CN114480297B - Serum type 1 Marek's disease virus MEQ monoclonal antibody, preparation method and application - Google Patents
Serum type 1 Marek's disease virus MEQ monoclonal antibody, preparation method and application Download PDFInfo
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Abstract
The invention discloses an antiserum type 1 Marek's disease virus MEQ protein monoclonal antibody, a preparation method and application. The MDV-1 specific MEQ protein monoclonal antibody is generated by a murine monoclonal antibody hybridoma cell strain MEQ-2A9-B12, the cell strain is preserved in the China general microbiological culture Collection center, and the preservation number is as follows: the monoclonal antibody has a subtype of IgG2b and a light chain type of Kappa, and has a heavy chain variable region amino acid sequence shown in SEQ ID No.3 and a light chain variable region amino acid sequence shown in SEQ ID No.4 and No CGMCC No.23023. The monoclonal antibody 2A9-B12 secreted by the invention can be used for IFA, western blot and other immunological detection analysis of different pathogenic strains of MDV-1, can also be used for identifying and distinguishing MDV-2 and MDV-3 strains, and is suitable for identifying and diagnosing different serotype MDV epidemic strains in clinical suspected cases. The invention solves the problem that the 'neck clamp' of the MDV-1MEQ protein-resistant specific monoclonal antibody is lacked in China at present, and provides a key core reagent for MD subsequent research and diagnostic reagent research and development.
Description
Technical Field
The invention relates to a serum type 1 Marek's disease virus (MEQ) monoclonal antibody, a preparation method and application, and belongs to the technical field of animal immunology.
Background
Marek's Disease (MD) is an important immunosuppressive disease and neoplastic disease caused by early infection of chicks by Marek's Disease Virus (MDV). The MDV produces serious immunosuppression after infecting a host early, is one of important factors with poor immunoprophylaxis effect of clinical poultry disease vaccines, and always troubles the healthy development of poultry industry in China for a long time. Also, MDV infection of natural hosts induces visceral tumors and mass deaths resulting in a direct annual economic loss of about $ 10-20 million from the poultry industry worldwide. The MDV induced host tumor can be effectively immune-prevented by using the vaccine, which is the first example in the world that the vaccine can be used for successfully preventing the tumorigenic virus from causing cancer, but in recent years, with the wide application of the MD vaccine in chicken flocks, under the long-term immune pressure selection, the toxicity of the MDV epidemic strain is continuously enhanced and varied, and the MD epidemic situation is still not continuously developed in the global range.
MDV shares 3 different serotypes: serotype 1 (MDV-1), serotype 2 (MDV-2) and serotype 3 (MDV-3, i.e. herpesvirus of turkeys HVT), of which only MDV-1 is pathogenic and tumorigenic. Depending on the pathogenicity, circulating strains of MDV-1 can be further classified into Mild (Mild MDV, mMDV), virulent (Virulent MDV, vMDV), very Virulent (Very viroent MDV, vMDV) and Very Virulent (Very viroent plus MDV, vv + MDV).
The MDV viral genome is a linear double-stranded DNA, about 180kb in length. The sequence mainly comprises a long Unique sequence area (UL) and a short Unique sequence area (US), and two inverted repeat sequence areas with completely identical sequences are respectively arranged on two sides of the two Unique sequence areas: the virus genes coded in the unique sequence region have high conservation with other herpes viruses, while the virus genes specific to MDV are mainly positioned in the inverted repeat region TR/IR.
Among the numerous viral genes encoded by MDV, the meq gene (MDV Ecorii-Q) is the most major proto-oncogene of MDV, and other genes such as pp38, vIL-8, ICP 4-related transcripts, and virally encoded telomerase RNA may also play important roles in MDV latent infection and tumorigenesis. The meq gene is a recognized tumorigenic gene at present and has a direct effect in the process of MD tumorigenesis and development. The full length of the MEQ gene sequence is 1020bp, the protein consisting of 339 amino acids is coded, the N end is a basic leucine chain (bZIP) structure, the C end is 2 proline-rich repetitive sequences, and the coded MEQ protein is similar to the structures of the tumor proteins FOS and JUN in a host proto-oncogene family, so the MEQ is considered to be an oncogenic protein and a transcription activating factor which are positioned in nucleoplasm, nucleolus and spirochete of a cell nucleus, promotes the anti-apoptosis by activating AP-1 and simultaneously inhibiting p53, and is the main proto-oncogenic protein in the MDV tumorigenesis process. The MEQ gene is a specific protein gene of the MDV-1, although the MEQ gene sequences among different strains are relatively conservative, the nucleotide homology is about 83.8 to 99.9 percent, and the amino acid homology is about 88.4 to 99.6 percent, but the MDV strains with different toxicity have certain difference on the amino acid sequence structure of the MEQ protein. At present, MDV presents a periodic and climbing-type continuously enhanced virulence trend, and the emergence of MDV epidemic strains with stronger virulence can cause that the existing vaccine can not provide effective immune protection, thereby causing larger economic loss. Therefore, the deep research on the structure and the function of the MEQ protein has important scientific significance for disclosing the current mechanism for enhancing the MDV-1 virulence.
MEQ is the MDV-1 specific protooncoprotein accepted by the academia at present, is usually abnormally and highly expressed in the viscera parenchymal tumor tissues and cells of the chicken infected by MDV, is an excellent MD diagnostic marker, and can be clinically used for differential diagnosis of poultry immunosuppressive diseases and neoplastic diseases caused by infection of Avian Leukemia Virus (ALV) or reticuloendotheliosis virus (REV) and the like. Therefore, the development of antibodies (monoclonal antibodies or polyclonal antibodies) specifically aiming at the MEQ protein has important scientific value and market application prospect in the differential diagnosis of the poultry immunosuppressive diseases and the neoplastic diseases, the identification of MDV infection and vaccine immunity, the separation identification and typing of MDV epidemic strains and the research of pathogenic and carcinogenic mechanisms of MDV.
Although the domestic scholars try to develop the antibody of the protein for many times, because of the factors of high GC content of the MEQ gene sequence, large protein, difficult artificial expression and purification and the like, china still cannot develop the practically available MEQ antibody and has no available commercial products in the market. At present, only the national animal health Organization (OIE) MDV reference laboratory of two organizations of American department of agriculture, poultry disease and tumor laboratory and English Pierburst institute successfully develops the monoclonal antibody of the protein internationally, but the monoclonal antibody is not commercialized, chinese scholars develop related researches, and the required antibody only depends on friendship of European and American scholars of the two organizations, so that the research requirements of the Chinese scholars are greatly limited. The development of MEQ mabs with proprietary intellectual property rights is imminent. The development of the MEQ monoclonal antibody can not only provide a key core reagent for the effective prevention and control of MD, but also provide technical support for guaranteeing the healthy development of poultry breeding industry in China.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a hybridoma cell strain MEQ-2A9-B12 and a monoclonal antibody 2A9-B12 for stably secreting the specific MEQ protein of the antiserum type 1 Marek's disease virus, a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
the method comprises the steps of comparing amino acid sequences of MEQ proteins of different virulence strains of a serum I Marek's disease virus MDV-1, selecting a conserved sequence region with hydrophilicity for polypeptide segmented synthesis, coupling the synthesized polypeptide to KLH protein to increase the immunogenicity of the KLH protein, immunizing a BALB/c mouse by using polypeptide-KLH coupled protein, establishing a monoclonal antibody hybridoma cell strain by using a cell fusion technology, simultaneously using a previously constructed MEQ gene editing-deleted GX0101 delta MEQ strain and a parent strain GX0101 thereof to perform indirect immunofluorescence experiment (IFA), performing cross screening and identification to obtain a monoclonal antibody hybridoma cell strain MEQ-2A9-B12 capable of specifically identifying the MDV-1MEQ protein, applying the stably secreted specific monoclonal antibody 2A9-B12 to immunological detection such as IFA and Western blot analysis (Western-blot), providing a core reagent for the subsequent structural and functional research of the MEQ protein, the rapid detection of MDV and the development of differential diagnosis reagent, and providing a new method for the screening, the preparation and identification of the monoclonal antibody.
A monoclonal antibody hybridoma cell strain secreting antiserum I-type Marek's disease virus MDV-1 specific MEQ protein is a murine monoclonal antibody hybridoma cell strain MEQ-2A9-B12 which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.23023.
The monoclonal antibody 2A9-B12 is secreted by the murine monoclonal antibody hybridoma cell strain meq-2A9-B12 as defined in claim 1; the light chain type of the monoclonal antibody 2A9-B12 is Kappa type, and the subtype is IgG2B.
The heavy chain variable region nucleotide sequence of the monoclonal antibody 2A9-B12 is shown as SEQ ID NO.1, and the light chain variable region nucleotide sequence is shown as SEQ ID NO. 2.
The heavy chain variable region amino acid sequence of the monoclonal antibody 2A9-B12 is shown as SEQ ID NO.3, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 4.
The preparation method of the murine monoclonal antibody hybridoma cell strain meq-2A9-B12 comprises the following steps:
(1) Comparing MEQ gene sequences of 4 serological I-type Marek's disease virus MDV-1 specific strains of different pathogenic types of a virulent strain GA, a super-virulent strain GX0101, md5 and a super-virulent strain 648A, analyzing hydrophilic regions and hydrophobic regions of MEQ protein, designing 4 hydrophilic immunogen polypeptides, and synthesizing amino acid sequences of 4 MEQ protein polypeptides, wherein the amino acid sequences are respectively as follows: SEQ ID NO.5 to SEQ ID NO.8;
(2) Respectively coupling the synthesized 4 MEQ protein polypeptides to KLH protein to obtain 4 polypeptide-KLH coupled proteins; mixing 4 kinds of polypeptide-KLH coupled protein in equal amount, immunizing BALB/c mouse with immunogen, preparing hybridoma cell by cell fusion technology to obtain hybridoma cell supernatant;
(3) Utilizing MDV-1 parent strain GX0101 and meq gene editing deletion strain GX0101 delta meq constructed based on CRISPR/Cas9 system, and combining indirect immunofluorescence experiment IFA to perform differential screening and cross identification on hybridoma cell supernatant obtained in the step (2);
obtaining hybridoma cells which only have specific staining reaction with parental strain GX0101 virus plaques and do not react with GX0101 delta meq, namely, the hybridoma cells are judged to be positive hybridoma cells and named as hybridoma cell meq-2A9;
(4) The positive hybridoma meq-2A9 was expanded and then subcloned by limiting dilution method to obtain 1 strong positive monoclonal hybridoma cell, which was named hybridoma cell strain meq-2A9-B12.
The step of immunizing BALB/c mouse with the polypeptide-KLH coupling protein in the step (2) is as follows: mixing and emulsifying the first non-injected mice with equivalent Freund complete adjuvant, immunizing 3 BALB/C female mice of 6 weeks old, 50 mu g/mouse, and injecting the mice at the back subcutaneous points; adding equivalent Freund incomplete adjuvant, mixing, emulsifying, injecting subcutaneously at back, 50 μ g/mouse, immunizing for 4 times, 3 weeks for each time, and collecting blood after 15d of 4-immunization to separate serum; and finally, performing hyperimmunization by adopting 4 mixed polypeptide-KLH coupled proteins without adjuvant, wherein each mixed polypeptide-KLH coupled protein is 50 mu g, and 3 days later, taking splenocytes of the mice for cell fusion.
The monoclonal antibody 2A9-B12 is applied to the typing and differential diagnosis of different serotype MDV strains.
The monoclonal antibody 2A9-B12 is applied to an immunological detection method.
The immunological detection method comprises but is not limited to indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA) or Western blot analysis (Western-blot).
The monoclonal antibody 2A9-B12 is applied to the research and development of immunological detection reagents and products.
The invention has the beneficial effects that:
(1) The invention provides 4 hydrophilic polypeptides aiming at amino acid sequence conserved regions of MEQ proteins of different virulence strains of serotype I Marek's disease virus MDV-1, the polypeptides are coupled to KLH proteins to prepare polypeptide-KLH coupled protein immunogens, and multiple antiserum separated after immunization of BALB/c mice can be specifically combined with MDV-1 virus plaques, so that the polypeptide-KLH coupled protein has good immunogenicity. The design, synthesis and verification of the polypeptides solve the problem that the prior domestic available 'neck clamp' of the monoclonal antibody of the anti-MEQ protein is unavailable due to the difficulty and high cost of in vitro prokaryotic expression or eukaryotic expression and purification of the MEQ protein, and provide possibility for successfully preparing the monoclonal antibody of the anti-MEQ protein.
(2) The invention utilizes the earlier stage GX0101 delta MEQ strain which is constructed based on the CRISPR/Cas9 system and is deleted by the MEQ gene editing and the parental strain GX0101 to carry out IFA staining cross screening on hybridoma cell supernatants, screens and identifies monoclonal antibody hybridoma cell strains for specifically identifying MEQ protein, the cross screening method has the advantages of intuition, rapidness, accuracy, strong specificity and the like, provides a brand new technical thought for the development of MDV encoded virus protein and other similar DNA virus protein monoclonal antibodies, and the method is not reported at home and abroad and has great innovation.
(3) The monoclonal antibody for resisting MEQ protein has strong specificity, can be simultaneously used for various immunological detections of different virulence separating strains of MDV-1 and IFA, western Blot and the like of MDV-1 vaccine strains, can effectively distinguish MDV-2 and MDV-3 (HVT) strains, and is used for typing and differential diagnosis of different serotype MDV strains.
(4) The monoclonal antibody prepared by the invention has high sensitivity, the subtype is IgG2b, the light chain is Kappa type, and the IFA titer can reach 1: 2.56 multiplied by 10 4 。
(5) The monoclonal antibody hybridoma cell strain established by the invention has stable antibody secretion capacity, can quickly and specifically identify MDV-1MEQ protein, lays a good foundation for solving the technical problem of quick detection of MDV immunology, and has wide market application prospect in MDV immunology diagnosis technology and reagent research and development.
Drawings
FIG. 1 shows the result of amino acid sequence alignment analysis of MEQ protein of different pathogenic MDV-1 strains.
Wherein CVI988 represents MDV-1 vaccine strain; GA, MDV-1 virulent strain; GX0101 and Md5, representing MDV-1 hyper-virulent strains; 648A, MDV-1 super virulent strain.
FIG. 2 shows the results of the hydropathic and hydrophobic analysis of MEQ proteins of different pathogenic MDV-1 strains.
Wherein: GA, MDV-1 virulent strain; GX0101 and Md5, representing MDV-1 hyper-virulent strains; 648A, MDV-1 super virulent strain.
FIG. 3 IFA staining results of supernatant from cross-screened hybridoma cells with GX0101 and GX 0101. Delta. Meq strains.
Wherein: GX0101, representing the MDV-1 parent strain; GX0101 Δ meq, indicating a meq gene editing deletion strain.
FIG. 4 shows the result of specific staining IFA identification of MEQ eukaryotic expression protein and monoclonal antibody 2A9-B12.
Wherein: 2A9-B12, which represents the MDV-1MEQ protein monoclonal antibody prepared by the invention; FD7, a positive control antibody for MEQ protein; EGFP, which represents the green fluorescent protein carried by the eukaryotic expression plasmid; MEQ, which represents a eukaryotically expressed MEQ protein.
FIG. 5 confocal results of cellular localization analysis of monoclonal antibody 2A9-B12 binding to MDV-1MEQ protein.
Wherein: DAPI, which represents a dye for nuclear staining, indicating the nucleus in blue; MDV-gB, representing incubation of MDV-gB antibody with DyLight 488 labeled goat anti-mouse IgG, indicating gB protein expressed in virus infected cells as green fluorescence; 2A9-B12 representing MEQ protein expressed in cells infected with virus, with red fluorescence, incubated with 2A9-B12 monoclonal antibody and DyLight 594 labeled goat anti-mouse IgG; merge, representing the overlapping condition of nucleus, gB protein and MEQ protein staining sites under the same visual field; enlarge, showing a map of the effect of the overlap of staining with magnification of the local field of view.
FIG. 6 shows the IFA and Western blot detection results of the monoclonal antibodies 2A9-B12 and MDV representative strains of different serotypes.
Wherein: a, represents the IFA detection result; b, representing a Western blot detection result; plaque, representing MDV virus plaques under normal light; 2A9-B12, which represents the MDV-1MEQ protein monoclonal antibody prepared by the invention; beta-actin, which represents the chicken's internal reference protein; GX0101, md5 and GA, representing MDV-1 strains of different pathotypes; CVI988, CVI988 vaccine strain for MDV-1; SB-1, which represents the vaccine strain of MDV-2; HVT, a vaccine strain for MDV-3.
Detailed Description
The following examples are given to illustrate the invention in detail and are not intended to limit the scope of the invention in any way.
Unless otherwise stated, the instruments and equipment referred to in the examples are conventional instruments and equipment; the related reagents are all conventional reagents sold in the market; the test methods involved are all conventional methods.
Example one design of hydrophilic polypeptide of Marek's disease virus serotype I MEQ protein and identification of immunogenicity
1. Comparison analysis of amino acid difference sites and hydrophilicity of MEQ protein of different pathogenic MDV-1 strains
The meq gene sequences of MDV-1 strains with different pathotypes (including virulent strain GA, super-virulent strain GX0101, md5 and super-virulent strain 648A) are downloaded from NCBI GenBank database. Gene sequence alignment analysis using DNAStar MegAlign software revealed that the MEQ protein sequences of 4 different pathotype MDV strains were highly conserved and differed from each other by only 11 amino acid positions (fig. 1 and table 1). The analysis result provides important basis for designing and preparing broad-spectrum MEQ protein monoclonal antibodies capable of identifying different pathogenic MDV-1 strains.
TABLE 1 amino acid difference sites of MEQ proteins of different pathogenic MDV-1 strains
The hydrophilicity of the MEQ proteins of the 4 different pathotype MDV-1 strains is compared and analyzed by using DNAMAN, DNAstar and ExPASY software, and the hydrophilicity is basically consistent (figure 2). Selecting a hydrophilic conserved region and designing synthetic polypeptides by avoiding 11 amino acid difference sites listed in Table 1, and respectively designing two polypeptides 26-70 and 81-113 in a region 26-113 of the hydrophilic region by avoiding the difference sites (71-80); designing a polypeptide 143-175 in the region of the hydrophilic region 141-183 avoiding the difference points 142 and 176; no sites of difference are found in the hydrophilic regions 285-315, and a polypeptide 285-315 is designed. The peptides are named as peptide 1, peptide 2, peptide 3 and peptide 4 (SEQ ID NO. 5-SEQ ID NO. 8) according to the sequence, and the polypeptide sequences are as follows:
peptide 1: GSTSRRKKRKSHDIPNPSKHPFPDGLSEEEKQKLERRRKRNRDA (SEQ ID NO. 5);
peptide 2: YVDKLHEECELQRANEHKEIRDLRTECTSL (SEQ ID NO. 6);
peptide 3: PVPEPPIPTPPPPSPDEPNAPHCGSQPPICTP (SEQ ID NO. 7);
peptide 4: LFTPSPPHPAPEPIPERILELTEDPEQDSLYS (SEQ ID NO. 8).
The above polypeptides are all synthesized and prepared by Gell Biochemical (Shanghai) Biometrics.
2. Analysis of immunogenicity of hydrophilic Polypeptides of MDV-1MEQ protein
Respectively coupling the synthesized 4 MEQ protein polypeptides to KLH protein, determining the content of the polypeptide-KLH coupled protein, mixing 4 polypeptide-KLH coupled proteins in equal amount, firstly, mixing and emulsifying without adding an equal amount of Freund complete adjuvant, immunizing 3 BALB/C female mice with the age of 6 weeks, and injecting 50 mu g of each female mouse with subcutaneous back; when the immune is strengthened, the same amount of Freund incomplete adjuvant is added for mixing and emulsifying, subcutaneous injection is carried out on the back, each dose is 50 mu g, 4 times of immunization are carried out, the interval is 3 weeks each time, and after 4 times of immunization, 4 groups of immunized mice are respectively subjected to tail breaking and blood collecting to separate serum 15 days after 4 times of immunization.
And (3) detecting the serum multi-antibody titer of the immune mice by using IFA. Briefly described, resuscitationGX0101 virus seed frozen in liquid nitrogen, rapidly dissolving in water bath at 37 deg.C, centrifuging at 1000rpm for 5min, discarding cell freezing solution, resuspending cells with 1mL of DMEM medium containing 1% (v/v) FBS (fetal bovine serum), inoculating 10 μ L of CEF monolayer (chicken embryo fibroblast) on 96-well cell culture plate, collecting 150 μ L of DMEM medium containing 1% (v/v) FBS (fetal bovine serum) maintaining solution, performing cell comparison with non-toxic CEF cells, and standing at 37 deg.C and 5% CO 2 Culturing in an incubator for 3-4 days; observing the virus-inoculated cells under a microscope to generate typical plaques, discarding maintenance liquid in a 96-well cell plate, adding precooled methanol to acetone (volume ratio is 1; discarding the blocking solution, washing for 3 times by using PBST solution, spin-drying, adding each diluted immune mouse serum (the volume ratio is 1; discarding the supernatant, washing with PBST solution for 3 times, spin-drying, adding FITC (fluorescein isothiocyanate) -labeled goat anti-mouse IgG (1: 500) secondary antibody diluent, 100 μ L/well, and placing in a 37 ℃ incubator for 30min; discarding the secondary antibody diluent, washing with PBST solution for 3 times, adding PBST solution 100 μ L/hole, and observing under an inverted fluorescence microscope.
The test results show that 3 mice all produced specific antibodies against viral plaques formed by MDV-infected CEF, and the specific IFA titer results are shown in Table 2.
TABLE 2 measurement of IFA titer of polypeptide-KLH-conjugated protein immunized mouse polyclonal antiserum
Note: + + +, strong positive; positive; -, negative
EXAMPLE II screening and characterization of anti-MDV-1 MEQ protein monoclonal antibodies
1. Cell fusion and establishment of hybridoma cell line
Mice # 2 with the highest IFA titer were selected for cell fusion. Before fusion, intraperitoneal injection is carried out on 4 polypeptide-KLH coupled proteins which are mixed in equal proportion without adding an adjuvant 50 mu g/mouse, after 3 days of hyperimmunization, mouse splenocytes are taken for cell fusion, the mouse splenocytes are taken and mixed according to the conventional method, after the SP20 cells and the hyperimmunized mouse splenocytes are respectively counted, the mixture is mixed according to the proportion of 1 2 After culturing in an incubator for about 7 to 10 days, a large hybridoma cell stack was grown, and then cell supernatants were extracted for IFA detection as described below.
2. Screening and identification of anti-MDV-1 MEQ protein monoclonal antibody
A deletion strain GX0101 delta meq and a parent strain GX0101 thereof are edited by a meq gene constructed in the early stage, the deletion strain is shown in the previously published documents of the present invention (Tenbine and the like, a Marek's disease virus super virulent strain proto-oncogene meq deletion strain is constructed by using a CRISPR/Cas9 gene editing technology and is identified, a virus bulletin 2020,36 (4): 675-684) respectively infects CEF single-layer cells, the cells are fixed after obvious virus plaques appear, the supernatant of the hybridoma cells and anti-FITC labeled goat anti-mouse IgG (1 500) secondary antibody diluent are respectively incubated for IFA detection and identification, and finally, the result is observed under an inverted fluorescence microscope.
As shown in fig. 3, after CEF cells are infected with GX0101 Δ meq and GX0101, typical and morphologically similar viral plaques are formed, and under a fluorescence microscope, it can be observed that the supernatant secreted by 1 strain of hybridoma (named: hybridoma meq-2 A9) is specifically combined with the viral plaques formed by CEF cells infected with GX0101, and under the indication of FITC labeled goat anti-mouse IgG (1.
Transferring the positive hybridoma cell meq-2A9 to a 24-well plate for amplification culture, after IFA retest is positive, carrying out subcloning on the hybridoma cell for 3 times by using a limiting dilution method, selecting a strong positive monoclonal hybridoma cell strain 1 with good state and vigorous growth after the last subcloning, and naming the strong positive monoclonal hybridoma cell strain as meq-2A9-B12, centrifugally collecting the cell after the amplification culture, carrying out resuspension by using a freezing medium (the volume ratio of DMSO to calf serum to DMEM is = 1: 5: 4), freezing and storing in liquid nitrogen, and simultaneously, reserving cell supernatant for storage at-20 ℃ for later use.
The mouse-derived monoclonal antibody hybridoma cell strain meq-2A9-B12 is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.23023, the preservation time is 2021 year, 9 month and 1 day, the preservation address is No.3 of the Xilu No.1 of Beijing Kogyo-sunward area, china academy of sciences and microbiology institute.
3. Specificity identification of 2A9-B12 monoclonal antibody
In order to verify the specificity of the supernatant secreted by the screened MEQ-2A9-B12 monoclonal hybridoma cell to MDV-1 strain MEQ protein, the full-length sequence of the MEQ gene is synthesized according to the genome sequence of GX0101 (GenBank accession number: JX 844666.1), a pEGFP-N1-MEQ eukaryotic expression plasmid is constructed according to Lipofectamine TM 2000 (Thermo Fisher) instruction, transfecting a 24-well plate 293T cell (with the confluence degree of about 80-90%) prepared in advance, fixing the cell by using a precooled methanol-acetone (volume ratio of 1: 1) solution after 48h transfection, standing at room temperature for 10min, discarding a cell fixing solution, washing a PBST buffer solution (PBS buffer solution containing 0.05% (v/v) Tween-20) for 3 times, spin-drying, adding a PBST blocking solution containing 5% (v/v) skimmed milk, standing at 200 μ L/well, and sealing at 37 ℃ for 30min; removing the sealing liquid, washing with PBST solution for 3 times, and spin-drying; positive control antibody FD7 specific to MEQ protein (as presented by Pirbright research institute, UK) and hybridoma cell supernatant of the MEQ-2A9-B12 monoclonal antibody of the invention were incubated, incubated at 37 ℃ for 30min in an incubator, and washed 3 times with PBST; adding secondary antibody diluent of goat anti-mouse IgG (1; PBST was added at 200. Mu.L/well and the result was observed under an inverted fluorescence microscope.
As shown in the result of FIG. 4, after 293T cells are transfected by pEGFP-N1-MEQ plasmid for 48h, most cells show obvious EGFP (enhanced green fluorescent protein) autofluorescence, meanwhile, the positive control monoclonal antibody FD7 is specifically combined with the 293T cells, and shows red fluorescence under the indication of sheep anti-mouse IgG marked by DyLight 594 and can be co-localized with the cell autofluorescence, which indicates that the constructed pEGFP-N1-MEQ plasmid can correctly express MEQ protein. Meanwhile, the cell supernatant of the monoclonal antibody hybridoma cell strain MEQ-2A9-B12 screened and identified by the invention also obtains a similar staining result, and the fact shows that the antibody secreted by the MEQ-2A9-B12 hybridoma cell strain is a monoclonal antibody with specificity aiming at the MDV-1MEQ protein.
4. Cell co-localization analysis of 2A9-B12 monoclonal antibody for recognition of MDV-1 strain
CEF monolayer cells were infected with MDV-1 strain GX0101, after typical viral plaques had appeared, the infected CEF cells were digested and transferred to confocal dedicated cell culture dishes to form a thin monolayer, after CEF adherence was fixed with a pre-cooled solution of methanol: acetone (volume ratio 1.
As shown in the results of fig. 5, in the immobilized CEF, the cytoplasm of a fraction of cells exhibited a specific green fluorescent stain (indicated by MDV-gB positive mab and DyLight 488 goat anti-mouse IgG secondary antibody), indicating that this fraction of cells was infected with virus; at the same time, the nuclei of these cells were observed to exhibit specific red fluorescent staining (indicated by meq-2A9-B12 cell supernatant and a DyLight 594-labeled goat anti-mouse IgG secondary antibody). In combination with the MDV-1 strain MEQ protein reported in the literature, the MEQ protein is mainly expressed in the nucleus of infected cells, so the results further confirm that the antibody secreted by the MEQ-2A9-B12 monoclonal antibody hybridoma cell strain is a monoclonal antibody specific to the MEQ protein.
5. 2A9-B12 monoclonal antibody ascites preparation and IFA titer determination
Female BALB/C mice were selected for delivery and injected intraperitoneally with 500. Mu.L of sterile paraffin to stimulate immune cells for promoting proliferation of monoclonal antibody hybridoma cells. After 7-10 days, meq-2A9-B12 hybridoma cells are collected by centrifugation and sterilizedPBS wash 2 times, about 3X 10 per mouse 6 ～6×10 6 Injecting the amount of each cell into abdominal cavity of mouse, observing the state of mouse day by day, extracting ascites after the abdominal cavity of mouse expands about 10 days, 8000r/min, centrifuging at 4 deg.C for 20min to remove oil and cell precipitate, collecting abdominal supernatant, and storing at-80 deg.C for use.
The single-antibody supernatant was diluted with PBS to 1.024 × 10 with a 2-fold gradient starting from 1 6 The CEF monolayer cells infected with GX0101 and showing significant viral plaques were fixed and the IFA detection was performed by incubating the monoclonal antibody ascites dilution and FITC-labeled goat anti-mouse IgG (1.
The result shows that the IFA titer of the ascites of the 2A9-B12 monoclonal antibody can reach 1: 2.56 multiplied by 10 4 。
6. Subtype identification of 2A9-B12 monoclonal antibody
The Monoclonal Antibody subtype was identified according to the Mouse Monoclonal Antibody Isotyping Kit (Mouse Monoclonal Antibody typing Kit) using the instructions. The results showed that the subtype of the 2A9-B12 monoclonal antibody was IgG2B and the light chain type was Kappa (Table 3).
TABLE 3 monoclonal antibody subtype identification
Note: positive; -, negative
7. Amplification and sequence analysis of 2A9-B12 monoclonal antibody variable region gene
PCR primers SEQ ID NO.9 to SEQ ID NO.12 for amplifying heavy chain variable region and light chain variable region were designed, respectively, based on the sequence characteristics of the murine monoclonal antibody (Table 4). The above primers were synthesized by Biotechnology engineering (Shanghai) Co., ltd.
TABLE 4 PCR primer sequence Listing for amplification of heavy and light chain genes of monoclonal antibodies
The gene products of the heavy chain and light chain variable regions of the monoclonal antibody 2A9-B12 are respectively obtained by PCR amplification, and are sent to the biological engineering (Shanghai) company Limited for sequencing after cloning.
Sequencing analysis results show that the gene sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 2A9-B12 are respectively shown in SEQ ID NO.1 and SEQ ID NO.2, and the deduced amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 2A9-B12 are respectively shown in SEQ ID NO.3 and SEQ ID NO. 4.
SEQ ID NO.1:
aggtcaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc ctgtccatca cttgcactgt ctctgggttt tcattaacca gctatggtgt acactgggtt cgccagcctc caggaaaggg tctggagtgg ctgggagtaa tatgggctgg tggaagcaca aattataatt cggctctcat gtccagactg agcatcagca aagacaactc caagagccaa gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatgtacta ctgtgccaga gacagggata acacctggtt tgcttactgg ggccaaggga ccacggtcac cgtctcctca SEQ ID NO.2:
gacattgagc tcacccagtc tccagcactc ccggctgcat ctccagggga gaaggtcact atcacctgca gtgccagctc aagtataagt tccaattact tgcattggta tcagcagaag ccaggattct cccctaaact cttgatttat aggacatcca atctggcttc tggagtccca gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaattgg caccatggag gctgaagatg ttgccactta ctactgccag cagggtagta gtataccact cacgttcggt gctggcacca agctggaaat caaacgg
SEQ ID NO.3:
Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala Arg Asp Arg Asp Asn Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
SEQ ID NO.4:
Asp Ile Glu Leu Thr Gln Ser Pro Ala Leu Pro Ala Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Ser Asn Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Phe Ser Pro Lys Leu Leu Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly Thr Met Glu Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ser Ile Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg
Application example I, indirect immunofluorescence assay (IFA) of MDV-1 specific MEQ protein
MDV strains of different serotypes and different pathotypes are selected, including MDV-1 strains (CVI 988, GA, md5 and GX 0101), MDV-2 strains (SB-1) and MDV-3 strains (HVT) are respectively inoculated with CEF monolayer cells to form typical virus plaque fixed cells, and 2A9-B12 monoclonal antibody ascites (1 10000) and FITC labeled goat anti-mouse IgG (1 500) secondary antibody diluent are respectively incubated for IFA detection as described before.
As shown in FIG. 6A, the results of the assay showed that viral plaques formed by CEF monolayers infected with MDV-1 strains (CVI 988, GA, md5, and GX 0101) of different pathotypes all exhibited specific fluorescent staining, whereas viral plaques formed by CEF monolayers infected with MDV-2 strain (SB-1) and MDV-3 strain (HVT) did not have specific green fluorescent staining. The results show that the 2A9-B12 monoclonal antibody prepared by the invention specifically recognizes the MDV-1 strain but does not recognize the MDV-2 and MDV-3 strains, and can be used for IFA specificity detection and identification of the MDV-1 strain.
Application example II, western blot analysis (Western blot) of MDV-1 specific MEQ protein
Respectively inoculating MDV strains of different serotypes and different pathotypes, including MDV-1 strains (CVI 988, GA, md5 and GX 0101), MDV-2 strains (SB-1) and MDV-3 strains (HVT) into CEF monolayer cells, digesting and collecting cell culture after forming typical virus plaques, simultaneously taking negative CEF cell samples without virus infection as a control, respectively extracting total cell proteins, respectively carrying out SDS-PAGE gel electrophoresis, then transferring the total cell proteins onto a nitrocellulose membrane, and sealing for 2 hours at room temperature by using PBST sealing solution containing 5% (v/v) skimmed milk; removing the blocking solution, and washing with PBST solution for 3 times; incubating ascites (1: 10000) of the 2A9-B12 monoclonal antibody, overnight at 4 ℃, and washing 3 times with PBST solution; HRP-labeled goat anti-mouse IgG (H + L) (1; and discarding the secondary antibody diluent, washing the PBST solution for 3 times, and finally, developing the color by using an AEC enzyme substrate kit to observe the result.
As shown in FIG. 6B, the results of the Western blot assay are completely consistent with the IFA results, i.e., CEF cell samples infected with different pathogenic MDV-1 strains (CVI 988, GA, md5 and GX 0101) all present specific reaction bands of MEQ protein, while CEF cell samples infected with MDV-2 strain (SB-1) and MDV-3 strain (HVT) and CEF negative control cell samples did not present any specific reaction bands. These results are consistent with the results reported in the previous document that the MEQ protein is a serotype specific viral protein encoded by MDV-1, and is consistent with the expected molecular weight size of the viral MEQ protein.
The result shows that the 2A9-B12 monoclonal antibody prepared by the invention can specifically identify the MDV-1 strain, has no non-specific reaction with the MDV-2 and MDV-3 strains, and can be used for Western blot specific detection and identification of the MDV-1 strain.
The present invention has been described in detail with reference to the above embodiments, and it will be understood by those skilled in the art that various changes, modifications and equivalents may be made in the specific parameters and steps of the above embodiments without departing from the spirit of the invention, and it is understood that various embodiments may be made without departing from the scope of the invention.
Sequence listing
<110> agricultural science institute of Henan province
<120> serum type 1 Marek's disease virus MEQ monoclonal antibody, preparation method and application
<130> preparation of monoclonal antibody
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aggtcaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc ctgtccatca 60
cttgcactgt ctctgggttt tcattaacca gctatggtgt acactgggtt cgccagcctc 120
caggaaaggg tctggagtgg ctgggagtaa tatgggctgg tggaagcaca aattataatt 180
cggctctcat gtccagactg agcatcagca aagacaactc caagagccaa gttttcttaa 240
aaatgaacag tctgcaaact gatgacacag ccatgtacta ctgtgccaga gacagggata 300
acacctggtt tgcttactgg ggccaaggga ccacggtcac cgtctcctca 350
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gacattgagc tcacccagtc tccagcactc ccggctgcat ctccagggga gaaggtcact 60
atcacctgca gtgccagctc aagtataagt tccaattact tgcattggta tcagcagaag 120
ccaggattct cccctaaact cttgatttat aggacatcca atctggcttc tggagtccca 180
gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaattgg caccatggag 240
gctgaagatg ttgccactta ctactgccag cagggtagta gtataccact cacgttcggt 300
gctggcacca agctggaaat caaacgg 327
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Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser
1 5 10 15
Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr Gly
20 25 30
Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly
35 40 45
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Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala Arg
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Thr Val Ser Ser
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Asp Ile Glu Leu Thr Gln Ser Pro Ala Leu Pro Ala Ala Ser Pro Gly
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Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Phe Ser Pro Lys Leu Leu
35 40 45
Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
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Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly Thr Met Glu
65 70 75 80
Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ser Ile Pro
85 90 95
Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg
100 105
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Ser Pro Ser Lys His Pro Phe Pro Asp Gly Leu Ser Glu Glu Glu Lys
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Gln Lys Leu Glu Arg Arg Arg Lys Arg Asn Arg Asp Ala
35 40 45
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<212> PRT
<213> Artificial sequence ()
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Tyr Val Asp Lys Leu His Glu Ala Cys Glu Glu Leu Gln Arg Ala Asn
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Glu His Leu Arg Lys Glu Ile Arg Asp Leu Arg Thr Glu Cys Thr Ser
20 25 30
Leu
<210> 7
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<213> Artificial sequence ()
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Pro Val Pro Glu Pro Pro Ile Cys Thr Pro Pro Pro Pro Ser Pro Asp
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Glu Pro Asn Ala Pro His Cys Ser Gly Ser Gln Pro Pro Ile Cys Thr
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Pro
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Leu Phe Thr Pro Ser Pro Pro His Pro Ala Pro Glu Pro Glu Arg Leu
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<210> 9
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aggtsmarct gcagsagtcw gg 22
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tgaggagacg gtgaccgtgg tcccttggcc cc 32
<210> 11
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<212> DNA
<213> Artificial sequence ()
<400> 11
gacattgagc tcacccagtc tcca 24
<210> 12
<211> 24
<212> DNA
<213> Artificial sequence ()
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ccgtttgatt tccagcttgg tgcc 24
Claims (8)
1. A monoclonal antibody hybridoma cell strain secreting antiserum I-type Marek's disease virus MDV-1 specific MEQ protein is characterized in that the monoclonal antibody hybridoma cell strain is a murine monoclonal antibody hybridoma cell strain MEQ-2A9-B12 which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.23023.
2. A monoclonal antibody 2A9-B12 against MDV-1 specific MEQ protein of serotype I Marek's disease virus, wherein said monoclonal antibody 2A9-B12 is secreted by the murine monoclonal antibody hybridoma cell line MEQ-2A9-B12 as defined in claim 1; the light chain type of the monoclonal antibody 2A9-B12 is Kappa type, and the subtype is IgG2B.
3. The monoclonal antibody 2A9-B12 of claim 2, wherein the heavy chain variable region nucleotide sequence of monoclonal antibody 2A9-B12 is shown as SEQ ID No.1 and the light chain variable region nucleotide sequence is shown as SEQ ID No. 2.
4. The monoclonal antibody 2A9-B12 of claim 2, wherein the heavy chain variable region amino acid sequence of monoclonal antibody 2A9-B12 is set forth in SEQ ID No.3 and the light chain variable region amino acid sequence is set forth in SEQ ID No. 4.
5. Use of the monoclonal antibody 2A9-B12 as claimed in claim 2 for typing and differential diagnosis of different serotype MDV strains for non-disease diagnostic purposes.
6. Use of the monoclonal antibody 2A9-B12 of claim 2 in an immunological detection method for non-disease diagnostic purposes.
7. The use of claim 6, wherein the immunological detection method comprises indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), or Western blot analysis (Western-blot).
8. Use of the monoclonal antibody 2A9-B12 of claim 2 in the preparation of an immunological detection reagent.
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