CN108866008B - Monoclonal antibody for resisting koi herpesvirus, cell strain and application thereof - Google Patents
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Abstract
The invention discloses a preparation method and application of a monoclonal antibody for resisting koi herpesvirus, wherein the monoclonal antibody for resisting koi herpesvirus prepared by the invention is prepared from a monoclonal antibody with a preservation number of CCTCC NO: the titer of the monoclonal antibody generated by the secretion of the mouse hybridoma cell strain of C201887 is 1: 1.28X 105(ii) a The hybridoma cell strain has high activity, can identify koi herpesvirus structural protein, and has strong specificity. And on the basis, a sandwich ELISA detection method for detecting koi herpesvirus is established, the minimum detection concentration is 3.923ng/mL, and the monoclonal antibody is utilized to establish an indirect sandwich ELISA kit for detecting koi herpesvirus, can be used for detecting large-scale koi herpesvirus, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a monoclonal antibody for resisting koi herpesvirus, a cell strain and an application thereof.
Background
The carp herpesvirus type 3, also called Koi Herpesvirus (KHV), is a pathogen causing Koi herpesvirus disease (KHVD) of carps, Koi carps and variants thereof, has a mortality rate of more than 80 percent, and causes huge economic loss to the carp and Koi breeding industries in various national regions all over the world. The cyprinus carpio herpesvirus disease is a fish disease determined at the end of the 20 th century and a disease seriously threatening the safety of cyprinus carpio and cyprinus carpio breeding industry, is classified as a disease which needs to be declared by the world animal health Organization (OIE), and is classified as a second type of epidemic disease in China. Since the first discovery of koi herpesvirus disease in the uk in 1996, the disease has been fulminating in germany, belgium, france, poland, japan, taiwan, vietnam, iran, and other countries.
The koi herpesvirus belongs to the order of herpesviridae, the family of herpesviridae, the genus cyprinid herpesviridae, is a double-stranded DNA virus with an envelope, the diameter of a virus particle is 167-200 nm, the genome is 295kbp in total length, 164 open reading frames are coded in total, and the koi herpesviridae is known to have the largest genome. To date, studies have been made on the functions associated with various structural proteins of koi herpesvirus. The result of identification by the biological mass spectrometry technology shows that the KHV contains 46 structural proteins, including 16 envelope proteins, 3 capsid proteins, 2 cortical proteins and 25 unknown proteins. The research on the immunogenic protein of CyHV-3 shows that structural proteins ORF25, ORF65, ORF81, ORF84, ORF148 and ORF149 have good immunogenicity, a DNA vaccine designed by taking ORF81 as a target gene shows higher immune protection rate in an experiment, and a DNA vaccine developed aiming at the protein ORF25 also proves that the DNA vaccine has good immune protection effect in a fish body experiment.
Because the separation of KHV cells has certain difficulty, the cell separation is not beneficial to the amplification of a large number of viruses and the preparation of virus antibodies, so that a serological detection method is difficult to develop, the conventional KHV detection mainly adopts a PCR method, but the PCR detection has the problems of omission and false positive, and is mostly only used for qualitative detection of the viruses. At present, no report related to the monoclonal antibody with strong specificity for resisting koi herpesvirus exists.
Disclosure of Invention
The invention aims to provide a cell strain secreting monoclonal antibodies against koi herpesvirus.
The invention also aims to provide a monoclonal antibody for resisting the koi herpesvirus.
Still another object of the present invention is to provide the use of the above cell strain and monoclonal antibody.
The technical scheme adopted by the invention is as follows:
a hybridoma cell strain D-IB7IB4 secreting monoclonal antibody against Koi herpesvirus is preserved in Wuhan university Collection with the preservation number of CCTCC NO. C201887.
A monoclonal antibody for resisting Koi herpesvirus is generated by the hybridoma cell strain with the preservation number of CCTCC NO. C201887.
Furthermore, the subtype of the monoclonal antibody is IgG2b type.
A kit of a monoclonal antibody for resisting Koi herpesvirus comprises the monoclonal antibody, a solid phase carrier coated with a polyclonal antibody for resisting Koi herpesvirus, a horseradish peroxidase-labeled goat anti-mouse antibody, an enzyme substrate reaction solution, a positive control substance, a negative control substance, a washing solution and a reaction termination solution.
A sandwich ELISA detection method for detecting koi herpesvirus comprises the following steps:
1) adding the sample to an ELISA (enzyme-Linked immuno sorbent assay) ELISA plate coated with a rabbit polyclonal antibody against Koi herpesvirus;
2) discarding liquid in the hole, diluting the monoclonal antibody, and adding the diluted monoclonal antibody to an enzyme label plate for incubation;
3) discarding liquid in the holes, and spin-drying and washing;
4) diluting a horse radish peroxidase-labeled goat anti-mouse antibody, and adding the diluted goat anti-mouse antibody to an enzyme label plate for incubation;
5) discarding liquid in the holes, and spin-drying and washing;
6) adding substrate reaction liquid of enzyme, and developing in dark;
7) adding a substrate termination solution of the enzyme, and measuring the virus concentration 1-5 minutes after terminating the reaction;
the above methods are not used for diagnosis and treatment of diseases.
Further, the dilution multiple in the step 2) is 3000-5000 times.
Further, the incubation condition in the step 2) is incubation for 0.5-2.5 hours at 34-39 ℃;
the monoclonal antibody is applied to preparation of a koi herpesvirus resisting kit.
The invention has the beneficial effects that:
1. the hybridoma cell strain obtained by screening can be used for preparing a large amount of monoclonal antibodies for resisting koi herpesviruses; the titer of the obtained monoclonal antibody is 1: 1.28X 105(ii) a Can identify the koi herpesvirus structural protein, has strong specificity and has the characteristics of rapidness and sensitivity.
2. The lowest detection concentration of the sandwich ELISA method for detecting the koi herpesvirus is 3.923 ng/mL. The detection method provides favorable conditions for inspection and quarantine of Koi herpesvirus by import and export inspection and quarantine organizations.
Drawings
FIG. 1 shows that the monoclonal antibody against Koi herpesvirus prepared by the invention can specifically react with protein in Koi herpesvirus.
FIG. 2 is an indirect immunofluorescence plot of virus-uninfected CCB cells.
FIG. 3 is an indirect immunofluorescence plot of virus infected CCB cells.
Detailed Description
A hybridoma cell strain D-IB7IB4 secreting monoclonal antibody against Koi herpesvirus is preserved in Wuhan university Collection with the preservation number of CCTCC NO. C201887.
A monoclonal antibody for resisting Koi herpesvirus is generated by the hybridoma cell strain with the preservation number of CCTCC NO. C201887.
Preferably, the subtype of the monoclonal antibody is IgG2b type.
A kit of a monoclonal antibody for resisting Koi herpesvirus comprises the monoclonal antibody, a solid phase carrier coated with a polyclonal antibody for resisting Koi herpesvirus, a horseradish peroxidase-labeled goat anti-mouse antibody, an enzyme substrate reaction solution, a positive control substance, a negative control substance, a washing solution and a reaction termination solution.
A sandwich ELISA detection method for detecting koi herpesvirus comprises the following steps:
1) adding the sample to an ELISA (enzyme-Linked immuno sorbent assay) ELISA plate coated with a rabbit polyclonal antibody against Koi herpesvirus;
2) discarding liquid in the hole, diluting the monoclonal antibody, and adding the diluted monoclonal antibody to an enzyme label plate for incubation;
3) discarding liquid in the holes, and spin-drying and washing;
4) diluting a horse radish peroxidase-labeled goat anti-mouse antibody, and adding the diluted goat anti-mouse antibody to an enzyme label plate for incubation;
5) discarding liquid in the holes, and spin-drying and washing;
6) adding substrate reaction liquid of enzyme, and developing in dark;
7) adding a substrate termination solution of the enzyme, and measuring the virus concentration 1-5 minutes after terminating the reaction;
the above methods are not used for diagnosis and treatment of diseases.
Preferably, the dilution multiple in the step 2) is 3000-5000 times.
Preferably, the dilution factor in step 2) is 4000.
Preferably, the incubation condition in the step 2) is incubation for 0.5-2.5 hours at 34-39 ℃;
the monoclonal antibody is applied to preparation of a koi herpesvirus resisting kit.
The present invention will be further described with reference to the following examples.
Example 1 preparation of monoclonal antibody against Koi herpesvirus
1. Preparation of antigens
CCB (common carp brain cell) is koi herpesvirus sensitive cell line, and when 80% of CCB cells are paved on a cell culture bottle, the virus titer is 106TCID50The KHV-GZ1301 virus strain is inoculated in CCB in a/ml manner, infected cells are placed in an incubator at the temperature of 22 ℃, and when the cells show obvious pathological effect, daily observation shows that: when a large amount of vacuoles appear in cells and the cells are fused, the cells are repeatedly frozen and thawed at the temperature of minus 80 ℃, the cells are cracked to release virus particles, virus suspension is collected, the virus suspension is purified by a 20-66% sucrose density gradient ultracentrifugation method, and the cryprinus carpiod is confirmed by electron microscope observationAfter herpesvirus storage of the samples at-80 ℃.
2. Immunization of mice
The mice used for immunization were female BALB/c mice of SPF grade 5 weeks old. And (3) injecting the purified koi herpesvirus serving as an antigen into each mouse subcutaneously for basic immunization, wherein the volume ratio of the purified virus (50 micrograms) to Freund's complete adjuvant is 1:1 mixing and injecting subcutaneously; after 2 weeks booster, the volume ratio of purified virus (50 μ g) to Freund's incomplete adjuvant was 1:1 mixing and injecting subcutaneously; then boosting for 1 time every 2 weeks, injecting subcutaneously, and injecting purified virus subcutaneously with 50 microgram/one; 2 weeks after the 4 th immunization, the mouse was subjected to an impact of purified virus (50. mu.g) on the abdominal cavity, and 3 days later, the mouse was sacrificed by cervical dislocation, and the spleen was removed under aseptic conditions to prepare immune splenocytes for cell fusion.
3. Cell fusion
Conventional cell fusion techniques: fusing splenocytes of an immune mouse and SP2/0 myeloma cells in a logarithmic growth phase according to the quantity ratio of 10:1, adding 1ml of preheated PEG, standing for 30-60s, slowly dripping 5ml of preheated DMEM culture solution along the tube wall, gradually increasing the speed, adding 15ml of preheated DMEM culture solution, and then adding 20ml of SP2/0 myeloma cell culture solution. The supernatant was discarded by centrifugation, the fused cells were added to HAT medium, and the mixed cell suspension was plated on a 96-well cell culture plate in an amount of 100 ul/well.
Preparing HAT culture medium: 50-fold concentrated HAT (2ml, GIBCO Co.) and special grade fetal bovine serum (20ml, GIBCO Co.) were added to high-glucose DMEM medium (80ml, hyclone Co.) and mixed.
4. Screening and cloning of hybridoma cells
After the fused cells are cultured for 10 days, collecting culture supernatant, and performing indirect ELISA screening by taking the koi herpesvirus purified by gradient centrifugation as an antigen. Positive and negative sera were used as controls. Cloning the screened positive hybridoma cell strain by a limiting dilution method. First, positive well viable cells were accurately counted, diluted 10-fold with DMEM medium, added to a 96-well cell plate on which cells had been plated, and cultured in a cell incubator at 37 ℃. And (3) selecting hybridoma cell holes which are positive in 2 times of continuous detection, and continuously cloning for 2-3 times by using a limiting dilution method.
The positive mouse hybridoma cell strain D-IB7IB4 has been preserved in the Wuhan university preservation center (CCTCC for short, the preservation number is CCTCC NO. C201887, and the address is the Wuhan university preservation center in the Wuhan district, Wuhan city, Hubei province) at 4 months and 18 days in 2018.
5. Induction of ascites
Taking 6-8 weeks old female BALB/c mice, and injecting sterile liquid paraffin 0.5 ml/mouse in the abdominal cavity; after 1 week, 1X 10 intraperitoneal injection7One positive hybridoma cell/one; 7-10 days after inoculating the hybridoma cells, observing that the abdomen of the mouse is obviously enlarged, pumping ascites, centrifuging at 3000rpm for 10min, collecting supernatant, and freezing at-80 ℃ for later use.
Example 2: characterization of monoclonal antibodies against koi herpesvirus
1. Identification of ascites titer of monoclonal antibody
The method comprises the following steps: and (3) identifying the ascites titer of the monoclonal antibody by adopting an indirect ELISA method.
As a result: the ascites titer of the monoclonal antibody is 1: 1.28X 105And the result shows that the hybridoma cell strain has the capacity of secreting high-titer antibodies.
2. Subtype identification of monoclonal antibodies
The method comprises the following steps: typing kit Using Southern Biotech in the United states (SBA Clonotyping)TMSystem/HRP) according to the specification, and identifying the Ig subtype of the monoclonal antibody according to the invention.
As a result: the subtype of the monoclonal antibody is IgG2b type.
3. Specificity analysis of monoclonal antibodies
Immunoblotting (western blotting): separating proteins of the purified koi herpesvirus through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferring the proteins on the gel to a nitrocellulose membrane (NC membrane with the aperture of 0.20 mu m) by an electrotransformation method, and sealing the gel in 0.01MPBS solution containing 10% of skimmed milk powder by mass and volume at 4 ℃ for overnight; after washing with PBS-T (in PBS solution containing 0.05% by volume of Tween-20), adding ascites (monoclonal antibody 1: 1000 diluted in PBS) of the mice, and incubating for 1h at 37 ℃; after washing with PBS-T (in PBS solution containing 0.05% by volume of Tween-20), horseradish peroxidase (HRP) -labeled goat anti-mouse IgG is used as a secondary antibody, and incubation is carried out at 37 ℃ for 1 h; after washing, color development was observed.
As a result: as shown in figure 1, the monoclonal antibody against Koi herpesvirus prepared by the invention can specifically react with protein in Koi herpesvirus, and the monoclonal antibody is specific to Koi herpesvirus.
Indirect immunofluorescence: the common carp brain cells (CCB cells) are subcultured into a cell culture plate, and after the cells grow to about 80% of monolayer confluency, the cells are infected with KHV GZ1301 strain viruses; after 5 days of virus infection, completely absorbing the culture medium in the cell culture plate, washing with 0.01mol/L PBS for 3 times, adding a methanol solution precooled at the temperature of minus 20 ℃ and incubating at the temperature of minus 20 ℃ for 30min, absorbing the methanol, and drying at the room temperature for 1 h; add 100. mu.L 1 per well: incubating a 1000-diluted mouse anti-KHV monoclonal antibody at 37 ℃ for 1h, washing PBST for 3 times, adding 100 mu L of goat anti-mouse IgG-FITC labeled fluorescent secondary antibody diluted at a ratio of 1:3000, and incubating at 37 ℃ for 1 h; PBST was washed 3 times, and finally, nuclear dye PI was added for 5min, and the results were observed under confocal microscope. The negative control was normal CCB cells not infected with virus.
As a result: as shown in FIGS. 2-3, the results of the indirect immunofluorescence experiments show that the prepared monoclonal antibody can enable CCB cells infected with KHVGZ1301 strain to generate specific fluorescence, and no fluorescence signal is observed in normal CCB cells not infected with virus, which indicates that the prepared monoclonal antibody can specifically recognize corresponding protein on KHV GZ1301 virions in infected CCB.
Example 3 ELISA kit for detecting Koi herpesvirus
The ELISA kit for detecting the koi herpesvirus comprises the following components: the monoclonal antibody prepared in example 1; a solid phase carrier coated with polyclonal antibody of anti koi herpesvirus; horse radish peroxidase-labeled goat anti-mouse antibody (purchased from sigma); a substrate reaction solution of an enzyme; a positive control; negative control; washing liquid; and (4) reaction terminating liquid.
Wherein, the preparation of rabbit polyclonal antibody against koi herpesvirus and the embedding of ELISA lath: using the purified koi herpesvirus recombinant expression protein as an antigen, and carrying out multi-point injection on the antigen to immunize rabbits for 4 times; and purifying the serum to obtain the polyclonal antibody for resisting the koi herpesvirus. The purified rabbit anti-koi herpesvirus polyclonal antibody was diluted to a concentration of 2. mu.g/ml with a coating buffer (0.05mol/L carbonate buffer, pH 8.2), and coated with a 96-well ELISA plate (Corning, USA) at 100. mu.L/well and stored at 4 ℃ for 1 month.
Reagent: preparation of 10 × PBST: NaCl: 80g, Na2HPO4·12H2O：29g，KH2PO4: 2g, KCl: 2g, Tween-20: 5ml, adding distilled water to 1000ml, and storing at 4 ℃; substrate reaction solution of enzyme: substrate color developing solution A, namely 13.6g of sodium acetate, 1.6g of citric acid, 0.3ml of 30% hydrogen peroxide and distilled water added to 500 ml; substrate color B solution which comprises 0.2g of disodium ethylene diamine tetraacetate, 0.95g of citric acid and 50ml of glycerol, 0.15g of TMB is dissolved in 3ml of DMSO, distilled water is added to 500ml, and when in use, equal amount of AB solution is measured according to the requirement and is uniformly mixed for use, and the substrate color B solution is prepared on site; washing liquid: the preparation method is consistent with the PBST preparation method; reaction termination solution: formulation 1M H with distilled water2SO4。
The positive control is purified koi herpesvirus solution, and the negative control is fish cell suspension, normal tissue homogenate or uninfected koi serum.
Example 4: sandwich ELISA detection method for detecting koi herpesvirus
The method comprises the following steps: 1. adding a sample to be detected into an ELISA plate coated with rabbit anti-KHV polyclonal antibody; diluting the purified virus (the concentration of the virus is 3mg/ml) with 0.01mol/l PBS at a ratio of 10 times to serve as a positive control, and taking the serum of the non-immunized normal koi as a negative control;
2. discarding liquid in the holes, drying, diluting a second antibody (namely the monoclonal antibody for resisting Koi herpesvirus prepared by the invention) by 5000 times with a diluent, adding the diluted antibody to an enzyme label plate, and incubating for 1.5 hours at 35 ℃;
3. discarding liquid in the hole, spin-drying, and washing with washing liquid for 3 times;
4. diluting a goat anti-mouse antibody marked by horseradish peroxidase according to a ratio of 1:5000, adding the diluted goat anti-mouse antibody to an enzyme label plate, and incubating for 1 hour at 37 ℃;
5. discarding liquid in the hole, spin-drying, and washing with washing liquid for 3 times;
6. adding substrate reaction liquid of enzyme, and developing in a dark place at 37 ℃ for 15-30 minutes;
7. adding 50 mu l of termination solution into each hole in sequence to terminate the reaction;
8. the optical density (OD value) of each well was measured at a wavelength of 450nm with a microplate reader within 5 minutes after the termination of the reaction.
Example 5: sandwich ELISA detection method for detecting koi herpesvirus
The method comprises the following steps: 1. adding a sample to be detected into an ELISA plate coated with rabbit anti-KHV polyclonal antibody; diluting the purified virus (the concentration of the virus is 3mg/ml) with 0.01mol/l PBS at a ratio of 10 times to serve as a positive control, and taking the serum of the non-immunized normal koi as a negative control;
2. discarding liquid in the holes, drying, diluting a second antibody (namely the monoclonal antibody for resisting Koi herpesvirus prepared by the invention) by 3000 times with a diluent, adding the diluted antibody to an enzyme label plate, and incubating for 0.5 hour at 39 ℃;
3. discarding liquid in the hole, spin-drying, and washing with washing liquid for 3 times;
4. diluting a goat anti-mouse antibody marked by horseradish peroxidase according to a ratio of 1:5000, adding the diluted goat anti-mouse antibody to an enzyme label plate, and incubating for 1 hour at 37 ℃;
5. discarding liquid in the hole, spin-drying, and washing with washing liquid for 3 times;
6. adding substrate reaction liquid of enzyme, and developing in a dark place at 37 ℃ for 15-30 minutes;
7. adding 50 mu l of termination solution into each hole in sequence to terminate the reaction;
8. the optical density (OD value) of each well was measured at a wavelength of 450nm with a microplate reader within 5 minutes after the termination of the reaction.
Example 6: sandwich ELISA detection method for detecting koi herpesvirus
The method comprises the following steps: 1. adding a sample to be detected into an ELISA plate coated with rabbit anti-KHV polyclonal antibody; diluting the purified virus (the concentration of the virus is 3mg/ml) with 0.01mol/l PBS at a ratio of 10 times to serve as a positive control, and taking the serum of the non-immunized normal koi as a negative control;
2. discarding liquid in the holes, drying, diluting a second antibody (namely the monoclonal antibody for resisting Koi herpesvirus prepared by the invention) by 4000 times with a diluent, adding the diluted antibody onto an enzyme label plate, and incubating for 1 hour at 37 ℃;
3. discarding liquid in the hole, spin-drying, and washing with washing liquid for 3 times;
4. diluting a goat anti-mouse antibody marked by horseradish peroxidase according to a ratio of 1:5000, adding the diluted goat anti-mouse antibody to an enzyme label plate, and incubating for 1 hour at 37 ℃;
5. discarding liquid in the hole, spin-drying, and washing with washing liquid for 3 times;
6. adding substrate reaction liquid of enzyme, and developing in a dark place at 37 ℃ for 15-30 minutes;
7. adding 50 mu l of termination solution into each hole in sequence to terminate the reaction;
8. the optical density (OD value) of each well was measured at a wavelength of 450nm with a microplate reader within 5 minutes after the termination of the reaction.
As a result: as shown in Table 1, the sandwich ELISA method of the present invention can specifically detect Koi herpesvirus, and the equation of the standard curve can be drawn as y (-6.91+77.79x)/(1+0.3x-0.12 x) according to the OD values corresponding to the standards in Table 12) And r is 0.99956. The OD value is more than or equal to 0.14, and the positive result is judged, so that the minimum detection concentration of the virus can be calculated to be 3.923ng/ml through an equation.
Table 1 test results of sandwich ELISA method for detecting Koi herpesvirus (OD value is more than or equal to 0.14 is positive)
Example 7 sensitivity and specificity test of Sandwich ELISA kit for detecting Koi herpesvirus
Grass Carp Reovirus (GCRV), spring carp virus (SVC), frog virus-3 (FV3) and 20 serum samples which are positive to KHV through PCR detection are detected by using the sandwich ELISA kit established by the invention.
As a result, the detection results of GCRV, SVC and FV3 were found to be negative (OD value <0.1), while the detection results of 20 KHV positive samples were found to be positive (OD value >0.5), indicating that the sandwich ELISA method has good specificity (Table 2).
TABLE 2 Sandwich ELISA specificity test results
OD450Positive > 0.14, OD450Is negative < 0.14
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (5)
1. A hybridoma cell strain D-IB7IB4 secreting monoclonal antibody against Koi herpesvirus is preserved in Wuhan university Collection with the preservation number of CCTCC NO. C201887.
2. A monoclonal antibody against Koi herpesvirus produced by the hybridoma cell strain with the preservation number of CCTCC NO. C201887 as claimed in claim 1.
3. The monoclonal antibody of claim 2, wherein the subtype of the monoclonal antibody is IgG2b type.
4. A kit of monoclonal antibody for resisting Koi herpesvirus is characterized in that the kit contains the monoclonal antibody of claim 2, a solid phase carrier coated with polyclonal antibody for resisting Koi herpesvirus, a horse radish peroxidase-labeled goat anti-mouse antibody, enzyme substrate reaction liquid, a positive control, a negative control, a washing liquid and a reaction termination liquid.
5. Use of the monoclonal antibody of any one of claims 2-3 in preparation of a kit for detecting Koi herpesvirus.
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| Detection of koi herpesvirus (KHV) using a monoclonal antibody against Cyprinus carpio IgM;Yingying Li et al;《Arch Virol》;20171231;第2381-2385页 * |
| Production of monoclonal antibody against ORF72 of koi herpesvirus isolated in Taiwan;Chien Tu et al;《Folia Microbiol》;20141231;第159-165页 * |
| 锦鲤疱疹病毒病免疫学检测方法-单克隆抗体技术研究进展;于慧等;《科学养鱼》;20160330(第3期);第54-55页 * |
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