaacc 1140

agcacagtgg aggcctgctt accactggaa ttaaccatga atgagagttg cctggcttcc 1200

agagagatct ctttgataac taacgggagt tgcctggcct ctggaaaggc ctcttttatg 1260

acggtcctgt gccttagcag catctatgag gacttgaaga tgtaccagat ggaattcaag 1320

gccatgaacg caaagctttt aatggatccc aagaggcaga tctttctgga tcaaaacatg 1380

ttgacagcta tcgatgagct gttacaggcc ctgaatttca acagtgtgac tgtgccacag 1440

aaatcctccc ttgaagagcc ggatttttat aaaactaaaa tcaagctctg catacttctt 1500

catgctttca gaattcgtgc ggtgaccatc gatagaatga tgagttatct gaattcttcc 1560

ggatcatcta caggatcatc tatatgggaa ctggagaaag atgtttatgt tgtagagttg 1620

gactggcacc ctgatgcccc cggagaaatg gtggtcctca cctgccatac ccctgaagaa 1680

gatgacatca cttggacctc agcgcagagc agtgaagtcc taggttctgg taaaactctg 1740

accatccaag tcaaagaatt tggagatgct ggccagtata cctgccataa aggaggcaag 1800

gttctgagcc gctcactcct gttgattcac aaaaaagaag atggaatttg gtccactgat 1860

atcttaaagg aacagaaaga atccaaaaat aagatctttc tgaaatgtga ggcaaagaat 1920

tattctggac gtttcacatg ctggtggctg acggcaatca gtactgattt gaaattcagt 1980

gtcaaaagta gcagaggctt ctctgacccc caaggggtga catgtggagc agtgacactt 2040

tcagcagaga gggtcagagt ggacaacagg gattataaga agtacacagt ggagtgtcag 2100

gaaggcagtg cctgcccctc tgccgaggag agcctaccca tcgaggtcgt ggtggatgct 2160

attcacaagc tcaagtatga aaactacacc agcagcttct tcatcagaga catcatcaaa 2220

ccagacccac ccacaaacct gcagctgaag ccattgaaaa attctcggca cgtggaggtc 2280

agctgggaat accccgacac ctggagcacc ccacattcct acttctccct gacattttgc 2340

gtacaggccc agggcaagaa caatagagaa aagaaagata gactctgcgt ggacaagacc 2400

tcagccaagg tcgtgtgcca caaggatgcc aagatccgcg tgcaagcccg agaccgctac 2460

tatagttcat cctggagcga ctgggcatct gtgtcctgcc atcatcacca tcaccattag 2520

gcggccgctc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc 2580

tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 2640

cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg 2700

tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa 2760

tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg 2820

gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 2880

ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 2940

cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 3000

ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 3060

tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 3120

gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 3180

ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 3240

gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt 3300

ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 3360

gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat 3420

ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg 3480

cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc 3540

gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta 3600

ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca 3660

ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct 3720

tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc 3780

gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc 3840

ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc 3900

gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg 3960

ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc 4020

atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac 4080

caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat 4140

caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc 4200

aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg 4260

aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg 4320

gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc 4380

gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc 4440

gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg 4500

accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa 4560

ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc 4620

tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat 4680

aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 4740

gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga 4800

gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 4860

cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 4920

aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 4980

agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 5040

ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 5100

ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 5160

tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 5220

tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 5280

gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 5340

ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 5400

tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 5460

agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5520

atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5580

acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 5640

actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 5700

tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggttttt 5760

ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 5820

tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 5880

gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 5940

tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 6000

ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 6060

taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 6120

cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 6180

gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 6240

gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 6300

tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 6360

gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 6420

ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 6480

ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 6540

cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 6600

ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 6660

gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 6720

ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 6780

ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 6840

tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 6900

ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 6960

cacctgacgt c 6971

Claims

1. A recombinant plasmid for expressing canine IL-12 is characterized in that the recombinant plasmid is an expression vector connected with a gene segment with a nucleotide sequence shown as SEQ ID NO. 7.

2. The recombinant plasmid of claim 1, wherein the expression vector is pcDNA3.1.

3. Use of the recombinant plasmid of claim 1 or 2 for the preparation of canine IL-12 protein.

4. A method for producing a cell line expressing canine IL-12 protein, which comprises introducing the recombinant plasmid according to claim 1 into a mammalian cell line, and subjecting the resulting cells to pressure screening; and screening and identifying the cells obtained by pressure screening to obtain the monoclonal cells.

5. The method according to claim 4, wherein the mammalian cell line is CHO-K1 cell line.

6. A cell line expressing canine IL-12 protein, which is prepared by the method of any one of claims 4 to 5.

7. Use of the cell line of claim 6 for the preparation of canine IL-12 protein.

8. A method for producing canine IL-12 protein, comprising culturing the cell line of claim 6 until the cell confluency reaches 90-96%, collecting the cells, lysing the cells, filtering off cell debris, and purifying the filtrate.

9. The canine IL-12 protein produced by the method of claim 8.

10. Use of the canine IL-12 protein of claim 9 in the preparation of a medicament for the treatment of canine diseases.