CN112921005B - Hybridoma cell strain, canine parvovirus VP2 protein monoclonal antibody produced by hybridoma cell strain and application of hybridoma cell strain - Google Patents
Hybridoma cell strain, canine parvovirus VP2 protein monoclonal antibody produced by hybridoma cell strain and application of hybridoma cell strain Download PDFInfo
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Abstract
The invention provides a hybridoma cell strain, a canine parvovirus VP2 protein monoclonal antibody produced by the hybridoma cell strain and application thereof, and belongs to the technical field of monoclonal antibody preparation, wherein the hybridoma cell strain 5A92B12 provided by the invention can be stably passaged and can continuously and stably secrete the monoclonal antibody of the canine parvovirus VP2 protein; after 10 generations of culture, the hybridoma cell strain 5A92B12 can still grow well and be stably passaged, and the supernatant titer of the culture solution can still reach 1 multiplied by 10 6 The above; the monoclonal antibody generated by the hybridoma cell strain and the canine parvovirus VP2 protein can be specifically identified, and the monoclonal antibody has the advantages of high specificity and high sensitivity, has the characteristic of inhibiting canine parvovirus replication, can be applied to detection and treatment of canine parvovirus diseases, and has good application prospects.
Description
Technical Field
The invention belongs to the technical field of monoclonal antibody preparation, and particularly relates to a hybridoma cell strain, a canine parvovirus VP2 protein monoclonal antibody produced by the hybridoma cell strain and application of the hybridoma cell strain.
Background
Canine parvovirus disease (Canine Parvovirus Disease) is an infectious disease which is mainly characterized by hemorrhagic enteritis and non-suppurative myocarditis and is caused by canine parvovirus (Canine Parvovirus, CPV) infection, and is mainly infected with puppies, and the infectious disease is fast in morbidity and high in mortality rate, so that the infectious disease becomes one of the most serious infectious diseases in the canine industry.
Canine parvovirus belongs to a single-stranded negative-strand DNA virus, has a genome size of about 5kb, and comprises two open reading frames (open reading frame, ORFs), the ORFs at the 3 'end encode nonstructural proteins NS1 and NS2, and the ORFs at the 5' end encode structural proteins VP1 and VP2; a hairpin structure is arranged at each end of the genome, wherein the 3 '-end is folded to form a Y-shaped structure, and the 5' -end is formed to form a U-shaped structure, which plays an important role in replication and transcription of viruses. VP2 gene 1755bp long and coded 584 amino acids are main proteins constituting viral capsid. VP2 protein is also the main immunity protein of parvovirus, can stimulate the organism to produce protective neutralizing antibodies, and is the first choice gene for preparing genetic engineering vaccine; in addition, VP2 gene plays a key role in receptor recognition and tissue tropism process, and has close relation with pathogenicity and hemagglutination characteristics of viruses.
Because canine parvovirus does not have a capsule membrane, the capsid protein has a compact structure and strong resistance to the external environment, and once infected, the canine parvovirus is difficult to thoroughly remove. Since the immune organs of puppies have not developed to be mature, the prophylactic effect of puppies by vaccine immunization is less than ideal. Thus, once puppies are infected with canine parvovirus, how to treat is important. At present, the treatment of canine parvovirus disease is mainly carried out by combining antiviral drugs with canine parvovirus hyperimmune serum, but most cases still take death as the final attribution.
Disclosure of Invention
In view of the above, the invention aims to provide a hybridoma cell strain, a canine parvovirus VP2 protein monoclonal antibody produced by the hybridoma cell strain and application of the hybridoma cell strain; the monoclonal antibody generated by the hybridoma cell strain and the canine parvovirus VP2 protein can be specifically identified, and the monoclonal antibody has the advantages of high specificity and high sensitivity, has the characteristic of inhibiting canine parvovirus replication, can be applied to detection and treatment of canine parvovirus diseases, and has good application prospects.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a hybridoma cell strain 5A92B12, the preservation number of which is CCTCC NO: C202110.
the invention provides application of the hybridoma cell strain 5A92B12 in preparing canine parvovirus VP2 protein monoclonal antibodies.
The invention provides a canine parvovirus VP2 protein monoclonal antibody, which is produced by the hybridoma cell strain 5A92B12.
Preferably, the amino acid sequence of the light chain variable region is shown as SEQ ID No.1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 2.
The invention provides application of the canine parvovirus VP2 protein monoclonal antibody in preparing a canine parvovirus disease detection kit.
The invention provides a canine parvovirus disease detection kit, which comprises the canine parvovirus VP2 protein monoclonal antibody and a detection reagent.
Preferably, the canine parvovirus disease detection kit is an ELISA detection kit, a Western-blot detection kit or an immunofluorescence detection kit.
Preferably, the detection reagent comprises canine parvovirus VP2 protein, wherein the canine parvovirus VP2 protein is used as a coating antigen, and the canine parvovirus VP2 protein monoclonal antibody is used as a detection competition antibody.
The invention provides application of the canine parvovirus VP2 protein monoclonal antibody in preparing medicaments for treating canine parvovirus diseases.
Preferably, the concentration of the canine parvovirus VP2 protein monoclonal antibody in the medicament is 0.5-1 mug.
The invention provides a hybridoma cell strain, a canine parvovirus VP2 protein monoclonal antibody produced by the hybridoma cell strain and application of the hybridoma cell strain; the monoclonal antibody generated by the hybridoma cell strain and the canine parvovirus VP2 protein can be specifically identified, and the monoclonal antibody has the advantages of high specificity and high sensitivity, has the characteristic of inhibiting canine parvovirus replication, can be applied to detection and treatment of canine parvovirus diseases, and has good application prospects.
The hybridoma cell strain provided by the inventionThe 5A92B12 can be stably passaged, and can continuously and stably secrete monoclonal antibodies against canine parvovirus VP2 protein; after 10 generations of culture, the hybridoma cell strain 5A92B12 can still grow well and be stably passaged, and the supernatant titer of the culture solution can still reach 1 multiplied by 10 6 The above.
The canine parvovirus VP2 protein monoclonal antibody provided by the invention can accurately detect the content of VP2 protein in an effective antigen component or an infected tissue sample in a canine parvovirus vaccine sample, and can be widely applied to vaccine and clinical detection; the monoclonal antibody has the capacity of inhibiting virus replication and can be used for treating canine parvovirus diseases.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis of recombinant expression of canine parvovirus VP2 protein, marker is a protein molecular weight standard (kDa);
FIG. 2 is a SDS-PAGE result of canine parvovirus VP2 protein monoclonal antibody, marker is a protein molecular weight standard (kDa);
FIG. 3 is a diagram showing the result of Westernblot of canine parvovirus VP2 protein monoclonal antibodies;
FIG. 4 shows the indirect ELISA for detecting the titer of canine parvovirus VP2 protein monoclonal antibody;
FIG. 5 shows the results of a virus inhibition experiment using a monoclonal antibody against VP2 protein of canine parvovirus, wherein 1 is canine parvovirus 10 -3 Diluting and inoculating F81 cells for 96 hours, wherein 2 is canine parvovirus 10 -3 Diluting and inoculating F81 cells, simultaneously adding 0.5 mug 5A92B12 monoclonal antibody to inhibit virus growth, 3 is canine parvovirus 10 -4 Diluting and inoculating F81 cells for 96 hours, wherein 4 is canine parvovirus 10 -4 Diluting and inoculating F81 cells, and simultaneously adding 0.5 mug of 5A92B12 monoclonal antibody to inhibit the growth of viruses; the scale bar in the figure is 400nm.
Description of biological preservation
The hybridoma cell strain 5A92B12 provided by the invention is preserved in China center for type culture collection, and the preservation number is CCTCC NO: c202110, the preservation date is 2021, 3 months and 25 days, and the preservation address is in the eight-path 299-No. Wuhan university school (the first side of the university of Wuhan) in Wuhan, wuhan City, hubei province, and the preservation center of Wuhan university.
Detailed Description
The invention provides a hybridoma cell strain 5A92B12, the preservation number of which is CCTCC NO: C202110. in the present invention, the hybridoma cell line 5A92B12 is preferably obtained by fusing spleen cells obtained by immunizing a mouse with VP2 antigen with SP2/0 cells and screening.
In the present invention, immunization of mice with VP2 antigen preferably includes a first immunization, a second immunization, a third immunization, a fourth immunization, and booster immunization. In the present invention, the first immunization is preferably performed by injecting mice after emulsifying the VP2 antigen in combination with Freund's complete adjuvant; in the invention, the volume ratio of the VP2 antigen to Freund's complete adjuvant is preferably 1:1; the injection site is preferably the back and the injection is preferably subcutaneous multipoint; the dose of the injection is preferably 50 μg. In the present invention, the second immunization is preferably performed 2 weeks after the first immunization, the third immunization is preferably performed 2 weeks after the second immunization, and the fourth immunization is preferably performed 2 weeks after the third immunization. In the present invention, the methods and dosages of the second immunization, the third immunization and the fourth immunization are consistent with those of the first immunization. In the present invention, the booster is preferably performed 3 days before cell fusion; the boost is preferably injected with a phosphate buffer containing 50 μ gVP2 antigen; the injection is preferably intraperitoneal.
After the boost, the spleen cells of the mice are collected. The method for collecting the spleen cells is not particularly limited, and the method for collecting the spleen cells is conventional in the art. In the present invention, collected spleen cells and SP2/0 cells were fused and selected to obtain hybridoma cells. In the present invention, the SP2/0 cells are preferably commercially available from the company Wohan Jin Kairui during the practice of the present invention. In the present invention, the ratio of the number of spleen cells to SP2/0 cells is preferably 5:1; the fusion is preferably performed using PEG 4000; the amount of PEG4000 is preferably 1ml of 45% PEG4000. The invention cultures after the cell fusion, and screens the cell strain for identifying the canine parvovirus VP2 protein, namely the hybridoma cell strain. In the present invention, the culture medium is HAT medium, the temperature of the culture is preferably 37 ℃, and the time of the culture is selected to be 2 weeks; the culture is preferably performed in 96-well plates. In the present invention, the cell lines recognizing canine parvovirus VP2 protein are preferably selected by detecting cell culture supernatants by an indirect ELISA method. In the present invention, it is preferable to subclone the cell lines recognizing canine parvovirus VP2 protein obtained by screening by limiting dilution.
The invention provides application of the hybridoma cell strain 5A92B12 in preparing canine parvovirus VP2 protein monoclonal antibodies. In the present invention, the hybridoma cell line 5A92B12 is obtained by intraperitoneal inoculation of a mouse. In the invention, the mice are preferably Balb/c mice of 6-8 weeks of age; the mice are preferably intraperitoneally injected with liquid paraffin in an amount of 0.5 mL/mouse one week prior to intraperitoneal inoculation. In the present invention, the number of cells inoculated per mouse is preferably 1X 10 5 ～1×10 6 A plurality of; the invention collects ascites preferably 5 days apart after said inoculation. After the ascites is obtained, centrifuging the ascites, and collecting supernatant; purifying the supernatant to obtain the canine parvovirus VP2 protein monoclonal antibody. In the present invention, the rotational speed of the centrifugation is preferably 12000 to 14000rpm, more preferably 13000rpm; the time of the centrifugation is preferably 25 to 35 minutes, more preferably 30 minutes. After the supernatant is obtained, the invention performs purification; the purification is preferably performed using Protein-Sepharose CL-4B: the upper column is preferably 20mM PBS buffer, the column chromatography eluent is preferably glycine buffer, the concentration of the glycine buffer is preferably 20mM, and the pH value is preferably 2.7.
The invention provides a canine parvovirus VP2 protein monoclonal antibody, the amino acid sequence of a light chain variable region of which is shown as SEQ ID No.1, which is specifically as follows:
DIQMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWKN。
the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.2, and is specifically as follows:
QVQLKESGAELVRPGASVTLSCKASVYTFTDYEMHWVKQTPVHGLEWIGAIDPETGGTAYNQNFKDKATLTADRSSSTAYMELR。
in the present invention, the canine parvovirus VP2 protein monoclonal antibody is preferably produced by the hybridoma cell line 5A92B12, and the specific method is described above.
The invention also provides application of the canine parvovirus VP2 protein monoclonal antibody in preparing a canine parvovirus disease detection kit. In the invention, the canine parvovirus VP2 protein monoclonal antibody can accurately detect the content of VP2 protein in effective antigen components or infected tissue samples in canine parvovirus vaccine samples. In the present invention, the detection is preferably achieved by ELISA detection, western-blot detection or immunofluorescence detection. In the invention, the canine parvovirus VP2 protein monoclonal antibody is preferably subjected to biological labeling or chemical labeling; the biomarker comprises an enzyme label, preferably a horseradish peroxidase label.
The invention provides a canine parvovirus disease detection kit, which comprises the canine parvovirus VP2 protein monoclonal antibody and a detection reagent. In the invention, the canine parvovirus disease detection kit is preferably an ELISA detection kit, a Western-blot detection kit or an immunofluorescence detection kit. The invention is not particularly limited in the type and specification of the detection reagent, and detection reagents required by ELISA detection, western-blot detection or immunofluorescence detection which are conventional in the art can be adopted. In the invention, the detection reagent in the ELISA detection kit preferably comprises canine parvovirus VP2 protein, wherein the canine parvovirus VP2 protein is used as a coating antigen, and the canine parvovirus VP2 protein monoclonal antibody is used as a detection competition antibody after being subjected to enzyme labeling.
The invention also provides application of the canine parvovirus VP2 protein monoclonal antibody in preparing medicaments for treating canine parvovirus diseases. In the invention, the concentration of the canine parvovirus VP2 protein monoclonal antibody in the medicament is preferably 0.5-1 mug. In the invention, the canine parvovirus VP2 protein monoclonal antibody can inhibit replication of canine parvovirus, and has the effect of treating canine parvovirus diseases. The invention has no special limitation on the formulation and auxiliary material composition of the medicament, and the canine parvovirus VP2 protein monoclonal antibody can be adopted.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Canine parvovirus isolation
1. Pretreatment of disease materials: collecting sample of canine intestine content suspected to be infected with canine parvovirus of certain pet hospital in Lanzhou, gansu province, adding suspension prepared by PBS solution according to the volume ratio of 1:10, centrifuging at 7000rpm for 15min, taking 500 mu L of supernatant, adding equal amount of chloroform, fully and uniformly mixing, centrifuging at 4000rpm for 15min, discarding precipitate, filtering and sterilizing the supernatant by a 0.22 mu m microporous filter membrane, and storing in a refrigerator at-20 ℃.
2. Virus isolation: inoculating F81 cells (purchased from vinca photobiotechnology Co., ltd., product number: HG-ATCCF 81) to the treated disease sample by synchronous inoculation, and inoculating 5% CO at 37deg.C 2 Culturing in an incubator, and observing cytopathic effect (CPE) day by day; continuous blind culture, CPE was observed on passage 4, designated CPV-LZ-1 isolate, initially demonstrating the presence of canine parvovirus in the disease.
3. Identification of hemagglutination characteristics: fresh 1% porcine red blood cell suspension was used, purchased from Beijing Anzhong Ruison technologies Co., ltd., cat: f7705-05, and the coagulability of the cultured 5-9-generation F81 cell culture of CPV-LZ-1 isolate was examined, and as a result, the coagulability was 2 in the cultured 5-9-generation cell culture 8 ～2 9 In between, it was demonstrated that the virus hemagglutination properties of the isolates were relatively stable.
4. Molecular biology identification: and (3) designing a specific primer for amplifying the VP2 gene according to the CPV sequence in GenBank, and connecting PCR amplification products with a cloning vector for sequence determination. The MegAlign software of the DNA STAR software package is adopted to compare the isolated strain with CPVP 2 genes registered by Genbank, and the result shows that the isolated strain has high homology with other reference strains, wherein the similarity of the isolated strain and the other reference strains is 98.8-99.9%. The amino acid sequence was further analyzed and the strain VP2 gene was such that amino acid 426 was Asn instead of Asp, and thus the isolate was of CPV-2a type.
Expression and purification of canine parvovirus VP2 protein
1. Gene cloning and expression vector construction
The canine parvovirus CPV-LZ-1 isolate isolated in example 1 was stored in the laboratory (replaced by a commercially available canine parvovirus type CPV-2a isolate) and primers were designed to amplify the fragment of interest as follows: the upstream primer CPV-VP2F: CGC (common gateway control)GGATCCATGAGTGATGGAGCAGTT (SEQ ID No. 3), the downstream primer CPV-VP2R: CCC (CCC)AAGCTTTTAATATAATTTTCTAGGTGC (SEQ ID No. 4); bamHI and HindIII enzyme cutting sites are respectively added at two ends of the primer; the size of the target fragment is 1755bp; through virus DNA extraction and cloning of target gene, VP2 gene is cloned to pMD18-T, positive clone is selected and sequenced. Positive clones with correct sequences are subjected to double digestion, cloned into pET-30a expression vector and transformed into Rosetta (DE 3) competent.
2. Expression and purification of recombinant proteins
Expanding and culturing bacterial liquid to OD (optical density) of 0.6-1 according to the volume ratio of 1:100, adding IPTG induction protein with the final concentration of 1mM, and collecting bacterial bodies according to the weight-volume ratio of 1:10 adding phosphate buffer solution containing 8mol/L urea to resuspend thalli and performing ultrasonic crushing; crushing and precipitating the supernatant and thalli, wherein the target protein is insoluble protein; the protein of interest was purified by HiTrapTALONcrude (GE) and detected by SDS-PAGE (FIG. 1).
Example 2
Establishment of hybridoma cell lines
1. Experimental materials
1. Antigen: expressing canine parvovirus VP2 protein as antigen with the prokaryotic expression system of example 1 above;
2. culture medium: hyclone DMEM medium is purchased from Thermo company; HAT is available from SIGMA company; HT is available from SIGMA company;
3. experimental animals: female Balb/c mice, 8-12 weeks old, are raised under SPF-level conditions;
4. other experimental materials: freund's complete adjuvant, freund's incomplete adjuvant, was purchased from SIGMA corporation; PEG4000 was purchased from MERCK; HRP-goat anti-mouse IgG antibodies were purchased from abcam corporation; the rest reagents are all domestic analytically pure products.
2. Method steps
1. Immunization of animals
(1) An exempt from: mixing VP2 antigen and Freund's complete adjuvant in equal volume, emulsifying by hand-held homogenizer at 3000rpm for 5min to fully emulsify, and subcutaneously injecting into back of mice at multiple points, wherein each Balb/c mouse has injection amount of 50 μg (antigen);
(2) And (2) avoiding: 2 weeks after the first immunization, the VP2 antigen is mixed with Freund's incomplete adjuvant in equal volume and fully emulsified, the back of the mice is subjected to subcutaneous multipoint injection, and the injection amount of each Balb/c mouse is 50 mug;
(3) Three-way: 2 weeks after the second immunization, the VP2 antigen and Freund's incomplete adjuvant are mixed in equal volume and fully emulsified, and the back subcutaneous multipoint injection is performed on immunized mice, wherein the injection amount of each Balb/c mouse is 50 mug;
(4) Four-exemption: 2 weeks after the third immunization, the VP2 antigen and Freund's incomplete adjuvant are mixed in equal volume and fully emulsified, and the back subcutaneous multipoint injection is performed on immunized mice, wherein the injection amount of each Balb/c mouse is 50 mug;
(5) Boosting: 3 days prior to cell fusion, a phosphate buffer containing 50 μ gVP2 antigen was intraperitoneally injected.
2. Preparation of hybridoma cells
Spleen cells and SP2/0 cells (purchased from Wuhan Jin Kairui company) were collected from mice according to a conventional method. After cell counting, the spleen cells of the mice were mixed with SP2/0 cell number ratio in a ratio of 5:1, and after centrifugation the cell pellet was fused with 1ml 45% PEG4000. After the fusion cells were suspended in HAT medium, 96-well plates were plated and cultured at 37 ℃. Two weeks after fusion, the cell culture supernatants were assayed by indirect ELISA and hybridoma cell lines recognizing canine parvovirus VP2 protein were selected. Subcloning the obtained positive clone strain by limiting dilution method.
3. Establishment of hybridoma cell lines
Obtaining 5 hybridoma cell lines aiming at the stable secretion monoclonal antibody of the canine parvovirus recombinant VP2 protein through 3 times of subcloning and indirect ELISA screening; among them, the best-effective 1 hybridoma cell line was designated as 5A92B12.
4. Injecting the hybridoma cell line into the abdominal cavity of a mouse, collecting and purifying ascites, then performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and detecting the titer of the obtained ascites as shown in figure 2, wherein the results are shown in table 1 and figure 4, and the results show that when the 5A92B12 monoclonal antibody is diluted to a concentration of 3.906ng/ml, the monoclonal antibody can still react with the coated antigen, the OD value can reach 0.9718, and the prepared monoclonal antibody has good specificity and sensitivity.
TABLE 1 results of determination of the titers of the A92B12 antibodies
5. Subculturing of hybridoma cell lines
The hybridoma cell line which stably secretes 5A9B12 is continuously cultured and passaged in a DMEM high-sugar culture medium containing 20 percent (volume percent) of fetal bovine serum, and after 10 generations of culture, the hybridoma cell line can still grow well and stably passaged, and the supernatant titer of a culture solution can still reach 1 multiplied by 10 6 The above.
The results show that the hybridoma cell line obtained by the invention can be stably passaged, and can continuously and stably secrete monoclonal antibodies against canine parvovirus VP2 protein.
Example 3
Large-scale preparation and identification of anti-canine parvovirus VP2 protein monoclonal antibody
1. Antibody preparation
Balb/c mice of 6-8 weeks old were selected, and 0.5mL liquid paraffin was intraperitoneally injected, 0.5mL each. One week later, hybridoma cells were inoculated in the abdominal cavity, and the number of inoculated cells per mouse was 1X 10 5 ～1×10 6 And each. After 5 days of interval, when the abdomen is obviously enlarged and touched by hands, the skin has tension, and the ascites can be collected by a needle head with the size of 9.
The ascites was centrifuged (13000 rpm,30 min) to remove cellular components and other sediments and the supernatant was collected. Purifying with Protein-Sepharose CL-4B, loading into 20mM PBS buffer solution, eluting with 20mM glycine buffer solution with pH value of 2.7 to obtain monoclonal antibody against canine parvovirus VP2 Protein, and subjecting to SDS-PAGE to obtain the final product with purity of 95% or higher, as shown in figure 2.
2. Identification of antibodies
1. Western validation of monoclonal antibodies
Canine parvovirus inactivated antigen was separated on 12% SDS-PAGE and protein acetate membranes (NC membranes) were applied after a treatment of 160mA for 90 min. NC membranes were blocked with 5% skim milk for 1h at room temperature. After 3 washes of PBST, monoclonal antibodies were added as primary antibodies and incubated for 1h at room temperature. After three washes of PBST, the goat anti-mouse IgGFc secondary antibody (1:5000) labeled with HRP was incubated for 1h at room temperature, and finally an immune response was detected by Easy See Western BlotKit (TRAN) (FIG. 3, wherein 5A9 is the 5A92B12 monoclonal antibody of the present invention, and the polyclonal antibody is mouse serum), and as a result, a target band at about 70kDa was found, indicating that the 5A92B12 monoclonal antibody used in the present invention was capable of specifically immunoreacting with canine parvovirus inactivated antigen.
2. Virus inhibition assay for monoclonal antibodies
Canine parvovirus was diluted by a double ratio and 10 selected in 12 well cell culture plates -1 ～10 -6 Diluted virus, each titer was inoculated with 2 well F81 cells simultaneously, after 2h infection, a group of cells was added with monoclonal antibody at a final concentration of 0.5. Mu.g, a group of cells was not added with antibody, and at 37℃ 5% CO 2 Culturing in an incubator, observing cytopathy day by day, and the result is shown in fig. 5, and the monoclonal antibody prepared by the invention has the capability of inhibiting virus replication.
3. Variable region sequence determination of monoclonal antibody for recognizing canine parvovirus VP2 protein
mRNA is extracted from 5A92B12 hybridoma cells, reverse transcribed into cDNA, and the variable region universal primer is used for heavy chainThe upstream primer of the variable region is as follows: 5'-TTAAAAGGTGTCCAGTGTGAAG-3' (SEQ ID No. 5), the heavy chain variable region downstream primer is: 5'-CCAGGTCACTGTCACTGGCTC-3' (SEQ ID No. 6); light chain variable region V L The upstream primer is 5'ATGAAGTTGCCTGTTAGGCTGTTG-3' (SEQ ID No. 7), and the downstream primer of the light chain variable region is 5'GATACAGTTGGTGCAGCATCAGCC-3' (SEQ ID No. 8). PCR amplification is performed with high fidelity enzymes, the PCR product fragments are inserted into a T vector for DNA sequencing, and the obtained sequences are translated into amino acid sequences of proteins. Variable region amino acid sequence of an antibody that recognizes VP2 protein 5a92B 12: the light chain is shown as SEQ ID No.1, and the heavy chain is shown as SEQ ID No. 2.
From the above examples, the hybridoma cell line 5A92B12 provided by the invention can stably passaged and can continuously and stably secrete monoclonal antibodies against canine parvovirus VP2 protein; the monoclonal antibody produced by the method can be specifically identified with the canine parvovirus VP2 protein, has the advantages of high specificity and high sensitivity, has the characteristic of inhibiting canine parvovirus replication, can be applied to detection and treatment of canine parvovirus diseases, and has good application prospect.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> the animal doctor institute of Lanzhou, china academy of agricultural sciences
<120> hybridoma cell strain, canine parvovirus VP2 protein monoclonal antibody produced by same and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 109
<212> PRT
<213> Artificial Sequence
<400> 1
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg
85 90 95
Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys Asn
100 105
<210> 2
<211> 84
<212> PRT
<213> Artificial Sequence
<400> 2
Gln Val Gln Leu Lys Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Thr Leu Ser Cys Lys Ala Ser Val Tyr Thr Phe Thr Asp Tyr
20 25 30
Glu Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile
35 40 45
Gly Ala Ile Asp Pro Glu Thr Gly Gly Thr Ala Tyr Asn Gln Asn Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Ala Asp Arg Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg
<210> 3
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 3
cgcggatcca tgagtgatgg agcagtt 27
<210> 4
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 4
cccaagcttt taatataatt ttctaggtgc 30
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 5
ttaaaaggtg tccagtgtga ag 22
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 6
ccaggtcact gtcactggct c 21
<210> 7
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 7
atgaagttgc ctgttaggct gttg 24
<210> 8
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 8
gatacagttg gtgcagcatc agcc 24
Claims (9)
1. A hybridoma cell strain 5a92B12, characterized by a preservation number of cctcrno: C202110.
2. the use of the hybridoma cell strain 5A92B12 according to claim 1 for preparing a canine parvovirus VP2 protein monoclonal antibody.
3. A canine parvovirus VP2 protein monoclonal antibody produced by hybridoma cell line 5a92B12 of claim 1.
4. Use of the canine parvovirus VP2 protein monoclonal antibody of claim 3 in preparing a canine parvovirus disease detection kit.
5. A canine parvovirus disease detection kit, comprising the canine parvovirus VP2 protein monoclonal antibody of claim 3 and a detection reagent.
6. The kit according to claim 5, wherein the canine parvovirus disease detection kit is an ELISA detection kit, a Western-blot detection kit or an immunofluorescence detection kit.
7. The kit according to claim 5, wherein the detection reagent comprises canine parvovirus VP2 protein, wherein the canine parvovirus VP2 protein is used as a coating antigen, and wherein the canine parvovirus VP2 protein monoclonal antibody is used as a detection competition antibody.
8. Use of the canine parvovirus VP2 protein monoclonal antibody of claim 3 in the preparation of a medicament for treating canine parvovirus disease.
9. The use according to claim 8, wherein the concentration of canine parvovirus VP2 protein monoclonal antibody in the medicament is 0.5-1 μg.
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| CN110964102A (en) * | 2018-09-28 | 2020-04-07 | 洛阳普泰生物技术有限公司 | Monoclonal antibody capable of simultaneously combining with canine, feline and mink parvoviruses, variable region sequence thereof, hybridoma cell strain and application |
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| WO2012164372A1 (en) * | 2011-06-01 | 2012-12-06 | Indian Immunologicals Limited | Recombinant anti-canine parvovirus antibody and uses thereof |
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| AR114112A1 (en) * | 2018-02-15 | 2020-07-22 | Seattle Genetics Inc | GLIPICAN 3 ANTIBODIES AND CONJUGATES THEREOF |
| CN109517061B (en) * | 2018-11-22 | 2021-03-19 | 暨南大学 | Lancet virus murine monoclonal antibody and preparation method and application thereof |
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| CN1844149A (en) * | 2005-04-07 | 2006-10-11 | 苏州大学 | Monoclonal antibody against human 4-1BBL and its use |
| CN111406069A (en) * | 2017-09-21 | 2020-07-10 | 祐和医药科技(北京)有限公司 | anti-CT L A4 antibodies and uses thereof |
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