CN116804186A - Anti-chicken infectious anemia virus monoclonal antibody hybridoma cell strain, monoclonal antibody, reagent or kit and application thereof - Google Patents
Anti-chicken infectious anemia virus monoclonal antibody hybridoma cell strain, monoclonal antibody, reagent or kit and application thereof Download PDFInfo
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- CN116804186A CN116804186A CN202310822501.7A CN202310822501A CN116804186A CN 116804186 A CN116804186 A CN 116804186A CN 202310822501 A CN202310822501 A CN 202310822501A CN 116804186 A CN116804186 A CN 116804186A
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Abstract
The invention belongs to the field of biotechnology detection for livestock, and particularly relates to a hybridoma cell strain of a monoclonal antibody against chicken infectious anemia virus, a monoclonal antibody, a reagent or a kit and application thereof. The invention provides a hybridoma cell strain of a monoclonal antibody against chicken infectious anemia virus, wherein the preservation number of the hybridoma cell strain CIAV Mab-VP2-4 is CGMCCNO:45614. The monoclonal antibody secreted by the hybridoma cell strain is convenient and efficient, has strong operability, and the indirect immunofluorescence kit prepared by the monoclonal antibody has higher specificity and sensitivity, and the popularization and the use of the method are beneficial to further ensuring the purity and the safety of the poultry virus live vaccine in China, and can also be used for clinical detection of CIAV, virus content measurement and epidemiological investigation.
Description
Technical Field
The invention belongs to the field of biotechnology detection for livestock, and particularly relates to a hybridoma cell strain of a monoclonal antibody against chicken infectious anemia virus, a monoclonal antibody, a reagent or a kit and application thereof.
Background
Chicken infectious anemia (Chicken Infectious Anemia, CIA) is an immunosuppressive disease caused by infection with chicken infectious anemia virus (Chicken Infectious Anemia Virus, CIAV), which is characterized mainly by chicken aplastic anemia and atrophy of whole body lymphoid tissues. Since the first isolation of chicken infectious anemia virus in japan, the virus has been isolated in almost all countries that own the poultry industry. Chickens are the only natural host of CIAV, are susceptible to chickens of various strains and ages, and have the morbidity of 20-60 percent generally; the mortality rate is generally 5-10%, and more than 60% can be achieved if the mortality rate is serious. Infection with CIAV alone or in combination with other pathogens will cause immunosuppression of the body, secondary to other diseases. The infectious anemia of chicken can be horizontally transmitted or vertically transmitted, and CIAV brings great economic loss to the world poultry industry.
CIAV is a single-stranded circular negative-strand DNA virus, and only one serotype is used, so that many studies on CIAV detection methods, such as etiology methods, immunological methods and molecular biological detection methods, are performed, and the sensitivity of the detection methods is different. The detection method which is incorporated into the national regulation is the chicken inspection method in the second good year edition of the beast pharmacopoeia of the people's republic of China. In the process of inspecting live vaccine of fowl origin, the method for inspecting CIAV antibody is mainly ELISA method. However, the above-mentioned legal method has drawbacks in that the detection of CIAVELISA antibody is carried out by obtaining chicken serum by a chicken assay, and the time and cost are high.
The ELISA is based on the detection of chicken infectious anemia virus, and monoclonal antibodies with high specificity and high sensitivity to chicken infectious anemia virus are obtained. Therefore, a high-specificity and high-sensitivity hybridoma cell strain for resisting the chicken infectious anemia virus is needed to realize high-sensitivity detection of the chicken infectious anemia virus, and a stable and efficient IFA (indirect immunofluorescence) method for detecting the chicken infectious anemia virus is established on the basis of obtaining the monoclonal antibody and is used for quality detection of a live avian vaccine and diagnosis of chicken infectious anemia virus epidemic disease.
Disclosure of Invention
The invention aims to provide a chicken infectious anemia virus resisting monoclonal antibody hybridoma cell strain, a monoclonal antibody, a reagent or a kit and application thereof, and an indirect immunofluorescence kit for detecting chicken infectious anemia virus, which is prepared from the monoclonal antibody secreted by the chicken infectious anemia virus resisting monoclonal antibody hybridoma cell strain, can simultaneously identify different strains of CIAV, and has good specificity and good sensitivity.
The invention provides a hybridoma cell strain of a monoclonal antibody against chicken infectious anemia virus, wherein the preservation number of the hybridoma cell strain CIAV Mab-VP2-4 is CGMCC NO.45614.
The invention provides the monoclonal antibody secreted by the chicken infectious anemia virus resistant monoclonal antibody hybridoma cell strain or the passage cell strain.
The invention provides a reagent or a kit for detecting chicken infectious anemia virus, which comprises the monoclonal antibody.
The invention provides an indirect immunofluorescence kit, which comprises the monoclonal antibody of claim 3, FITC-labeled goat anti-mouse antibody, sample diluent and sample washing liquid.
Preferably, the sample diluent comprises 9-11 mM phosphate buffer with a pH of 7.2-7.4.
Preferably, the sample washing solution comprises 9-11 mM phosphate buffer solution with pH value of 7.2-7.4.
The invention provides application of the chicken infectious anemia virus resistant monoclonal antibody hybridoma cell strain, the monoclonal antibody, the reagent, the kit or the indirect immunofluorescence kit in the safety detection of the live virus vaccine.
The invention provides application of the monoclonal antibody hybridoma cell strain for resisting the chicken infectious anemia virus or the monoclonal antibody in the technical scheme in preparation of a reagent or a kit for detecting the chicken infectious anemia virus.
The invention has the beneficial effects that: the invention provides a hybridoma cell strain of a monoclonal antibody for resisting chicken infectious anemia virus, a monoclonal antibody, a reagent or a kit and application thereof. The invention provides a hybridoma cell strain of a monoclonal antibody against chicken infectious anemia virus, wherein the preservation number of the hybridoma cell strain CIAV Mab-VP2-4 is CGMCC NO.45614. The chicken infectious anemia virus resistant monoclonal antibody hybridoma cell strain is prepared based on chicken infectious anemia virus VP2 protein, the chicken infectious anemia virus VP2 protein is an important immunogenic protein of chicken infectious anemia virus, the monoclonal antibodies prepared by the chicken infectious anemia virus resistant monoclonal antibody hybridoma cell strain are relatively conserved among different strains of chicken infectious anemia virus, different strains of chicken infectious anemia virus can be identified, and the kit prepared by the monoclonal antibodies does not cross react with viruses such as chicken Egg Drop Syndrome Virus (EDSV) and the like, and has good specificity and sensitivity. The kit can be used for detecting exogenous viruses of chicken infectious anemia viruses in avian live vaccines, and can also be used for clinical identification, virus content measurement and epidemiological investigation of chicken infectious anemia.
Evidence of biological preservation
The hybridoma cell strain CIAVMab-VP2-4 is preserved in China general microbiological culture collection center (CGMCC) in 2023, 5 and 17 days, and the preservation number is CGMCC No.45614, and the preservation unit address is: beijing Chaoyang area North Chenxi Lu No. 1, 3.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a graph showing the protein amount expressed in example 2; wherein M represents a Marker;1 represents the uninduced control (BL 21); 2 represents IPTG induction (BL 21);
FIG. 2 is a graph showing the protein expression level of example 2; m represents Marker;1 represents the supernatant after sonication; 2 represents post-ultrasound precipitation;
FIG. 3 example 2 protein purification scheme; m represents Marker;1 represents a protein before purification; 2 represents a purified protein;
FIG. 4 is a diagram of the detection result of CIAVAV1550 strain detected by the indirect immunofluorescence kit of example 4;
FIG. 5 is a graph showing the detection results of CIAV Cux-1 strain by using the indirect immunofluorescence kit of example 4;
FIG. 6 is a graph showing the detection result of EDSV (127 strains) by using the indirect immunofluorescence kit of example 4;
FIG. 7 is a graph showing the results of the detection of ALV (RAV-1 strain) by the indirect immunofluorescence kit of example 4;
FIG. 8 is a graph showing the results of detection of ILTV (ILT/13 strain) by the indirect immunofluorescence kit of example 4;
FIG. 9 is a diagram showing the results of detection of ARV (Reo 1133 strain) by the indirect immunofluorescence kit of example 4.
Detailed Description
The invention provides a hybridoma cell strain of a monoclonal antibody against chicken infectious anemia virus, wherein the preservation number of the hybridoma cell strain CIAV Mab-VP2-4 is 45614.
In the present invention, the screening method of the chicken infectious anemia virus resistant monoclonal antibody hybridoma cell strain preferably comprises: constructing recombinant expression plasmids from the chicken infectious anemia virus VP2 protein to obtain VP2 protein; the VP2 protein is used for immunizing a mouse to obtain an immunized mouse spleen cell suspension; the immunized mouse spleen cell suspension is fused with SP2/0 cells, cultured and screened to obtain hybridoma cell strains.
The invention constructs the coding gene of the chicken infectious anemia virus VP2 protein into a recombinant expression plasmid; the original vector applied in constructing the recombinant expression plasmid comprises pET28a. The invention prefers VP2 protein to be added with the gene sequences of enzyme cutting sites EcoRI and XhoI, and then restriction enzymes EcoRI and XhoI are utilized to carry out enzyme cutting, purification and recovery to obtain a first enzyme cutting product; preferably, restriction enzymes EcoRI and XhoI are used for carrying out enzyme digestion on the original vector pET30a to obtain a second enzyme digestion product; in the present invention, the recombinant expression plasmid is preferably obtained by ligating the first cleavage product and the second cleavage product with DNALationkit.
After the recombinant expression plasmid is obtained, the recombinant expression plasmid is preferably transformed into BL21 strain for induced expression to obtain an expression product; and purifying the expression product to obtain VP2 protein. The method for purifying the expression product is not particularly limited, and a conventional method can be adopted. The concentration of VP2 protein prepared by the method is more than 0.5mg/mL, the purity is more than 85%, and the protein is named CIAV-VP2. The chicken infectious anemia virus VP2 protein is an immunogen, the length of the chicken infectious anemia virus VP2 protein is 648bp, the chicken infectious anemia virus VP2 protein is highly conserved, and the detection sensitivity of the indirect immunofluorescence kit for detecting chicken infectious anemia virus is improved.
After VP2 protein is obtained, the VP2 protein is used for immunizing mice to obtain immunized spleen cell suspension of the mice. The VP2 protein is preferably emulsified with Freund's complete adjuvant to immunize mice. The immunized mice of the present invention preferably include primary immunization, secondary immunization, third immunization, fourth immunization, and impact immunization in that order. The dose of VP2 protein immunized mice according to the invention is preferably 55-65. Mu.g, more preferably 60. Mu.g, per mouse. In the present invention, the time interval of the primary immunization, the secondary immunization, the third immunization and the fourth immunization is preferably 14 days, and the time interval of the impact immunization from cell fusion is preferably 3 to 5 days, more preferably 3d. The impact immunity is preferably the intraperitoneal injection immunity of immunogen VP2 protein.
After obtaining immunized mice, the present invention separates spleen cells from the immunized mice in vivo and prepares a spleen cell suspension. The spleen cell suspension of the immunized mice is fused with myeloma cells (SP 2/0 cells) to obtain fused cells. The ratio of the cell number of the spleen cell suspension and the myeloma cells is (1-2): (1-2), more preferably 1:1. The cell fusion according to the invention preferably uses the 50% PEG method.
The cell number ratio of the spleen cell suspension to the myeloma cells can be set during fusion, so that the cell fusion efficiency can be improved.
After obtaining the fusion cells, the invention cultures and screens the fusion cells to obtain hybridoma cell strains. The fusion cells of the invention are preferably cultured in HAT medium; after the culture, hybridoma cell lines were selected using ELISA plates. The hybridoma cells secrete monoclonal antibodies against chicken infectious anemia viruses. After obtaining the hybridoma cell strain, the invention preferably utilizes an indirect immunofluorescence detection method to screen monoclonal antibody titer of the hybridoma cell strain. The hybridoma cell strain preferably selected in the invention is subjected to subcloning culture and cryopreservation, and simultaneously meets two conditions, namely, the hybridoma cell strain has good reactivity with full viruses of different strains of CIAV; secondly, the secreted monoclonal antibodies have high titers. According to the invention, the mouse spleen cells with high secreted monoclonal antibody titer are selected for fusion, so that the specificity and sensitivity of monoclonal antibody detection are improved.
The invention provides the monoclonal antibody secreted by the chicken infectious anemia virus resistant monoclonal antibody hybridoma cell strain or the passage cell strain. The invention preferably uses mice to inject hybridoma cell strains of monoclonal antibodies against chicken infectious anemia virus, then collects ascites, and uses Protein-A affinity purification to obtain CIAV monoclonal antibodies.
The invention provides a reagent or a kit for detecting chicken infectious anemia virus, which comprises the monoclonal antibody.
The invention provides an indirect immunofluorescence kit, which comprises the monoclonal antibody, FITC-labeled goat anti-mouse antibody, sample diluent and sample washing liquid.
In the present invention, the sample dilution preferably includes 9 to 11mM phosphate buffer having a pH of 7.2 to 7.4; more preferably 10mM phosphate buffer pH 7.2.
In the invention, the sample washing liquid comprises 9-11 mM phosphate buffer solution with the pH value of 7.2-7.4; more preferably 10mM phosphate buffer pH 7.2.
The invention provides application of the chicken infectious anemia virus resistant monoclonal antibody hybridoma cell strain, the monoclonal antibody, the reagent, the kit or the indirect immunofluorescence kit in the safety detection of the live virus vaccine. In the invention, the fowl virus live vaccine preferably comprises one or more of fowl pox live vaccine, fowl Marek's disease type I and III bivalent live vaccine, fowl newcastle disease live vaccine, fowl encephalomyelitis, fowl pox bivalent live vaccine, fowl infectious bronchitis live vaccine, fowl infectious laryngotracheitis recombinant fowl pox virus genetic engineering vaccine and newcastle disease live vaccine; more preferably, the vaccine is a chicken pox live vaccine, chicken Marek's disease type I and III bivalent live vaccine, chicken newcastle disease live vaccine, avian encephalomyelitis bivalent live vaccine, chicken infectious bronchitis live vaccine, chicken infectious laryngotracheitis recombinant chicken pox virus genetic engineering vaccine and chicken newcastle disease live vaccine. The chicken pox live vaccine is preferably chicken pox live vaccine quail attenuated strain. The chicken Marek's disease type I and III bivalent live vaccine is preferably chicken Marek's disease type I and III bivalent live vaccine 814 strain+HVT Fc-126 clone strain or chicken Marek's disease I, III bivalent live vaccine CVI988+FC126 strain. The chicken newcastle disease live vaccine is preferably a chicken newcastle disease live vaccine Clone30 strain. The avian encephalomyelitis and chicken pox bivalent live vaccine is preferably an avian encephalomyelitis and chicken pox bivalent live vaccine YBF02 strain and a quail attenuated strain; the live vaccine for the infectious bronchitis is preferably a live vaccine H120 strain for the infectious bronchitis; the chicken newcastle disease live vaccine is preferably a chicken newcastle disease live vaccine CS2 strain. When the kit is used for detecting the safety of the live virus vaccine for the poultry, if specific green fluorescence does not appear, the live virus vaccine for the poultry is proved to be safe without infecting chicken infectious anemia virus.
The method for detecting chicken infectious anemia virus by using the indirect immunofluorescence kit preferably comprises the following steps: inoculating a sample to be detected, performing fluorescent staining and judging the result. In the present invention, the sample to be tested is inoculated preferably into MDCC-MSB1 cells (Marek's disease lymphoblastic-like cell line) for adsorption and culture. The time of the adsorption according to the present invention is preferably 50 to 70 minutes, more preferably 60 minutes. The temperature of the culture according to the present invention is preferably 37℃and the time of the culture is preferably 5 to 7 days, more preferably 6 days. The inoculation of the sample to be detected according to the invention has the effect that the CIAV is incubated and proliferated on MSB1 cells. In the present invention, the fluorescent staining preferably includes immobilization, primary antibody addition, washing, fluorescent secondary antibody staining and washing. The primary antibody of the invention is preferably a monoclonal antibody of chicken infectious anemia virus. The fluorescent secondary antibody is preferably FITC-labeled goat anti-mouse IgG. In the invention, when the result is determined that specific green fluorescence appears in the visual field of the inoculated hole, and when the specific green fluorescence is amplified to 200-400 times, the cell nucleus and cytoplasm of the infected cell are colored, and the hole CIAV detection is determined to be positive; when the inoculated wells did not exhibit specific green fluorescence, the wells were judged to be negative for CIAV detection. The color development principle is as follows: in the IFA method, if the sample to be detected contains CIAV, the monoclonal antibody and the secondary antibody are respectively combined on the sample to be detected, and green specific fluorescence is visible under a fluorescence microscope because the secondary antibody carries FITC fluorescence.
The monoclonal antibody secreted by the hybridoma cell strain of the anti-chicken infectious anemia virus monoclonal antibody is based on VP2 protein as an important immunogenic protein, is highly conserved among different strains of chicken infectious anemia virus, can identify the different strains of chicken infectious anemia virus, and does not cross react with viruses such as chicken Egg Drop Syndrome Virus (EDSV). The kit has good specificity.
The invention provides application of the monoclonal antibody hybridoma cell strain for resisting the chicken infectious anemia virus or the monoclonal antibody in the technical scheme in preparation of a reagent or a kit for detecting the chicken infectious anemia virus.
The IFA detection method based on the monoclonal antibody technology, which is established by the invention, can be used for detecting CIAV viruses without cross reaction to other viruses, is convenient and efficient, has strong operability and higher specificity and sensitivity. The popularization and the use of the technical scheme of the invention are beneficial to further ensuring the purity and the safety of the poultry virus live vaccine in China, further improving the vaccine quality, and can also be used for clinical detection of CIAV, virus content measurement and epidemiological investigation.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
EXAMPLE 1CIAVVP2 protein sequence analysis
Different CIAV strains only have one serotype, and different CIAV strain reference sequences are downloaded from NCBI, so that analysis shows that CIAVVP2 protein has higher homology in different strains. VP2 of CIAV Hebei2 strain is selected for comprehensive secondary analysis and antigenicity analysis, and the result shows that CIAVVP2 protein is highly conserved and has extremely low antigenicity difference, and can be used as an alternative fragment of expressed protein. Based on the VP2 sequence of CIAV Hebei2 strain (Genbank accession number: VP2 sequence of MH 186139.1), the nucleotide fragment (648 bp) of full-length VP2 protein is selected, and sequence synthesis is carried out after codon optimization, ecoRI (GAATTC) and XhoI (CTCGAG) enzyme cutting sites are added at the 5 'and 3' ends for cloning the vector according to the secondary structure analysis and antigenicity of the protein sequence. The sequence after addition of the cleavage site was synthesized in Beijing Liuhua large protein research and development center Co., ltd (see SEQ ID NO: 1).
SEQ ID NO. 1CIAVVP2 protein recombinant epitope:
GAATTCATGCACGGGAACGGCGGACAACCGGCCGCTGGGGGCAGTGAATCGGCGCTTAGCCGAGAGGGGCAACCTGGGCCCAGCGGAGCCGCGCAGGGGCAAGTAATTTCAAATGAACGCTCTCCAAGAAGATACTCCACCAGGACCATCAACGGTGTTCAGGCCACCAACAAGTTCACGGCCGTCCCCAACCCCTCACTGCAGAGAGATCCGGATTGGTATCGCTGGAATTACAATCACTCTATCGCTGTGTGGCTGCGCGAATGCTCGCGCTCCCACGCTAAGATCTGCAACTGCGGACAATTCAGAAAACACTGGTTTCAAGAATGTGCCGGACTTGAGGACCGATCAACCCAAGCCTCCCTCGAAGAAGCGATCCTGCGACCCCTCCGAGTACAGGGTAAGCGAGCTAAAAGAAAGCTTGATTACCACTACTCCCAGCCGACCCCGAACCGCAAGAAGGTGTATAAGACTGTGAGATGGCAAGACGAGCTCGCAGACCGAGAGGCCGATTTTCCGCCTTCAGAAGAGGACGCTGGCACCAGCTCAAGCGAAGTCGACGAAGATATAAATTTCGACATCGGAGGAGACAGCGGTATCGTAGACGAGCTTTTAGGAAGGCCTTTCACAACCCCCGCCCCGGTACGTATAGTGCTCGAG。
note that: the EcoRI and XhoI cleavage sites are underlined.
EXAMPLE 2 construction and expression purification of recombinant expression plasmids
1. Construction of recombinant expression plasmids
EcoRI and Xho I cleavage sites were introduced at the 5 'and 3' ends of SEQ ID NO. 1 of example 1, respectively, (cleavage sites were added at both ends of SEQ ID NO. 1 by sequence synthesis) for the preparation of recombinant expression plasmids.
The sequence of SEQ ID NO:1 and plasmid pET28a, to which EcoRI and XhoI cleavage sites were added, were double-digested with restriction enzymes EcoRI and XhoI, respectively, and the fragment recovered by purification and the digested product of the expression vector were ligated with the DNA Ligation Kit to give a recombinant expression plasmid, which was transformed into competent cells (BL 21).
Small amount of expression of CIAV-VP2 recombinant protein
BL21 clones were selected to 1.5mL containing kanamycin resistance after transformation identified as positive by PCRCulturing in a sexual LB liquid medium at 37 ℃ and a rotating speed of 200 r/min; OD of culture to LB culture solution 600 =0.6 to 0.8. The mass concentration of kanamycin in LB liquid medium was 50. Mu.g/mL. IPTG was added to the cultured bacterial liquid to induce, and the final concentration of IPTG in the cultured bacterial liquid was 0.5mM. The induction temperature is 37 ℃, the rotating speed is 200r/min, and the time is 2h. 1mL of the resulting bacterial solution was centrifuged at 12000r/min for 1min, the supernatant was discarded, and the pellet was blown off with 50. Mu.L of 10mM Tris-HCl (pH 8.0) solution (the amount of the buffer added depends on the amount of the cells), and 2 Xloading buffer was added in an equal volume to the buffer, and the pellet was kept at 100℃for 5min, followed by electrophoresis detection. The results of the electrophoresis detection before and after the bacterial liquid induction are shown in FIG. 1, and according to FIG. 1, the specific target band of the recombinant CIAV VP2 protein appears at the position of about 28KD, which indicates that the CIAVVP2 protein expression is successful.
Large scale expression of CIAV-VP2 recombinant proteins
And (3) identifying the transformed BL21 obtained in the step (1) by adopting PCR, and culturing the BL21 identified as positive. Transferring the bacterial liquid obtained by culture into 250mL of kanamycin-resistant LB liquid culture medium according to the volume ratio of 1:50, and shake-culturing at 37 ℃ and the rotating speed of 200r/min until the OD of the LB liquid culture medium obtained by culture is reached 600 =0.6 to 0.8. The mass concentration of kanamycin in LB liquid medium was 50. Mu.g/mL. And adding IPTG into the LB liquid medium obtained by culture for induction. The final concentration of IPTG in the LB liquid medium was 0.5mM. The IPTG induction temperature was 37℃and induction time was 3h. And (3) carrying out centrifugal bacteria collection on the obtained product, wherein the centrifugal rotating speed is 8000r/min, and the centrifugal time is 6min. Centrifuging and discarding the supernatant to obtain thalli; the obtained thalli are subjected to ultrasonic bacteria breaking, and the specific process is as follows: the obtained cells were subjected to ultrasonic disruption with a power of 500W after being blown off with 30ml of 10mM Tris-HCl (pH 8.0) solution, and subjected to ultrasonic disruption 180 times each for 5s at an interval of 5s, followed by the next ultrasonic disruption.
The electrophoresis detection is carried out on the ultrasonic crushed product, and the specific process is as follows: taking 100 mu L of the ultrasonic bacterial suspension, centrifuging for 10min at the rotating speed of 12000r/min, retaining 50 mu L of supernatant and the obtained precipitate after centrifugation, and blowing off the obtained precipitate by using 50 mu L of 10mM Tris-HCl (pH 8.0) solution. SDS-PAGE was performed on the supernatant and the solution obtained after the pellet was blown off, respectively, and as a result, a large amount of the target protein was detected in the pellet (see "2" in FIG. 2), indicating that the recombinant expression form was inclusion body expression (FIG. 2).
The CIAV-VP2 protein expressed in the bacterial cell precipitate is purified by the following method: suspending the precipitate obtained by ultrasonic centrifugation in 20-30 ml of 10mM Tris-HCl (pH 8.0) solution, and standing for 10min; centrifuging at 12000r/min for 10min, and transferring supernatant into another tube for storage. For precipitation, re-suspending the precipitate with 20-30 mL of 10mM Tris-HCl (pH 8.0) solution, and standing for 10min; centrifuging at 12000r/min for 10min, and discarding supernatant to obtain first precipitate; for the first precipitate, the above steps of re-suspending and centrifuging were repeated once to obtain a second precipitate. And adding a small amount of 10mM Tris-HCl (pH 8.0) solution into the second precipitate to resuspend the precipitate, adding 5-10 mL10mM Tris-HCl (pH 8.0) solution containing 8M urea to dissolve protein, centrifuging for 10min at 12000r/min, collecting the supernatant, taking 50 mu L of sample, and carrying out SDS-PAGE electrophoresis detection, wherein the detection results before and after protein purification are shown in FIG. 3. Purified protein concentration >0.5mg/mL, purity >85% was estimated by SDS-PAGE gel scanning analysis using BSA (bovine serum albumin) as a standard, and designated CIAV-VP2.
EXAMPLE 3 preparation of monoclonal antibodies
1. Immunization of mice
After the CIAV-VP2 purified protein and Freund's complete adjuvant are emulsified according to the volume ratio of 1:1, 4 SPFBALB/c female mice are subcutaneously injected (namely, primary immunization) according to the amount of 60 mu g CIAV-VP2 protein/mouse. And 2 weeks, 4 weeks and 6 weeks after the primary immunization, a booster immunization is injected subcutaneously, with an immunization amount of 30 mug per mouse, with a primary immunization and a primary booster immunization interval 14d, a primary booster immunization and a secondary booster immunization interval 14d, and a secondary booster immunization and a tertiary booster immunization interval 14d, respectively. 10 days after the third booster immunization, the orbit was bled and serum titers were measured by indirect ELISA. Mice with high serum titers (ELISA antibody titers of 1:12800) were selected and immunized by intraperitoneal injection with 50. Mu.g of immunogen for one time, 3 days after immunization, for cell fusion.
2. Cell fusion assay
And (3) taking the spleen of the mice immunized in the step (1) in a sterile manner, preparing spleen cell suspension, taking equal amount of the immune spleen cell suspension and SP2/0 cells, and carrying out cell fusion by adopting a conventional 50% PEG method. The fused cells obtained after fusion were placed in 5 96-well plates, and selectively cultured in HAT medium (purchased from Sigma).
3. Cloning and screening of hybridoma cells
Cultures from step 2 were screened in 96-well plates by ELISA plates coated with CIAV-VP2 protein and His tag protein, respectively.
(1) The screening method for the cultures in the 96-well plates in step 2 by ELISA plates coated with CIAV-VP2 proteins is as follows:
1) ELISA plates were coated. The CIAV-VP2 protein purified in example 2 was diluted with sodium carbonate-sodium bicarbonate buffer at pH 9.6 to a final concentration of 2. Mu.g/mL. 100 mu L of diluted CIAV-VP2 protein is added to each hole of the ELISA plate, and the ELISA plate is subjected to overnight at the temperature of 4 ℃; the cells were then washed 3 times with PBST (PBS containing 0.05% Tween-20).
2) And (5) sealing. After coating the ELISA plates, they were blocked with PBS containing milk at a mass concentration of 2%. ELISA plates were incubated with 200. Mu.L of PBS containing 2% milk per well in a incubator at 37℃for 2h and washed 3 times with PBST (PBS containing 0.05% Tween-20).
3) And (5) incubating the primary antibody. After blocking, hybridoma cell culture supernatant obtained after cell fusion in step 2, negative control (SP 2/0 culture supernatant), blank control (PBS), and positive control (CIAV positive serum was 1000-fold diluted with PBS) were added as primary antibodies, and incubated at 37℃for 1h in incubator.
4) And (5) washing. After incubation of the primary antibody, the ELISA plate of step (3) was washed 3 times with PBST (PBS containing 0.05% Tween-20).
5) And (5) incubating the secondary antibody. After washing, goat anti-mouse IgG/HRP diluted 20000 times with PBS was added as secondary antibody to ELISA plates, and incubated at 37℃for 1h at 100. Mu.L/well.
6) And (5) washing. ELISA plates after incubation of the secondary antibodies in step (5) were washed 3 times with PBST (PBS containing 0.05% Tween-20).
7) And (5) developing. Adding color development solution (citric acid buffer solution containing 1%A solution and 10% B solution, solution A containing TMB prepared by DMSO to 1% by mass concentration, solution B containing H0.1% by mass concentration) 2 O 2 Aqueous solution) of 100. Mu.L/well, the development time was about 5 minutes.
8) Each well was terminated by adding 50. Mu.L of a stop solution (containing 2M sulfuric acid).
9) And (5) reading. Absorbance was measured at two wavelengths (450 nm,630 nm) and the data was recorded.
(2) The screening method for the culture in the 96-well plate through the His tag protein coated ELISA plate is the same as that of the CIAV-VP2 protein.
Through detection, 3ELISA positive hybridoma cell strains of 1# and 3# and 4# are selected for further screening. The test results are shown in Table 1: the high absorbance value for the target protein and the low value for the tag protein indicate that the hybridoma cell strain has high titer. As can be seen from Table 1, the titer of the 4# hybridoma cell line was the highest.
TABLE 1 ELISA screening results for hybridoma cell lines
4. Indirect immunofluorescence assay
And (3) taking the supernatant of 3 positive hybridoma cell strains (1#, 3# and 4#) obtained by ELISA screening in the step (3) as a primary antibody, screening a cell strain with good reactivity between the 4# and full viruses of different CIAV strains by adopting an indirect immunofluorescence method, and the cell strain is named CIAV VP2 protein hybridoma cell strain CIAV Mab-VP2-4. Selecting a cell strain with positive immunofluorescence detection, and subcloning by adopting a limiting dilution method until the positive rate of the cell clone antibody reaches 100%. And (3) performing expansion culture on the positive cell strain with the positive rate reaching 100% after subcloning, and then preserving the positive cell strain in liquid nitrogen.
The indirect immunofluorescence detection method comprises the following steps:
(1) Positive virusPreparation of the plates: the CIAV different strains (Table 2) virus solutions were diluted to 100TCID with 1640 culture solution containing 2% new born calf serum 50 0.1mL, inoculating chicken lymphocyte MDCC-MSB1 with the sample to be tested, inoculating the sample with toxic cells (each well contains 100 TCID) 50 Virus) was plated onto polylysine treated 96-well cell plates, each strain was inoculated with 4 wells, 100 μl/well. Meanwhile, MDCC-MSB1 cells are used for comparison, and after a period of time, the cells are fixed for 15min by cold methanol, and fluorescent staining is carried out.
TABLE 2 information table of different strains of CIAV and strains for control
The above strains are from the national collection center for veterinary microorganisms (toxin) species, CVCC, (please see the national institute for veterinary medicine, the national institutes of culture preservation, the national institutes of microorganisms, the national institutes of culture, the national institutes of veterinary microorganisms, the first edition of the Chinese and English contract, 1992, pages 132, 135 and 143). The Chicken Infectious Anemia Virus (CIAV) AV1550 strain is found in the Chinese veterinary medicine information net, the strain preservation and the bacterial (toxic) species retrieval. Cux-1 strain was maintained by the laboratory and was a vaccine strain, kong Yibo, zhang Xingxiao, jiang Shijin, etc. (CAV nucleic acid vaccine and preliminary study of immune effects; the Committee of the society of livestock and poultry disease, shandong society of livestock and poultry disease, the first academic conference of the Committee of the society of livestock and poultry disease, 2009:163-166). Avian Leukemia Virus (ALV) RAV-1 strain was from the Chinese veterinary drug administration (Mao Yaqing, wang Jia, wu Tao, wang Zhe, jiang Taozhen, expression of avian leukemia Virus p27 protein in E.coli and preparation of polyclonal antibodies, journal of veterinary medicine, 2013, 11, 61-66).
(2) Dyeing
1) Fixing. After the positive virus plate is prepared, discarding the cell culture solution of the 96-well plate, adding 250 mu LPBS to each well, lightly washing the cell surface for 1 time, discarding PBS as much as possible, adding 100 mu L of cold methanol to each well, standing at room temperature for 10-15 min, discarding the methanol, and naturally airing for 2-5 min.
2) Primary antibodies (monoclonal antibodies) are added. After fixation of the 96-well plate, the cell surface was washed 1 time with PBS (pH 7.2), supernatants of hybridoma cell lines (1#, 3# and 4#) producing the monoclonal antibodies to be examined were diluted 10-fold with PBS, and added to CIAV virus positive cell plates (washed 1 time with PBS before use), 50. Mu.L per well, and protected from light at 37℃for 1 hour.
3) And (5) washing. After primary antibody was applied to the 96-well plate, the monoclonal antibodies in the plate wells were discarded, washed 5 times with PBS, 0.3mL of wash solution was added to each well, and the washing was performed with gentle shaking.
4) And (5) staining by using a fluorescent secondary antibody. The wash was discarded as much as possible, 50. Mu.L of fluorescent-labeled goat anti-mouse IgG diluted with PBS was added to each well, and the mixture was protected from light at 37℃for 1 hour. The volume ratio of the fluorescence-labeled goat anti-mouse IgG after PBS dilution is 1:100-1:200.
5) And (5) washing. The method is the same as 3).
6) And (5) observation and result judgment. The cells had intact cell morphology as observed under a fluorescent inverted microscope with blue excitation light (wavelength 490 nm). When specific green fluorescence appears in the visual field of the inoculation hole and is amplified to 200-400 times, and the cell nucleus and cytoplasm of the infected cell are colored, the CIAV detection is judged to be positive. When the inoculated wells did not exhibit specific green fluorescence, the visual field was darkened, demonstrating that the cells were not infected, and the wells were judged to be negative for CIAV detection. The results show that: the 4# hybridoma cell strain has good reactivity with the whole virus of different strains of CIAV.
5. Identification of hybridoma cells
(1) Culture characteristics
Using 1640 culture solution containing 10% -15% of fetal bovine serum at 37 ℃ and 5% of CO 2 Culturing in an incubator, and examining the cell morphology of the hybridoma cell line by a microscope, wherein the cell morphology is consistent, and the cell morphology is consistent, which indicates that the cell state is good.
(2) Purity test
According to the current method of annex of ' Chinese animal pharmacopoeia ' (compiled by Chinese veterinary drug dictionary Committee), chinese people's republic of China, animal pharmacopoeia, 2020 edition, chinese agricultural press 2020, the invention is abbreviated as ' Chinese animal pharmacopoeia ', and the results of sterility test, mycoplasma test and exogenous virus test are all in accordance with the regulations.
(3) Nuclear examination
The hybridoma cells cultured for 24 hours were examined for chromosome number by colchicine method, and whether the chromosome characteristics were in accordance with the staining characteristics of the hybridoma cells was observed, and the results were in accordance with expectations.
(4) Preparation of ascites BALB/C mice of 8-10 weeks old were intraperitoneally injected with pristane, each 0.5mL. 7-10 days later mice were intraperitoneally injected with hybridoma cell strain 10, which is a 4# hybridoma cell strain 6 ～10 7 After 7-10 days, observing the state of the mice, extracting ascites of the mice when the abdomen of the mice is obviously enlarged and the action is inconvenient, centrifuging for 10min at 3000r/min, taking the supernatant, and storing at-40 ℃. If ascites is regenerated, the ascites can be collected again after 2-3 days, and the ascites is affinity purified by Protein-A to obtain the mouse anti-CIAV monoclonal antibody.
(5) Determination of ascites titer
The ascites of the mice injected with the 4# hybridoma cell line was diluted to 1:16000 in a double ratio from 1:100, the fluorescent antibody titer of the ascites was measured according to the "4 indirect immunofluorescence detection method" in example 3, the fluorescent antibody titer of the ascites of the hybridoma cell line 4# was 1:2000, and the hybridoma cell line was named CIAVVP2 protein hybridoma cell line CIAVMab-VP2-4, with a preservation number of CGMCC No.45614.
Example 4 Indirect immunofluorescence kit for detecting CIAV (cytokine induced killer) and application
The kit comprises a monoclonal antibody of anti-CIAV secreted by hybridoma cell strain CIAV Mab-VP2-4, a commercial FITC-labeled goat anti-mouse antibody (purchased from Sigma company, cat# F9006), a sample diluent and a washing solution. The dilutions and washes were each 10mM Phosphate Buffer (PBS) pH 7.2.
The kit comprises the following steps and judgment standards for detecting chicken infectious anemia viruses:
1. sample inoculation
100 mu L of the sample to be detected is inoculated to a full-grown chicken lymphocyte MDCC-MSB1, and after 2-3 days of culture at 37 ℃, toxic cells are paved on a 96-well cell plate treated by polylysine.
2. Fluorescent staining and result determination
(1) Fixing: after 1h, the cell culture solution of the 96-well cell plate is discarded, the cell surface is lightly washed 1 time by adding 0.3mLPBS (pH 7.2) to each well, PBS is discarded as much as possible, then 0.2mL of cold methanol is added to each well, the mixture is kept at room temperature for 15min, the methanol is discarded, and the mixture is naturally dried (5 min).
(2) Adding an antibody: after natural air drying, the cell surface was washed 1 time with PBS (pH 7.2), and then 50. Mu.L IAV monoclonal antibody was added to each well and allowed to act at 37℃for 1 hour. The added CIAV monoclonal antibody is diluted with PBS (pH 7.2-7.4) at a dilution volume ratio of 1:100.
(3) Washing: the CIAV monoclonal antibody was discarded, washed 5 times with PBS (pH 7.2), 0.3mL of wash solution was added to each well, and the wash was gently swirled.
(4) And (5) staining by using a fluorescent secondary antibody. The wash was discarded as much as possible, 50. Mu. LFITC-labeled goat anti-mouse IgG was added to each well, and the mixture was allowed to act at 37℃for 1 hour. The added FITC-labeled goat anti-mouse IgG is diluted with PBS (pH 7.2-7.4) at a volume ratio of 1:100.
(5) Washing: the method is the same as (3).
(6) Observation and determination: the cells had intact cell morphology as observed under a fluorescent inverted microscope with blue excitation light (wavelength 490 nm). When specific green fluorescence appears in the visual field of the inoculated hole and the amplification is 200-400 times, the cell nucleus and cytoplasm of the infected cell are colored, and the hole CIAV detection is judged to be positive. When the inoculated wells did not exhibit specific green fluorescence, the visual field was darkened, demonstrating that the cells were not infected, and the wells were judged to be negative for CIAV detection.
3. Specificity test of kit
The specificity of the indirect immunofluorescence method was determined by examining the cell staining of the infectious laryngotracheitis virus (ILTV) of chickens, avian Leukemia Virus (ALV), avian infectious laryngotracheitis virus (ILTV), avian orthoreovirus (ARV) of table 2 using the indirect immunofluorescence kit according to the established procedures of "1. Sample inoculation" and "2. Fluorescence staining and result determination" in example 4.
The detection results show that the method can specifically identify and detect different CIAV strains (fig. 4-5), the reaction results are positive, and the reaction results with EDSV (fig. 6), ALV (fig. 7), ILTV (fig. 8) and ARV (fig. 9) are negative, so that the established method has good specificity, and the indirect immunofluorescence kit can be applied to the specific detection of CIAV.
4. Sensitivity test of kit
Dilution of CIAV (AV 550 strain) to 80TCID 50 100 μl, based on which a double dilution to 40TCID was performed 50 /100μL、20TCID 50 /100μL、10TCID 50 /100μL、5TCID 50 /100μL、2.5TCID 50 /100μL、1.25TCID 50 /100μL、0.625TCID 50 100. Mu.L. The above 8 dilutions of samples were inoculated with a single layer of MDCC-MSB1, 100. Mu.L/well, 4 replicates each. Set 1640 group simultaneously as negative control. The CIAV was tested according to the procedure and criteria for "1. Sample inoculation" and "2. Fluorescent staining and results determination" in example 4.
Results CIAV detection results for each dose gradient were that when CIAV infection dose was 5TCID or greater 50 The result is positive when the virus is present.
The detection method of Cux-1 strain is the same as that of AV1550 strain, and the detection result is the same when CIAV infection dose is more than or equal to 5TCID 50 The result is positive when the virus is present.
The result shows that the lowest detection limit of CIAV pollution of the kit is 5TCID 50 。
5. Preliminary application of kit
The kit is applied to exogenous virus inspection of the avian live vaccines, 10 important species of the avian live vaccines produced by domestic production enterprises are selected in the experiment, CIAV detection is carried out according to the kit of the patent, and serological inspection of CIAV pollution is carried out on the selected avian live vaccines according to three parts of Chinese animal pharmacopoeia 2020 edition. PBS group was also set as negative control, and CIAV (Cux-1 strain) infected group as positive control. As can be seen from Table 3, the results of the serological test and the test using the kit are consistent, and the selected live vaccine for poultry has no CIAV pollution, the negative control is CIAV test negative, and the positive control is CIAV test positive.
TABLE 3 detection results of domestic Enterprise vaccine Using the kit of the present invention
In summary, the anti-CIAV monoclonal antibody disclosed by the invention is based on CIAVVP2 protein as an important immunogenic protein of CIAV, and is prepared by highly conserving between different strains of CIAV, and can be used for identifying different strains of CIAV at the same time without cross reaction with viruses such as chicken Egg Drop Syndrome Virus (EDSV). The kit has good specificity. The kit can be used for exogenous virus detection (cytocheck method) of CIAV in avian virus live vaccine, and can also be used for clinical detection and epidemiological investigation of CIAV.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (8)
1. The hybridoma cell strain of the monoclonal antibody for resisting the chicken infectious anemia virus is characterized in that the preservation number of the hybridoma cell strain CIAV Mab-VP2-4 is CGMCC NO.45614.
2. The monoclonal antibody secreted by the hybridoma cell strain or the passage cell strain of the chicken infectious anemia virus resistant monoclonal antibody according to claim 1.
3. A reagent or kit for detecting chicken infectious anemia virus, comprising the monoclonal antibody of claim 2.
4. An indirect immunofluorescence kit, comprising the monoclonal antibody of claim 3, a FITC-labeled goat anti-mouse antibody, a sample diluent, and a sample wash.
5. The indirect immunofluorescence kit of claim 4, wherein the sample diluent comprises 9-11 mM phosphate buffer at a pH of 7.2-7.4.
6. The indirect immunofluorescence kit of claim 4, wherein the sample wash solution comprises 9-11 mM phosphate buffer at a pH of 7.2-7.4.
7. Use of the chicken infectious anemia virus resistant monoclonal antibody hybridoma cell line of claim 1 or the monoclonal antibody of claim 2 or the reagent or kit of claim 3 or the indirect immunofluorescence kit of any one of claims 4-6 for detecting the safety of live avian virus vaccines.
8. Use of the monoclonal antibody hybridoma cell strain against chicken infectious anemia virus according to claim 1 or the monoclonal antibody according to claim 2 for preparing a reagent or a kit for detecting chicken infectious anemia virus.
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