Patent Description:
Tumor is a new organism formed by the clonal abnormal proliferation of a certain cell in a local tissue that has lost the normal regulation of its growth at the gene level under the action of various carcinogenic factors. These new organisms are mostly space-occupying massive protrusions, so they are also called neoplasms.

In recent years, many new methods for treating tumors have appeared. At present, common and effective treatment methods include radiotherapy, chemotherapy, surgery, immunotherapy, and so on. However, there are still some problems, such as serious adverse reactions of radiotherapy and chemotherapy, high risk of surgery, and poor effect of immunotherapy on solid tumors.

In the pending Chinese patent application No. <CIT> previously filed by the applicant, an anti-tumor drug composition is disclosed, which comprises proteins IL12, GMCSF and IL2, wherein the mass ratio of IL12 protein: GMCSF protein: IL2 protein is <NUM>-<NUM>:<NUM>-<NUM>:<NUM>-<NUM>. The composition in the application plays a very good role in disease state control for a variety of solid tumor patients, and some patients can even achieve complete remission. During the treatment, the patient's body is less irritated and the adverse reactions are mild, and thus the patient's quality of life is greatly improved. However, since the composition of three proteins is used in the application, in practical application, it is necessary to prepare the components one by one, and then mix them in proportion. Consequently, there are problems such as complex preparation process, difficult quality control, and high cost.

<CIT> discloses a protein heterodimer comprising a first polypeptide chain and a second polypeptide chain different from said first polypeptide chain, wherein said first polypeptide chain comprises IL12a, and said second polypeptide chain comprises IL12b. Said protein heterodimer further comprises IL2 or a functional fragment thereof, GMCSF or a functional fragment thereof, and one or more targeting moieties, whereby said IL2 or the functional fragment thereof is located in said first polypeptide chain or in said second polypeptide chain, said GMCSF or the functional fragment thereof is located in said first polypeptide chain or in said second polypeptide chain, and said one or more targeting moieties are each independently located in said first polypeptide chain or in said second polypeptide chain.

Further, <CIT> relates to a protein molecule, comprising IL12a or a functional fragment thereof, IL12b or a functional fragment thereof, a first factor and a second factor, wherein the first factor and the second factor are each independently selected from the group consisting of: IL2, GMCSF, IL7, IL15, IL21 and FLT3L; and the IL12a or the functional fragment thereof, the IL12b or the functional fragment thereof, the first factor and the second factor are located in the same polypeptide chain.

Furthermore, the scientific publication of <NPL>, is concerned with the application of antibody-cytokine fusion proteins in cancer therapy focusing on IL-<NUM>, IL-<NUM> and GM-CSF and inter alia discloses an antibody-cytokine fusion protein of IL-<NUM> and IL-<NUM> fused to anti-EpCAM IgG.

Moreover, <CIT> refers to a cytokine fusion protein comprising a first cytokine, which is IL-<NUM> in a heterodimeric or single-chain form in which the p35 and p45 subunit(s) of IL-<NUM> are linked by a disulfide bond, and a second cytokine, selected from IL-<NUM> and GM-CSF, wherein said second cytokine is covalently bonded at the amino terminus or carboxyl terminus of said p35 or p40 subunit.

Likewise, the scientific publication of <NPL>, describes the fusion cytokine IL-<NUM>-GM-CSF which shows enhanced and synergistic anticancer immune responses compared to the individual cytokines.

<CIT> discloses a bioactive IL-<NUM> fusion protein which comprises IL-<NUM> p35 subunit and IL-<NUM> p40 subunit joined by a polypeptide linker, and which may further comprise monomers which are individuall bioactive (e.g., IL-<NUM>, GMCSF).

Further, the scientific publication of <NPL>, is concerned with the application of an NHS-IL-<NUM> immunocytokine composed of two IL-<NUM> heterodimers fused to a NHS76 antibody to target a cytokine to the regions of tumor necrosis.

Furthermore, the scientific publication of <NPL>, describes the application of GM-CSF along with either IL-<NUM> or IL-<NUM> and irradiated tumor cells for increasing the survival rate of rats suffering from <NUM> brain tumors.

Moreover, the scientific publication of <NPL>, discloses the in-situ administration of cytokine combinations to treat tumours in mice, wherein particularly the triple combination of IL-<NUM>, GMCSF and IL-<NUM> was tested, in further combination with chitosan.

Further, <CIT> relates to a cytokine combination comprising at least three cytokines selected from the group consisting of IL12 or a functional variant thereof, GMCSF or a functional variant thereof, FLT3L or a functional variant thereof, IL2 or a functional variant thereof, IL15 or a functional variant thereof, IL21 or a functional variant thereof and IL7 or a functional variant thereof, for treating tumors and/or for preventing tumor recurrence or metastasis.

Therefore, there is a need in the art for a single-component anti-tumor protein drug capable of better controlling or even completely alleviating the disease states of solid tumors, so as to reduce the preparation processes, improve quality control and reduce drug costs while achieving good curative effects.

In view of the shortcomings in the prior art, one objective of the present invention is to provide a fusion protein capable of treating animal tumors with low cost, high efficiency and simplicity.

The above objective is achieved by the following solutions:
In a first aspect, the present invention provides a fusion protein for treating animal tumors, wherein the fusion protein comprises IL12, IL2 and GMCSF peptide fragments, wherein the IL12, IL2 and GMCSF peptide fragments are obtained from felines or canines, and wherein the fusion protein is set forth in SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, or SEQ ID NO: <NUM>. In the amino acid sequence of the fusion protein, the order of connections among the peptide fragments is changeable, such as IL12, IL2 and GMCSF, or IL12, GMCSF and IL2. The three peptide fragments can be directly linked. The IL12 peptide fragment consists of two subunits, IL12a and IL12b.

Through the technical solution, a fusion protein that has a good inhibitory effect on various animal solid tumors can be obtained, and the fusion protein is capable of shrinking tumors, or even causing same to disappear; is capable of better inhibiting the metastasis of malignant tumors; and improves the quality of life of patients to a large extent.

The IL2 and GMCSF peptide fragments may also be linked with DiaNHS76F8 peptide fragment. Accordingly, the fusion protein is: cIL12bIL12aIL2GMCSF (ID SEQ NO: <NUM>), cIL12bIL12aGMCSFIL2 (ID SEQ NO: <NUM>), fIL12bIL12aIL2GMCSF (ID SEQ NO: <NUM>), fIL12bIL12aGMCSFIL2 (ID SEQ NO: <NUM>), cIL12bIL12aIL2DiaNHS76F8GMCSF (ID SEQ NO: <NUM>) or fIL12bIL12aIL2DiaNHS76F8GMCSF (ID SEQ NO: <NUM>).

The DiaNHS76F8 peptide fragment has a capability of enhancing tumor targeting. Through the technical solution, the tumor targeting effect of the fusion protein can be further enhanced.

Further preferably, the tumor includes one or more selected from the group consisting of melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, liver cancer, bone cancer, pancreas cancer, skin cancer, head or neck cancer, oral cancer, nasopharynx cancer, malignant melanoma of skin or eye, uterine cancer, ovarian cancer, rectum cancer, anus cancer, stomach cancer, testicle cancer, uterus cancer, fallopian tube cancer, endometrium cancer, uterine cervix cancer, vagina cancer, vulva cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophagus cancer, small intestine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethra cancer, penis cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system neoplasm, primary central nervous system lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T cell lymphoma, trunk sarcoma and basal cell tumor. In some preferred embodiments, the tumors are trunk sarcoma, basal cell tumor and breast cancer.

In a second aspect, there is provided a composition for treating animal tumors, wherein the composition comprises the fusion protein according to the aforementioned first aspect and a chitosan solution. Preferably, the composition comprises from <NUM> vol% to <NUM> vol% of fusion protein solution and from <NUM> vol% to <NUM> vol% of chitosan solution. Further preferably, the composition comprises from <NUM> vol% to <NUM> vol% of fusion protein solution and from <NUM> vol% to <NUM> vol% of chitosan solution. Further preferably, the composition comprises <NUM> vol% of fusion protein solution and <NUM> vol% of chitosan solution.

Through the technical solution, a composition that can be directly injected into animal tumors for treatment can be obtained. Without being bound by any theory, in the composition, chitosan mainly acts as a thickener, which increases the viscosity of the solution while slowing down the release rate of the fusion protein, thereby prolonging the effective acting time of the fusion protein as the main therapeutic component.

Further preferably, the chitosan solution is a solution containing from <NUM> wt% to <NUM> wt% of chitosan, further preferably a solution containing from 2wt% to <NUM> wt% of chitosan, and further preferably a solution containing from <NUM> wt% to <NUM> wt% of chitosan. In a specific embodiment, in the chitosan solution, the content of the chitosan can be <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt% or any range or any value therebetween, including but not limited to integral values or decimal values.

By adjusting the content range of chitosan, the viscosity of the composition and the release rate of the fusion protein can be adjusted, so that targeted treatment can be carried out.

Further preferably, the fusion protein solution is from <NUM> vol% to <NUM> vol% and the chitosan solution is from <NUM> vol% to <NUM> vol%, and more preferably, the fusion protein solution is <NUM> vol% and the chitosan solution is <NUM> vol%. In some embodiments, in the composition, the fusion protein solution can be <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, or any range or any value therebetween. The chitosan solution can be <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, <NUM> vol%, or any range or any value therebetween.

By adjusting the relative volume ratio of the fusion protein solution to the chitosan solution, the viscosity of the composition and the release rate of the fusion protein can be adjusted, so that targeted treatment can be carried out.

In a third aspect, the present invention provides the fusion protein according to the aforementioned first aspect for use as a medicament, particularly for use in treating animal tumors.

In a fourth aspect, there is provided the composition according to the aforementioned second aspect for use as a medicament, particularly for use in in treating animal tumors.

Through the above technical solutions, the present invention obtains the following beneficial effects:.

Some preferred embodiments of the present invention will be described in detail hereinafter with reference to the accompanying drawings. Those of ordinary skill in the art will understand that these accompanying drawings are for illustrative purpose only and are not intended to limit the scope of the present invention in any way.

Some specific embodiments of the present invention will be described hereinafter with reference to the accompanying drawings and the examples. Those of ordinary skill in the art will understand that these examples are provided only to illustrate how the solutions of the present invention can be implemented, and not to limit the scope of the present invention in any way.

The present invention provides a fusion protein for treating animal tumors, wherein the fusion protein comprises IL12, IL2 and GMCSF peptide fragments.

In the fusion protein, IL <NUM> is interleukin-<NUM> (IL-<NUM>), which is a cytokine with a wide range of biological activities and is mainly produced by activated inflammatory cells. The coding region of the IL12 comprises two subunits, IL12a and IL12b, which can be linked with a T2A sequence. IL12 is a cytokine responsible for regulating T cells in the immune system and produced by activated T cells, which plays an important role in immune response, anti-virus infection, and the like of a body. At present, IL12 has been approved for the treatment of certain cancers, including metastatic melanoma and renal cell carcinoma. However, at present, there are some shortcomings in the application of IL12 alone: IL12 may cause general symptoms such as fever, vomiting, and the like, and may also lead to disorders of water and salt metabolism and abnormal functions of kidney, liver, heart, lung, and the like; and the most common and serious consequence is capillary leakage syndrome, which makes patients have to suspend treatment. Therefore, combined administration is usually needed to alleviate adverse reactions.

In the fusion protein, IL2 is interleukin-<NUM>. IL2 has a relatively strong anti-tumor activity (stronger than IL12) and relatively slight side effects. However, in a large number of experiments conducted by the inventor, there are still many problems when the IL2 is used alone. For example, IL2 is dose-dependent, and a relatively high dose of IL2 is required in the course of treatment, which leads to a high cost and is easy to cause anemia, thrombocytopenia, and the like.

In the fusion protein, GMCSF is granulocyte-macrophage colony stimulating factor, which stimulates proliferation and differentiation of myeloid stem cells to mature granulocytes, and can promote differentiation of myeloid stem cells to common progenitor cells of granulocytes (including neutrophile granulocytes and eosinophilic granulocytes), erythroid cells, megakaryocytes, myelomonocytes and monocytes, and promote proliferation and maturation of hematopoietic stem cells of the above series. However, the defects lie in a non-obvious effect on basophilic granulocytes and a little effect on erythroid advanced hematopoietic cells.

For the above reasons, the applicant tried to combine various cytokines to reduce the toxic and side effects while utilizing the effective synergistic effect. Through a lot of screening work, the inventor finally found that combining the above three cytokines, i.e. IL12, IL2 and GMCSF can achieve very significant tumor treatment effects while avoiding their respective shortcomings. Although the current mechanism is still unclear, the inventor has observed that there is a very good synergistic effect among IL12, IL2 and GMCSF, which is manifested in that the combination stimulates the immune system of diseased animals to recognize and kill tumor cells, so as to inhibit malignant tumors, and thus shrink tumors, or even cause same to disappear, and at the same time, almost no side effects are observed.

On the basis of this discovery, the inventor completed the contents of the pending application (Chinese Patent Application No. <CIT>) previously filed by the applicant. In addition, the inventor also realized that when IL12, IL2 and GMCSF were prepared into a composition, it was necessary to prepare cells separately, and culture them to obtain their respective proteins and finally mix the proteins. This brought about the problems of complex preparation process, difficult control of process quality and high cost. On this basis, the inventor proposed that IL2, IL2 and GMCSF were prepared into a fusion protein to solve these problems. The present invention is made based on the discovery.

The preferred embodiments of the present invention are further described in detail hereinafter with reference to the specific embodiments. Those of ordinary skill in the art will understand that these illustrative examples are for illustrative purpose only and are not intended to limit the present invention in any way.

Reagents: DMEM medium, <NUM> medium and fetal bovine serum were purchased from Life Technologies; CDM4HEK293 serum-free medium was purchased from Thermo; cell culture flasks and culture plates were purchased from Corning; Puromycin was purchased from Chemicon; restriction enzymes were purchased from Takara and NEB; ligase was purchased from NEB; DNA polymerase was purchased from Takara; plasmid extraction kit and gel recovery kit were purchased from Omega Biotech; primer synthesis was completed by Sangon Biotech (Shanghai) Co. ; and gene synthesis and sequencing were completed by Life Technologies. IL12 and IL2 ELISA kits were purchased from Thermo; GMCSF ELISA kit was purchased from Sigma; and chitosan (Protosan G <NUM>) was purchased from NovaMatrix. Recombinant canine IL12, GMCSF and IL2 proteins and recombinant feline IL12, GMCSF and IL2 proteins were purchased from Novus Biologicals.

The coding region of the canine IL12 gene was synthesized and contained two subunits, IL12a (Genbank Accession No.: NM_001003293) and IL12b (Genbank Accession No.: NM_001003292), which were linked with a T2A sequence. The synthesized gene had BamHI and XhoI restriction sites at the two ends respectively, and then was digested with BamHI and XhoI enzymes. The system was as follows: <NUM>µg of IL12 plasmid, <NUM>µL of enzyme digestion buffer, <NUM>µL of BamHI, <NUM>µL of XhoI, supplemented with water to a final volume of <NUM>µL. The mixture was stood at <NUM> for <NUM> hours. The EP tube was taken out, and <NUM>µL of <NUM>× loading buffer was added, and then the resulting mixture was subjected to <NUM>% agarose gel electrophoresis. After the electrophoresis, the IL12 gene segment was recovered for later use.

Expression vector pLentis-CMV-MCS-IRES-PURO was digested. The system was as follows: <NUM>µg of plasmid, <NUM>µL of enzyme digestion buffer, <NUM>µL of BamHI, <NUM>µL of XhoI, supplemented with water to a final volume of <NUM>µL. The mixture was stood at <NUM> for <NUM> hours. The EP tube was taken out, <NUM>µL of <NUM>× loading buffer was added, and then the resulting mixture was subjected to <NUM>% agarose gel electrophoresis. After the electrophoresis, the vector segment was recovered for later use.

The pLentis-CMV-MCS-IRES-PURO was ligated with IL12. The system was as follows: <NUM>µL of pLentis-CMV-MCS-IRES-PURO, <NUM>µL of IL12, <NUM>µL of ligase buffer, <NUM>µL of T4 DNA ligase, and <NUM>µL of water. The mixture was placed at room temperature to ligate for <NUM> hours. Then, the ligation system was transformed into competent Escherichia coli cells. On the second day, a bacterial colony was picked from the transformed plate, inoculated into LB medium and cultured overnight at <NUM> in a shaker. The plasmids were extracted from the cultured bacteria by using the plasmid extraction kit. Enzyme digestion was conducted to determine whether the segment was successfully ligated into the vector, and then the correct vector was sent for sequencing, and it was confirmed that the construction was successful. The expression vector was pLentis-CMV-IL12-PGK-PURO.

A virus containing the regulatory vector was prepared, and the method was as follows: <NUM>. cultured 293FT cells were digested, after counting, <NUM>×<NUM> cells/well were spread onto a <NUM>-cm culture dish, and the volume of the culture solution was <NUM>; <NUM>. on the next night, the cell status was observed and if the cell status was good, the cells could be transfected; chloroquine was added to the culture plate to a final concentration of <NUM>, one test tube was taken and added with sterilized water and the following plasmids (<NUM>µg of pMD2. G + <NUM>µg of pSPAX2 + <NUM>µg of pLentis-CMV-IL12-PGK-PURO) with a total volume of <NUM>µL, then added with <NUM>µL of <NUM> CaCl<NUM>, mixed evenly, and finally added with <NUM>µL of <NUM>×HBS; the test tube was oscillated while dropwise adding these components; after dropwise adding was completed, the mixture was quickly added into the cell culture wells, and shaken gently and mixed evenly; <NUM>. on the third morning, the cell status was observed, and the medium was replaced by <NUM> of fresh DMEM medium; <NUM>. on the fifth morning, the cell status was observed; the supernatant in the culture dish was collected, filtered with a <NUM> filter, then placed in a high-speed centrifuge tube, and centrifuged at <NUM> for <NUM> hours; the supernatant was carefully discarded, the liquid was absorbed with absorbent paper as much as possible, and then the precipitate was re-suspended with <NUM>µL of HBSS, dissolved for <NUM> hours, then packed into small tubes, and stored at -<NUM>.

The <NUM> cells were transfected with the virus by the following method: the cultured <NUM> cells were digested, and seeded in a <NUM>-well plate at a concentration of <NUM> cells/well, with a culture volume of <NUM>; after <NUM> hours, <NUM>µL of the virus containing the regulatory vector was added, and the culture was continued in an incubator for an additional <NUM> hours, then the supernatant was discarded, and the medium was replaced by a fresh medium to continue the culture; after the cells were full, they were transferred out to a culture flask, and puromycin at a concentration suitable for the cells was added to continue the culture, the medium was replaced every two days, and the concentration of the puromycin was kept at <NUM>µg/ml; after screening for one week, the surviving cells were the cells stably expressing the regulatory protein, and named <NUM> (IL12).

The coding region of the canine GMCSF (Genbank Accession No.: NM_001003245) gene was synthesized. The synthesized gene had BamHI and XhoI restriction sites at the two ends respectively, and then was digested with BamHI and XhoI enzymes. The system was as follows: <NUM>µg of GMCSF plasmid, <NUM>µL of enzyme digestion buffer, <NUM>µL of BamHI, <NUM>µL of XhoI, supplemented with water to a final volume of <NUM>µL. The mixture was stood at <NUM> for <NUM> hours. The EP tube was taken out, and <NUM>µL of <NUM>× loading buffer was added, and then the resulting mixture was subjected to <NUM>% agarose gel electrophoresis. After the electrophoresis, the GMCSF gene segment was recovered for later use.

Expression vector pLentis-CMV-MCS-IRES-PURO was digested. The system was as follows: <NUM>µg of plasmid, <NUM>µL of enzyme digestion buffer, <NUM>µL of BamHI, <NUM>µL of XhoI, supplemented with water to a final volume of <NUM>µL. The mixture was stood at <NUM> for <NUM> hours. The EP tube was taken out, <NUM>µL of <NUM>× loading buffer was added, and then the resulting mixture was subjected to <NUM>% agarose gel electrophoresis. After electrophoresis, the vector segment was recovered for later use.

The pLentis-CMV-MCS-IRES-PURO was ligated with GMCSF. The system was as follows: <NUM>µL of pLentis-CMV-MCS-IRES-PURO, <NUM>µL of GMCSF, <NUM>µL of ligase buffer, <NUM>µL of T4 DNA ligase, and <NUM>µL of water. The mixture was placed at room temperature to ligate for <NUM> hours. Then, the ligation system was transformed into competent Escherichia coli. On the second day, a bacterial colony was picked from the transformed plate , inoculated into LB medium and cultured overnight at <NUM> in a shaker. The plasmids were extracted from the cultured bacteria by using the plasmid extraction kit. Enzyme digestion was conducted to determine whether the segment was successfully ligated into the vector, and then the correct vector was sent for sequencing, and it was confirmed that the construction was successful. The expression vector was pLentis-CMV-GMCSF-PGK-PURO.

A virus containing the regulatory vector was prepared, and the method was as follows: <NUM>. cultured 293FT cells were digested, after counting, <NUM>×<NUM> cells/well were spread onto a <NUM>-cm culture dish, and the volume of the culture solution was <NUM>; <NUM>. on the next night, the cell status was observed and if the cell status was good, the cells could be transfected; chloroquine was added to the culture plate to a final concentration of <NUM>, one test tube was taken and added with sterilized water and the following plasmids (<NUM>µg of pMD2. G + <NUM>µg of pSPAX2 + <NUM>µg of pLentis-CMV-GMCSF-PGK-PURO) with a total volume of <NUM>µL, then added with <NUM>µL of <NUM> CaCl<NUM>, mixed evenly, and finally added with <NUM>µL of <NUM>×HBS; the test tube was oscillated while dropwise adding these components; after dropwise adding was completed, the mixture was quickly added into the cell culture wells, and shaken gently and mixed evenly; <NUM>. on the third morning, the cell status was observed, and the medium was replaced by <NUM> of fresh DMEM medium; <NUM>. on the fifth morning, the cell status was observed; the supernatant in the culture dish was collected, filtered with a <NUM> filter, then placed in a high-speed centrifuge tube, and centrifuged at <NUM> for <NUM> hours; the supernatant was carefully discarded, the liquid was absorbed with absorbent paper as much as possible, and then the precipitate was re-suspended with <NUM>µL of HBSS, dissolved for <NUM> hours, then packed into small tubes, and stored at -<NUM>.

The <NUM> cells were transfected with the virus by the following method: the cultured <NUM> cells were digested, and seeded in a <NUM>-well plate at a concentration of <NUM> cells/well, with a culture volume of <NUM>; after <NUM> hours, <NUM>µL of the virus containing the regulatory vector was added, and the culture was continued in an incubator for an additional <NUM> hours, then the supernatant was discarded, and the medium was replaced by a fresh medium to continue the culture; after the cells were full, they were transferred out to a culture flask, and puromycin at a concentration suitable for the cells was added to continue the culture; the medium was replaced every two days, and the concentration of the puromycin was kept at <NUM>µg/ml; after screening for one week, the surviving cells were the cells stably expressing the regulatory protein, and named <NUM> (GMCSF).

The coding region of the canine IL2 (Genbank Accession No.: NM_001003305) gene was synthesized. The synthesized gene had BamHI and XhoI restriction sites at the two ends respectively, and then was digested with BamHI and XhoI enzymes. The system was as follows: <NUM>µg of IL2 plasmid, <NUM>µL of enzyme digestion buffer, <NUM>µL of BamHI, <NUM>µL of XhoI, supplemented with water to a final volume of <NUM>µL. The mixture was stood at <NUM> for <NUM> hours. The EP tube was taken out, and <NUM>µL of <NUM>× loading buffer was added, and then the resulting mixture was subjected to <NUM>% agarose gel electrophoresis. After the electrophoresis, the IL2 gene segment was recovered for later use.

Expression vector pLentis-CMV-MCS-IRES-PURO was digested. The system was as follows: <NUM>µg of plasmid, <NUM>µL of enzyme digestion buffer, <NUM>µL of BamHI, <NUM>µL of XhoI, supplemented with water to a final volume of <NUM>µL. The mixture was stood at <NUM> for <NUM> hours. The EP tube was taken out, and <NUM>µL of <NUM>× loading buffer was added, and then the resulting mixture was subjected to <NUM>% agarose gel electrophoresis. After the electrophoresis, the vector segment was recovered for later use.

The pLentis-CMV-MCS-IRES-PURO was ligated with IL2. The system was as follows: <NUM>µL of pLentis-CMV-MCS-IRES-PURO, <NUM>µL of IL2, <NUM>µL of ligase buffer, <NUM>µL of T4 DNA ligase, and <NUM>µL of water. The mixture was placed at room temperature to ligate for <NUM> hours. Then, the ligation system was transformed into competent Escherichia coli cells. On the second day, a bacterial colony was picked from the transformed plate, inoculated into LB medium and cultured overnight at <NUM> in a shaker. The plasmids were extracted from the cultured bacteria by using the plasmid extraction kit. Enzyme digestion was conducted to determine whether the segment was successfully ligated into the vector, then the correct vector was sent for sequencing, and it was confirmed that the construction was successful. The expression vector was pLentis-CMV-IL2-PGK-PURO.

A virus containing the regulatory vector was prepared, wherein the method was as follows: <NUM>. cultured 293FT cells were digested, after counting, <NUM>×<NUM> cells/well were spread onto a <NUM>-cm culture dish, and the volume of the culture solution was <NUM>; <NUM>. on the next night, the cell status was observed and if the cell status was good, the cells could be transfected; chloroquine was added to the culture plate to a final concentration of <NUM>, one test tube was taken and added with sterilized water and the following plasmids (<NUM>µg of pMD2. G + <NUM>µg of pSPAX2 + <NUM>µg of pLentis-CMV-IL2-PGK-PURO) with a total volume of <NUM>µL, then added with <NUM>µL of <NUM> CaCl<NUM>, mixed evenly, and finally added with <NUM>µL of <NUM>×HBS; the test tube was oscillated while dropwise adding these components; after dropwise adding was completed, the mixture was quickly added into the cell culture wells, and shaken gently and mixed evenly; <NUM>. on the third morning, the cell status was observed, and the medium was replaced by <NUM> of fresh DMEM medium; <NUM>. on the fifth morning, the cell status was observed; the supernatant in the culture dish was collected, filtered with a <NUM> filter, then placed in a high-speed centrifuge tube, and centrifuged at <NUM> for <NUM> hours; the supernatant was carefully discarded, the liquid was absorbed with absorbent paper as much as possible, and then the precipitate was re-suspended with <NUM>µL of HBSS, dissolved for <NUM> hours, then packed into small tubes, and stored at -<NUM>.

The <NUM> cells were transfected with the virus by the following method: the cultured <NUM> cells were digested, and seeded in a <NUM>-well plate at a concentration of <NUM> cells/well, with a culture volume of <NUM>; after <NUM> hours, <NUM>µL of the virus containing the regulatory vector was added, and the culture was continued in an incubator for an additional <NUM> hours, then the supernatant was discarded, and the medium was replaced by a fresh medium to continue the culture; after the cells were full, they were transferred out to a culture flask, and puromycin at a concentration suitable for the cells was added to continue the culture; the medium was replaced every two days, and the concentration of the puromycin was kept at <NUM>µg/ml; after screening for one week, the surviving cells were the cells stably expressing the regulatory protein, and named <NUM> (IL2).

The protein molecule cIL12bIL12aIL2GMCSF had a secretory signal peptide at the front end thereof and was added with <NUM>*His at the back end thereof to facilitate purification. The DNA sequence corresponding to the gene was synthesized, and the BamHI or XhoI site in the sequence was mutated to a degenerate codon. The front and back ends of the synthesized sequence were respectively provided with BamHI and XhoI restriction sites. The synthesized plasmid carrying the target gene was digested and the system was as follows: <NUM>µg of plasmid, <NUM>µl of enzyme digestion buffer, <NUM>µl of BamHI, <NUM>µl of XhoI, supplemented with water to a final volume of <NUM>µl. The mixture was stood at <NUM> for <NUM> hours. The EP tube was taken out, and <NUM>µl of <NUM>× loading buffer was added, and then the resulting mixture was subjected to <NUM>% agarose gel electrophoresis. After the electrophoresis, the segment was recovered for later use.

The vector pLentis-CMV-MCS-IRES-PURO was digested in an EP tube and the system was as follows: <NUM>µg of pLentis-CMV-MCS-IRES-PURO plasmid vector, <NUM>µl of enzyme digestion buffer, <NUM>µl of BamHI, <NUM>µl of XhoI, supplemented with water to a final volume of <NUM>µl. The mixture was stood at <NUM> for <NUM> hours. The EP tube was taken out, and <NUM>µl of <NUM>× loading buffer was added, and then the resulting mixture was subjected to <NUM>% agarose gel electrophoresis. After the electrophoresis, the pLentis-CMV-MCS-IRES-PURO vector segment was recovered for later use.

The cIL12bIL12aIL2GMCSF was ligated with the pLentis-CMV-MCS-IRES-PURO. The system was as follows: <NUM>µl of pLentis-CMV-MCS-IRES-PURO vector segment, <NUM>µl of gene segment, <NUM>µl of ligase buffer, <NUM>µl of T4 DNA ligase, and <NUM>µl of water. The mixture was placed at room temperature to ligate for <NUM> hours. Then, the ligation system was transformed into competent Escherichia coli cells. On the second day, a bacterial colony was picked from the transformed plate, inoculated into LB medium and cultured overnight at <NUM> in a shaker. The plasmids were extracted from the cultured bacteria by using the plasmid extraction kit. Enzyme digestion was conducted to determine whether the segment was successfully ligated into the vector, then the correct vector was sequenced, and it was confirmed that the construction was successful. Expression vector pLentis-CMV-cIL12bIL12aIL2GMCSF-IRES-PURO was obtained.

Cultured 293A cells were digested, and seeded in a <NUM>-well plate at a concentration of <NUM> cells/well, with a culture volume of <NUM>. After <NUM> hours, <NUM>µl of virus expressing the above target gene was added, and the culture was continued in an incubator for an additional <NUM> hours, then the supernatant was discarded, and the medium was replaced by a fresh medium to continue the culture. After the cells were full, they were transferred out to a culture flask, and puromycin at a final concentration of <NUM>µg/ml was added to continue the culture. The medium was replaced every two days, and the concentration of the puromycin was kept. After screening for one week, the surviving cells were the cells stably expressing the protein, and named 293A-cIL12bIL12aIL2GMCSF.

The constructed cIL12bIL12aIL2GMCSF-expressing cells 293A-cIL12bIL12aIL2GMCSF were sub-cultured into a <NUM>-cm culture dish. After the cells were full, the medium was replaced by <NUM> of CDM4HEK293 to continue the culture for an additional <NUM> days, and then the supernatant was collected, filtered by a <NUM> filter, and ultra-filtered and concentrated with AMICON ULTRA-<NUM> of <NUM> kd. The concentrated protein solution obtained was purified with nickel-chelated magnetic beads (Beaver Biosciences Inc. ), and the operation flow was carried out according to the instruction. The purified protein solution obtained was ultra-filtered with an AMICON ULTRA-<NUM> ultrafiltration tube, and the buffer was replaced by PBS. The protein concentration of the protein solution finally obtained was detected with an IL12p70 ELISA kit. After the protein concentration was adjusted to <NUM>µg/µl by PBS, the protein solution was packed and then stored at -<NUM>.

The protein molecule cIL12bIL12aGMCSFIL2 had a secretory signal peptide at the front end thereof and was added with <NUM>*His at the back end thereof to facilitate purification. The DNA sequence corresponding to the gene was synthesized, and the BamHI or XhoI site in the sequence was mutated to a degenerate codon. The front and back ends of the synthesized sequence were respectively provided with BamHI and XhoI restriction sites. The synthesized plasmid carrying the target gene was digested and the system was as follows: <NUM>µg of plasmid, <NUM>µl of enzyme digestion buffer, 1µl of BamHI, <NUM>µl of XhoI, supplemented with water to a final volume of <NUM>µl. The mixture was stood at <NUM> for <NUM> hours. The EP tube was taken out, and <NUM>µl of <NUM>× loading buffer was added, and then the resulting mixture was subjected to <NUM>% agarose gel electrophoresis. After the electrophoresis, the segment was recovered for later use.

The cIL12bIL12aGMCSFIL2 was ligated with the pLentis-CMV-MCS-IRES-PURO. The system was as follows: <NUM>µl of pLentis-CMV-MCS-IRES-PURO vector segment, <NUM>µl of gene segment, <NUM>µl of ligase buffer, <NUM>µl of T4 DNA ligase, and <NUM>µl of water. The mixture was placed at room temperature to ligate for <NUM> hours. Then, the ligation system was transformed into competent Escherichia coli cells. On the second day, a bacterial colony was picked from the transformed plate, inoculated into LB medium and cultured overnight at <NUM> in a shaker. The plasmids were extracted from the cultured bacteria by using the plasmid extraction kit. Enzyme digestion was conducted to determine whether the segment was successfully ligated into the vector, then the correct vector was sequenced, and it was confirmed that the construction was successful. Expression vector plentis-CMV-cIL12bIL12aGMCSFIL2-IRES-PURO was obtained.

Cultured 293A cells were digested and seeded in a <NUM>-well plate at a concentration of <NUM> cells/well, with a culture volume of <NUM>. After <NUM> hours, <NUM>µl of virus expressing the above target gene was added, and the culture was continued in an incubator for an additional <NUM> hours, then the supernatant was discarded, and the medium was replaced by a fresh medium to continue the culture. After the cells were full, they were transferred out to a culture flask, and puromycin at a final concentration of <NUM>µg/ml was added to continue the culture. The medium was replaced every two days, and the concentration of the puromycin was kept. After screening for one week, the surviving cells were cells stably expressing the protein, and named 293A-cIL12bIL12aGMCSFIL2.

The constructed cIL12bIL12aGMCSFIL2-expressing cells 293A-cIL12bIL12aGMCSFIL2 were sub-cultured into a <NUM>-cm culture dish. After the cells were full, the medium was replaced by <NUM> of CDM4HEK293 to continue the culture for an additional <NUM> days, and then the supernatant was collected, filtered by a <NUM> filter, and ultra-filtered and concentrated with AMICON ULTRA-<NUM> of <NUM> kd. The concentrated protein solution obtained was purified with nickel-chelated magnetic beads (Beaver Biosciences Inc. ), and the operation flow was carried out according to the instruction. The purified protein solution obtained was ultra-filtered with an AMICON ULTRA-<NUM> ultrafiltration tube, and the buffer was replaced by PBS. The protein concentration of the protein solution finally obtained was detected with an IL12p70 ELISA kit. After the protein concentration was adjusted to <NUM>µg/µl by PBS, the protein solution was packed and then stored at -<NUM>.

The protein molecule fIL12bIL12aIL2GMCSF had a secretory signal peptide at the front end thereof and was added with <NUM>*His at the back end thereof to facilitate purification. The DNA sequence corresponding to the gene was synthesized, and the BamHI or XhoI site in the sequence was mutated to a degenerate codon. The front and back ends of the synthesized sequence were respectively provided with BamHI and XhoI restriction sites. The synthesized plasmid carrying the target gene was digested and the system was as follows: <NUM>µg of plasmid, <NUM>µl of enzyme digestion buffer, 1µl of BamHI, <NUM>µl of XhoI, supplemented with water to a final volume of <NUM>µl. The mixture was stood at <NUM> for <NUM> hours. The EP tube was taken out, and <NUM>µl of <NUM>× loading buffer was added, and then the resulting mixture was subjected to <NUM>% agarose gel electrophoresis. After the electrophoresis, the segment was recovered for later use.

The fIL12bIL12aIL2GMCSF was ligated with the pLentis-CMV-MCS-IRES-PURO. The system was as follows: <NUM>µl of pLentis-CMV-MCS-IRES-PURO vector segment, <NUM>µl of gene segment, <NUM>µl of ligase buffer, <NUM>µl of T4 DNA ligase, and <NUM>µl of water. The mixture was placed at room temperature to ligate for <NUM> hours. Then, the ligation system was transformed into competent Escherichia coli cells. On the second day, a bacterial colony was picked from the transformed plate, inoculated into LB medium and cultured overnight at <NUM> in a shaker. The plasmids were extracted from the cultured bacteria by using the plasmid extraction kit. Enzyme digestion was conducted to determine whether the segment was successfully ligated into the vector, then the correct vector was sequenced, and it was confirmed that the construction was successful. Expression vector pLentis-CMV-fIL12bIL12aIL2GMCSF-IRES-PURO was obtained.

Cultured 293A cells were digested and seeded in a <NUM>-well plate at a concentration of <NUM> cells/well, with a culture volume of <NUM>. After <NUM> hours, <NUM>µl of virus expressing the above target gene was added, and the culture was continued in an incubator for an additional <NUM> hours, then the supernatant was discarded, and the medium was replaced by a fresh medium to continue the culture. After the cells were full, they were transferred out to a culture flask, and puromycin at a final concentration of <NUM>µg/ml was added to continue the culture. The medium was replaced every two days, and the concentration of the puromycin was kept. After screening for one week, the surviving cells were the cells stably expressing the protein, and named 293A-fIL12bIL12aIL2GMCSF.

The constructed fIL 12bIL 12aIL2GMCSF -expressing cells 293A-fIL12bIL12aIL2GMCSF were sub-cultured into a <NUM>-cm culture dish. After the cells were full, the medium was replaced by <NUM> of CDM4HEK293 to continue the culture for an additional <NUM> days, and then the supernatant was collected, filtered by a <NUM> filter, and ultra-filtered and concentrated with AMICON ULTRA-<NUM> of <NUM> kd. The concentrated protein solution obtained was purified with nickel-chelated magnetic beads (Beaver Biosciences Inc. ), and the operation flow was carried out according to the instruction. The purified protein solution obtained was ultra-filtered with an AMICON ULTRA-<NUM> ultrafiltration tube, and the buffer was replaced by PBS. The protein concentration of the protein solution finally obtained was detected with an IL12p70 ELISA kit. After the protein concentration was adjusted to <NUM>µg/µl by PBS, the protein solution was packed and then stored at -<NUM>.

The protein molecule fIL12bIL12aGMCSFIL2 had a secretory signal peptide at the front end thereof and was added with <NUM>*His at the back end thereof to facilitate purification. The DNA sequence corresponding to the gene was synthesized, and the BamHI or XhoI site in the sequence was mutated to a degenerate codon. The front and back ends of the synthesized sequence were respectively provided with BamHI and XhoI restriction sites. The synthesized plasmid carrying the target gene was digested and the system was as follows: <NUM>µg of plasmid, <NUM>µl of enzyme digestion buffer, 1µl of BamHI, <NUM>µl of XhoI, supplemented with water to a final volume of <NUM>µl. The mixture was stood at <NUM> for <NUM> hours. The EP tube was taken out, and <NUM>µl of <NUM>× loading buffer was added, and then the resulting mixture was subjected to <NUM>% agarose gel electrophoresis. After the electrophoresis, the segment was recovered for later use.

The vector pLentis-CMV-MCS-IRES-PURO was digested in an EP tube and the system was as follows: <NUM>µg of pLentis-CMV-MCS-IRES-PURO plasmid vector, <NUM>µl of enzyme digestion buffer, <NUM>µl of BamHI, <NUM>µl of XhoI, supplemented with water to a final volume of <NUM>µl. The mixture was stood at <NUM> for <NUM> hours. The EP tube was taken out, and <NUM>µl of <NUM>× loading buffer, was added, and then the resulting mixture was subjected to <NUM>% agarose gel electrophoresis. After the electrophoresis, the pLentis-CMV-MCS-IRES-PURO vector segment was recovered for later use.

The fIL12bIL12aGMCSFIL2 was ligated with the pLentis-CMV-MCS-IRES-PURO. The system was as follows: <NUM>µL of pLentis-CMV-MCS-IRES-PURO vector segment, <NUM>µl of gene segment, <NUM>µl of ligase buffer, <NUM>µl of T4 DNA ligase, and <NUM>µl of water. The mixture was placed at room temperature to ligate for <NUM> hours. Then, the ligation system was transformed into competent Escherichia coli cells. On the second day, a bacterial colony was picked from the transformed plate, inoculated into LB medium and cultured overnight at <NUM> in a shaker. The plasmids were extracted from the cultured bacteria by using the plasmid extraction kit. Enzyme digestion was conducted to determine whether the segment was successfully ligated into the vector, then the correct vector was sequenced, and it was confirmed that the construction was successful. Expression vector pLentis-CMV-fIL12bIL12aGMCSFIL2-IRES-PURO was obtained.

Cultured 293A cells were digested and seeded in a <NUM>-well plate at a concentration of <NUM> cells/well, with a culture volume of <NUM>. After <NUM> hours, <NUM>µl of virus expressing the above target gene was added, and the culture was continued in an incubator for an additional <NUM> hours, then the supernatant was discarded, and the medium was replaced by a fresh medium to continue the culture. After the cells were full, they were transferred out to a culture flask, and puromycin at a final concentration of <NUM>µg/ml was added to continue the culture. The medium was replaced every two days, and the concentration of the puromycin was kept. After screening for one week, the surviving cells were the cells stably expressing the protein, and named 293A-fIL12bIL12aGMCSFIL2.

The constructed fIL12bIL12aGMCSFIL2-expressing cells 293A-fIL12bIL12aGMCSFIL2 were sub-cultured into a <NUM>-cm culture dish. After the cells were full, the medium was replaced by <NUM> of CDM4HEK293 to continue the culture for an additional <NUM> days, and then the supernatant was collected, filtered by a <NUM> filter, and ultra-filtered and concentrated with AMICON ULTRA-<NUM> of <NUM> kd. The concentrated protein solution obtained was purified with nickel-chelated magnetic beads (Beaver Biosciences Inc. ), and the operation flow was carried out according to the instruction. The purified protein solution obtained was ultra-filtered with an AMICON ULTRA-<NUM> ultrafiltration tube, and the buffer was replaced by PBS. The protein concentration of the protein solution finally obtained was detected with an IL12p70 ELISA kit. After the protein concentration was adjusted to <NUM>µg/µl by PBS, the protein solution was packed and then stored at -<NUM>.

The protein molecule cIL12bIL12aIL2DiaNHS76F8GMCSF had a secretory signal peptide at the front end thereof and was added with <NUM>*His at the back end thereof to facilitate purification. The DNA sequence corresponding to the gene was synthesized, and the BamHI or XhoI site in the sequence was mutated to a degenerate codon. The front and back ends of the synthesized sequence were respectively provided with BamHI and XhoI restriction sites. The synthesized plasmid carrying the target gene was digested and the system was as follows: <NUM>µg of plasmid, <NUM>µl of enzyme digestion buffer, 1µl of BamHI, <NUM>µl of XhoI, supplemented with water to a final volume of <NUM>µl. The mixture was stood at <NUM> for <NUM> hours. The EP tube was taken out and <NUM>µl of <NUM>× loading buffer was added, and then the resulting mixture was subjected to <NUM>% agarose gel electrophoresis. After the electrophoresis, the segment was recovered for later use.

The vector pLentis-CMV-MCS-IRES-PURO was digested in an EP tube and the system was as follows: <NUM>µg of pLentis-CMV-MCS-IRES-PURO plasmid vector, <NUM>µl of enzyme digestion buffer, <NUM>µl of BamHI, <NUM>µl of XhoI, supplemented with water to a final volume of <NUM>µl. The mixture was stood at <NUM> for <NUM> hours. The EP tube was taken out and <NUM>µl of <NUM>× loading buffer was added, and then the resulting mixture was subjected to <NUM>% agarose gel electrophoresis. After the electrophoresis, the pLentis-CMV-MCS-IRES-PURO vector segment was recovered for later use.

The cIL12bIL12aIL2DiaNHS76F8GMCSF was ligated with the pLentis-CMV-MCS-IRES-PURO. The system was as follows: <NUM>µl of pLentis-CMV-MCS-IRES-PURO vector segment, <NUM>µl of gene segment, <NUM>µl of ligase buffer, <NUM>µl of T4 DNA ligase, and <NUM>µl of water. The mixture was placed at room temperature to ligate for <NUM> hours. Then, the ligation system was transformed into competent Escherichia coli cells. On the second day, a bacterial colony was picked from the transformed plate, inoculated into LB medium and cultured overnight at <NUM> in a shaker. The plasmids were extracted from the cultured bacteria by using the plasmid extraction kit. Enzyme digestion was conducted to determine whether the segment was successfully ligated into the vector, then the correct vector was sequenced, and it was confirmed that the construction was successful. Expression vector: pLentis-CMV-cIL12bIL12aIL2DiaNHS76F8GMCSF-IRES-PURO was obtained.

Cultured 293A cells were digested and seeded in a <NUM>-well plate at a concentration of <NUM> cells/well, with a culture volume of <NUM>. After <NUM> hours, <NUM>µl of virus expressing the above target gene was added, and the culture was continued in an incubator for an additional <NUM> hours, then the supernatant was discarded, and the medium was replaced by a fresh medium to continue the culture. After the cells were full, they were transferred out to a culture flask, and puromycin at a final concentration of <NUM>µg/ml was added to continue the culture The medium was replaced every two days, and the concentration of the puromycin was kept. After screening for one week, the surviving cells were the cells stably expressing the protein, and named 293A-cIL 12bIL 12aIL2DiaNHS 76F8GMCSF.

The constructed cIL12bIL12aIL2DiaNHS76F8GMCSF-expressing cells 293A-cIL12bIL12aIL2DiaNHS76F8GMCSF were sub-cultured into a <NUM>-cm culture dish. After the cells were full, the medium was replaced by <NUM> of CDM4HEK293 to continue the culture for an additional <NUM> days, and then the supernatant was collected, filtered by a <NUM> filter, and ultra-filtered and concentrated with AMICON ULTRA-<NUM> of <NUM> kd. The concentrated protein solution obtained was purified with nickel-chelated magnetic beads (Beaver Biosciences Inc. ), and the operation flow was carried out according to the instruction. The purified protein solution obtained was ultra-filtered with an AMICON ULTRA-<NUM> ultrafiltration tube, and the buffer was replaced by PBS. The protein concentration of the protein solution finally obtained was detected with an IL12p70 ELISA kit. After the protein concentration was adjusted to <NUM>µg/µl by PBS, the protein solution was packed and then stored at -<NUM>.

The protein molecule fIL12bIL12aIL2DiaNHS76F8GMCSF had a secretory signal peptide at the front end thereof and was added with <NUM>*His at the back end thereof to facilitate purification. The DNA sequence corresponding to the gene was synthesized, and the BamHI or XhoI site in the sequence was mutated to a degenerate codon. The front and back ends of the synthesized sequence were respectively provided with BamHI and XhoI restriction sites. The synthesized plasmid carrying the target gene was digested and the system was as follows: <NUM>µg of plasmid, <NUM>µl of enzyme digestion buffer, 1µl of BamHI, <NUM>µl of XhoI, supplemented with water to a final volume of <NUM>µl. The mixture was stood at <NUM> for <NUM> hours. The EP tube was taken out and <NUM>µl of <NUM>× loading buffer was added, and then the resulting mixture was subjected to <NUM>% agarose gel electrophoresis. After the electrophoresis, the segment was recovered for later use.

The fIL12bIL12aIL2DiaNHS76F8GMCSF was ligated with the pLentis-CMV-MCS-IRES-PURO. The system was as follows: <NUM>µl of pLentis-CMV-MCS-IRES-PURO vector segment, <NUM>µl of gene segment, <NUM>µl of ligase buffer, <NUM>µl of T4DNA ligase, and <NUM>µl of water. The mixture was placed at room temperature to ligate for <NUM> hours. Then, the ligation system was transformed into competent Escherichia coli cells. On the second day, a bacterial colony was picked from the transformed plate, inoculated into LB medium and cultured overnight at <NUM> in a shaker. The plasmids were extracted from the cultured bacteria by using the plasmid extraction kit. Enzyme digestion was conducted to determine whether the segment was successfully ligated into the vector, then the correct vector was sequenced, and it was confirmed that the construction was successful. Expression vector: pLentis-CMV-fIL12bIL12aIL2DiaNHS76F8GMCSF-IRES-PURO was obtained.

Cultured 293A cells were digested and seeded in a <NUM>-well plate at a concentration of <NUM> cells/well, with a culture volume of <NUM>. After <NUM> hours, <NUM>µl of virus expressing the above target gene was added, and the culture was continued in an incubator for an additional <NUM> hours, then the supernatant was discarded, and the medium was replaced by a fresh medium to continue the culture. After the cells were full, they were transferred out to a culture flask, and puromycin at a final concentration of <NUM>µg/ml was added to continue the culture. The medium was replaced every two days, and the concentration of the puromycin was kept. After screening for one week, the surviving cells were the cells stably expressing the protein, and named 293A-fIL12bIL12aIL2DiaNHS76F8GMCSF.

The constructed fIL12bIL12aIL2DiaNHS76F8GMCSF-expressing cells 293A-fIL12bIL12aIL2DiaNHS76F8GMCSF were sub-cultured into a <NUM>-cm culture dish. After the cells were full, the medium was replaced by <NUM> of CDM4HEK293 to continue the culture for an additional <NUM> days, and then the supernatant was collected, filtered by a <NUM> filter, and ultra-filtered and concentrated with AMICON ULTRA-<NUM> of <NUM> kd. The concentrated protein solution obtained was purified with nickel-chelated magnetic beads (Beaver Biosciences Inc. ), and the operation flow was carried out according to the instruction. The purified protein solution obtained was ultra-filtered with an AMICON ULTRA-<NUM> ultrafiltration tube, and the buffer was replaced by PBS. The protein concentration of the protein solution finally obtained was detected with an IL12p70 ELISA kit. After the protein concentration was adjusted to <NUM>µg/µl by PBS, the protein solution was packed and then stored at -<NUM>.

The purified cIL12bIL12aIL2GMCSF fusion protein solution was mixed with <NUM>% chitosan in a volume ratio of <NUM>: <NUM>, and then packed in <NUM>/vial. The mixture was mixed just before use. Three vials of the reagent were injected intratumorally each time, and the intratumoral injection was carried out once every <NUM> days for three consecutive times. The size of the tumor was recorded as follows:
Note: "c" indicated that the fusion protein was a canine protein, which was the same in the whole text.

A deer dog, <NUM> years old, had sarcoma in the right foreleg (see <FIG>) in a size of <NUM>*<NUM>*<NUM>. As shown in Table <NUM>, after intratumoral injection for three times, the tumor area was decreased by <NUM>%. The tumor volume was decreased by about <NUM>% (see <FIG>). After administration, the body temperature was normal, and no other adverse reactions were observed.

The purified cIL12bIL12aGMCSFIL2 fusion protein solution was mixed with <NUM>% chitosan in a volume ratio of <NUM>: <NUM>, and then packed in a volume of <NUM>/vial. The mixture was mixed just before use. Three vials of the reagent were injected intratumorally each time, and the intratumoral injection was carried out once every <NUM> days for three consecutive times. The size of the tumor was recorded as follows:.

A samoyed dog, <NUM> years old, suffered from thyroid cancer, and the size of the tumor was about <NUM>*<NUM>. As shown in Table <NUM>, after intratumoral injection for three times, the tumor area was decreased by <NUM>%. After administration, the body temperature was normal, and no other adverse reactions were observed.

The purified fIL12bIL12aIL2GMCSF fusion protein solution was mixed with <NUM>% chitosan in a volume ratio of <NUM>: <NUM>, and then packed in <NUM>/vial. The mixture was mixed just before use. Two vials of the reagent were injected intratumorally each time, and the intratumoral injection was carried out once every <NUM> days for three consecutive times. The size of the tumor was recorded as follows:.

A mixed-breed cat, <NUM> years old, suffered from breast basal cell carcinoma, and the size of the tumor was <NUM>*<NUM>. As shown in Table <NUM>, after intratumoral injection for three times, the tumor area was decreased by <NUM>%. After administration, the body temperature was normal, and no other adverse reactions were observed.

The purified fIL12bIL12aGMCSFIL2 fusion protein solution was mixed with <NUM>% chitosan in a volume ratio of <NUM>: <NUM>, and then packed in <NUM>/vial. The mixture was mixed just before use. One vial of the reagent was injected intratumorally each time, and the intratumoral injection was carried out once every <NUM> days for three consecutive times. The size of the tumor was recorded as follows:.

A mixed-breed cat, <NUM> years old, suffered from squamous cell carcinoma in a tongue root, and the size of the tumor was <NUM>*<NUM>. As shown in Table <NUM>, after intratumoral injection for three times, the tumor area was decreased by <NUM>%. After administration, the body temperature was normal, and no other adverse reactions were observed.

The purified cIL12bIL12aIL2DiaNHS76F8GMCSF fusion protein solution was mixed with <NUM>% chitosan in a volume ratio of <NUM>: <NUM>, and then packed in <NUM>/vial. The mixture was mixed just before use. Two vials of the reagent were injected intratumorally each time, and the intratumoral injection was carried out once every <NUM> days for three consecutive times. The size of the tumor was recorded as follows:.

A mixed-breed dog, <NUM> years old, suffered from breast cancer, and the size of the tumor was <NUM>*<NUM>. As shown in Table <NUM>, after intratumoral injection for three times, the tumor area was decreased by <NUM>%. After administration, the body temperature was normal, and fever or other adverse reactions were not observed.

The purified fIL12bIL12aIL2DiaNHS76F8GMCSF fusion protein solution was mixed with <NUM>% chitosan in a volume ratio of <NUM>: <NUM>, and then packed in <NUM>/vial. The mixture was mixed just before use. Two vials of the reagent were injected intratumorally each time, and the intratumoral injection was carried out once every <NUM> days for three consecutive times. The size of the tumor was recorded as follows:.

A mixed-breed cat, <NUM> years old, suffered from breast cancer, and the size of the tumor was <NUM>*<NUM>. As shown in Table <NUM>, after intratumoral injection for three times, the tumor area was decreased by <NUM>%. After administration, the body temperature was normal, and fever or other adverse reactions were not observed.

Claim 1:
A fusion protein for treating animal tumors, wherein the fusion protein comprises IL12, IL2 and GMCSF peptide fragments, wherein the IL12, IL2 and GMCSF peptide fragments are obtained from felines or canines, and wherein the fusion protein is set forth in SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, or SEQ ID NO: <NUM>.