CN116735873B - Application of monoclonal antibody specifically binding to canine parvovirus VP2 protein in detection reagent - Google Patents
Application of monoclonal antibody specifically binding to canine parvovirus VP2 protein in detection reagent Download PDFInfo
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- CN116735873B CN116735873B CN202310997808.0A CN202310997808A CN116735873B CN 116735873 B CN116735873 B CN 116735873B CN 202310997808 A CN202310997808 A CN 202310997808A CN 116735873 B CN116735873 B CN 116735873B
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/015—Parvoviridae, e.g. feline panleukopenia virus, human Parvovirus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an application of a monoclonal antibody specifically binding with canine parvovirus VP2 protein in a detection reagent, wherein the monoclonal antibody contains a polypeptide named V H Heavy chain variable region of (2) and designated V L Light chain variable region of (V) H And V L Are each composed of a complementarity determining region and a framework region; the complementarity determining region consists of CDR1, CDR2 and CDR 3; v of the monoclonal antibody H The amino acid sequences of CDR 1-CDR 3 are shown as SEQ ID No. 1-3; v of the monoclonal antibody L The amino acid sequences of CDR 1-CDR 3 are shown as SEQ ID No. 4-6. According to the invention, the VP2 protein with high conservation is obtained through insect baculovirus system expression, the specific monoclonal antibody is obtained by immunizing mice, the specificity is good, and a foundation is laid for establishing a more accurate and faster canine parvovirus detection method.
Description
Technical Field
The invention relates to the technical field of rapid biological detection of animal epidemic diseases, in particular to application of a monoclonal antibody specifically combined with canine parvovirus VP2 protein in a detection reagent.
Background
Canine parvovirus (canine parvovirus, CPV), a member of the parvoviridae genus, is one of the major pathogens causing severe gastroenteritis in puppies, particularly in non-immunized puppies or puppies without maternal antibodies, are more susceptible to canine parvovirus disease. The virus was first observed in 1977 from the feces of dogs with hemorrhagic enteritis, was isolated in 1978 from dogs with enteritis, and was subsequently discovered in dogs worldwide. The sick dogs are mainly characterized by severe vomiting, diarrhea and significant reduction of white blood cells, and the disease can occur all the year round. Since the outbreak of canine parvovirus disease, the canine industry and the economic animal industry are seriously affected due to the high morbidity and mortality of canine parvovirus disease, and the survival of wild animals is endangered. There is currently no effective drug for the treatment of CPV infections, and for diseased animals, symptomatic treatments such as the use of antibacterial agents to prevent secondary bacterial infections, and measures to prevent dehydration, heart failure, poisoning, etc. are available. Therefore, aiming at the early prevention, early discovery and early treatment of canine parvovirus, which are important in controlling canine parvovirus transmission, the development of researches on the aspect of CPV infection diagnosis and control is very important.
CPV is an encysted virus capable of infecting vertebrates and insects, and parvoviral particles consist of viral capsids and coated single-stranded linear DNA, the molecular mass of the virus is 5.5X10 3 ~6.2×10 3 The full length of the genome is about 5200 nucleotides. There are two major open reading frames on the complementary strand of the genome. VP1, VP2 and VP3 are encoded by the second ORF, and these three proteins are assembled in a proportion to form canine parvovirus capsids, which are expressed late in viral infection. VP2 is the primary protective antigenic protein of CPV and plays an important role in determining host range and viral invasion. The VP2 gene is 1755nt in length, encodes the main epitope of CPV and can induce the organism to produce neutralizing antibodies.
The method for detecting the canine parvovirus comprises a plurality of detection means such as virus separation and identification, electron microscope observation, PCR technology, hemagglutination and hemagglutination inhibition test, immunofluorescence test, enzyme-linked immunosorbent assay, immunochromatography test and the like. The virus separation and identification method can also affect sensitive cells due to other substances or viruses in the disease materials, pollute the cell growth environment, cause cytopathy, affect judgment, and is long in time consumption and only suitable for laboratories. Electron microscopy requires high precision instruments and specialized technicians to process and observe samples, which requires more time and effort than other methods of detection, which are not suitable for large-scale detection of samples and are not widely used in clinical settings. The PCR technology is used as a molecular biology detection method, and the designed primer is utilized to conveniently and rapidly identify suspected CPV disease materials. However, the conventional PCR technology cannot detect a large number of samples at the same time, and cross contamination is easy to occur in the operation process, so that the sample detection result is wrong. The blood coagulation and blood coagulation inhibition test is one of the rapid diagnosis methods, but the defects of low sensitivity, low positive detection rate and non-uniform positive standard of blood coagulation inhibition test by serum samples usually occur when the fecal samples are used for detection experiments, so the blood coagulation inhibition test cannot be widely applied to clinical diagnosis. The method has the characteristics of strong specificity, high sensitivity and high speed, is an immunofluorescence antibody detection technology, and is a detection method for detecting canine parvovirus in a common laboratory. However, the problem of nonspecific staining of the method is not solved at present, and compared with other detection methods, the objectivity of the result judgment of the method is insufficient, and the technical method is relatively complex for a common laboratory.
The inconvenience limits the wide application of the canine parvovirus VP2 gene in clinical detection, and the preparation of the canine parvovirus VP2 protein specific antibody has important significance for clinically and accurately detecting canine parvovirus.
Monoclonal antibodies (monoclonal antibodies, MAb) have been widely used in the diagnosis and treatment of diseases due to their high specificity and high homogeneity, and anti-CPV MAb has become one of the main tools for the treatment and diagnosis of canine parvovirus disease. The specific monoclonal antibody is screened by hybridoma cell technology, and a high-quality raw material is provided for establishing an ELISA method or an immunochromatography method and is used for detecting canine parvovirus antigen or antibody in canine clinical samples.
Disclosure of Invention
In order to solve the problems, the invention provides an application of a monoclonal antibody specifically binding to canine parvovirus VP2 protein in a detection reagent, which specifically comprises the following technical scheme:
in a first aspect, the present invention provides the use of a monoclonal antibody which specifically binds canine parvovirus VP2 protein, said monoclonal antibody comprising the designation V, in a detection reagent H Heavy chain variable region of (2) and designated V L Light chain variable region of (V) H And V L Are each composed of a complementarity determining region and a framework region; the complementarity determining region consists of CDR1, CDR2 and CDR 3;
v of the monoclonal antibody H The amino acid sequence of CDR1 of (1) is shown as SEQ ID No. 1;
v of the monoclonal antibody H The amino acid sequence of CDR2 of (2) is shown as SEQ ID No. 2;
v of the monoclonal antibody H The amino acid sequence of CDR3 of (1) is shown as SEQ ID No. 3;
v of the monoclonal antibody L The amino acid sequence of CDR1 of (1) is shown as SEQ ID No. 4;
v of the monoclonal antibody L The amino acid sequence of CDR2 of (2) is shown as SEQ ID No. 5;
v of the monoclonal antibody L The amino acid sequence of CDR3 of (B) is shown in SEQ ID No. 6.
In a second aspect, the detection reagent is used to detect canine parvovirus antigen or antibody.
Preferably, the detection reagent is a sandwich ELISA antigen detection kit, or a competition ELISA antibody detection kit, or a colloidal gold detection test strip, or a fluorescent microsphere detection test strip, or a latex microsphere detection test strip.
In a third aspect, the monoclonal antibody V H The amino acid sequence of (2) is shown as SEQ ID No. 7 in the sequence table;
the monoclonal antibodyV of antibody L The amino acid sequence of (2) is shown as SEQ ID No. 8 in the sequence table;
v encoding the monoclonal antibody H The nucleotide sequence of (2) is shown as SEQ ID No. 9 in the sequence table;
v encoding the monoclonal antibody L The nucleotide sequence of (2) is shown as SEQ ID No. 10 in the sequence table.
In a fourth aspect, the monoclonal antibody is any one of the following:
(a) V of the monoclonal antibody H And V L Connecting the obtained single-chain antibody;
(b) A fusion antibody comprising the single chain antibody of (a);
(c) V of the monoclonal antibody H And V L Fab of (d);
(d) V of the monoclonal antibody H And V L Is a whole antibody of (a).
In a fifth aspect, the monoclonal antibody is a murine monoclonal antibody.
The embodiment of the invention has the following advantages:
the VP2 protein with high conservation is obtained through insect baculovirus system expression, the specific monoclonal antibody is obtained by immunizing a mouse, and the prepared sandwich ELISA antigen detection kit and fluorescent microsphere detection test strip have no obvious cross reaction on canine distemper virus, canine adenovirus type 1, canine adenovirus type 2, canine coronavirus and canine parainfluenza virus, have good specificity, and lay a foundation for establishing a more accurate and faster canine parvovirus detection method.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the scope of the invention.
FIG. 1 is a SDS-PAGE analysis of purified canine parvovirus VP2 protein provided by the embodiment of the invention;
FIG. 2 is a SDS-PAGE analysis of a monoclonal antibody of the VP2 protein of the purified canine parvovirus provided by the embodiment of the invention;
FIG. 3 is a diagram showing analysis of nucleotide sequence homology of a heavy chain variable region of a monoclonal antibody of canine parvovirus VP2 protein provided by the embodiment of the invention;
FIG. 4 is a nucleotide sequence homology analysis chart of a light chain variable region of a monoclonal antibody of canine parvovirus VP2 protein provided by the embodiment of the invention;
FIG. 5 is an analysis chart of amino acid sequence homology of a heavy chain variable region of a monoclonal antibody of canine parvovirus VP2 protein provided by the embodiment of the invention;
FIG. 6 is an analysis chart of amino acid sequence homology of a monoclonal antibody light chain variable region of canine parvovirus VP2 protein provided by the embodiment of the invention.
Detailed Description
Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 preparation of canine parvovirus VP2 protein
The embodiment provides a preparation method of canine parvovirus VP2 protein, which comprises the following specific steps:
step one, construction of recombinant vector
According to CPV-VP2 sequence (M19296.1) logged in GenBank, codons are optimized to better adapt to insect cell expression, an optimized CPV-VP2 gene is synthesized and connected to a pFastBac ™ 1 vector, bamHI and SalI enzyme cutting sites are selected, the synthesized recombinant plasmid is transformed into DH10Bac engineering bacteria, the three-antibody screening medium plate is coated, blue and white bacterial colonies are grown on the medium after 48 hours, white bacterial colonies are picked and streaked on the three-antibody screening medium plate again, large and round white bacterial colonies are obtained through twice purification, the bacterial colonies are cultured in the three-antibody screening medium, and recombinant plasmids, namely, recombinant shuttle vector Bacmid DNA, are extracted and transfected into insect cells.
Step two, preparation of recombinant plasmid
The sf9 cells were cultured in a 27℃incubator until the cell density reached 8.0X10 5 Taking 1 mug recombinant shuttle vector Bacmid DNA from cells/ml, and passing through Cellfectin ® II Reagent was transfected into sf9 cells. No contamination was observed within 24 hours after transfection, lesions were observed after 96 hours, culture supernatant was collected, centrifuged at 500rpm for 5 minutes to pellet cells and cell debris, and the supernatant was taken as the P1 generation baculovirus. At this time, the virus titer is usually 1X 10 6 ~1×10 7 pfu/ml. The P1 generation virus can be stored at 4deg.C for a short period of time or at-80deg.C for a long period of time after being split-packed in 2% FBS-containing culture solution. Subsequently, the supernatant of sf9 cells was continuously infected with the P1-generation virus to obtain a P2-generation virus, and the P2-generation virus cells and the supernatant were subjected to SDS-PAGE analysis to determine whether the target protein had been normally expressed. And the like, carrying out blind transmission for three generations to obtain the P3 generation virus, and freezing and storing the virus in a refrigerator at the temperature of minus 80 ℃.
Step three, purity identification of recombinant protein
Purifying the recombinant protein obtained in the second step by affinity chromatography, and performing SDS-PAGE electrophoresis on the purified recombinant protein to determine the purity of the protein, wherein the method comprises the following specific steps of:
preparing 12% of separating gel and 5% of concentrated gel. Carefully injecting the separating gel liquid into a gap of a glass plate with the height of 1.5mm, sealing the top with pure water, standing at room temperature for 20min, discarding pure water, adding concentrated gel liquid on the gap, adding a comb with the height of 1cm, carefully inserting the comb, avoiding mixing bubbles in the gel, and solidifying at room temperature for 10min. After the colloid is solidified, fixing the gel in an electrophoresis tank, and adding electrophoresis liquid. Mu.l of the sample was added to 1/4 of the 5 Xloading buffer and mixed well, and boiled at 100℃for 10min. Each well was charged with 20. Mu.l of 80V, and after 20min the voltage was adjusted to 120V. And after 50min, taking out the colloid after the bromophenol blue reaches the bottom of the separating colloid, putting the colloid into coomassie brilliant blue dye solution, oscillating and dyeing for 2h, washing the colloid with clear water, removing the redundant dye solution, then changing the decolorizing solution for decolorizing, changing the decolorizing solution every 30min until the protein strips are clear, and analyzing the protein strips. As shown in FIG. 1, it can be seen that the target band appears near 65kDa, the band is clear, no impurity band is present, and the purification requirement is met.
EXAMPLE 2 preparation of monoclonal antibodies to canine parvovirus VP2 protein
The embodiment provides a preparation method of a monoclonal antibody of canine parvovirus VP2 protein, which comprises the following specific steps:
step one, animal immunization
The canine parvovirus VP2 recombinant protein prepared and purified by the baculovirus expression system in example 1 is used as an immunogen, and is mixed with Freund's complete adjuvant in an equivalent amount, and is fully emulsified. The first immunization dose was 50. Mu.g per BALB/C mouse, followed by 50. Mu.g per mouse by subcutaneous multipoint immunization with Freund's incomplete adjuvant emulsified protein in the same way. Each immunization interval is 2-3 weeks. Collecting blood from mouse tail tip at day 7 after 3 rd immunization, standing at 4deg.C for 30min to separate out serum, diluting the serum with PBS, and treating with the same method as the mouse serum, experimental group and control group OD 450nm The maximum serum dilution ratio is the serum titer of the mice when the ratio is greater than 2.1. Mice were selected for high titers and boosted by intraperitoneal injection of 50. Mu.g of an emulsion of antigen and Freund's incomplete adjuvant in an equal volume of PBS (200. Mu.l). Mice were sacrificed 3 days later and cell fusion was performed.
Step two, cell fusion
The boosted mice were eyeballs were collected and serum was isolated as a positive control. After the mice were sacrificed by cervical fracture, the mice were immersed in 75% ethanol for 10min and fixed on an dissecting table in an ultra clean bench. The abdomen skin was lifted with sterilized forceps, small pieces of skin were cut upward from below the abdomen with sterile scissors, the skin and peritoneum were separated, other viscera were carefully pulled apart, and the spleen was gently removed and placed in a petri dish containing 20ml of incomplete DMEM broth. The spleen cells were blown out in a petri dish by puncturing the spleen from the top with a syringe filled with 20ml of incomplete DMEM broth, penetrating the spleen, gently pushing the syringe, repeating several times until the spleen no longer discolored, and filtering the spleen cells with a filter screen. Counting SP2/0 and spleen cells, mixing the spleen cells with SP2/0 according to the cell number ratio of 8:1, reversing and mixing uniformly, and centrifuging at 1000rpm for 4min; beating the precipitated cells in a water bath at 37 ℃ to uniformly distribute the cells at the bottom of the tube, standing for 1min, adding 1ml of PEG1450 into the centrifuge tube within 1min, standing for 1min, and adding 1ml of incomplete DMEM culture solution preheated at 37 ℃ along the tube wall within 30s to terminate the cell fusion reaction; extending the gun head under the liquid level, adding 1ml of incomplete DMEM for 1min, and repeating the steps until 20ml of incomplete DMEM culture solution is added; slowly adding 30ml of incomplete DMEM culture medium, centrifuging at 800rpm for 4min, discarding supernatant, adding 50ml of incomplete DMEM culture medium, centrifuging at 800rpm for 4min, discarding supernatant, adding HAT culture medium, gently blowing up cells, adding 96-well cell culture plate (200 μl/well), and marking; an indirect ELISA assay was performed 7 days later.
Step three, screening positive hybridoma cells
The hybridoma cell supernatant was taken and screened for positive hybridoma cells by indirect ELISA. Selecting OD 450nm The values were high and only wells of single cell pellet, medium was discarded, 200 μl HT medium was added, cells were blown and counted, about 200 cells were plated in half 96 well plates, the remaining cells were passaged to 48 well plates for continued expansion culture, and frozen. After 7d, carrying out indirect ELISA identification on the monoclonal cells, subcloning again by adopting the method, and selecting OD after 3 times of subcloning 450nm Higher value single cell pellet, as described aboveCloning was performed, if there was a cell well OD 450nm Higher values, cell-free pellet well OD 450nm The value is not higher than that of the negative control, and the strain is considered to be a hybridoma cell strain capable of secreting monoclonal antibodies of canine parvovirus VP2 protein.
Step four, preparation of monoclonal antibody ascites
Injecting the cell strain obtained in the step three into the abdominal cavity of a mouse, culturing the mouse, and extracting ascites from the abdominal cavity of the mouse for purification. The specific operation steps are as follows:
mice were intraperitoneally injected with 500. Mu.l Freund's incomplete adjuvant, and after 24 hours, about 1X 10 was taken 7 The hybridoma cells were injected into the abdominal cavity of the mice, and after 7d, ascites were collected. The antibody was purified using a commercial antibody purification kit, specifically as follows: centrifuging the ascites at 10000rpm for 10min, collecting supernatant, and collecting 20 μl to obtain sample; 60 μl 1M Tris-HCl (pH=9.0) was added to the centrifuge tube; filtering Binding buffer and the partition buffer for standby by using a filter with the diameter of 0.45 mu m; ascites diluted twice by Binding buffer; filling 10ml Binding buffer with a syringe, connecting the syringe to a purification column, removing bubbles, slowly pushing a piston, and removing the storage liquid; sucking 10ml Binding buffer at a flow rate of 1ml/min, and balancing the column; sucking diluted ascites by the injector at a flow rate of 0.2ml/min to combine the antibody with the column; absorbing 10ml Binding buffer, washing out unbound antibody until the effluent liquid is colorless; the antibody bound to the column was eluted by pipetting 5ml Elution buffer and added dropwise to the above Tris-HCl added centrifuge tube, 8 drops/tube. Samples were prepared by sampling 20. Mu.l of each tube for later use, and the purified monoclonal antibodies were identified.
EXAMPLE 3 characterization of monoclonal antibodies to canine parvovirus VP2 protein
The present example provides characterization of monoclonal antibodies to canine parvovirus VP2 protein, including concentration determination, purity characterization, sensitivity characterization, specificity characterization, and subclass characterization:
1. concentration determination of monoclonal antibody to canine parvovirus VP2 protein
And (3) carrying out concentration measurement on the purified monoclonal antibody by adopting a nucleic acid protein concentration measuring instrument, wherein the concentration of the monoclonal antibody is 14mg/ml.
2. Purity identification of monoclonal antibody of canine parvovirus VP2 protein
SDS-PAGE analysis was performed on the purified monoclonal antibodies. As shown in FIG. 2, 2 distinct bands appeared around 25kDa and 50kDa, with an antibody light chain at 25kDa and an antibody heavy chain at 50kDa, with no other bands and purity meeting the expected requirements.
3. Sensitivity identification of monoclonal antibodies to canine parvovirus VP2 protein
Sensitivity identification was performed using an indirect ELISA method. The method comprises the following specific steps: the canine parvovirus VP2 recombinant protein in the first embodiment is diluted to 0.5 mug/ml by using a coating liquid, an ELISA plate is coated according to 100 mug/hole, the washing liquid is used for washing 3 times, and finally the drying is performed. Subjecting the purified monoclonal antibody to gradient dilution, 100 μl/well, incubation at 37deg.C for 1h, washing with washing solution for 3 times, adding HRP-labeled goat anti-mouse IgG diluted 1:5000 times, reacting at 37deg.C for 30min, washing for 3 times, adding substrate color development solution, 50 μl/well, stopping reaction with concentrated sulfuric acid, and measuring OD of each well with enzyme-labeled instrument 450nm And reading the value. At the same time, the culture supernatant of SP2/0 cells is used as a negative control, and monoclonal antibody OD 450nm The value was 2.1 times higher than that of the negative control, and positive was judged. As shown in Table 1, the purified monoclonal antibody was positive even after 51200-fold dilution, and had high sensitivity.
TABLE 1 monoclonal antibody sensitivity identification
4. Specific identification of monoclonal antibodies to canine parvovirus VP2 protein
Specificity was identified by indirect ELISA. The method comprises the following specific steps: and (3) respectively diluting the inactivated canine distemper virus, canine adenovirus type 1, canine adenovirus type 2, canine coronavirus and canine parainfluenza virus to 1 mug/ml by using a coating liquid, coating an ELISA plate according to 100 mug/hole, washing for 3 times by using a working washing liquid, and finally beating to dryness. Diluting the purified monoclonal antibody 10000 times as a singleAnti, 100 μl/well, incubation at 37deg.C for 1 hr, washing with washing solution for 3 times, adding HRP-labeled goat anti-mouse IgG diluted 1:5000 times, reacting for 30min at 37deg.C, washing for 3 times, adding substrate chromogenic solution, 50 μl/well, stopping reaction with concentrated sulfuric acid, and measuring OD of each well with enzyme-labeled instrument 450nm And reading the value. At the same time, the culture supernatant of SP2/0 cells was used as a negative control, and the OD of the monoclonal antibody was used 450nm The value was less than 2.1 times that of the negative control, and was judged as negative. As shown in Table 2, the purified monoclonal antibodies did not react specifically with canine distemper virus, canine adenovirus type 1, canine adenovirus type 2, canine coronavirus and canine parainfluenza virus, indicating that the purified monoclonal antibodies have good specificity.
Table 2 monoclonal antibody specificity identification
5. Monoclonal antibody subclass identification of canine parvovirus VP2 protein
The subclass of monoclonal antibodies to canine parvovirus VP2 protein was detected using an ELISA kit for murine monoclonal antibody class/subclass identification. The results show that the monoclonal antibody of canine parvovirus VP2 protein has a heavy chain of IgG1 type and a light chain of kappa chain.
EXAMPLE 4 monoclonal antibody variable region Gene PCR amplification and sequence identification of canine parvovirus VP2 protein
The embodiment provides a monoclonal antibody variable region gene PCR amplification and sequence identification of canine parvovirus VP2 protein, which comprises the following specific steps:
step one, culturing hybridoma secreting canine parvovirus VP2 protein monoclonal antibody by using RPMI 1640 complete culture medium at 37 ℃ under the condition of 5% carbon dioxide to ensure that the number of the cells reaches 1 multiplied by 10 7 Total RNA in the cells was then extracted using the total RNA extraction kit (purchased from Tiangen).
Step two, designing the specificity upstream and downstream universal primers of the murine heavy chain antibody genes and the light chain antibody genes.
Heavy chain upstream primer: TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC the number of the individual pieces of the plastic,
heavy chain downstream primer: AGGTSMARCTGCAGSAGTCWGG.
Light chain upstream primer 1: CCGTTTGATTTCCA GCTTGGTGCC the number of the individual pieces of the plastic,
light chain upstream primer 2: CCGTTTTATTTCCAGCTTGGTCCC the number of the individual pieces of the plastic,
light chain upstream primer 3: CCGTTTTATTTCCAACTTTGTCCC the number of the individual pieces of the plastic,
light chain upstream primer 4: CCGTTTCAGCTCCAGCTTGGTCCC the number of the individual pieces of the plastic,
light chain downstream primer: GACATTGAGCTCACCCAGTCTCCA.
And thirdly, amplifying the heavy chain and the light chain by using an RT-PCR kit, connecting the heavy chain and the light chain to a pMD18-T cloning vector, and carrying out gene sequencing.
The variable region gene sequence and the amino acid sequence of the monoclonal antibody of the canine parvovirus VP2 protein are as follows:
1. the monoclonal antibody heavy chain variable region nucleotide sequence of canine parvovirus VP2 protein (SEQ ID No. 9):
CAGCTGCAGGAGTCTGGACCTGCGCTGCTGAAGCCTGGAGCTTCAGTGCAGATATCCTGTACGACTCCAGGATACACGATGACAGACTACCACATGAACTGCGTGCAGCAGAGCGATGGAAACAGCGTTGAGTGGATTGCAGATATTGCAAGCAAGCAATGCTCTACTAACTACAACCAGAAGATCAAGCGCAACGCCACATTGAGTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCAGCACGCAGACATCTGAGCACTCTGCAGTCTATTACTGTGCAAGATGGGACTACTATAGTAACTCGTCGGTGCACTACTGCGCGCAAGGCAGCACTCTCACAGTCTCCTCA; as shown in FIG. 3, the results of homology analysis between the heavy chain variable region gene Sequence of the monoclonal antibody and the heavy chain variable region of the mouse immunoglobulin (Sequence ID: KU 257020.1) are shown.
2. Monoclonal antibody light chain variable region nucleotide sequence of canine parvovirus VP2 protein (SEQ ID No. 10):
AGCGTGAGCATTGGCCAGCCGCCGAGCATTAGCTGCACAAGCAGGCAGAGCCTGCTGCATAGCGATGCCAAAACCTATCTGAACTGGCTGCGACAGAGCACGCGGCACAGGACGAAACGCGTGATCTATCTGGTGAGCAAACTGGATAGCGGCGTGCCGGATCGCTTTACCGGCAGCGGCAGCGGCGCCGATTTCGCCCTGAAAATTAGCAGCGTGGACGCGGAAGATCTGGGCATGTATTATTGCTGGCAGCGCACCGAGTTTCCGATTACCTTTGCCAGCGCCACCAAACTG; as shown in FIG. 4, the results of homology analysis between the monoclonal antibody light chain variable region gene Sequence and the mouse immunoglobulin light chain variable region (Sequence ID: KY 038040.1) are shown.
3. The monoclonal antibody heavy chain variable region amino acid sequence of canine parvovirus VP2 protein (SEQ ID No. 7):
QLQESGPALLKPGASVQISCTTPGYTMTDYHMNCVQQSDGNSVEWIADIASKQCSTNYNQKIKRNATLSVDKSSSTAYMELSTQTSEHSAVYYCARWDYYSNSSVHYCAQGSTLTVSS; as shown in FIG. 5, the results of homology analysis between the amino acid Sequence of the heavy chain variable region of the monoclonal antibody and the heavy chain variable region of the mouse immunoglobulin (Sequence ID: AML 31669.1) were shown.
Monoclonal antibody V of canine parvovirus VP2 protein of the embodiment of the invention H And V L Are each composed of a complementarity determining region and a framework region; the complementarity determining regions consist of CDR1, CDR2 and CDR 3. As shown in Table 3, V is a monoclonal antibody against VP2 protein of canine parvovirus H Is a complementary determining region amino acid sequence of (a).
TABLE 3V of monoclonal antibodies to canine parvovirus VP2 protein H Amino acid sequence of Complementarity Determining Region (CDRs)
4. Amino acid sequence of monoclonal antibody light chain variable region of canine parvovirus VP2 protein (SEQ ID No. 8):
SVSIGQPPSISCTSRQSLLHSDAKTYLNWLRQSTRHRTKRVIYLVSKLDSGVPDRFTGSGSGADFALKISSVDAEDLGMYYCWQRTEFPITFASATKL; as shown in FIG. 6, the results of the analysis of the homology between the amino acid Sequence of the monoclonal antibody light chain variable region and the mouse immunoglobulin light chain variable region (Sequence ID: AQQ 81286.1) are shown. As shown in Table 4, V is a monoclonal antibody against VP2 protein of canine parvovirus L Is a complementary determining region amino acid sequence of (a).
TABLE 4V of monoclonal antibodies to canine parvovirus VP2 protein L Amino acid sequence of Complementarity Determining Region (CDRs)
Example 5 preparation of canine parvovirus fluorescent microsphere detection test strip
The preparation method of the canine parvovirus fluorescent microsphere detection test strip of the embodiment comprises the following steps:
step one, preparation of time-resolved fluorescence microsphere marked canine parvovirus VP2 protein monoclonal antibody complex
1. Dilution of time-resolved fluorescent microspheres: time-resolved fluorescent microspheres with a particle size of 200nm were sonicated for 5min, and 100. Mu.l of microspheres were added to 900. Mu.l of MES (50 mmol/L, pH 6.0). Centrifuging at 16000rpm for 10min, removing supernatant, adding 1ml MES heavy suspension microsphere, centrifuging at 16000rpm for 10min again, removing supernatant, and adding MES heavy suspension microsphere;
2. activation of microspheres: weighing 20mg of NHS and EDC respectively, dissolving with a labeling buffer solution, and preparing in situ, namely 20mg/ml of NHS and EDC; adding 10 μl of NHS into the cleaned microsphere, and rapidly mixing; then 5 μl EDC is added into the microspheres, and the microspheres are quickly mixed and incubated for 20min at room temperature;
3. washing to remove residual EDC: centrifuging the activated microsphere at 16000rpm for 10min, removing supernatant, and adding phase 1ml MES to resuspend microsphere; centrifuging at 16000rpm for 10min, discarding supernatant, and adding 1ml MES to resuspend microsphere;
4. coupling of time-resolved fluorescent microspheres with antibodies: adding 0.02mg of canine parvovirus VP2 protein monoclonal antibody, adding activated microspheres, quickly and uniformly mixing, and incubating for 2 hours at room temperature;
5. closing: adding a blocking solution (10% BSA) into the same volume as the measured microspheres, and incubating for 1h at room temperature;
6. removing unbound antibody: centrifuge at 16000rpm for 10min, discard supernatant and add 1ml MES to resuspend microspheres. Repeating twice to remove unbound antibody;
7. re-suspending and re-dissolving: finally, 1ml (0.02M Tris-HCl+20% sucrose+20% trehalose, pH 8.0) of the microspheres are used for resuspension, namely the canine parvovirus VP2 protein monoclonal antibody complex marked by the time-resolved fluorescent microspheres, and the microspheres are placed at 4 ℃ for standby.
Step two, preparation of chicken IgY antibody-microsphere labeled compound
1. Dilution of time-resolved fluorescent microspheres: step 1;
2. activation of microspheres: step one, 2;
3. washing to remove residual EDC: step one, 3;
4. coupling of time-resolved fluorescent microspheres with antibodies: adding 0.01mg of chicken IgY, adding activated microspheres, quickly and uniformly mixing, and incubating for 2 hours at room temperature;
5. closing: step one, 5;
6. removing unbound antibody: step one, 6;
7. re-suspending and re-dissolving: finally, 1ml (0.02M Tris-HCl+20% sucrose+20% trehalose, pH 8.0) is used for resuspension of the microsphere, namely the chicken IgY antibody-microsphere labeled compound, and the microsphere is placed at 4 ℃ for standby.
Step three, preparation of conjugate release pad
1. Pretreatment of conjugate release pad: preparing a 0.02M Tris-HCl binding pad treatment solution containing 5% of sucrose, 5% of trehalose, 0.5% of Chinese medicine Tween-20, 0.1% of casein and pH8.0, putting a glass cellulose membrane into the prepared binding pad treatment solution, requiring the liquid to submerge the paper, shaking at 60rpm on an orbital shaker for 30min, dehydrating at 1200rpm for 14min, and drying at 40 ℃ overnight.
2. Uniformly spraying the prepared time-resolved fluorescence microsphere labeled canine parvovirus VP2 protein monoclonal antibody complex and chicken IgY antibody-microsphere labeled complex on a conjugate release pad by using a gold-labeled gold-spraying instrument, spraying 2.5 mu l of the conjugate release pad every 1cm, then placing the conjugate release pad in a 40 ℃ environment for 16 hours, taking out the conjugate release pad, and preserving the conjugate release pad in a dry environment for later use.
Step four, preparation of sample absorption pad
1. Sample absorbent pad treatment fluid: A0.1M Tris-HCl sample pad treatment solution containing 0.8% BSA (IgG Free), 0.1% casein, 0.6% triton-100, 0.03% PC-300, pH8.0 was prepared.
2. The sample pad was placed in the sample pad treatment solution prepared as described above, requiring the liquid to flood the paper, then shaking at 60rpm for 30min, dehydrating at 1200rpm for 14min on an orbital shaker, and oven-drying at 40 ℃ overnight.
Step five, preparation of nitrocellulose membrane
1. The time-resolved fluorescent microsphere marked canine parvovirus VP2 protein monoclonal antibody complex is coated on a nitrocellulose membrane to form a detection line T, and the chicken IgY antibody-microsphere marked complex is coated on the nitrocellulose membrane to form a quality control line C.
2. The coating process comprises the following steps: diluting the time-resolved fluorescence microsphere marked canine parvovirus VP2 protein monoclonal antibody complex to 0.5mg/ml by using 0.05mol/L phosphate buffer solution with pH7.2, and coating the complex on a detection line T on a nitrocellulose membrane by using a gold mark film-dividing instrument, wherein the coating amount is 1.0 mu L/cm; the chicken IgY antibody-microsphere labeled complex was diluted to 20. Mu.g/ml with 0.01mol/L phosphate buffer at pH7.2, and coated on a quality control line C on a nitrocellulose membrane with a gold-labeled film marker in an amount of 1.0. Mu.l/cm. And (5) drying the coated nitrocellulose membrane for 16 hours at 40 ℃ for standby.
Step six, assembling all the components
Sequentially adhering a sample absorption pad, a conjugate release pad, a nitrocellulose membrane and a water absorption pad on a PVC base plate; the conjugate release pad is covered by the sample absorption pad from the initial end, the nitrocellulose membrane is covered by the conjugate release pad from the initial end in 1/4 area, the tail end of the nitrocellulose membrane is connected with the initial end of the water absorption pad, the initial end of the sample absorption pad is aligned with the initial end of the PVC bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC bottom plate; the nitrocellulose membrane is provided with a detection line T and a quality control line C which are strip-shaped belts perpendicular to the length of the test strip; the detection line T is positioned at one side close to the tail end of the conjugate release pad; the control line C is located on the side of the end remote from the conjugate release pad. Cutting the test paper strip into small strips with the width of 4.05mm by a machine, putting the small strips into a specially-made plastic card shell, sealing the small strips by an aluminum foil bag, and storing the small strips for 12 months at the temperature of 2-30 ℃.
Example 6 specific identification of canine parvovirus fluorescent microsphere detection test strips
The canine parvovirus fluorescent microsphere test strip prepared in example 5 was used to detect the assay specificity of inactivated canine parvovirus, inactivated canine distemper virus, canine adenovirus type 1, canine adenovirus type 2, canine coronavirus, and canine parainfluenza virus. As shown in Table 5, the inactivated canine parvovirus is detected to be positive, the detection results of the other five samples are all negative, no cross reaction exists, and the specificity of the test strip is good.
TABLE 5 specificity identification of canine parvovirus fluorescent microsphere detection test strips
Example 7 preparation of canine parvovirus sandwich ELISA antigen detection kit
The preparation method of the canine parvovirus sandwich ELISA antigen detection kit of the embodiment comprises the following steps:
1. the canine parvovirus sandwich ELISA antigen detection kit of the embodiment comprises the following components: 1) The monoclonal antibody coated with the canine parvovirus VP2 protein is used for capturing a solid-phase carrier ELISA plate of canine parvovirus antigen; 2) Horseradish peroxidase-labeled canine parvovirus VP2 protein monoclonal antibody; 3) A positive antigen; 4) A negative antigen; 5) A substrate color development solution TMB; 6) Wash solution PBST; 7) And (5) stopping liquid is dilute sulfuric acid.
2. The preparation method of the canine parvovirus sandwich ELISA antigen detection kit comprises the following steps:
1) The monoclonal antibody coated with the canine parvovirus VP2 protein is used for capturing a solid-phase carrier ELISA plate of canine parvovirus antigen: after the CB coating solution and the monoclonal antibody are uniformly mixed, 100 mu L/hole is added into an ELISA plate, the coating solution is coated for 16 hours at 4 ℃ overnight, the coating solution is discarded and the plate is washed by a washing solution, 200 mu L/hole of 1% BSA blocking solution is added into the room temperature, the mixture is blocked for 2 hours, 2% sucrose solution is added into the mixture after the blocking solution is discarded, 200 mu L/hole is added, and the mixture is incubated for 1 hour at room temperature, thus obtaining the canine parvovirus VP2 protein coated monoclonal antibody ELISA plate.
2) Monoclonal antibodies to horseradish peroxidase-labeled canine parvovirus VP2 protein were prepared: preparing a solution containing 0.06M sodium periodate and 0.16M glycol, adding horseradish peroxidase solid powder to be marked, adding purified monoclonal antibody to be marked, uniformly mixing, transferring into a dialysis bag, dialyzing with 0.05M carbonate, and dialyzing at 4deg.C for 16-18 hr; taking out the dialyzed antibody solution, adding (5 mg/ml) sodium borohydride solution, standing at room temperature for 2h, loading into PBS with dialysis solution of 0.2M in a dialysis bag again, dialyzing at 4deg.C for 16-18h, taking out the dialyzed solution, adding high-quality glycerol, and packaging for storage.
3) Positive antigen preparation: inactivated canine parvovirus.
4) Negative antigen was prepared: cell culture broth without canine parvovirus.
5) Substrate color development solution TMB: commercial substrate color development solutions.
6) Preparation of wash PBST: A0.01M PBS pH7.2 solution containing 0.1% Tween-20.
7) Preparation of stop solution 2M H 2 SO 4 : adding 28ml sulfuric acid into 900ml deionized water, and fixing volume to 1000ml to obtain 2M H 2 SO 4 。
After assembling the components, the canine parvovirus sandwich ELISA antigen detection kit of the embodiment is obtained.
Example 8 characterization of canine parvovirus sandwich ELISA antigen detection kit
1. Specific identification of the kit in the embodiment of the invention
And detecting the analysis specificity of the inactivated canine distemper virus, the canine adenovirus type 1, the canine adenovirus type 2, the canine coronavirus and the canine parainfluenza virus by using the established sandwich ELISA antigen detection kit. The results show that the detection results of the five samples are all negative, and have no cross reaction, and the method has good specificity. As shown in table 6.
Table 6 kit specific identification
2. The sensitivity identification method of the kit provided by the embodiment of the invention
Inactivated canine parvovirus VP2 protein is diluted to 1000ng/ml, 500ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml, 31.25ng/ml, 15.625ng/ml, 7.812ng/ml, 3.906ng/ml with an established sandwich ELISA antigen detection kit. The results show that the sensitivity of the kit in the embodiment of the invention reaches 7.812ng/ml. As shown in table 7.
Table 7 kit sensitivity identification
3. The kit of the embodiment of the invention identifies the coincidence rate
And confirming 30 clinical sample backgrounds by a virus separation identification-indirect immunofluorescence method, detecting by using an established sandwich ELISA antigen detection kit, and comparing the coincidence rate of the two methods. The results show that the coincidence rate of 11 positive samples and 19 negative samples in 30 samples is 100 percent. As shown in table 8.
Table 8 kit compliance identification
Therefore, the sandwich ELISA antigen detection kit prepared by the monoclonal antibody of the canine parvovirus VP2 protein has high sensitivity, good specificity and high coincidence rate, and lays a foundation for establishing a more accurate and faster canine parvovirus detection method.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting it, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.
Claims (6)
1. The use of a monoclonal antibody specifically binding to canine parvovirus VP2 protein in a detection reagent, wherein the monoclonal antibody comprises a polypeptide designated V H Heavy chain variable region of (2) and designated V L Light chain variable region of (V) H And V L Are each composed of a complementarity determining region and a framework region;
the complementarity determining region consists of CDR1, CDR2 and CDR 3;
v of the monoclonal antibody H The amino acid sequence of (2) is shown as SEQ ID No. 7 in the sequence table;
v of the monoclonal antibody L The amino acid sequence of (2) is shown as SEQ ID No. 8 in the sequence table;
v of the monoclonal antibody H The amino acid sequence of CDR1 of (1) is shown as SEQ ID No. 1;
v of the monoclonal antibody H The amino acid sequence of CDR2 of (2) is shown as SEQ ID No. 2;
v of the monoclonal antibody H The amino acid sequence of CDR3 of (1) is shown as SEQ ID No. 3;
v of the monoclonal antibody L The amino acid sequence of CDR1 of (1) is shown as SEQ ID No. 4;
v of the monoclonal antibody L The amino acid sequence of CDR2 of (2) is shown as SEQ ID No. 5;
v of the monoclonal antibody L The amino acid sequence of CDR3 of (B) is shown in SEQ ID No. 6.
2. The use according to claim 1, wherein the detection reagent is for detecting canine parvovirus antigen or antibody.
3. The use according to claim 1, wherein the detection reagent is a sandwich ELISA antigen detection kit, or a competition ELISA antibody detection kit, or a colloidal gold detection test strip, or a fluorescent microsphere detection test strip, or a latex microsphere detection test strip.
4. The use according to claim 1, wherein,
v encoding the monoclonal antibody H The nucleotide sequence of (2) is shown as SEQ ID No. 9 in the sequence table;
v encoding the monoclonal antibody L The nucleotide sequence of (2) is shown as SEQ ID No. 10 in the sequence table.
5. The use of claim 1, wherein the monoclonal antibody is any one of the following:
(a) V from the monoclonal antibody of claim 1 H And V L Connecting the obtained single-chain antibody;
(b) A fusion antibody comprising the single chain antibody of (a);
(c) V comprising the monoclonal antibody of claim 1 H And V L Fab of (d);
(d) V comprising the monoclonal antibody of claim 1 H And V L Is a whole antibody of (a).
6. The use according to any one of claims 1 to 5, wherein the monoclonal antibody is a murine monoclonal antibody.
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