CN115725511A - Hybridoma cell strain R2McAb2A1, monoclonal antibody secreted by same and application - Google Patents
Hybridoma cell strain R2McAb2A1, monoclonal antibody secreted by same and application Download PDFInfo
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Abstract
The invention discloses a hybridoma cell strain R2McAb2A1, wherein the hybridoma cell R2McAb2A1 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation date of 2022 years, 7 months and 27 days, and the preservation number is CCTCC NO: C2022205. The invention also discloses a monoclonal antibody secreted by the hybridoma cell strain R2McAb2A1. The invention also discloses application of the monoclonal antibody in preparation of a rabbit hemorrhagic disease virus type2 detection kit. The invention successfully prepares a hybridoma cell strain R2McAb2A1 by a hybridoma technology, and a monoclonal antibody secreted by the hybridoma cell strain and rabbit hemorrhagic disease virus type2 (RHDV 2) VP60 protein generate specific immunoreaction; and through indirect ELISA test, westernblot test and hemagglutination inhibition test, the monoclonal antibody has high titer and good specificity, and can effectively identify rabbit hemorrhagic disease virus type 1 (RHDV 1) strains and RHDV2 strains.
Description
Technical Field
The invention relates to the technical field of immunity, in particular to a hybridoma cell strain R2McAb2A1, a monoclonal antibody secreted by the same and application of the monoclonal antibody.
Background
Rabbit Hemorrhagic Disease Virus (RHDV) belongs to the family of Caliciviridae (Caliciviridae) and the genus Rabbit virus (logvirus), and is an infectious disease which causes acute hemorrhagic disease in adult rabbits after infection, namely Rabbit viral hemorrhagic disease (RHD), also called Rabbit fever disease. The rabbit fever comprises classical rabbit fever and rabbit fever type2, and mainly causes bleeding of the respiratory system of the rabbits, congestion, swelling, bleeding and liver necrosis of parenchymal organs. Rabbit hemorrhagic disease type2 (RHDV 2), i.e., lepidoptera 2. The exploration of the rapid detection and monitoring method technology of classical rabbit plague and rabbit plague type2 has important practical significance, thereby becoming an important research direction and content for preventing and controlling the rabbit plague at present.
The RHDV2 is reported to be different from the classical RHDV strain, has wider infection range, can not only infect all lagomorphs, but also infect and cause death of young rabbits within 30d, and has quick transmission and high death rate. Therefore, as the biggest rabbit breeding country and rabbit meat export country in China, the research of developing the RHDV2 specific diagnosis method is very urgent.
The RHDV virion capsid is composed of 180 chemically identical units, each monomer being the major capsid protein VP60 with a molecular weight of about 60 kD. VP60 can be assembled to form virus-like particles RHDV VLPs, which are virus immunoprotective antigens and play an important role in the immune response of animal organisms to induce virus infection. Research shows that the difference of different subtype VP60 proteins of RHDV also determines the difference of the different subtype VP60 proteins in heredity, antigen and epidemiological diversity and immunological response. However, VP60 genes of isolates of different subtypes of RHDV are highly conserved, and the homology of nucleotides and amino acids is over 90%, so that the difficulty in research of differential diagnosis methods is increased. The same is true between the classical RHDV (RHDV 1) and RHDV2 strains, which are difficult to identify effectively in practical production.
Disclosure of Invention
The invention aims to solve the problem that the RHDV1 type and the RHDV2 type of rabbit hemorrhagic disease virus are difficult to effectively distinguish in the prior art, and provides a hybridoma cell strain R2McAb2A1, a monoclonal antibody secreted by the hybridoma cell strain and application of the monoclonal antibody.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a hybridoma cell strain R2McAb2A1, wherein the hybridoma cell R2McAb2A1 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation date of 2022 years, 7 months and 27 days, and the preservation number is CCTCC NO: C2022205.
The invention also provides the monoclonal antibody secreted by the hybridoma cell strain R2McAb2A1.
Preferably, the monoclonal antibody is a specific monoclonal antibody of rabbit hemorrhagic disease virus type 2VP60 protein, and the amino acid sequence of the rabbit hemorrhagic disease virus type 2VP60 protein is shown as SEQ ID N0.1.
The invention also provides application of the monoclonal antibody in preparation of a rabbit hemorrhagic disease virus type2 detection kit.
The invention has the following beneficial effects: the invention successfully prepares a hybridoma cell strain R2McAb2A1 by a hybridoma technology, and the secreted monoclonal antibody generates specific immunoreaction with rabbit hemorrhagic disease virus type2 (RHDV 2) VP60 protein; the detection of indirect ELISA test, westernblot test and hemagglutination inhibition test proves that the monoclonal antibody has high titer and good specificity, and can effectively identify rabbit hemorrhagic disease virus type 1 (RHDV 1) strains and RHDV2 strains.
Drawings
FIG. 1 is the expression and purification diagram of recombinant proteins pET-32a-R1VP60 and pET-32a-R2VP 60;
FIG. 2 is a Western blot identification chart of recombinant proteins pET-32a-R1VP60 and pET-32a-R2VP 60;
FIG. 3 is a diagram showing the determination of ascites titer of monoclonal antibody secreted by hybridoma cell R2McAb2A 1;
FIG. 4 is a diagram showing the purification and identification of monoclonal antibodies secreted by hybridoma R2McAb2A 1;
FIG. 5 is an electron micrograph of RHDV1 and RHDV2 virions;
FIG. 6 is a Werternblot identification of monoclonal antibodies secreted by hybridoma cells R2McAb2A 1;
FIG. 7 is a chromosome map of SP20 cells and hybridoma cells R2McAb2A 1;
FIG. 8 is a graph showing the results of hemagglutination inhibition tests on RHDV2 hemagglutination and monoclonal antibodies secreted from hybridoma cell R2McAb2A1.
The hybridoma cell strain R2McAb2A1 related in the invention is preserved in China Center for Type Culture Collection (CCTCC) and has the address of university in Wuhan, wuhan and China; the preservation date is 2022 years, 7 months and 27 days, the preservation number is CCTCC NO: C2022205, and the hybridoma cell strain is classified and named as a hybridoma cell strain R2McAb2A1.
Detailed Description
The invention is further described with reference to the following figures and specific examples.
Example 1 establishment of hybridoma cell line R2McAb2A1 and preparation of monoclonal antibody
RHDV1 in this example is RHDV1 SCH04 strain (NCBI accession number: KX 844830), and RHDV2 is RHDV2SCCN03 strain (NCBI accession number: MW 178245). RHDV1 SCH04, RHDV2SCCN03, and SP2/0 myeloma cells were obtained from Sichuan university of agriculture animal quarantine laboratories, and 8 female SPF-grade BALB/c mice, about 6-7 weeks old, were purchased from Duoduoshu laboratory animals Co.
The HRP-labeled mouse anti-HIS monoclonal antibody, the HRP-labeled goat anti-rabbit IgG, the HRP-labeled rabbit anti-mouse IgG, the Ni-NTAHis labeled Protein purification kit and the Protein G pre-loaded gravity column are purchased from Shanghai GmbH of biological engineering; the SDS-PAGE gel preparation kit is purchased from Beijing Solaibao science and technology Limited; PEG 1500, 50 XHAT and 50 XHT were purchased from Sigma; mouse monoclonal antibody subclass identification kit was purchased from Proteintech.
Prokaryotic expression and purification of pET-32a-R1VP60 and pET-32a-R2VP60
1.1 designed RHDV1 and RHDV 2VP60 amplification primers by SnapGene according to the VP60 gene sequences of RHDV1 SCH04 strain (accession number: KX 844830) and RHDV2SCCN03 strain (accession number: MW 178245) in NCBI, and added with SalI and HindIII restriction sites at the upstream and downstream primers respectively. The primer sequence R1-VP60-F, R1-VP60-R, R-VP 60-F, R2-VP60-R is shown in SEQ ID N0.3 to SEQ ID N0.6 in sequence.
1.2 RHDV1 and RHDV2 virus RNA extraction, reverse transcription
Respectively grinding liver tissues of rabbits infected with RHDV1 and RHDV2 and died of illness, repeatedly freezing and thawing for three times, adding physiological saline, transferring to a centrifugal tube of 1.5mL, extracting total RNA of the tissues, and storing cDNA obtained by reverse transcription at-20 ℃ for later use.
1.3 PCR amplification of RHDV 1VP60 gene fragment and RHDV 2VP60 gene fragment
And (3) respectively using the obtained cDNA of the RHDV1 and RHDV2 virus RNA as a template, and respectively using the corresponding upstream and downstream primers in the step 1.1 to amplify the VP60 genes of the RHDV1 and the RHDV 2. After the reaction, the PCR product was detected by 1% agarose gel electrophoresis. And (3) PCR reaction system: 2 XTaq PCR MasterMix 25. Mu.L, upstream and downstream primers 2. Mu.L each, cDNA 2. Mu.L, ddH 2 O19. Mu.L. PCR reaction procedure: pre-denaturation at 94 ℃ for 3min; 30 cycles of 94 ℃ 30s,55 ℃ 20s,72 ℃ 2min; 10min at 72 ℃. And respectively recovering VP60 PCR products of the RHDV1 and the RHDV2, and storing at-20 ℃ for subsequent enzyme digestion ligation tests.
1.4 Construction of prokaryotic expression vectors of pET-32a-R1VP60 and pET-32a-R2VP60
And (3) carrying out double enzyme digestion on the VP60 PCR products of the RHDV1 and the RHDV2 purified and recovered in the step 1.3 and the expression vector PET-32a by using Sal I and Hind III respectively, and carrying out enzyme digestion for 3-5h at the temperature of 37 ℃. The cleavage system is shown in Table 1.
TABLE 1 double digestion reaction System
The RHDV 1VP60 gene fragment and the RHDV 2VP60 gene fragment which are recovered by enzyme digestion are respectively connected with the pET-32a carrier part by using T4 DNA ligase, and the connection system is shown in table 2.
TABLE 2 prokaryotic expression vector ligation System
Ligation was carried out at 16 ℃ for 12h. 10. Mu.L of the two ligation products were used to transform DH 5. Alpha. Competent cells, respectively. Colonies grown on the transformed LB ampicillin plates were selected and inoculated in LB ampicillin liquid medium for about 12 hours. And (3) respectively taking the bacteria liquid after the amplification culture as a template to carry out PCR amplification, wherein the reaction procedure and the system are the same as the VP60 amplification procedure and the system in the step 1.3. And sequencing the bacteria liquid with positive PCR result. The recombinant plasmids with correct sequencing are named pET-32a-R1VP60 and pET-32a-R2VP60 respectively.
The recombinant plasmids pET-32a-R1VP60, pET-32a-R2VP60 and pET-32a empty vectors are transformed into BL21 competent cells according to a conventional method, single colonies are respectively selected and inoculated in an LB liquid culture medium containing Amp, shaking culture is carried out at 37 ℃ until OD600nm is about 0.5, IPTG with the final concentration of 1mmol/L is added, and continuous culture and induction are carried out for about 6 hours. And (3) centrifugally collecting thalli, performing ultrasonic disruption in PBS, centrifuging the disrupted product for 10min at 8000r/min, collecting supernatant and precipitate, and finally performing solubility analysis on the recombinant protein through SDS-PAGE electrophoresis. Purifying the two recombinant proteins according to the specification of the Ni-NTAHis tag protein purification kit, and collecting eluent; and (3) carrying out purity identification on the purified protein after SDS-PAGE and Coomassie brilliant blue staining and decoloring. Finally, carrying out amino acid sequencing on the purified recombinant protein, wherein the amino acid sequence of the recombinant protein expressed by pET-32a-R2VP60 is shown as SEQ ID N0.1; the amino acid sequence of the recombinant protein expressed by pET-32a-R1VP60 is shown as SEQ ID N0.2.
The results show that the recombinant proteins expressed by pET-32a-R1VP60 and pET-32a-R2VP60 are in the form of inclusion bodies. After dissolving and precipitating by using Binding buffer containing 8M urea, two recombinant proteins are purified by a nickel column affinity chromatography method and are concentrated and renatured by using an ultrafiltration tube. SDS-PAGE analysis showed that both recombinant proteins were 79kD in size, as shown in FIG. 1, and FIG. 1-A shows the expression of the pET-32a-R1VP60 recombinant protein, where M: pre-stained protein marker;1: pET-32a-R1VP60 thallus precipitation; 2: pET-32a-R1VP60 thalli supernatant; 3: pET-32a no-load thallus precipitation; 4: pET-32 a-unloaded cell supernatant. FIG. 1-B shows the purification of pET-32a-R1VP60 recombinant protein, wherein M: pre-stained protein marker;1: the purified pET-32a-R1VP60 protein. FIG. 1-C shows the expression of the pET-32a-R2VP60 recombinant protein, where M: pre-stained protein marker;1: pET-32a no-load thallus supernatant; 2: pET-32a no-load thallus precipitation; 3: pET-32a-R2VP60 thalli supernatant; 4: pET-32a-R2VP60 bacterial precipitation. FIG. 1-D shows the purification of pET-32a-R2VP60 recombinant protein, where M: pre-stained protein marker;1: the purified pET-32a-R2VP60 protein. The electrophoresis result shows that the pET-32a-R1VP60 and pET-32a-R2VP60 prokaryotic expression recombinant protein conforms to the expected size and has good purification effect.
Westernblot identification of pET-32a-R1VP60 and pET-32a-R2VP60 recombinant proteins
And (3) identifying the antigenicity of the pET-32a-R1VP60 and pET-32a-R2VP60 recombinant proteins prepared in the step 1 by using a Western blot test. Performing SDS-PAGE electrophoresis on the purified recombinant protein, then transferring the protein to a polyvinylidene fluoride membrane, sealing the protein for 2h at normal temperature by using 5% skimmed milk powder, then incubating the protein for 2h by using a mouse anti-HIS antibody (1. The results of Westrnbot are shown in FIG. 2, where M: pre-stained protein marker;1: pET-32a-R1VP60 protein; 2: pET-32a-R2VP60 protein. Westrn blot results show that both recombinant proteins of pET-32a-R1VP60 and pET-32a-R2VP60 can react with an anti-HIS antibody of a mouse marked by HRP, and show that both recombinant proteins are successfully expressed.
3. Immunization of mice
The concentration of the pET-32a-R2VP60 recombinant protein after purification is measured by using a nucleic acid protein instrument, and the concentration of the pET-32a-R2VP60 recombinant protein is adjusted to be 0.5mg/mL by using PBS for standby. 8 female SPF-grade BALB/c mice were divided into 5 groups and immunized by injecting pET-32a-R2VP60 recombinant protein. The control group was 3, and was not immunized, and was continuously fed with a feeder layer for cell fusion. The experimental group of mice was immunized and the immunization procedure is shown in table 3.
TABLE 3 Experimental group mice immunization procedure
4. Establishment of Indirect ELISA method
Respectively taking the pET-32a-R1VP60 and pET-32a-R2VP60 recombinant proteins prepared in the step 1 as antigen coatings, and optimizing conditions such as the optimal antigen coating concentration, the optimal dilution ratio of serum to be detected and the like by using a chessboard method, wherein rabbit anti-RHDV 2 serum and RHDV positive serum which are immunized by virus tissue inactivated vaccine are respectively used as primary antibody, and goat anti-rabbit IgG is marked by HRP as secondary antibody. When P/N is more than or equal to 2.1, the positive judgment standard is adopted. Coating the antigen at 4 ℃ overnight, wherein the coating concentration of the recombinant proteins pET-32a-R1VP60 and pET-32a-R2VP60 is 18 mu g/mL, the dilution of the serum to be detected is 1: 320. the secondary antibody dilution was 1: the P/N value can reach the highest value at 8000. The results show that both the pET-32a-R1VP60 and the pET-32a-R2VP60 recombinant protein can react with the corresponding RHDV positive serum.
The rabbit anti-RHDV 2 serum and the RHDV positive serum after the immunization of the virus tissue inactivated vaccine are prepared by adopting a conventional method: the rabbits of 2 months age were immunized with virus tissue inactivated with 0.3% formaldehyde (liver tissue/normal saline ratio 1/10), and 3 times of immunization with subcutaneous multipoint injection on the back was performed, each time at 2mL, once in 15 days.
5. Cell fusion and monoclonal screening
And (4) collecting serum by tail-off blood collection after the mice in the step (3) are subjected to tertiary immunization, using the ELISA method reaction conditions established in the step (4), replacing the secondary antibody with HRP-labeled goat anti-mouse IgG diluted by 1. Cell fusion was performed using conventional methods. The fused cells were resuspended using 20% HAT medium and plated onto two 96-well plates. Change of medium according to color change and change of medium after about 15 days, change of medium to 20% HT medium. And when the hybridoma cells grow to about 1/3 of the bottom of the hole, sucking cell supernatants to perform positive cell strain screening and antibody titer detection, wherein the screening standard is a specific monoclonal antibody cell strain combined with the pET-32a-R2VP60 recombinant protein but not combined with the pET-32a-R1VP60 recombinant protein. Selecting a positive cell strain meeting the conditions for subcloning, and finally screening out a hybridoma cell strain capable of stably secreting the specific RHDV2 monoclonal antibody, which is named as R2McAb2A1.
6. Preparation, titer determination and purification of monoclonal antibody McAb ascites
Selecting BALB/c mice for 6-8 weeks according to a conventional method to prepare monoclonal antibody ascites, performing sensitization by injecting paraffin into the abdominal cavity, and injecting hybridoma cells R2McAb2A1 prepared in the step 5 after about one week to prepare ascites. After about 15 days, when the abdomen of the mouse swells, ascites is sucked, and the ascites is centrifuged to obtain a clear ascites antibody according to the following ratio of 1:10 gradient dilution the titer was determined with reference to the ELISA protocol in step 5. The ELISA detection result is shown in FIG. 3, and the ascites titer of the hybridoma cell R2McAb2A1 monoclonal antibody can reach 1. The prepared ascites fluid was purified by Protein G pre-packed gravity column, and the purity and size were analyzed by SDS-PAGE electrophoresis after purification. The electrophoresis results are shown in FIG. 4, where M: prestained protein marker;1: an unpurified monoclonal antibody; 2: the purified monoclonal antibody, lane 2, yielded two proteins with molecular weights of about 25kD and 50kD, respectively, corresponding to the heavy and light chains of the monoclonal antibody, demonstrating the higher purity of the monoclonal antibody.
7. Westernblot identification of monoclonal antibodies
Purifying the RHDV1 and RHDV2 virus particles by using a sucrose density gradient centrifugation method, carrying out virus negative staining on the purified virus particles, and observing whether the virus particles exist under an electron microscope. The purified RHDV1, RHDV2, pET-32a-R1VP60 and pET-32a-R2VP60 were subjected to SDS-PAGE electrophoresis and then subjected to western blot analysis on a polyvinylidene fluoride membrane, wherein the primary antibody in the assay was the ascites monoclonal antibody (1. As shown in fig. 5, the purified virus liquid is observed under a projection electron microscope, and a plurality of virus particles can be observed under the microscope, wherein fig. 5-a shows RHDV1 virus particles; FIG. 5-B is RHDV2 virions; the purified virus liquid is proved to contain virus particles and can be used for subsequent Westernblot tests. Westernblot results are shown in FIG. 6, and the monoclonal antibody produced by hybridoma cell R2McAb2A1 can react with native RHDV2 virions and pET-32a-R2VP60 protein, and target protein bands appear at about 60kD and 79kD respectively, which are consistent with expected sizes; meanwhile, the monoclonal antibody does not react with RHDV1 virus particles and pET-32a-R1VP60, and the monoclonal antibody generated by the hybridoma cell R2McAb2A1 is proved to have good specificity.
Example 2 subclass identification of specific RHDV2 monoclonal antibodies and hybridoma chromosome analysis
1. Subclass identification and stability analysis of monoclonal antibodies
The hybridoma cell line R2McAb2A1 cultured in a 96-well cell plate in example 1 was passaged and culture supernatants were collected for 30 consecutive passages, and after measurement by the ELISA method established in example 1, it was found to stably secrete antibodies and to have a substantially stable titer. The ascites 1 purified in example 1 was diluted with 100000 and subclassed with a mouse monoclonal antibody subclass identification kit, and the results showed that the monoclonal antibody secreted from the hybridoma cell line R2McAb2A1 was of IgG3 subclass and the light chain was of Kappa type.
2. Chromosome number analysis
Taking hybridoma cells and SP20 cells which grow well and are in a logarithmic growth phase, adding colchicine to a final concentration of 0.2mg/mL, culturing at 37 ℃ for about 2 hours, centrifuging to remove cell supernatant, adding preheated 0.075mol/L KCL hypotonic solution for about 5mL, and uniformly blowing and beating for 20min in a water bath at 37 ℃. After centrifuging again to remove the supernatant, a fixing solution (methanol: glacial acetic acid = 3:1) is added for fixation, and finally, after flaking and giemsa staining, the shape and the number of chromosomes are observed under a mirror. As a result, as shown in FIG. 7, 7-A is the chromosome of SP20 cell; in the figure, 7-B is the chromosome of the R2McAb2A1 hybridoma, the chromosome number of the fused cell R2McAb2A1 is obviously more than that of the SP20 cell, about 100 cells are obtained, the chromosome number accords with the chromosome number characteristic of the hybridoma after the mouse spleen cell is fused with the SP20 cell, and the R2McAb2A1 cell is proved to be the fused cell.
Example 3 hemagglutination and hemagglutination inhibition assay
The liver of the rabbit infected with RHDV2 and died of illness is treated according to the prior art, the liver is ground and repeatedly frozen and thawed for three times, then the supernatant is centrifuged and taken as RHDV2 suspension, 1 percent of goose fresh red blood cells are used as an indication system, and the detection of the RHDV2 agglutination inhibition effect of the ascites McAb (prepared in example 1) prepared according to the HA and HI test operation in GB/T14926.54 is carried out. Meanwhile, the liver grinding fluid of healthy rabbits is used as RHDV2 negative control. The results are shown in FIG. 8, where the HA titer of RHDV2 virus for 4 units of the working antigen in 8-A is 2 5 The results show that virus particles exist in the ground liver tissue homogenate and the RHDV2 virus can agglutinate fresh goose erythrocytes; in FIG. 8-B, the ascites McAb purified from R2McAb2A1 can completely inhibit RHDV2 agglutinated goose red blood cells, and the HI titer is as high as 2 10 。
In conclusion, the expressed pET-32a-R2VP60 recombinant protein is used as immunogen, pET-32a-R2VP60 and pET-32a-R1VP60 recombinant protein are used as coating antigen to establish an indirect ELISA screening method, the conventional hybridoma technology is adopted to prepare the hybridoma of RHDV2, about 12 hybridoma cells are fused, 91.7% of the hybridoma cells are cell strains secreting RHDV1 and RHDV2 facultative monoclonal antibodies, and the hybridoma cell strain R2McAb2A1 is finally screened out through the indirect ELISA method, cloning and subcloning. Western blot test and hemagglutination inhibition test show that the monoclonal antibody secreted by the hybridoma cell strain R2McAb2A1 is only specifically combined with the RHDV2 virus particles, is not combined with the RHDV1 virus particles, and has good specificity to the RHDV2 virus particles. Therefore, the hybridoma cell strain R2McAb2A1 prepared by the invention and the monoclonal antibody secreted by the hybridoma cell strain can be applied to preparation of a rabbit hemorrhagic disease virus type2 detection kit, and can effectively identify the rabbit hemorrhagic disease virus type 2.
The present specification and figures are to be regarded as illustrative rather than restrictive, and it is intended that all such alterations and modifications that fall within the true spirit and scope of the invention, and that all such modifications and variations are included within the scope of the invention as determined by the appended claims without the use of inventive faculty.
Claims (4)
1. A hybridoma cell strain R2McAb2A1 is characterized in that the hybridoma cell R2McAb2A1 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation date of 2022 years, 7 months and 27 days and the preservation number of CCTCC NO: C2022205.
2. A monoclonal antibody secreted by the hybridoma cell line R2McAb2A1 of claim 1.
3. The monoclonal antibody of claim 2, wherein the monoclonal antibody is a specific monoclonal antibody of rabbit hemorrhagic disease virus type 2VP60 protein, and the amino acid sequence of the rabbit hemorrhagic disease virus type 2VP60 protein is shown as SEQ ID N0.1.
4. Use of the monoclonal antibody of claim 2 or 3 in the preparation of a rabbit hemorrhagic disease virus type2 detection kit.
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