CN113480642B

CN113480642B - anti-African swine fever virus CD2v protein monoclonal antibody, preparation method and application

Info

Publication number: CN113480642B
Application number: CN202110919068.XA
Authority: CN
Inventors: 王爱萍; 牛艳; 杨伟如; 陈玉梅; 周景明; 张守涛; 刘燕凯; 刘红亮; 丁培阳; 祁艳华; 梁超; 朱习芳; 马红芳
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2021-08-11
Filing date: 2021-08-11
Publication date: 2022-06-24
Anticipated expiration: 2041-08-11
Also published as: CN113480642A

Abstract

The invention discloses an African swine fever virus CD2v protein-resistant monoclonal antibody, a preparation method and application, and belongs to the technical field of genetic engineering. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the light chain variable region is shown as SEQ ID NO: 2, respectively. The amino acid sequence of the epitope specifically recognized by the monoclonal antibody is shown as SEQ ID NO: 5, respectively. The monoclonal antibody or the kit containing the monoclonal antibody is applied to preparation of a reagent for detecting or assisting in diagnosis of African swine fever virus or African swine fever virus CD2v protein. The monoclonal antibody disclosed by the invention can specifically recognize and bind to a linear B cell epitope region of the CD2v protein, and provides a valuable tool for further researching the biological function of the CD2v protein.

Description

anti-African swine fever virus CD2v protein monoclonal antibody, preparation method and application

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an anti-African swine fever virus CD2v protein monoclonal antibody, a preparation method and application.

Background

African Swine Fever Virus (ASFV) is a large and complex DNA virus that is the causative agent of African Swine Fever (ASF) and is the only known member of the genus Asfarviridae. ASF is a highly contagious hemorrhagic disease in domestic pigs, with morbidity and mortality rates as high as 100%. ASFV has multiple natural hosts and repositories, which further add to the potential threat of ASF. In view of the great risk and threat of such a cross-border animal disease, it has been placed in the world animal health Organization (OIE) terrestrial animal health code and is defined as an animal disease that must be reported to the OIE.

Due to the global economic importance of ASFV, since the end of the 1960 s, attempts have been made to develop an effective, safe vaccine to protect pigs against ASFV infection. However, due to the complex structure of ASFV and limited understanding of the function of viral proteins, a safe and effective vaccine against ASF has not been developed worldwide, and control of african swine fever is strictly dependent on animal quarantine. Therefore, the kit is of great importance for detection and prevention and control of ASFV.

ASFV has a large linear double-stranded DNA (dsDNA) genome of 170-194kb, encoding more than 150 proteins. Although as many as 68 structural proteins have been identified from virions, the function of most proteins is unclear. ASFV virions have a unique multilayer structure, and intracellular virions are composed of nucleosides (first layer), an inner nucleocapsid (second layer), an inner membrane (third layer), and a capsid (fourth layer). The extracellular virion has an additional outer envelope (fifth layer). Both intracellular and extracellular ASFV virions are infectious. The structural proteins constituting the virus particle are distributed in these multilayer structures and play various roles in viral infection. The identification of antigenic properties of viral structural proteins is essential for understanding the interaction between virus and host, and is crucial for improving serological diagnosis of ASFV and designing subunit vaccines or viral vector vaccines. At present, ASFV has 25 genotypes, and the genetic diversity and antigenic diversity of some genes are one of the main reasons for the lack of cross-protective vaccines against this lethal swine disease. The diversity of antigenic structural proteins may also have a tremendous impact on the reliable diagnosis of ASFV infection.

In order to deeply research the role of antigenic structural proteins in virus infection and realize accurate diagnosis of ASFV virus infection, more monoclonal antibodies capable of recognizing different structural proteins and different epitopes of ASFV are still needed in the field, and a new direction and a new idea are provided for preventing and treating ASF. The CD2v protein is encoded by ASFV EP402R gene, expresses transmembrane protein for ASFV late stage, and has N-terminal signal peptide and single transmembrane structural domain. Can cause erythrocyte adsorption phenomenon in infected animal cells, and plays an important role in the process of virus transmission and replication. Therefore, the ASFV CD2v protein is a good target, and has good application prospect when being used for ASFV detection and vaccine development.

Disclosure of Invention

The invention aims to provide a monoclonal antibody against African swine fever virus CD2v protein, a preparation method and application thereof, so as to solve the problems existing in the prior art, the monoclonal antibody can specifically recognize and bind to a linear B cell epitope region of CD2v protein, and provides a valuable tool for further researching the biological function of the CD2v protein.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides an anti-African swine fever virus CD2v protein monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 1, and the light chain variable region is shown as SEQ ID NO: 2, respectively.

The monoclonal antibody against the African swine fever virus CD2v protein can specifically recognize glycosylated CD2v protein, and further can specifically recognize glycosylated CD2v protein¹³³VKYTNESILEYNWNNSNI¹⁵⁰A linear B cell epitope region.

It is obvious to those skilled in the art that, based on the linear B cell epitope specifically disclosed in the present invention, one or more amino acid additions, deletions, substitutions, and other modifications can be made by conventional protein engineering methods to obtain conservative variants or fragments thereof, while still maintaining specific binding to the VKYTNESILEYNWNNSNI sequence of the CD2v protein of african swine fever virus.

The invention also provides an epitope specifically recognized by the monoclonal antibody, and the amino acid sequence of the epitope is shown as SEQ ID NO: 5, respectively.

The invention also provides a gene for coding the monoclonal antibody, and the gene sequence for coding the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO: 3, the gene sequence of the variable region of the light chain of the monoclonal antibody is shown as SEQ ID NO: 4, respectively.

The invention also provides an expression vector containing the gene.

The invention also provides a host cell containing the expression vector.

The invention also provides a preparation method of the monoclonal antibody, which comprises the steps of culturing the host cell and expressing the monoclonal antibody of the anti-African swine fever virus CD2v protein.

The invention also provides a kit containing the monoclonal antibody.

Preferably, the monoclonal antibody in the kit is used as a coating antibody or a labeled antibody.

The invention also provides application of the monoclonal antibody or the kit in preparation of a reagent for detecting or assisting in diagnosis of African swine fever virus.

The invention also provides application of the monoclonal antibody or the kit in preparation of a reagent for detecting or assisting in diagnosis of African swine fever virus CD2v protein.

The invention discloses the following technical effects:

the invention discloses an African swine fever virus CD2v protein monoclonal antibody, which is prepared by expressing a CD2v protein in CHO cells by using a lentivirus expression system, then taking the purified CD2v protein as an immunogen and immunizing a BALB/c mouse by an immunological method. The monoclonal antibody can specifically recognize and bind to the 133-155 specific linear B cell epitope region of the CD2v protein, has strong specificity and good stability, and provides a valuable tool for further researching the biological function of the CD2v protein. The detection reagent or the kit for detecting the African swine fever virus or the CD2v protein thereof disclosed by the invention can specifically detect cells infected by ASFV, so that the reagent or the kit has strong specificity, and provides an experimental basis for clinical application.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows the results of fluorescence detection of lentiviral packaging; a: fluorescence detection results of pLVX-CD2v/HEK293T after 24h of transfection; b: fluorescence detection results of pLVX-CD2v/HEK293T after 48h of transfection;

FIG. 2 is a screening of CHO cell line stably expressing African swine fever virus CD2v protein; a: CHO/CD2v cells under an inverted fluorescence microscope; b: FACS detection of the fluorescence rate of the CHO/CD2v cell line;

FIG. 3 shows the purification and identification of CD2v protein; a is the purification result of the CD2v protein, wherein M: marker; 1: cell culture supernatant; 2: flow through; 3: washing impurities; 4: eluting; b and C are respectively SDS-PAGE and Western Blot to identify glycosylation of the purified CD2v protein, wherein M is Marker; 1: recombinant ASFV-CD2v protein; 2: PNGase F-treated CD2v protein; 3-4: 1-2 corresponding Western-blot results;

FIG. 4 is the results of the mouse multiple antisera titers;

FIG. 5 shows the results of purification of ascites of monoclonal antibodies; m: marker; 1: 4B11 before purification of ascites; 2: 4B11 purifying ascites; 3: 5F7 before purification of ascites; 4: 5F7 purifying ascites; 5: before purification of 5H4 ascites; 6: purifying ascites with 5H 4; 7: before 8D5 ascites purification; 8: 8D5 ascites after purification; 9: 12B4 before purification of ascites; 10: 12B4 purifying ascites;

FIG. 6 shows the titer of different monoclonal antibodies after purification;

FIG. 7 shows the results of identification of monoclonal antibodies by IFA;

FIG. 8 shows the result of identifying monoclonal antibody by blocking ELISA;

FIG. 9 shows the results of ELISA assays for conjugated polypeptide and positive serum reactivity; a: coupling the ELISA detection results of the reactivity of the polypeptide and ASFV positive pig serum; b: ELISA detection results of the reactivity of the conjugated polypeptide and the serum of the CD2v positive mouse;

FIG. 10 shows the results of ELISA assays for reactivity of conjugated polypeptides and CD2v monoclonal antibody.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

EXAMPLE 1 selection and preparation of immunogens

The CD2v protein is the main structural protein of ASFV, can cause erythrocyte adsorption phenomenon in infected animal cell, and plays an important role in the virus transmission and replication process. The preparation of the anti-ASFV CD2v protein monoclonal antibody and the identification of the CD2v protein epitope have important significance for the prevention and detection of the disease. Therefore, the CD2v protein is used as immunogen in the present invention. Specifically, the preparation of the immunogen comprises the following steps:

1. a signal peptide, a CD2v protein extracellular region sequence and a coding region gene corresponding to a 6 XHis tag are optimized and synthesized according to CHO cell codon preference by referring to sequence information of a China/2018/Anhui XCGQ strain (GenBank: AYW34030.1) published on GenBank, and are cloned into a pLVX-IRES-ZsGreen1 lentiviral vector to construct eukaryotic recombinant expression pLVX-CD2v-IRES-ZsGreen1, which is called pLVX-CD2v for short. Packaging of lentiviruses was performed in 293T cells, briefly: 293T cells were placed in Opti-MEM complete medium, cultured at 37 ℃ for 2h, and pLVX-CD2v 0.4.4. mu.g, pMD2.G 0.6. mu.g, psPAX 21. mu.g were added to 200. mu.L jet-

In buffer, vortex for 5s, place in a clean bench for 10min, add 293T cells and mix gently; 6h after transfection, the transfection complex was replaced by Opti-MEM medium, and the medium was left at 37 ℃ for further culture; fluorescence was observed over 48h to determine the effect of this transfection. Continuously observing the growth state of the cells, collecting culture supernatant, namely the lentivirus suspension, when more than 80 percent of the cells are broken and shed, and centrifuging at 12000r/min for 10min for later use. The lentivirus packaging results are shown in FIG. 1. Adding the collected lentivirus suspension into a growth density of 1 × 10 at a volume ratio of 1:1⁵In the CHO cells of/mL, the cell culture medium is serum-free, SMM CHO-SI expression culture medium containing 4mM L-glutamine is mixed and cultured for 24h, the culture medium with the same volume is supplemented, the culture is continued for 24h, and the fluorescence is observed to confirm whether the transduction is successful. CHO cells are transduced and CHO/CD2v positive cells are screened, wells with high green fluorescent protein expression and bright fluorescence intensity are selected, enlarged culture is carried out, centrifugation is carried out at 1000r/min for 10min, supernatant is discarded, and PBS is used for resuspending the cells. PBS-treated cells were filtered through nylon mesh and then loaded onto BD FACS Aria III, positive cells were screened by green fluorescence signal, and then cells positive to ZsGreen1 (ZsGreen1+) were sorted and collected in 96-well cell plates at 1 cell per well and cultured at 37 ℃. The obtained ZsGreen1 positive monoclonal cells were subcloned 3 times until the positive rate of ZsGreen1 approached 100%, and the obtained positive clonal cells were named CHO/CD2v (as shown in FIG. 2).

2. Expression and purification of CD2v protein

(1) Mass culture of cells: CHO/CD2v cells were inoculated into 50mL shake flasks, cultured at 120rpm, and the growth density of the cells was determined by cell counting. Expanding the cells in a 50mL shake flask into a 500mL shake flask for high-density culture when the cell culture density is kept unchanged, counting the cells every day, starting adding the SMS CHO-SUPI culture medium additive solution when the cell density is more than 1.5 times of the initial density, adding the SMS CHO-SUPI culture medium additive solution in an amount of 1.5%, adding the SMS CHO-SUPI culture medium additive solution every day, stopping adding the SMS CHO-SUPI culture medium additive solution when the cell density is kept unchanged or the death rate of the cells starts to increase, collecting cell suspension, and centrifuging to collect supernatant liquid.

(2) The culture supernatant expressing the CD2v protein was collected and centrifuged at 12000r/min for 10 min. The centrifuged supernatant was filtered through a 0.45 μm filter head and then placed in an ice box for further use.

(3) Sucking 2mL of Ni-NTA filler by a liquid shifter, adding the Ni-NTA filler to the installed protein purification column, and carefully adding an upper gasket after the Ni filler is settled; opening the control valve to allow 20% ethanol to flow out, and continuing to wash the column with 10mL of deionized water;

(4) the flow rate was stabilized by adjusting the control valve of the protein purification apparatus, and after the flow rate was adjusted, the column was washed with 10mL or more of the equilibration buffer at a rate of 1 mL/min.

(5) And (3) dropwise adding the cell culture supernatant filtered by the filter membrane into the well-balanced column in batches, 5mL each time, adjusting the control valve to ensure that the flow rate does not exceed 1mL/min, quickly collecting the filtrate and repeatedly loading the filtrate for 2-3 times, collecting the filtrate of the last time, and standing at-20 ℃ for later use.

(6) The column was washed with washing buffer to remove contaminating proteins.

(7) Finally, adding 10mL of elution buffer solution into the column to elute the target protein; adjusting the control valve to reduce the speed of liquid flowing through as much as possible; the eluted liquid was collected in 1.5mL EP tubes, 1mL per tube, the concentration of protein in each tube was measured with a microplate reader and labeled, and the tube was left at-20 ℃ for future use.

(8) After the purification was completed, 20mL of ddH was used₂The nickel column was washed with O, then once more with NaOH solution (0.1mol/L), with ddH₂And O, cleaning the nickel column again, and finally storing the nickel column in 20% ethanol and storing in a refrigerator at 4 ℃.

(9) Adding 5 × Loading Buffer into the cell culture supernatant before purification and the eluate, impurity washing solution and eluate collected after purification, boiling for 10min, and analyzing the purification result by 12% SDS-PAGE.

As shown in FIG. 3, the CD2v protein obtained by elution has dispersed target bands at about 55-110KDa, the CD2v protein is subjected to enzyme digestion for 1h at 37 ℃ by glycosidase PNGase F under a non-denaturing condition, and the results of SDS-PAGE and Western-blot analysis show that the CD2v protein has different degrees of glycosylation, which indicates that the dispersed target bands at about 55-110KDa are CD2v protein and have higher purity.

EXAMPLE 2 preparation of monoclonal antibodies

1. Animal immunization

(1) Adding Freund's complete adjuvant into immunogen CD2v protein, emulsifying for first immunization;

(2) immunizing 2 female BALB/c mice of 4-8 weeks old by a back subcutaneous multipoint injection method, wherein the immunization dose is 10 mu g/mouse;

(3) emulsifying the immune antigen with Freund's incomplete adjuvant at intervals of 3 weeks, and performing booster immunization on BALB/c mice by the same method and dosage;

(4) after three weeks, tail vein blood collection is carried out to determine the specific antibody titer aiming at the CD2v protein, a mouse with higher titer (shown in figure 4) is selected, and the BALB/c mouse is subjected to super-strong immunity by using immunogen without adjuvant through a tail vein injection method 3-4 days before cell fusion, wherein the immunizing dose is 20 mu g/mouse.

2. Cell fusion and monoclonal antibody preparation

The method of polyethylene glycol is adopted, and the spleen cells of the immunized mice and the mouse myeloma cells SP2/0 are mixed according to the cell number of 8: 1, and screening the fused cells by using an HAT selective medium; positive hybridoma cells were initially selected by indirect ELISA 12 days after fusion using CD2v protein as the coating antigen.

The indirect ELISA method comprises the following steps:

(1) diluting the CD2v protein into a coating solution with the concentration of 2 mug/mL by using CBS solution to coat the ELISA plate, sealing at the temperature of 4 ℃ overnight at a hole of 100 mug/mL;

(2) diluting hybridoma supernatant (primary antibody) with 5% skimmed milk 2 times, sequentially adding into enzyme labeling plate at 50 μ l/well, and incubating at 37 deg.C for 30min, wherein the positive control is ASFV pig positive serum;

(3) discarding the primary antibody, washing the plate by PBST, cleaning and drying;

(4) diluted HRP-labeled goat anti-mouse IgG (secondary antibody) was added to the reaction wells at 50 μ l/well. Incubating at 37 ℃ for 30 min;

(5) discarding the secondary antibody, washing with PBST, and patting dry;

(6) adding 100 mul of TMB color developing solution prepared in situ into each hole, and reacting for 15min in a dark room;

(7) add 50. mu.l of 2M H per well₂SO₄Terminating the reaction;

(8) microplate reader for reading OD of each well₄₅₀The value is obtained.

3. Subcloning of hybridoma cells by limiting dilution method

The above positive hybridoma cells were diluted to about 1.5cells/ml with 10% FBS in RPMI1640 complete medium (1640/10), 100. mu.l per well was added to a 96-well plate pre-plated with 100. mu.l feeder cells, and the plate was placed at 37 ℃ in 5% CO₂Culturing for 6-8 days in an incubator; further screening positive hybridoma cells by an indirect ELISA method; performing subcloning for 2-3 times until obtaining hybridoma cell strain stably secreting anti-CD 2V protein monoclonal antibody, obtaining target hybridoma cell, performing expanded culture on the screened positive monoclonal antibody, and performing 1-2 × 10 cell number⁶Freezing and storing in a tube.

4. Stability identification of monoclonal hybridoma cell strain

Continuously culturing the established monoclonal hybridoma cell strain for 3 months and repeatedly freezing and thawing by liquid nitrogen so as to identify the stability of the hybridoma cell; the results show that the monoclonal hybridoma cell strain has good stability.

5. Method for preparing monoclonal antibody by in vivo induced ascites

Female BALB/c mice were selected and intraperitoneally injected with 500. mu.l of sterile paraffin for one week, and the resulting monoclonal hybridoma cells were again intraperitoneally injected at an injection rate of 2X 10⁵After one week, ascites is extracted after the abdomen of the mouse is enlarged, the supernatant is centrifuged, and the ascites is purified by ammonium caprylate method.

EXAMPLE 3 purification and characterization of antibodies

1. The saturated ammonium sulfate precipitation method is used for purifying the antibody and the operation method is as follows:

(1) 5ml of monoclonal antibody ascites is taken, 5ml of PBS buffer solution is added, 2.5ml of saturated ammonium sulfate solution is added dropwise to obtain 20% ammonium sulfate solution, stirring is carried out while adding, and after complete mixing, standing is carried out for 30 min.

(2)8000r/min, centrifuging for 20min, and discarding the precipitate to remove fibrin.

(3) Adding 12.5ml saturated ammonium sulfate solution into the supernatant, mixing well, standing for 30 min.

(4)8000r/min, centrifuging for 20min, and discarding the supernatant.

(5) Dissolving the precipitate in 10ml PBS buffer solution, adding 5ml saturated ammonium sulfate solution to obtain 33% ammonium sulfate solution, mixing, and standing for 30 min.

(6)8000r/min, centrifuging for 20min, and discarding supernatant to remove albumin.

(7) And repeating the step 5, 2-3 times.

(8) The precipitate was dissolved in 5ml of PBS buffer, and the solution was placed in a dialysis bag, dialyzed against PBS buffer at 4 ℃ and changed 4 times.

(9)8000r/min, centrifuging for 20min, discarding precipitate to obtain supernatant as purified antibody (shown in figure 5), measuring antibody concentration, packaging, and storing at-20 deg.C.

2. Monoclonal antibody potency assay

The indirect ELISA assay was performed with reference to example 2, with a slight difference in primary antibody: diluting the purified monoclonal antibody with 5% skimmed milk at a ratio of 1:1000 by 2 times, sequentially adding into an enzyme label plate at 50 μ l/well, and incubating at 37 deg.C for 30min, wherein the positive control is ASFV pig positive serum; the other steps were carried out as described in example 2, and the results of ELISA detection (shown in FIG. 6) showed that the titers of monoclonal antibodies 4B11 and 8D5 were 1: 2.56X 10⁵(ii) a The titers of the monoclonal antibodies 5H4 and 12B4 are 1: 5.12X 10⁵The monoclonal antibody 5F7 has the potency of 1:1.024 × 10⁶。

The invention also sequences the monoclonal antibody 5F7 with higher titer, and the result is as follows:

the heavy chain variable region amino acid sequence is shown as SEQ ID NO: 1, and the following components:

QESGPDLVKPSQSLSLTCTVTGYSITSSYMPGTGSGNFQETNWNGWATYTTVVALTTTHLSKVESLSLETHPRTSSSCNGILGLLRIQPHITVQDMVTTALPIGAKGPRSPSPQ

the nucleotide sequence is shown as SEQ ID NO: 3, showing:

caggagtcaggacctgacctggtgaaaccttctcagtcactttcactcacctgcactgtcactggctactccatcaccagtagttatatgcctggcactggatccggcaatttccaggaaacaaactggaatggatgggctacatacactacagtggtagcactgactacaacccatctctcaaaagtcgaatctctatcactcgagacacatccaagaaccagttcttcctgcaatggaattctgggactactgaggatacagccacatattactgtgcaagatatggtaactacggctttgcctattggggccaagggaccacggtcaccgtctcctcaa

the variable region amino acid sequence of the light chain is shown as SEQ ID NO: 2, as shown in the figure:

LPVRLLVLDVLDSGASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHINGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPVDVRWRHQAGNQTGWCCTNCIHLPT

the nucleotide sequence is shown as SEQ ID NO: 4, and (2) is as follows:

ttgcctgttaggctgttggtgctggatgttctggattccggtgcttccagcagtgatgttgtgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagagccttgtacacattaatggaaacacctatttacattggtacctgcagaagccaggccagtctccaaagctcctgatctacaaagtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatttctgctctcaaagtacacatgttcctgtggacgttcggtggaggcaccaagctggaaatcaaacgggctggtgctgcaccaactgtatccatcttcccacc

3. results of IFA identification of reactivity of monoclonal antibody with CD2v protein

Reconstruction of pTrip-CD2v-IRES-puro recombinant vector transduction of HEK293T cells, and the five different monoclonal antibodies were incubated, and FITC-conjugated goat anti-mouse IgG antibody was added.

As a result, all nuclei were stained blue with DAPI staining solution as shown in FIG. 7. Wherein fluorescence signals were detected in all wells incubated with anti-CD 2v monoclonal antibody and no fluorescence signals were detected in negative control and blank control wells. IFA results prove that the monoclonal antibody screened by the screening experiment has better reactivity with the ASFV CD2v protein transiently expressed by 293T cells.

4. Blocking ELISA for identifying reactivity of monoclonal antibody and CD2v protein

Purifying the CD2v proteinCoating in an ELISA plate, sealing according to the indirect ELISA step, and washing the plate; in blocking ELISA, an ASFV positive pig serum sample is diluted by PBS buffer solution, added into the ELISA plate, and incubated for 30min at 37 ℃; then diluting the purified 5 monoclonal antibodies with PBS buffer solution, adding the diluted monoclonal antibodies into an ELISA plate, and incubating for 30min at 37 ℃; finally adding goat anti-mouse IgG/HRP secondary antibody diluted by 1:5000, and incubating for 30min at 37 ℃; adding a developing solution for developing for 5-10 min, and adding 2mol/L_H2SO₄Stopping the reaction; reading the absorbance value at 450nm, recording and analyzing data; and (4) judging a result: blocking rate (percent inhibition/PI) (negative serum OD)₄₅₀Value-serum OD to be detected₄₅₀Value)/negative serum OD₄₅₀Value X100%.

As shown in fig. 8, the mean OD values of all the monoclonal antibodies obtained from the negative and positive sera were significantly different, with the PI value of monoclonal antibody 12B4 being greater than 80% and the PI values of the other monoclonal antibodies being between 52.87% and 72.99%. The polyclonal serum of the African swine fever virus can block the combination of all monoclonal antibodies.

Example 4 identification of epitope recognized by monoclonal antibody

1. Design of overlapping polypeptides

The overlapping polypeptide method is adopted to design and synthesize 15 segments of polypeptide, which covers the whole extracellular region, and adjacent polypeptides overlap by 6 amino acids. The amino acid sequence of the polypeptide fragment is shown in Table 1.

TABLE 1 polypeptide sequences

2. ELISA detection result of coupling polypeptide and positive serum reactivity

The ELISA plate is coated with the polypeptide coupled with BSA, and the reactivity of the monoclonal antibody and the polypeptide is identified by indirect ELISA detection of ASFV positive pig serum, CD2v positive mouse serum and the polypeptide, and the results are shown in FIG. 9, wherein the polypeptides P2, P3, P4, P10 and P12 have strong reaction with the ASFV positive pig serum and also react with the CD2v positive mouse serum.

3. Identification of monoclonal antibody recognition epitope

The polypeptide is coated on an enzyme label plate, indirect ELISA detection is carried out on the CD2v monoclonal antibody and the polypeptide, the reactivity of the monoclonal antibody and the polypeptide is identified, and the result is shown in figure 10, the polypeptide P10 can be identified by the monoclonal antibody 4B 11; polypeptide P12 (amino acid sequence SEQ ID NO: 5, i.e., VKYTNESILEYNWNNSNI) was recognized by monoclonal antibodies 5H4, 5F 7.

Example 6 application of monoclonal antibody in preparation of African swine fever virus detection reagent or kit

The embodiment provides an African swine fever virus detection reagent, which comprises the anti-African swine fever virus CD2v protein monoclonal antibody provided by the invention.

The African swine fever virus HLJ/18 strain infects primary alveolar macrophages of pigs, and is prepared into a monolayer cell reaction plate after methanol fixation treatment. The monolayer cell reaction plate infected by the ASFV HLJ/18 strain is provided by Harbin veterinary institute of Chinese academy of agricultural sciences.

1. The method for detecting the African swine fever infected cells by the IPMA method comprises the following specific steps:

(1) the monolayer cell reaction plate prepared above was taken out of the refrigerator, equilibrated to room temperature, and washed 1 time with PBST.

(2) The detection reagent (containing the African swine fever virus CD2v protein monoclonal antibody provided by the invention) is used as a primary antibody, and 5% of skim milk 1:1000, and setting the positive control as ASFV pig positive serum and the negative control as anti-PCV 2 Cap protein monoclonal antibody, wherein the sample adding amount is 50 μ l per well. Incubate at 37 ℃ for 30 min.

(3) PBST was washed 3 times with HRP-labeled goat anti-mouse IgG as secondary antibody. HRP-labeled goat anti-porcine IgG was added to the positive control wells as a secondary antibody. Incubate at 37 ℃ for 30 min.

(4) PBST was washed 3 times, added with AEC substrate color developing solution, developed for 10min at room temperature, and placed under an optical microscope to observe the dyeing result.

And (4) judging a result: typical reddish brown staining was observed for cells infected with African swine fever virus, and no reddish brown staining was observed for uninfected cells and negative controls.

The result shows that the monoclonal antibody of the African swine fever virus CD2v protein can specifically detect cells infected by ASFV.

2. IFA detection method can be performed with reference to IPMA detection method, and the secondary antibody is slightly different, and a FITC-labeled goat-anti-mouse secondary antibody is added at a ratio of 1:500 dilution and addition to corresponding wells and other steps were performed with reference to IPMA. No chromogenic substrate was required and the results were observed under a fluorescent microscope.

And (4) judging a result: typical green fluorescence was observed for cells infected with African swine fever virus, and for uninfected cells and negative controls there was no fluorescence.

The results are shown in FIG. 9, which shows that the monoclonal antibody against African swine fever virus CD2v protein can specifically detect ASFV infected cells.

Meanwhile, the monoclonal antibody of the invention can also be used in an ELISA detection reagent or an ELISA detection kit, such as in example 2, or the monoclonal antibody of the invention is labeled with a label and then used in the ELISA detection reagent or the kit to detect ASFV or ASFV CD2v protein; the monoclonal antibody of the invention can also be used as a primary antibody in western blotting to detect ASFV or ASFV CD2v protein, as in example 3; or the monoclonal antibody of the invention is used as a gold-labeled antibody or a capture antibody for immunochromatography test paper to detect ASFV or ASFV CD2v protein.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

<110> Zhengzhou university

<120> African swine fever virus-resistant CD2v protein monoclonal antibody, preparation method and application

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 114

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln Ser Leu Ser Leu

1 5 10 15

Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Ser Tyr Met Pro Gly

20 25 30

Thr Gly Ser Gly Asn Phe Gln Glu Thr Asn Trp Asn Gly Trp Ala Thr

35 40 45

Tyr Thr Thr Val Val Ala Leu Thr Thr Thr His Leu Ser Lys Val Glu

50 55 60

Ser Leu Ser Leu Glu Thr His Pro Arg Thr Ser Ser Ser Cys Asn Gly

65 70 75 80

Ile Leu Gly Leu Leu Arg Ile Gln Pro His Ile Thr Val Gln Asp Met

85 90 95

Val Thr Thr Ala Leu Pro Ile Gly Ala Lys Gly Pro Arg Ser Pro Ser

100 105 110

Pro Gln

<210> 2

<211> 143

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Leu Pro Val Arg Leu Leu Val Leu Asp Val Leu Asp Ser Gly Ala Ser

1 5 10 15

Ser Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser

20 25 30

Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val

35 40 45

His Ile Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly

50 55 60

Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly

65 70 75 80

Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser

100 105 110

Gln Ser Thr His Val Pro Val Asp Val Arg Trp Arg His Gln Ala Gly

115 120 125

Asn Gln Thr Gly Trp Cys Cys Thr Asn Cys Ile His Leu Pro Thr

130 135 140

<210> 3

<211> 342

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

caggagtcag gacctgacct ggtgaaacct tctcagtcac tttcactcac ctgcactgtc 60

actggctact ccatcaccag tagttatatg cctggcactg gatccggcaa tttccaggaa 120

acaaactgga atggatgggc tacatacact acagtggtag cactgactac aacccatctc 180

tcaaaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc ctgcaatgga 240

attctgggac tactgaggat acagccacat attactgtgc aagatatggt aactacggct 300

ttgcctattg gggccaaggg accacggtca ccgtctcctc aa 342

<210> 4

<211> 429

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ttgcctgtta ggctgttggt gctggatgtt ctggattccg gtgcttccag cagtgatgtt 60

gtgatgaccc aaactccact ctccctgcct gtcagtcttg gagatcaagc ctccatctct 120

tgcagatcta gtcagagcct tgtacacatt aatggaaaca cctatttaca ttggtacctg 180

cagaagccag gccagtctcc aaagctcctg atctacaaag tttccaaccg attttctggg 240

gtcccagaca ggttcagtgg cagtggatca gggacagatt tcacactcaa gatcagcaga 300

gtggaggctg aggatctggg agtttatttc tgctctcaaa gtacacatgt tcctgtggac 360

gttcggtgga ggcaccaagc tggaaatcaa acgggctggt gctgcaccaa ctgtatccat 420

cttcccacc 429

<210> 5

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Val Lys Tyr Thr Asn Glu Ser Ile Leu Glu Tyr Asn Trp Asn Asn Ser

1 5 10 15

Asn Ile

Claims

1. The monoclonal antibody against African swine fever virus CD2v protein comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 1, and the light chain variable region is shown as SEQ ID NO: 2, respectively.

2. The gene encoding the monoclonal antibody of claim 1, wherein the gene sequence encoding the heavy chain variable region of said monoclonal antibody is as set forth in SEQ ID NO: 3, the gene sequence of the variable region of the light chain of the monoclonal antibody is shown as SEQ ID NO: 4, respectively.

3. An expression vector comprising the gene of claim 2.

4. A host cell comprising the expression vector of claim 3.

5. A method for producing the monoclonal antibody of claim 1, comprising the step of culturing the host cell of claim 4 to express a monoclonal antibody against African swine fever virus CD2v protein.

6. A kit comprising the monoclonal antibody of claim 1.

7. The kit of claim 6, wherein the monoclonal antibody is a coating antibody or a labeled antibody.

8. Use of the monoclonal antibody according to claim 1 or the kit according to claim 7 for the preparation of a reagent for the detection or the auxiliary diagnosis of African swine fever virus.

9. Use of the monoclonal antibody according to claim 1 or the kit according to claim 7 for the preparation of a reagent for detecting african swine fever virus CD2v protein.