A genetically engineered chimeric antibody against canine distemper virus and its application

Abstract

The present invention discloses a genetically engineered chimeric antibody against canine distemper virus and its application. The genetically engineered chimeric antibody against canine distemper virus of the present invention includes a mouse-canine chimeric antibody or a mouse-giant panda chimeric antibody; the heavy chain amino acid sequence of the mouse-canine chimeric antibody is as shown in SEQ ID NO: 6, and the light chain amino acid sequence is as shown in SEQ ID NO: 8; the heavy chain amino acid sequence of the mouse-giant panda chimeric antibody is as shown in SEQ ID NO: 10, and the light chain amino acid sequence is as shown in SEQ ID NO: 12. This genetically engineered chimeric antibody exhibits good activity in neutralizing CDV virus, can be directly applied to the treatment of canine distemper disease in canids and giant pandas, and is of great significance for promoting the development of canine-derived and giant panda-derived monoclonal antibody drugs.

Description

Technical Field

[0001] The invention relates to the technical field of antibody engineering, and in particular to a genetically engineered chimeric antibody against canine distemper virus and its application. Background Art

[0002] Canine distemper virus (CDV) belongs to the family Paramyxoviridae and the genus Morbillivirus. CDV infection often causes systemic clinical symptoms in the respiratory tract, digestive tract, urinary tract, lymphatic system, nervous system, skin, etc. It can infect many hosts, including Canidae, Muscidae, Mustelidae, Ursidae, Felidae, etc. There is cross-species infection and transmission in wild animals, and there is also cross-infection between different species in domestic animals. With an incidence rate of 100% and a mortality rate of more than 80%, CDV has become one of the important infectious diseases that endanger the dog breeding industry and economic animal breeding industry.

[0003] At present, most vaccines used to prevent the disease are attenuated vaccines, but many dog, mink, fox and other farms still suffer from the disease after using attenuated vaccines. Analysis of the reasons is related to maternal antibodies, vaccine titer, usage methods and virus mutations. Animals immunized with attenuated vaccines have not been fully protected, and many immune animal groups have suffered outbreaks of canine distemper, causing heavy losses. There is currently no specific drug for canine distemper virus infection, and symptomatic treatment, supportive therapy and specific therapy are often used in clinical practice. Among them, specific therapy generally uses canine distemper virus monoclonal antibodies and canine distemper virus antiserum in the early stage. However, the preparation cost of high-immune serum of canine distemper virus is high, it is easy to spread other viruses, and the antibody neutralization titer is uneven, making it difficult to standardize. The anti-canine distemper virus monoclonal antibodies used clinically are mouse monoclonal antibodies. Since mouse monoclonal antibodies are easily immune-rejected when injected into other source animals, the therapeutic effect is reduced, which affects clinical application.

[0004] At present, the canine distemper virus monoclonal antibodies and antiserum on the market have strong immunogenicity, and there will be side effects during the treatment process that reduce the efficacy, limiting their clinical application. Therefore, the preparation of monoclonal antibodies with low immunogenicity, high affinity and strong specificity is more conducive to promoting the large-scale clinical application of antibody drugs.

[0005] At the same time, canine distemper is also extremely harmful to species such as giant pandas. Currently, there is no specific drug for the treatment of canine distemper in giant pandas, and there is an urgent need for therapeutic drugs for canine distemper diseases in giant pandas. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a genetically engineered chimeric antibody against canine distemper virus and its application. The heavy chain and light chain variable region sequences in the genetically engineered chimeric antibody against canine distemper virus of the present invention are provided by the canine distemper virus H protein monoclonal antibody 9-7B, and the light chain and heavy chain constant region sequences are provided by canine-derived antibodies or giant panda-derived antibodies. The genetically engineered chimeric antibody can specifically identify canine distemper virus and has the advantages of high affinity and low immunogenicity to canine distemper virus.

[0007] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0008] The present invention provides a genetically engineered chimeric antibody against canine distemper virus, wherein the genetically engineered chimeric antibody comprises a mouse-dog chimeric antibody or a mouse-giant panda chimeric antibody; the heavy chain amino acid sequence of the mouse-dog chimeric antibody is shown in SEQ ID NO: 6, and the light chain amino acid sequence is shown in SEQ ID NO: 8; the heavy chain amino acid sequence of the mouse-giant panda chimeric antibody is shown in SEQ ID NO: 10, and the light chain amino acid sequence is shown in SEQ ID NO: 12.

[0009] Furthermore, the mouse-dog chimeric antibody and the mouse-giant panda chimeric antibody both include the variable regions of mouse antibodies;

[0010] The mouse-dog chimeric antibody also includes the constant region of the canine antibody, and the constant region of the canine antibody includes the heavy chain constant region and the light chain constant region.

[0011] The amino acid sequence of the heavy chain constant region of the canine antibody is shown in SEQ ID NO: 13.

[0012] The amino acid sequence of the light chain constant region of the canine antibody is shown in SEQ ID NO: 14;

[0013] The mouse-giant panda chimeric antibody also includes the constant region of the giant panda-derived antibody, and the constant region of the giant panda-derived antibody includes the heavy chain constant region and the light chain constant region.

[0014] The amino acid sequence of the heavy chain constant region of the giant panda-derived antibody is shown in SEQ ID NO: 15.

[0015] The amino acid sequence of the giant panda-derived antibody light chain constant region is shown in SEQ ID NO:16.

[0016] Furthermore, the variable region of the murine antibody includes a heavy chain variable region and a light chain variable region,

[0017] The amino acid sequence of the heavy chain variable region of the mouse antibody is shown in SEQ ID NO: 2.

[0018] The amino acid sequence of the light chain variable region of the murine antibody is shown in SEQ ID NO:4.

[0019] Furthermore, the mouse-dog chimeric antibody and the mouse-giant panda chimeric antibody both include a KOZAK sequence and a signal peptide sequence;

[0020] The nucleotide sequence of the KOZAK sequence is shown in SEQ ID NO: 17; the nucleotide sequence of the signal peptide sequence is shown in SEQ ID NO: 18.

[0021] The present invention also provides a gene encoding the genetically engineered chimeric antibody against canine distemper virus, the nucleotide sequence encoding the heavy chain of the mouse-dog chimeric antibody is shown in SEQ ID NO: 5, and the nucleotide sequence encoding the light chain of the mouse-dog chimeric antibody is shown in SEQ ID NO: 7;

[0022] The nucleotide sequence encoding the heavy chain of the mouse-giant panda chimeric antibody is shown in SEQ ID NO:9, and the nucleotide sequence encoding the light chain of the mouse-giant panda chimeric antibody is shown in SEQ ID NO:11.

[0023] The present invention also provides a method for preparing the genetically engineered chimeric antibody, comprising the following steps:

[0024] (1) connecting the mouse antibody heavy chain variable region and the dog / giant panda antibody heavy chain constant region, adding the KOZAK sequence and the signal peptide sequence, connecting the mouse antibody light chain variable region and the dog / giant panda antibody light chain constant region, adding the KOZAK sequence and the signal peptide sequence, and performing CHO codon optimization to obtain a chimeric antibody heavy chain and a chimeric antibody light chain;

[0025] (2) constructing the chimeric antibody heavy chain and the chimeric antibody light chain into expression vectors of the CHO expression system respectively, and performing fusion transformation to obtain a mouse-canine chimeric antibody plasmid or a mouse-giant panda chimeric antibody plasmid;

[0026] (3) The mouse-dog chimeric antibody plasmid and the mouse-giant panda chimeric antibody plasmid are respectively transfected into CHO cell lines, high-expression cell lines are screened, monoclonal cell lines are screened, and the cell lines are subjected to high-density expression and purification to obtain mouse-dog chimeric antibodies or mouse-giant panda chimeric antibodies.

[0027] Furthermore, in step (1), the heavy chain variable region or the light chain variable region is located at the N-terminus of the heavy chain constant region or the light chain constant region; and the KOZAK sequence and the signal peptide sequence are added to the N-terminus of the heavy chain variable region or the light chain variable region.

[0028] Furthermore, in step (2), the expression vector is PXC17.4.

[0029] The present invention also provides an application of the genetically engineered chimeric antibody in the preparation of a product for preventing and treating canine distemper virus, wherein the concentration of the mouse-dog chimeric antibody is 1-2 mg / mL and / or the concentration of the mouse-giant panda chimeric antibody is 3-4 mg / mL.

[0030] Furthermore, the concentration of the mouse-dog chimeric antibody is 1.5 mg / mL and / or the concentration of the mouse-giant panda chimeric antibody is 3.5 mg / mL.

[0031] Beneficial effects of the present invention:

[0032] 1. The heavy chain and light chain variable region sequences in the anti-canine distemper virus genetic engineering chimeric antibody of the present invention are provided by the canine distemper virus H protein monoclonal antibody hybridoma cell 9-7B strain, and the light chain and heavy chain constant region sequences are provided by canine-derived antibodies or giant panda-derived antibodies. Canine distemper virus H protein monoclonal antibody 9-7B is obtained by immunizing balb / c mice with recombinant H protein expressed by the CHO system as an immunogen and screening by hybridoma technology. It has excellent biological properties, not only has high affinity for canine distemper H protein and canine distemper virus, but also has good neutralizing effect on canine distemper vaccine virus and Asian type 1 strains prevalent in my country.

[0033] 2. The amino acid sequence and nucleotide sequence of the variable regions of the heavy chain and light chain of the anti-canine distemper virus antibody 9-7B of the present invention provide support for the construction of a genetically engineered chimeric antibody against canine distemper virus with high affinity and low immunogenicity.

[0034] 3. The present invention assembles the variable region sequence of the anti-canine distemper virus antibody 9-7B with the constant regions of canine and giant panda origin to obtain a genetically engineered antibody against canine distemper virus, which exhibits good activity in neutralizing CDV virus against canine distemper vaccine virus and wild strains. It can be applied to the research on canine and giant panda origin of canine distemper virus monoclonal antibodies, and is of great significance to promoting the development of canine and giant panda origin monoclonal antibody drugs.

[0035] 4. In the present invention, the production technology used is mature, the operation is simple, and the preparation cycle is short. The recombinant canine and giant panda antibodies produced by this technology have the advantages of high specificity and purity, stable repeatability, and low production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the SDS-PAGE identification diagram of mouse-canine chimeric antibody;

[0037] Figure 2 This is a diagram of Western Blot identification results of mouse-dog chimeric antibodies;

[0038] Figure 3This is the result of indirect immunofluorescence experiment identification of mouse-dog chimeric antibody;

[0039] Figure 4 This is a diagram showing the results of the neutralization activity identification of mouse-canine chimeric antibodies;

[0040] Figure 5 This is the SDS-PAGE identification diagram of mouse-giant panda chimeric antibody;

[0041] Figure 6 This is the Western Blot identification result of mouse-giant panda chimeric antibody;

[0042] Figure 7 This is the result of indirect immunofluorescence experiment identification of mouse-giant panda chimeric antibody;

[0043] Figure 8 This is a diagram showing the results of the neutralization activity identification of the mouse-giant panda chimeric antibody;

[0044] Fig. 9 This is a diagram of the physiological state of a dog infected with canine distemper virus;

[0045] Fig.10 This is a diagram of the physiological state of a dog infected with canine distemper virus after treatment. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0047] Example 1 Preparation of recombinant CHO cells expressing anti-canine distemper virus mouse-dog chimeric antibodies and mouse-giant panda chimeric antibodies

[0048] Construction of recombinant expression plasmid

[0049] Step 1: Immunize mice with CDV H protein expressed by CHO eukaryotic expression system, and obtain mouse hybridoma monoclonal cell 2-2B with neutralizing effect after screening. Use 1640 complete medium to culture hybridoma cells secreting CDV H protein monoclonal antibody at 37°C and 5% carbon dioxide until the cell number reaches 1×10 7 Total RNA was extracted from cells using a total RNA extraction kit (purchased from Tiangen). Reverse transcription was performed according to the following reaction system and conditions: 5 μg RNA, 82 μL Oligo(dT), DEPC H 2 0 to 14 μL, put it in 65℃ for 5min, and immediately put it in an ice bath. Take out the sample, add 2 μL 10×RT buffer, 1 μL Rnase Inhibitor, 2 μL dNTP (10mM), 1 μL MLV. Set the program to 42℃ for 60min, 75℃ for 10min amplification. The reaction product is cDNA.

[0050] Step 2: Design specific upstream and downstream universal primers for mouse heavy chain antibody gene and light chain antibody gene, and perform PCR amplification using cDNA as template. The PCR system is 50 μL: cDNA 3 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL, 2× Phanta Max Buffer 25 μL, dNTP Mix (10 mM each) 1 μL, upstream primer (10 μM) 2 μL, downstream primer (10 μM) 2 μL, ddH 2 O 16μL. PCR conditions were: 95℃ pre-denaturation for 3 minutes, 95℃ denaturation for 15 seconds, 56℃ annealing for 15 seconds, 72℃ extension for 30 seconds, a total of 35 cycles; and finally 72℃ extension for 5 minutes. The PCR product was subjected to 1% gel electrophoresis, and the expected bands were cut and sent to the company for sequencing to obtain the heavy chain and light chain variable region gene sequences of the monoclonal antibody 9-7B.

[0051] Step 3: The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 9-7B is shown in SEQ ID No: 1;

[0052] The amino acid sequence encoding the heavy chain variable region of monoclonal antibody 9-7B is shown in SEQ ID No: 2;

[0053] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 9-7B is shown in SEQ ID No: 3;

[0054] The amino acid sequence of the light chain variable region encoded by monoclonal antibody 9-7B is shown in SEQ ID No:4.

[0055] Step 4: Variable region amino acid sequence and homology analysis

[0056] The heavy chain and light chain gene sequences were compared and analyzed in the NCBI database. The analysis results showed that the nucleotide sequence of the light chain variable region gene of the monoclonal antibody 9-7B had the highest homology with the mouse immunoglobulin kappa chain variable region (Sequence ID: U60464.1), with a homology of 332 / 337 and a homology percentage of 99%. The amino acid sequence of the light chain variable region of the monoclonal antibody had the highest homology with the mouse immunoglobulin kappa chain variable region amino acid sequence (Sequence ID: AAB03599.18), with a homology of 109 / 112 and a homology percentage of 97%.

[0057] The nucleotide sequence of the heavy chain variable region gene of the monoclonal antibody has the highest homology with the mouse immunoglobulin heavy chain variable region (Sequence ID: AB734740.1), with a homology of 334 / 362 and a homology percentage of 92%. The amino acid sequence of the heavy chain variable region of the monoclonal antibody has the highest homology with the mouse immunoglobulin heavy chain variable region amino acid sequence (Sequence ID: BAM75797.1), with a homology of 106 / 119 and a homology percentage of 89%. The results of homology analysis of the gene sequence and amino acid sequence encoding the heavy chain and light chain variable regions of the monoclonal antibody showed that no sequence identical to that of the monoclonal antibody 9-7B was found.

[0058] The sequences of the heavy chain variable region and light chain variable region of monoclonal antibody 9-7B were analyzed to obtain its CDR region.

[0059] Among them, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in Table 1.

[0060] Table 1 Amino acid sequence of heavy chain variable region CDR region

[0061] name sequence CDR-H1 GFSLTNYG CDR-H2 IWRGGGT CDR-H3 AKSGPYYSGEGFAY

[0062] The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in Table 2.

[0063] Table 2 Amino acid sequences of the light chain variable region CDR region

[0064] name sequence CDR-L1 QSLLNSRTRKNN CDR-L2 WAS CDR-L3 KQS

[0065] Step 5: Construction of mouse-dog chimeric antibody and mouse-giant panda chimeric antibody plasmids

[0066] The heavy and light chain constant region sequences of canine and giant panda antibodies were queried and determined from the IMGT official website.

[0067] The sequenced mouse antibody 9-7B heavy chain variable region sequence and canine antibody heavy chain constant region sequence (as shown in SEQ ID NO: 13) (NCBI GenBank: AF354266.1) were connected, and KOZAK and signal peptide sequences were added. The antibody 9-7B light chain variable region sequence and canine antibody light chain constant region sequence (as shown in SEQ ID NO: 14) (NCBI GenBank: CP050603.1) were connected, and KOZAK and signal peptide sequences were added. CHO codon optimization was performed (the nucleotide sequence of the mouse-canine chimeric antibody heavy chain gene is shown in SEQ ID No: 5, and its amino acid sequence is shown in SEQ ID No: 6, and the nucleotide sequence of the mouse-canine chimeric antibody light chain gene is shown in SEQ ID No: 7, and its amino acid sequence is shown in SEQ ID No: 8), and gene synthesis was directly performed at Qingke Biotechnology Co., Ltd.

[0068] The sequenced mouse antibody heavy chain variable region sequence was connected to the giant panda antibody heavy chain constant region (as shown in SEQ ID NO: 15) (NCBI GenBank: AAX73307.1), KOZAK and signal peptide sequences were added, the antibody light chain variable region sequence was connected to the giant panda antibody light chain constant region sequence (as shown in SEQ ID NO: 16) (NCBI GenBank: EFB14759.1), KOZAK and signal peptide sequences were added, and CHO codon optimization was performed (the nucleotide sequence of the mouse-giant panda chimeric antibody heavy chain gene is shown in SEQ ID No: 9, and its amino acid sequence is shown in SEQ ID No: 10, and the nucleotide sequence of the mouse-giant panda chimeric antibody light chain gene is shown in SEQ ID No: 11, and its amino acid sequence is shown in SEQ ID No: 12), and gene synthesis was directly performed at Qingke Biotechnology Co., Ltd.

[0069] The chimeric antibody heavy chain and light chain were constructed into the expression vector PXC17.4 of the CHO expression system respectively, and fusion transformation was performed using the principle of homologous recombination. The obtained plasmids were identified by single colony PCR and sent for sequencing. The bacterial solution with correct sequencing was shaken in large quantities, and the plasmid was extracted using an endotoxin-free plasmid extraction kit.

[0070] Example 2 Establishment and screening of mouse-canine chimeric antibody and mouse-giant panda chimeric antibody plasmid stably transfected CHO cell lines

[0071] Step 1: Processing plasmid:

[0072] (1) Extract the plasmid according to the instructions of the endotoxin-free extraction kit and linearize it with PVUⅠ enzyme. The enzyme digestion system is: PVUⅠ enzyme 2μL, plasmid 2μg, 10×K buffer 4μL, 1% 0.1BSA 4μL, add ddH 2Dose 40 μL and digest in a 37°C water bath for 3 hours.

[0073] (2) Add 40 μL of isopropanol and 5 μL of 5M NaCL to 40 μL of linearized enzyme digestion product, place at -20°C overnight for precipitation, centrifuge at room temperature at 12000 rpm for 15 min, discard the supernatant, wash twice with pre-cooled 80% ethanol, centrifuge at 4°C 12000 rpm / 5 min each time, dry at 37°C, add 10 μL of sterilized ddH 2 O to dissolve and place at 37℃ for 1h to dissolve.

[0074] Step 2: Screening of high-expressing cell lines:

[0075] (1) Resuscitate CHO protocells. When the number of cells is sufficient and in good condition, count them and use 2×10 6 The CHO primary cells were centrifuged to remove the culture medium, washed twice with PBS, and then 100 μL of electroporation buffer was added. The constructed plasmid was added at a ratio of light chain: heavy chain = 1:1 for electroporation. The cells were resuspended in CHO CD04 culture medium without glutamine and added to a 6-well plate. The plates were placed at 37°C and 5% CO 2 Cultivate on a shaking incubator.

[0076] (2) After culturing for 48 h, when the cells are in good condition, they are cultured in a medium containing 15 μM MSX. At the same time, the limiting dilution method is used to screen high-expressing cell lines. The cells in the cell pool are diluted to 2×10 3 The cells were plated into 96-well plates, and the cell growth was observed on the 7th day. The culture medium was added at the same time. The supernatant of the cell line with a confluence of 80% was detected by dot hybridization. The positive cell lines were expanded to 48-well plates, and then gradually expanded to 6-well plates and cultured in a shaker.

[0077] (3) After one generation of culture in a 6-well plate, the supernatant was subjected to a neutralization experiment to detect whether it had a neutralization effect. At the same time, the supernatant was diluted in multiples for a second dot hybridization screening to screen for cell lines with a higher reaction to HRP-labeled sheep anti-dog IgG (H+L).

[0078] Step 3: Screening of monoclonal cell lines:

[0079] (1) The obtained highly reactive positive cell lines were subcultured to screen and obtain monoclonal cells. First, the culture medium for monoclonal cell culture was prepared and QuaMono TMPlus component A (Cat. A11602A) and component B (Cat. A11602B) were fully mixed in proportion (5 mL B was added for every 500 mL A), and 1% glutamine was added to obtain an AB mixture.

[0080] (2) Use AB mixed solution to dilute the cell line to 0.5 cells / 120 μL. Spread 5 plates and calculate the total cell volume. Use an 8-channel pipette to absorb the above cell solution and spread it on a 96-well plate at 120 μL / well. Let it stand for 12 hours; observe the cell culture plate under a microscope, mark the wells of single cells, place it in an incubator for static culture, and perform further observation, marking and detection.

[0081] (3) Between the 7th and 8th day after plating, add 100 μL of QuaMono to each well. TM Plus component C (Cat.A11602C) promotes rapid proliferation of clones. When the cell density reaches 80%, take the supernatant for dot hybridization and neutralization experiments to screen out monoclonal cell lines with high response and neutralization effects.

[0082] Step 4: High-density expression of cell lines:

[0083] (1) The high-yield cell lines screened were divided into 1×10 6 The density of cells / mL was inoculated in 50 mL of 15 μm MSX medium in a 250 mL cell shake flask and incubated in 5% CO 2 , cultured in a shaker at 37°C and 120 rpm, samples were taken every day to count the cell density, and the survival rate was calculated by trypan blue staining.

[0084] (2) On days 1, 3, and 5, 5% Feed A and 1% Feed B were added to the culture medium. On days 7 and 9, 7.5% Feed A and 1% Feed B were added to the culture medium. The cells were cultured until the cell density was between 1 and 2 × 10 7 cells / mL, and the supernatant was collected when the cell survival rate dropped to about 80%. Centrifugation was performed to remove cells, and the collected supernatant was filtered with a 0.45nm filter membrane and purified with a Protein A column. The purified mouse-dog chimeric antibody and mouse-giant panda chimeric antibody were verified.

[0085] Example 3 Identification of anti-canine distemper virus mouse-dog chimeric antibodies

[0086] 1. SDS-PAGE and Western Blot Identification of Mouse-Canine Chimeric Antibodies Against Canine Distemper Virus

[0087] The cell line was expressed and purified in large quantities, and the expression level of mouse-dog chimeric antibodies could reach 120 mg / L. The purified antibodies were used to prepare samples and identified by reducing and non-reducing SDS-PAGE.

[0088] The results are as follows Figure 1 As shown, the identification results show that the cell line can secrete the heavy chain and light chain of the mouse-canine chimeric antibody, and the heavy chain and light chain of the mouse-canine chimeric antibody can be correctly folded and combined.

[0089] Western blot was performed using rabbit anti-dog IgG-HRP antibody as the secondary antibody. The results after color development were as follows: Figure 2 The assembled complete antibody can specifically bind to the secondary antibody, and a band can be seen at 55kDa, indicating that the heavy chain of the antibody specifically reacts with the secondary antibody, indicating that the expressed mouse-dog chimeric antibody is successful and has normal biological activity.

[0090] 2. ELISA identification of mouse-dog chimeric antibodies against canine distemper virus

[0091] Indirect ELISA detection: CDV-H protein antigen was coated on the ELISA plate at 4℃ overnight, washed 3 times, and patted dry; blocked with blocking solution at 37℃ for 1h, washed 3 times, and patted dry; purified antibody was diluted in a gradient ratio starting from 1:100 and added to the ELISA plate, incubated at 37℃ for 1h, washed 3 times, and patted dry; rabbit anti-dog IgG-HRP was added, incubated at 37℃ for 1h, washed 5 times, and patted dry; TMB color development solution was added to develop at 37℃ for 10min, terminated with 2M sulfuric acid, and the results were observed.

[0092] The results are shown in Table 3. The initial concentration of the mouse-dog chimeric antibody was 1 mg / mL. When the mouse-dog chimeric antibody was diluted to 102400 times, the P / N was greater than 2.76, indicating that the ELISA titer of the mouse-dog chimeric antibody was 1:102400. The chimeric antibody specifically binds to the CDVH protein.

[0093] Table 3 ELISA identification results

[0094]

[0095]

[0096] 3. Identification of anti-canine distemper virus mouse-dog chimeric antibody by indirect immunofluorescence assay

[0097] Dilute canine distemper virus CDV (America-1 genotype) to 200TCID50, add 100μL of the diluted virus solution to an equal volume of chimeric antibody, mix well, incubate at 37℃ for 1 hour, take 100μL of the mixture and inoculate 4 wells of a 96-well cell culture plate with an equal volume of Vero-slam cell suspension. Set up a virus control group and a normal cell control group, inoculate 4 wells each. Set up at 37℃, 5% CO 2 After culturing in the incubator for 4 days, an indirect immunofluorescence test was performed. The primary antibody was the mouse canine distemper virus H protein monoclonal antibody 9-7B, and the secondary antibody was a goat anti-mouse FITC fluorescent secondary antibody.

[0098] The results are as follows Figure 3 The experimental wells where the mouse-dog chimeric antibody reacted with the virus showed no green fluorescence, the virus group had obvious green fluorescence, and the cell group had no green fluorescence. This showed that the mouse-dog chimeric antibody had neutralized the canine distemper virus, causing no lesions in the cells, and therefore had no specific reaction with the antibody. This proved that the mouse-dog chimeric antibody could be correctly folded and assembled into an active antibody protein.

[0099] 4. Identification of the neutralizing activity of mouse-dog chimeric antibodies against canine distemper virus

[0100] The chimeric antibody was diluted to a concentration of 1 mg / mL, and after a 2-fold dilution, an equal volume of CDV virus dilution solution of 200 TCID50 / 0.5 mL was added. At the same time, a virus group and a cell control group were set up. The virus group was the supernatant of CHO cells that were not electroporated with any plasmids. The cells were incubated at 37°C for 1 hour. The antibody and virus mixture was added to the 96-well plate of Vero-slam cells, with 4 replicates for each group. After gently mixing, the cells were placed in a 37°C 5% CO 2 In a constant temperature incubator, observe the results every day and count the results after 5 days.

[0101] The results are as follows Figure 4 As shown, the cell group did not have lesions, while the virus group had obvious lesions, and the control was established. The neutralization titer of the mouse-dog chimeric antibody reached 1:256, and the results showed that the antibody was a neutralizing antibody, which could effectively bind to CDV to block its infection of cells, had a certain therapeutic effect, and could be added with a pharmaceutically acceptable carrier to prepare a drug for treating canine distemper virus infection.

[0102] Example 4 Identification of anti-canine distemper virus mouse-giant panda chimeric antibodies

[0103] 1. SDS-PAGE and Western Blot Identification of Anti-Canine Distemper Virus Mouse-Giant Panda Chimeric Antibodies

[0104] The cell line was expressed and purified in large quantities, and the expression level of mouse-giant panda chimeric antibody could reach 600 mg / L. The purified antibody was used to prepare samples and identified by reducing and non-reducing SDS-PAGE.

[0105] The results are as follows Figure 5 As shown, the identification results show that the cell line can secrete the heavy chain and light chain of the mouse-giant panda chimeric antibody, and the heavy chain and light chain of the mouse-giant panda chimeric antibody can be correctly folded and combined.

[0106] Western blot identification was performed. Because there was no secondary antibody specifically targeting giant pandas, and the heavy chains of giant pandas and dogs had a high homology, rabbit anti-dog IgG-HRP antibody was used as the secondary antibody.

[0107] The results are as follows Figure 6 As shown, a band of the antibody at 55 kDa can be seen, indicating that the heavy chain part of the antibody reacts specifically with the secondary antibody, indicating that the expressed mouse-giant panda chimeric antibody is successful and has normal biological activity.

[0108] 2. ELISA identification of mouse-giant panda chimeric antibodies against canine distemper virus

[0109] Indirect ELISA detection: The method is the same as Example 3.

[0110] The results are shown in Table 4. The initial concentration of the mouse-giant panda chimeric antibody was 1 mg / mL. When the chimeric antibody was diluted to 25,600 times, the P / N was greater than 2.28, indicating that the ELISA titer of the mouse-giant panda chimeric antibody was 1:25,600. The mouse-giant panda chimeric antibody specifically binds to the CDV H protein.

[0111] Table 4 ELISA identification results

[0112]

[0113] 3. The indirect immunofluorescence test identification steps of anti-canine distemper virus mouse-giant panda chimeric antibodies are the same as those in Example 3.

[0114] The results are as follows Figure 7 As shown, it shows that the mouse-giant panda chimeric antibody has neutralized the canine distemper virus and caused no cell lesions, so it has no specific reaction with the 9-7B antibody. It proves that the mouse-giant panda chimeric antibody can be correctly folded and assembled into an active antibody protein.

[0115] 4. Identification of the neutralizing activity of mouse-giant panda chimeric antibodies against canine distemper virus

[0116] The steps are the same as in Example 3.

[0117] The results are as follows Figure 8As shown, the control was established, and the neutralization effect of the mouse-giant panda chimeric antibody reached 1:128, which could effectively bind to CDV to block its infection of cells, indicating that the mouse-giant panda chimeric antibody expressed in the mammalian system retained the neutralization activity of the original MAb against CDV.

[0118] Example 5 Preparation of canine distemper virus prevention and treatment product

[0119] The mouse-dog chimeric antibody and / or the mouse-giant panda chimeric antibody are dried (wherein the concentration of the mouse-dog chimeric antibody is 1.5 mg / mL and the concentration of the mouse-giant panda chimeric antibody is 3.5 mg / mL) to obtain mouse-dog chimeric antibody and / or mouse-giant panda chimeric antibody powder, i.e., a product for preventing and treating canine distemper virus.

[0120] Example 6 Therapeutic test of canine distemper virus prevention and treatment product

[0121] If you encounter a dog with canine distemper, Fig. 9 As shown in the figure, the sick dog had obvious clinical symptoms of canine distemper, depression, purulent discharge from the eyes, diarrhea, and watery feces, and was diagnosed with canine distemper. The canine distemper virus prevention and treatment product was used for treatment, and 1 mg / kg was injected continuously for 3 days.

[0122] After treatment, such as Fig.10 As shown, the dog recovered to a normal physiological state, indicating that the mouse-dog chimeric antibody constructed in this case has a good therapeutic effect against canine distemper.

[0123] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A genetically engineered chimeric antibody against canine distemper virus, characterized in that: The genetically engineered chimeric antibody is a mouse-canine chimeric antibody or a mouse-giant panda chimeric antibody; the heavy chain amino acid sequence of the mouse-canine chimeric antibody is shown in SEQ ID NO: 6, and the light chain amino acid sequence is shown in SEQ ID NO: 8; The heavy chain amino acid sequence of the mouse-giant panda chimeric antibody is shown in SEQ ID NO: 10, and the light chain amino acid sequence is shown in SEQ ID NO:

12.

2. The genetically engineered chimeric antibody according to claim 1, characterized in that: The mouse-dog chimeric antibody and the mouse-giant panda chimeric antibody both include the variable region of the mouse antibody; The mouse-dog chimeric antibody also includes the constant region of the canine antibody, and the constant region of the canine antibody includes the heavy chain constant region and the light chain constant region. The amino acid sequence of the heavy chain constant region of the canine antibody is shown in SEQ ID NO:

13. The amino acid sequence of the light chain constant region of the canine antibody is shown in SEQ ID NO: 14; The mouse-giant panda chimeric antibody also includes the constant region of the giant panda-derived antibody, and the constant region of the giant panda-derived antibody includes the heavy chain constant region and the light chain constant region. The amino acid sequence of the heavy chain constant region of the giant panda-derived antibody is shown in SEQ ID NO:

15. The amino acid sequence of the giant panda-derived antibody light chain constant region is shown in SEQ ID NO:

16.

3. The genetically engineered chimeric antibody according to claim 2, characterized in that: The variable region of the mouse antibody includes a heavy chain variable region and a light chain variable region, The amino acid sequence of the heavy chain variable region of the mouse antibody is shown in SEQ ID NO:

2. The amino acid sequence of the light chain variable region of the murine antibody is shown in SEQ ID NO:

4.

4. The genetically engineered chimeric antibody according to claim 1, characterized in that: The mouse-dog chimeric antibody and the mouse-giant panda chimeric antibody both include a KOZAK sequence and a signal peptide sequence; The nucleotide sequence of the KOZAK sequence is shown in SEQ ID NO: 17; the nucleotide sequence of the signal peptide sequence is shown in SEQ ID NO:

18.

5. A gene encoding a genetically engineered chimeric antibody against canine distemper virus as claimed in claim 1, characterized in that: The nucleotide sequence encoding the heavy chain of the mouse-canine chimeric antibody is shown in SEQ ID NO: 5, and the nucleotide sequence encoding the light chain of the mouse-canine chimeric antibody is shown in SEQ ID NO: 7; The nucleotide sequence encoding the heavy chain of the mouse-giant panda chimeric antibody is shown in SEQ ID NO:9, and the nucleotide sequence encoding the light chain of the mouse-giant panda chimeric antibody is shown in SEQ ID NO:

11.

6. A method for preparing a genetically engineered chimeric antibody as claimed in claim 1, characterized in that: The following steps are involved: (1) connecting the murine antibody heavy chain variable region described in claim 3 and the canine / giant panda antibody heavy chain constant region described in claim 3, adding a KOZAK sequence and a signal peptide sequence, connecting the murine antibody light chain variable region described in claim 3 and the canine / giant panda antibody light chain constant region described in claim 3, adding a KOZAK sequence and a signal peptide sequence, and performing CHO codon optimization to obtain a chimeric antibody heavy chain and a chimeric antibody light chain; (2) constructing the chimeric antibody heavy chain and the chimeric antibody light chain into expression vectors of the CHO expression system respectively, performing fusion transformation, and obtaining a mouse-dog chimeric antibody plasmid or a mouse-giant panda chimeric antibody plasmid; (3) The mouse-dog chimeric antibody plasmid and the mouse-giant panda chimeric antibody plasmid are respectively transfected into CHO cell lines, high-expression cell lines are screened, monoclonal cell lines are screened, and the cell lines are subjected to high-density expression and purification to obtain mouse-dog chimeric antibodies or mouse-giant panda chimeric antibodies.

7. The preparation method according to claim 6, characterized in that: In the step (1), the heavy chain variable region or the light chain variable region is located at the N-terminus of the heavy chain constant region or the light chain constant region; the KOZAK sequence and the signal peptide sequence are added to the N-terminus of the heavy chain variable region or the light chain variable region.

8. The preparation method according to claim 6, characterized in that: In the step (2), the expression vector is PXC17.

4.

9. Use of the genetically engineered chimeric antibody according to claim 1 in the preparation of a product for preventing and treating canine distemper virus, characterized in that: In the product, the concentration of the mouse-dog chimeric antibody is 1-2 mg / mL and / or the concentration of the mouse-giant panda chimeric antibody is 3-4 mg / mL.

10. The use according to claim 9, characterized in that: The concentration of the mouse-dog chimeric antibody is 1.5 mg / mL and / or the concentration of the mouse-giant panda chimeric antibody is 3.5 mg / mL.

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