CN110894216A

CN110894216A - Porcine epidemic diarrhea virus epitope peptide, monoclonal antibody and application

Info

Publication number: CN110894216A
Application number: CN201911149815.5A
Authority: CN
Inventors: 宋勤叶; 王亚文; 郭海勇; 李丽敏; 徐瑞涛; 逯继成; 孙泰然
Original assignee: Hebei Agricultural University
Current assignee: Hebei Agricultural University
Priority date: 2019-11-21
Filing date: 2019-11-21
Publication date: 2020-03-20
Anticipated expiration: 2039-11-21
Also published as: CN110894216B

Abstract

The invention relates to the technical field of cell engineering and immunology, and particularly discloses a porcine epidemic diarrhea virus epitope peptide, a monoclonal antibody and application. The antigen comprises epitope polypeptide shown in SEQ ID No.1, and a monoclonal antibody corresponding to the antigen is prepared by adopting a hybridoma cell technology. The monoclonal antibody provided by the invention can be specifically combined with PEDV recombinant S protein and PEDV, and can effectively identify PEDV variant strains and classical strains.

Description

Porcine epidemic diarrhea virus epitope peptide, monoclonal antibody and application

Technical Field

The invention relates to the technical field of cell engineering and immunology, in particular to a porcine epidemic diarrhea virus epitope peptide, a monoclonal antibody and application.

Background

Porcine Epidemic Diarrheal Virus (PEDV) is a member of the genus Alphacoronavirus of the subfamily Coronaviridae (Coronaviridae) of the order Nidovirales (Nidovirales), and is a enveloped, single-stranded, positive-strand RNA virus. PEDV is the pathogen of Porcine Epidemic Diarrhea (PED), mainly causes pig vomiting, diarrhea and dehydration death, has the most serious harm to the suckling piglets of 1-2 weeks old, has the fatality rate of 90-100 percent, and causes serious loss to the pig industry.

Since the first occurrence of PED in china in 1973, with the widespread use of oil-adjuvanted inactivated vaccines prepared from the strain PEDV CV777 nationwide, the disease has occurred mainly in a local epidemic or sporadic form. However, in recent years, PED begins to frequently burst and become epidemic, which leads to the death of newborn suckling piglets in large quantities. Analysis of viral genome sequence and molecular genetic evolution shows that the S gene of PEDV is mutated. PEDV variant strains and classical strains are currently prevalent in swine herds in countries such as china, korea, japan, and the united states, where PEDV variant virulent strains are dominant strains causing severe diarrhea in suckling piglets. When PED occurs, the identification of the type of PEDV is of great significance to the prevention and treatment of epidemic situations. Gene sequencing is an effective way for identifying the strain type, but the method has the problems of complex operation, long consumed time, high cost and the like. Although fluorescent quantitative pcr (qPCR) can identify variant and classical strains of PEDV, serological detection technology is an important means for qPCR clinical investigation of infection status of PEDV and scientific research, however, serological detection reagents capable of identifying classical and virulent variant strains of PEDV are still lacking at present. Therefore, research on related detection reagents is urgently needed, and a rapid, simple and effective identification and detection method for the porcine epidemic diarrhea virus variant strain and the classical strain is established.

Disclosure of Invention

Aiming at the technical problems existing in the existing identification of classical strains and variant virulent strains of porcine epidemic diarrhea viruses, the invention provides an epitope peptide of porcine epidemic diarrhea viruses, a monoclonal antibody and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

an epitope peptide of porcine epidemic diarrhea virus, the amino acid sequence of which is shown in SEQ ID No. 1.

Compared with the prior art, the epitope contained in the porcine epidemic diarrhea virus epitope peptide (Pep) provided by the invention has difference between the porcine epidemic diarrhea virus variant strain and the classical strain and strong antigenicity, the serum antibodies of mice immunized by the epitope peptide are all positive, the ELISA antibody titer is more than 1:51200 and can be higher than 1:10 at most⁵。

The invention also provides application of the epitope peptide in preparation of a reagent for identifying classical strains and variant strains of porcine epidemic diarrhea viruses.

The epitope peptide provided by the invention is adopted to carry out animal immunity, and the epitope peptide is combined with a hybridoma monoclonal antibody technology to obtain a monoclonal antibody which can be specifically combined with PEDV recombinant S protein and PEDV and is used for identifying classical strains and variant strains of porcine epidemic diarrhea viruses.

The invention also provides an antigen conjugate which comprises the antigen epitope peptide and a carrier protein, wherein the carrier protein is hemocyanin (KLH), Bovine Serum Albumin (BSA), Ovalbumin (OVA) or Human Serum Albumin (HSA).

The antigen conjugate provided by the invention couples the epitope peptide with the carrier protein, the carrier protein stimulates the helper T cell to further induce the immune reaction of the B cell, the sufficient immune reaction is aroused by the synergistic effect of the carrier protein and the epitope peptide, and the titer of an ELISA antibody is improved.

The invention also provides a preparation method of the PEDV-resistant hybridoma cell strain, which comprises the following steps: and (3) adopting the antigen conjugate to carry out animal immunity, fusing the obtained B lymphocyte with myeloma cells, selecting positive clone, and carrying out subcloning to obtain the anti-PEDV hybridoma cell strain.

The preparation method provided by the invention has the advantages that after B lymphocytes obtained after animal immunization are fused with myeloma cells, cell cloning can be observed in the 10 th day, and after two times of screening by using an indirect ELISA method, the PEDV-resistant hybridoma cell strain is obtained, and the ascites titer induced by the PEDV-resistant hybridoma cell strain can reach 10⁶。

Further, the animal immunization process is as follows: BALB/c mice were injected with the antigen three times with an interval of 14d and re-intraperitoneally injected 2-4d before cell fusion for booster immunization. And (4) taking blood from eye sockets 8-12 days after the third immunization, detecting the titer of the specific antibody of the serum by using indirect ELISA, and selecting mice with high titer before fusion for boosting the immunity.

Further, the antigen injection amount of the three times of immunization is 25-35 mu g/mouse; the injection amount of the antigen for boosting immunity is 45-55 mug/mouse.

Further, ELISA antibody titers were higher than 1:10⁵The splenic B lymphocytes of the mice were fused with myeloma cells.

Furthermore, an antigen conjugate is used as an envelope antigen to screen positive clones, and then PEDV recombinant S protein is used as an envelope antigen to subclone the positive clones to obtain the anti-PEDV hybridoma cell strain, so that the antibody secreted by the anti-PEDV hybridoma cell strain can be specifically combined with the PEDV S protein.

The invention also provides a porcine epidemic diarrhea virus monoclonal antibody which is produced by the PEDV-resistant hybridoma cell strain.

Furthermore, the subtype of the monoclonal antibody is IgG2b, and the light chain is a kappa chain, so that the monoclonal antibody can be better specifically combined with the S protein of PEDV and PEDV.

Furthermore, the monoclonal antibody is an HRP labeled antibody, has strong specific binding activity with the S protein of PEDV, and can be used as a necessary experimental material for an enzyme-labeled detection technology of the antigen and the antibody based on the monoclonal antibody.

The invention also provides application of the monoclonal antibody in preparation of a reagent for identifying classical strains and variant strains of porcine epidemic diarrhea viruses.

The monoclonal antibody provided by the invention has good biological activity, can be specifically combined with PEDV recombinant S protein and PEDV, can be used for identifying PEDV variant virulent strains and classical strains, and is simple, convenient, rapid and effective.

Drawings

FIG. 1 is a graph of serum antibody titers of immunized mice in the examples of the invention;

FIG. 2 is a graph showing the results of the titer of ascites antibodies induced by hybridoma cell line A according to the present invention;

FIG. 3 is an SDS-PAGE pattern of hybridoma cell line A according to an embodiment of the invention;

FIG. 4 is a Western-blot diagram of a hybridoma cell strain A according to an embodiment of the invention;

FIG. 5 is an indirect immunofluorescence chart corresponding to the monoclonal antibody produced by hybridoma cell line A in the example of the present invention;

FIG. 6 is a graph of indirect immunofluorescence corresponding to a negative control;

FIG. 7 is an immunofluorescence plot of monoclonal antibodies produced by hybridoma cell line A diluted 1:400 times against PEDV QY 2016;

FIG. 8 is an immunofluorescence chart of the monoclonal antibody produced by the hybridoma cell line A diluted 1:400 times against PEDV CV 777;

fig. 9 is a graph showing the binding activity of PEDV recombinant S protein of HRP-labeled monoclonal antibody according to example of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1

The epitope peptide is used for preparing a monoclonal antibody for identifying classical strains and variant strains of porcine epidemic diarrhea viruses.

According to the S protein amino acid sequences of PEDV variant strains and classical strains (shown in the table 1), four epitope peptides are synthesized: IGENQGVNSTWYCAGRHPTAS, FVSHIRGGHG, PAHMSEHSVVG and LQNHTSTEYFVSS, however, the latter three have poor antigenicity, are difficult to use in animal immunization, are not favorable for obtaining antibodies, and have strong antigenicity of IGENQGVNSTWYCAGRHPTAS. The epitope peptide is synthesized and prepared by Suzhou Qiangyao biotechnology limited.

TABLE 1

Note: classical strains include: CV777, DR13, LZC and attenuated CV 777; variant strains include: HBMC2012, ZJCZ4, USA/NC/2013/35140, USA/Colorado/2013 and GD-A.

The epitope peptide is coupled with the carrier protein hemocyanin to obtain an antigen conjugate Pep-KLH which is used as an immunogen for preparing monoclonal antibodies, and meanwhile, the epitope peptide is coupled with bovine serum albumin to obtain an antigen conjugate Pep-BSA which is used as a detection antigen for screening the antibodies.

Example 2

A preparation method of anti-PEDV hybridoma cell strains comprises the steps of adopting the antigen conjugate Pep-KLH to carry out animal immunization, fusing obtained B lymphocytes with myeloma cells, screening positive clones by taking the antigen conjugate Pep-BSA as an envelope antigen, then subcloning the positive clones by taking PEDV recombinant S protein as the envelope antigen, and obtaining the anti-PEDV hybridoma cell strains, wherein the specific steps are as follows:

s1: 4 BALB/c mice with the age of 7 weeks are selected to be injected with the antigen conjugate Pep-KLH, the injection amount is 30 mu g/mouse, three times of immunization are carried out, the immunization interval time is 14d, 10d after the third immunization, blood is taken from the orbit, the specific antibody titer of serum is detected by indirect ELISA, the result is shown in figure 1, and the ELISA antibody titer selected before fusion is higher than 1:10⁵The mice are injected with the antigen conjugate Pep-KLH to strengthen the immunity, and the injection amount is 50 mu g/mouse。

S2: SP2/0 myeloma cells in good state were fused with splenic B lymphocytes of mice 3d after boosting, under the mediation of polyethylene glycol (PEG 1500). 10mL of fetal bovine serum and 5mL of IMDM were added to the fused cell pellet, mixed well, and 5mL of feeder cells (mouse thymocytes) and 25mL of sterilized semisolid culture medium (IMDM containing 2.2% methylcellulose and 1% HAT) were added thereto, and after mixing well, the mixture was transferred to cell culture dishes, each of which was transferred in an amount of 1.5 mL. The cell culture dish is put into a wet box and placed in an incubator for culturing for 10 days, and the growth condition of cell clones is observed every day.

S3: and (3) after 10 days of cell fusion, observing the appearance of cell clones, selecting 93 cell monoclonals, culturing in a 96-hole cell culture plate paved with feeder cells (mouse thymocytes) in advance, when the hybridoma cell clones in the holes grow to 1/4 of the area of the bottom of the holes, taking supernatant of the hybridoma cells, firstly using an antigen conjugate Pep-BSA as a coating antigen, carrying out two times of primary screening on the cell clones by an ELISA method, then using PEDV recombinant S protein as the coating antigen on positive clones obtained by secondary screening, and screening the positive clones to obtain the anti-PEDV hybridoma cell strains. ELISA main steps: coating each hole of the ELISA plate with 2 mu g/mL of antigen conjugate Pep-BSA or PEDV recombinant S protein, and incubating overnight at 4 ℃; adding 5% defatted milk powder PBST, and sealing at 37 deg.C for 2 hr; 100. mu.L of cell culture supernatant diluted 1:4 times with PBS was added, and incubated at 37 ℃ for 1 hour while setting negative control (SP2/0 myeloma cell culture supernatant), blank control (PBS), and positive control (1: 1000-fold diluted positive serum). After washing for 3 times, adding PBS to dilute 20000 times of HRP-goat anti-mouse IgG, 100 mu L/hole, and incubating for 1h at 37 ℃; washing for 3 times, adding 100 μ L/hole of color developing solution, and developing for 5 min; after stopping the reaction by adding 50. mu.L of stop solution to each well, the OD values were measured at two wavelengths (450nm and 630 nm). Diluting the obtained supernatant of positive cells by 1:50 times, and performing antibody type identification according to the instruction of a mouse monoclonal antibody class/subclass identification ELISA kit (Thermo Scientific), when OD is obtained_450nmThe positive reaction is judged to be more than or equal to 0.2.

Example 3

A preparation method of an anti-PEDV hybridoma cell strain comprises the steps of adopting the antigen conjugate Pep-KLH to carry out animal immunization, fusing obtained B lymphocytes with myeloma cells, selecting positive clones, and carrying out subcloning to obtain the anti-PEDV hybridoma cell strain, wherein the preparation method comprises the following specific steps:

s1: 4 BALB/c mice of 8 weeks old are selected to be injected with an antigen conjugate Pep-KLH, the injection amount is 25 mug/mouse, three times of immunization are carried out, the immunization interval time is 14 days, 12 days after the third immunization, blood is taken from eye sockets, the specific antibody titer of serum is detected by indirect ELISA, and the ELISA antibody titer is selected to be higher than 1:10⁵The mice were injected intraperitoneally with the antigen conjugate Pep-KLH for boosting, and the injection amount was 45. mu.g/mouse.

S2: SP2/0 myeloma cells in good state were fused with splenic B lymphocytes of mice 3d after boosting, under the mediation of polyethylene glycol (PEG 1500). 10mL of fetal bovine serum and 5mL of IMDM were added to the fused cell pellet, mixed well, and 5mL of feeder cells (mouse thymocytes) and 25mL of sterilized semisolid culture medium (IMDM containing 2.2% methylcellulose and 1% HAT) were added thereto, and after mixing well, the mixture was transferred to cell culture dishes, each of which was transferred in an amount of 1.5 mL. The cell culture dish is put into a wet box and placed in an incubator for culture for 14d, and the growth condition of cell clones is observed every day in the process.

S3: 14d after cell fusion, cell cloning can be observed, 93 cell monoclonals are selected to be cultured in a 96-hole cell culture plate paved with feeder cells (mouse thymocytes) in advance, when the hybridoma cell clones in the holes grow to 1/3 of the area of the bottom of the holes, supernatant of the hybridoma cells is taken, an antigen conjugate Pep-BSA is firstly used as a coating antigen, the cell clones are preliminarily screened twice by an ELISA method, positive clones obtained by secondary screening are then screened, and PEDV recombinant S protein is used as the coating antigen to obtain the anti-PEDV hybridoma cell strain. The ELISA was performed as described in example 2.

Example 4

s1: 8w of 4 BALB/c mice are selected to be injected with the antigen conjugate Pep-KLH, the injection amount is 35 mu g/mouse, three times of immunization are carried out, the immunization interval is 14d, 8d after the third immunization, blood is taken from the orbit, the specific antibody titer of serum is detected by indirect ELISA, the result is shown in figure 1, and the ELISA antibody titer before fusion is selected to be higher than 1:10⁵The mice are injected with the antigen conjugate Pep-KLH to strengthen the immunity, and the injection amount is 55 mu g per mouse.

S2: SP2/0 myeloma cells in good state were fused with splenic B lymphocytes of mice 3d after boosting, under the mediation of polyethylene glycol (PEG 1500). 10mL of fetal bovine serum and 5mL of IMDM were added to the fused cell pellet, mixed well, and 5mL of feeder cells (mouse thymocytes) and 25mL of sterilized semisolid culture medium (IMDM containing 2.2% methylcellulose and 1% HAT) were added thereto, and after mixing well, the mixture was transferred to cell culture dishes, each of which was transferred in an amount of 1.5 mL. The cell culture dish is put into a wet box and placed in an incubator for culture for 12 days, and the growth condition of cell clones is observed every day.

S3: and 12d after cell fusion, observing cell clones, selecting 93 cell monoclonals, culturing in a 96-hole cell culture plate paved with feeder cells (mouse thymocytes) in advance, taking supernatant of the hybridoma cells when the hybridoma cell clones in the holes grow to 1/4 of the area of the bottom of the holes, firstly using an antigen conjugate Pep-BSA as a coating antigen, carrying out two times of primary screening on the cell clones by an ELISA method, then using PEDV recombinant S protein as the coating antigen on positive clones obtained by the secondary screening, and screening the positive clones to obtain the anti-PEDV hybridoma cell strains. The ELISA was performed as described in example 2.

Example 5

s1: selecting4 BALB/c mice of 8 weeks old are taken and injected with an antigen conjugate Pep-KLH, the injection amount is 25 mug/mouse, three times of immunization are carried out, the immunization interval is 14 days, 12 days after the third immunization, blood is taken from the orbit, the specific antibody titer of serum is detected by indirect ELISA, and the ELISA antibody titer before fusion is selected to be higher than 1:10⁵The mice were injected intraperitoneally with the antigen conjugate Pep-KLH for boosting, and the injection amount was 45. mu.g/mouse.

Example 6

A porcine epidemic diarrhea virus monoclonal antibody is produced from the four PEDV-resistant hybridoma cell lines A-D obtained in examples 2-5 by the following specific method.

The monoclonal antibody is prepared by adopting an in vivo induced ascites method. Taking female BALB/c mice (the age is more than or equal to 12 weeks), and injecting 0.5mL of sterilized paraffin oil into the abdominal cavity of each mouse; 10-14 days later, the hybridoma cell line A5X 10 was injected into the abdominal cavity⁶One/one; after 7-10 days, the ascites is extracted with a syringe at 8000r/minCentrifuging for 10min, collecting supernatant, purifying ascites fluid according to the IgG monoclonal antibody purification kit, measuring antibody titer by ELISA method described in example 2 to obtain ascites fluid containing monoclonal antibody, packaging, and freezing at-80 deg.C.

The hybridoma cell lines A to D and the classes, subclasses and types of the antibodies produced by the hybridoma cell lines A to D were identified, and the results are shown in Table 2. The antibody secreted by the other strains is IgG2b, except that the antibody secreted by D is IgG2 a. B secretes the antibody light chain as lambda chain, and the antibody light chains of other cells are all kappa. Wherein the ascites titer induced by the A hybridoma cells can reach 10⁶(as shown in fig. 2).

TABLE 2

To better illustrate the properties of the monoclonal antibodies provided in the examples of the present invention, Western blot assay and indirect immunofluorescence assay (IFA) were performed on the monoclonal antibody-containing ascites fluid prepared from hybridoma cell line A in example 6. The related Vero-81 cells are preserved in an infectious disease laboratory of the university of agriculture in North and Hei; E.Coli BL21 expressing PEDV recombinant N protein is prepared and stored in the infectious disease laboratory of North river agricultural university; PEDV HBMC2012 strain (GenBank ID: JX163294) is separated, identified and stored in the infectious disease laboratory of North and Hebei agricultural university.

Western blot assay:

performing SDS-PAGE electrophoresis on PEDV recombinant N protein, S protein and E.coli BL21/pET-32a induced and expressed lysate, transferring the protein onto a PVDF membrane by a semi-dry transfer method, sealing, taking ascites diluted by 1:500 times as a western blot, and detecting the specific binding activity of the antibody and the S protein. The results of SDS-PAGE and western blot are shown in FIGS. 3 and 4, respectively. In the figure, 1 is a protein molecular weight standard; 2 is truncated PEDV recombinant N protein; 3 is truncated PEDV recombinant S protein; and 4, an induced expression lysate of E.coli BL21/pET-32a, and a Western blot result shows that a clear band (about 38kDa) with an expected size appears after an ascites monoclonal antibody induced by the anti-PEDV hybridoma cell strain A reacts with the PEDV recombinant S protein, and no target band with an expected size can be seen after the reaction with the PEDV recombinant N protein and the lysate induced and expressed by E.coli BL21/pET-32a, so that the monoclonal antibody can be specifically combined with the PEDV recombinant S protein.

Indirect immunofluorescence assay (IFA):

vero-81 cells were plated in 96-well cell culture plates at 1.6X 10⁴One/well, when cells reached 90% confluence (about 48h), washed once with PBS; the cells were infected with 100 μ L of the variant PEDV HBQY2012, at an MOI of 0.5; and meanwhile, a negative serum control is set. Place the cell culture plate at 37 ℃ in 5% CO₂Incubate for 1h, discard the virus solution, wash 1 time with 100 μ L PBS (0.01mol/L, pH7.4) per well; 200. mu.L of MEM holding solution was added to each well and placed in 5% CO at 37 ℃₂Incubate for 24h, discard the maintenance medium, and wash each well 2 times with 280. mu.L PBS. Cells were fixed with ice-cold absolute methanol at-20 ℃ for 10min at 50. mu.L/well, methanol was discarded and each well was washed 1 time with 280. mu.L PBS. Adding ascites diluted by 1:400 times, reacting at 37 ℃ for 1h, discarding reaction solution, adding PBS of 280 mu L into each well for washing for 2 times, adding goat anti-swine FITC labeled fluorescent antibody diluted by 1:500 times into each well for 100 mu L, reacting at 37 ℃ for 45min, discarding reaction solution, adding PBS of 280 mu L into each well for washing for 2 times, adding bisbenzamide (10 mu g/mL) of 50 mu L into each well for dyeing at room temperature for 20-30min, and adding PBS of 280 mu L/well for washing for 2 times. And (4) observing by a fluorescence microscope, wherein a positive result shows green fluorescence, and a negative result shows no signal. As shown in FIGS. 5 and 6, the monoclonal antibody prepared in example 6 was able to specifically bind to the virus in PEDV-infected Vero-81 cells, emitting specific green fluorescence in the cytoplasm (FIG. 5), while the negative control cells not infected with the virus did not emit green fluorescence (FIG. 6).

At the same time, Vero-81 cells were infected with PEDV QY2016 and CV777 at 0.5MOI, respectively, 5% CO at 37 deg.C₂After culturing in an incubator for 48 hours, ascites diluted by 1:300, 1:400, 1:500 and 1:1000 times is added respectively, and the ascites obtained in this example 6 is evaluated as to whether two strains can be distinguished by detecting the virulent strain QY2016 and the attenuated strain CV777 by IFA. The results are shown in FIGS. 7 and 8, in which case, the results are shown in FIG. 7 for QY2016 (1: 400-fold dilution of monoclonal antibody) and in FIG. 8 for CV777 attenuated strain (1: 400-fold dilution of monoclonal antibody). From the above results, no significant fluorescence signal was observed when ascites were reacted with the CV777 attenuated strain (FIG. 8), indicating that ascites prepared in example 6 can distinguish the PEDV variant strain QY2016 from the classical strain represented by CV 777.

In addition, the monoclonal antibody obtained in example 6 was HRP-labeled, and the activity thereof was detected. Add 50. mu.L of labeling buffer to 500. mu.L of ascites fluid obtained in example 6, and mix well according to the HRP labeling kit; adding 50 mu L of HRP enzyme, repeatedly blowing and sucking, fully and uniformly mixing, and reacting for 3 hours at room temperature in a dark place; adding 60 mu L of reaction termination solution, fully and uniformly mixing, and standing for 1h at room temperature in a dark place; adding 660 mu L of marker preservation solution, fully and uniformly mixing to obtain the monoclonal antibody marked by the HRP, and preserving at the temperature of minus 20 ℃.

And (3) coating an enzyme label plate with 10 mu g/mL PEDV recombinant S protein, adding 100 mu L of HRP-labeled monoclonal antibody diluted by times (1: 100-1: 12800) into each hole, and setting the HRP-goat anti-mouse IgG diluted by times as a negative control. Incubating at 37 deg.C for 1h, washing plate, adding substrate TMB, developing at 37 deg.C in dark for 15min, terminating reaction, and measuring OD with enzyme-labeling instrument_450nmLight absorption value. When the average OD value of the experimental wells is more than or equal to 2 times the average OD value of the negative wells, the HRP-labeled monoclonal antibody is judged to have the binding activity. The result is shown in fig. 9, the titer of the HRP-labeled monoclonal antibody is 1:3200, which indicates that the HRP-labeled monoclonal antibody has good binding activity with the PEDV recombinant S protein. The monoclonal antibodies produced by the anti-PEDV hybridoma cell lines obtained in examples 3-5 of the present invention had comparable effects to the monoclonal antibody obtained in example 6.

According to the data, the monoclonal antibody provided by the invention can be specifically combined with PEDV recombinant S protein and PEDV, and the monoclonal antibody can be used for effectively identifying PEDV variant strains and classical strains.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

SEQUENCE LISTING

<110> university of agriculture in Hebei

<120> porcine epidemic diarrhea virus epitope peptide, monoclonal antibody and application

<130>20191028

<160>1

<170>PatentIn version 3.5

<210>1

<211>21

<212>PRT

<213> Artificial Synthesis

<400>1

Ile Gly Glu Asn Gln Gly Val Asn Ser Thr Trp Tyr Cys Ala Gly Arg

1 5 10 15

His Pro Thr Ala Ser

20

Claims

1. The epitope peptide of the porcine epidemic diarrhea virus is characterized by comprising the following components in percentage by weight: the amino acid sequence is shown in SEQ ID No. 1.

2. Use of the epitope peptide of claim 1 for preparing a reagent for identifying classical strains and variant strains of porcine epidemic diarrhea virus.

3. An antigen conjugate, comprising: comprising the epitope peptide according to claim 1 and a carrier protein, wherein the carrier protein is hemocyanin, bovine serum albumin, ovalbumin or human serum albumin.

4. A preparation method of a PEDV-resistant hybridoma cell strain is characterized by comprising the following steps: adopting the antigen conjugate of claim 3 to carry out animal immunization, fusing the obtained B lymphocyte with myeloma cells, selecting positive clone, and then carrying out subcloning to obtain the anti-PEDV hybridoma cell strain.

5. The PEDV-resistant hybridoma cell of claim 4The preparation method of the cell strain is characterized by comprising the following steps: ELISA antibody titers were higher than 1:10⁵The splenic B lymphocytes of the mice were fused with myeloma cells.

6. The method for preparing a PEDV hybridoma cell line according to claim 4, wherein the method comprises the following steps: and screening positive clones by taking the antigen conjugate as an envelope antigen, and subcloning the positive clones by taking the PEDV recombinant S protein as the envelope antigen to obtain the anti-PEDV hybridoma cell strain.

7. A monoclonal antibody against porcine epidemic diarrhea virus, which is characterized in that: the monoclonal antibody is produced by the anti-PEDV hybridoma cell line prepared according to any one of claims 4 to 6.

8. The porcine epidemic diarrhea virus monoclonal antibody of claim 7, wherein: the subtype of the monoclonal antibody is IgG2b, and the light chain is a kappa chain.

9. Use of the monoclonal antibody of claim 7 or 8 in the preparation of a reagent for identifying classical and variant strains of porcine epidemic diarrhea virus.