Marker and kit for screening foot-and-mouth disease virus persistently infected cattle and application of marker and kit

Abstract

The invention provides a marker and a kit for screening foot-and-mouth disease virus persistently infected cattle and application of the marker and the kit, and belongs to the technical field of virus immunological detection. The marker consists of CD21 + B cells in bovine peripheral blood and IgA (Immunoglobulin A) in bovine oropharyngeal fluid. The number of CD21 + B cells in bovine peripheral blood and the level of IgA in oropharyngeal fluid are detected at the same time 28 days after foot-and-mouth disease virus infection, the proportion of the CD21 + B cells in the bovine peripheral blood continuously infected by FMDV is remarkably lower than that of the CD21 + B cells in the peripheral blood of the recovered bovine, and meanwhile the level of the IgA in the bovine OPF continuously infected by FMDV is remarkably higher than that of the IgA in the recovered bovine OPF. Therefore, the cattle continuously infected by the FMDV can be more effectively screened out. The ROC analysis result shows that the AUC is 1.000 (the confidence interval of 95%, 0.782-1.000) at 35 dpc, and the sensitivity and specificity of diagnosis both reach 100%. Therefore, the invention provides an effective technical means for screening FMDV persistently infected cattle.

Description

Technical Field

[0001] The present invention relates to a marker for screening cattle persistently infected with foot-and-mouth disease virus, and also relates to a kit for screening cattle persistently infected with foot-and-mouth disease virus and its application. The present invention belongs to the technical field of virus immunology detection. Background Art

[0002] Foot-and-mouth disease (FMD) is an acute, highly contagious vesicular disease of even-toed ungulates caused by infection with foot-and-mouth disease virus (FMDV), which seriously affects agricultural production and trade in affected areas. Its pathogen, FMDV, is an icosahedral, non-enveloped, single-stranded positive-sense RNA virus belonging to the family Picornaviridae. Seven serotypes of FMDV (A, O, C, SAT1, SAT2, SAT3, and Asia1) and multiple lineages and subtypes within the serotypes are prevalent in different countries or regions around the world, showing poor cross-protection activity and high genetic variability. Due to its wide host range, low minimum infectious dose, rapid replication, high level of viral excretion, and diverse modes of transmission, FMD is a disease that is difficult to control and eradicate, and the subclinical persistent infection that occurs after acute infection in ruminants further complicates the situation. Persistent FMDV infection is often referred to as the FMDV carrier state, and the World Organization for Animal Health (WOAH) defines an FMDV carrier as an animal that still has infectious virus in the oropharynx 28 days after infection.

[0003] Clinical studies have shown that the incidence of persistent FMDV infection is greater than 50%, even in vaccinated cattle that are completely protected from clinical disease. Although some studies have failed to demonstrate natural transmission of FMDV carriers to cattle, there is evidence that oropharyngeal fluids from FMDV carriers are Persistently infected African buffalo have been observed to transmit the virus to Previous studies have shown that persistent FMDV infection is mainly related to host immune factors. Therefore, studying the immunological characteristics of FMDV carriers under immune pressure has important guiding significance for the clinical prevention and control of FMD and the development of new vaccines.

[0004] FMDV infection usually causes a rapid, strong and persistent antibody response, and rapid clearance of circulating viruses is of great significance. A comparative study of the humoral immune response of FMDV persistently infected cattle and recovered cattle found that there was no significant difference in systemic antibody levels between FMDV persistently infected cattle and recovered cattle, but FMDV persistently infected cattle had a strong antibody response in the infected area, indicating that during the FMDV persistent infection period, the in vivo distribution of B cells between FMDV persistently infected cattle and recovered cattle may be different. Although the bovine nasopharyngeal mucosal epithelium has been identified as the replication site of foot-and-mouth disease virus persistent infection, peripheral blood mononuclear cells (PBMCs) have the advantages of easy access, easy preparation and ability to represent the body's immune status. scRNA-seq was used to comprehensively and deeply reveal the specific molecular characteristics of specific immune cells in PBMCs of FMDV persistently infected cattle and recovered cattle, thereby explaining the in vivo dynamic distribution characteristics of immune cells in different disease states and the corresponding immunological characteristics. This provides a new technical means for the rapid differentiation of FMDV persistently infected cattle. Summary of the invention

[0005] One of the purposes of the present invention is to provide a marker for screening cattle persistently infected with foot-and-mouth disease virus and its application;

[0006] The second object of the present invention is to provide a kit for screening cattle persistently infected with foot-and-mouth disease virus and its application.

[0007] In order to achieve the above object, the present invention adopts the following technical means:

[0008] In one aspect, the present invention provides a marker for screening for persistent infection of cattle with foot-and-mouth disease virus, wherein the marker is CD21 in the peripheral blood of cattle. + B cell and IgA composition in bovine oropharyngeal fluid.

[0009] Preferably, CD21 in the peripheral blood of cattle is detected 28 days after foot-and-mouth disease virus infection. + B cell numbers and IgA levels in oropharyngeal fluid, and CD21 in peripheral blood of cattle persistently infected with FMDV + The proportion of B cells was significantly lower than that of CD21 in the peripheral blood of recovered cattle. + The proportion of B cells and the level of IgA in OPF of cattle with persistent FMDV infection were significantly higher than those in OPF of recovered cattle, which can more effectively screen out cattle with persistent FMDV infection.

[0010] Furthermore, the present invention also proposes the use of the marker in the preparation of a reagent for screening cattle persistently infected with foot-and-mouth disease virus.

[0011] Furthermore, the present invention also proposes a method for detecting CD21 in bovine peripheral blood. + Application of reagents for B cell number and IgA expression level in bovine oropharyngeal fluid in the preparation of a kit for screening cattle persistently infected with foot-and-mouth disease virus.

[0012] In another aspect, the present invention provides a kit for screening cattle for persistent infection with foot-and-mouth disease virus, wherein the kit contains a protein for detecting CD21 in the peripheral blood of cattle. + Reagents for detecting B cell numbers and IgA expression levels in bovine oropharyngeal fluid.

[0013] Among them, preferably, the method for detecting CD21 in bovine peripheral blood + B cell count was determined by flow cytometry to detect CD21 + The reagent for B cells, the reagent for detecting the expression level of IgA in bovine oropharyngeal fluid is a reagent for detecting the expression level of IgA by ELISA method.

[0014] Preferably, CD21 is detected by flow cytometry. + B cell reagents include anti-bovine CD21 monoclonal antibody, active dye eFluor TM 780 and a staining solution, wherein the staining solution is DPBS or PBS buffer containing 2% v / v fetal bovine serum and 0.09% w / v sodium azide, or the staining solution is DPBS or PBS buffer containing 2% w / v BSA and 0.09% w / v sodium azide; the reagent for detecting the expression level of IgA by ELISA method includes 146s FMDV / O / BY / 2010 particles, PBS blocking solution containing 1% w / v BSA, PBST washing solution, IgA-HRP monoclonal antibody, TMB color developing solution and 0.2M H2SO4.

[0015] Preferably, CD21 is detected by flow cytometry. + When B cells were isolated, the following steps were followed: 1) thawed frozen PBMCs and washed with RPIM-1640 medium, then resuspended in RPIM-1640 medium containing 10% v / v fetal bovine serum and placed at 37°C for at least one hour, centrifuged to remove cell debris, and counted the cells; the staining step was performed after the freshly isolated PBMCs were counted; 2) Staining: 1×10 6 PBMCs were seeded into 96-well U-bottom plates and incubated with 5% v / v bovine serum for 20 min to inhibit Fc receptors, washed twice with PBS, and then incubated with the vital dye eFluor 40 diluted in PBS. TMThe cells were stained with 780 for 30 min, washed twice with staining solution, and stained with anti-bovine CD21 monoclonal antibody containing fluorescent label for 30 min. All staining steps were performed on ice. Finally, the cells were washed twice with staining solution and resuspended in staining solution. The data were collected by flow cytometer CytExpert and analyzed by FlowJo software.

[0016] Among them, preferably, when detecting the IgA expression level by ELISA method, the following steps are followed: a 96-well plate is directly coated with 2ug / mL sucrose gradient purified 146s FMDV / O / BY / 2010 particles in a carbonate / bicarbonate buffer at pH: 9.6, and the coated plate is first blocked with PBS containing 1% w / v BSA and incubated with bovine oropharyngeal fluid at 37°C for 1 hour, washed 5 times with PBST and drained, and then incubated with a monoclonal antibody of IgA-HRP, washed 5 times with PBST, and TMB colorimetric solution was added to each well for reaction for 10 to 30 minutes, 0.2M H2SO4 was added to stop the reaction, and read in a BioRad450 ELISA plate reader.

[0017] Among them, preferably, the sucrose gradient purified 146s FMDV / O / BY / 2010 particles are prepared by the following method: the FMDV / O / BY / 2010 virus propagated by BHK-21 is repeatedly frozen and thawed three times at -80, first centrifuged at 4,000g at 4°C for 30min, and then centrifuged at 10,000g at 4°C for 30min to remove cell debris; then the virus solution is concentrated 50 times, and then centrifuged at 10,000g at 4°C for 30min, and the precipitate is discarded. Ultracentrifuged at 36,000rpm at 4°C for 2.5h, the precipitated virus is soaked in NET buffer, and placed at 4°C overnight. The NET buffer is 0.05M containing 0.1M NaCl and 0.004M EDTA. Tris, pH 8.0; resuspend the precipitate and grind on ice for 5 to 6 hours; centrifuge at 10,000g, 30min, 4℃ to remove impurities and discard the precipitate; prepare 15% w / w and 45% w / w sucrose solutions with NET buffer, filter with 0.22um filter membrane, first add 15% w / w sucrose solution to the sucrose tube, then inject 45% w / w sucrose solution to the bottom of the tube, prepare a sucrose pad with a sucrose density gradient preparation instrument, so that the sucrose density gradient is: 15% w / w-25% w / w-35% w / w-45% w / w, add the virus solution to the sucrose pad, centrifuge at 36,000rpm, 4℃ for 2.5h; use a 1ml pipette to continuously take 500ul equal portions of sucrose solution into continuous EP tubes, measure OD260, and draw a curve chart based on the measured absorbance values ​​to determine and calculate the distribution and content of virus 146S, and virus 146S is located near the highest peak.

[0018] Furthermore, the present invention also proposes the use of the kit in preparing a reagent for screening cattle persistently infected with foot-and-mouth disease virus.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a marker for screening cattle with persistent foot-and-mouth disease virus infection, wherein the marker is a marker of CD21 in the peripheral blood of cattle. + The present invention performed ROC analysis on these two markers, calculated the area under the curve (AUC) and its 95% confidence interval (95% CI), as well as the sensitivity and specificity of the diagnosis. The results showed that the ROC curve of the combination of these two indicators showed higher diagnostic accuracy. At 35dpc, the AUC was 1.000 (95% confidence interval, 0.782-1.000), and the sensitivity and specificity of the diagnosis reached 100%. This result shows that the simultaneous detection of CD21 in peripheral blood + The number of B cells and the level of IgA in OPF can more accurately screen cattle for persistent infection with FMDV. Therefore, the invention provides an effective technical means for screening cattle for persistent infection with FMDV. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of cell clustering and cell type proportion analysis of control cattle (HC), FMDV persistently infected cattle (CR) and recovered cattle (NCR);

[0022] Among them, A represents the eight major immune cell lineages identified, namely B cells, CD8+T cells, CD4+T cells, γδT cells, NKT-like cells, NK cells, proliferating T cells and myeloid cells; B represents the relative proportion of each type of immune cell among the three groups;

[0023] Figure 2 To investigate the differences in molecular characteristics and in vivo distribution of B cells in control cattle (HC), FMDV persistently infected cattle (CR) and recovered cattle (NCR);

[0024] Among them, A is the KEGG analysis result of differentially expressed genes in B cells of persistently infected cattle compared with recovered cattle; B is the expression of representative genes PRDM1, XBP1 and IRF4 related to B cell development and activation, and chemokine receptor CXCR4; C is the level of IgG secreting cells in lymph nodes and tonsils around the nasopharynx;

[0025] Figure 3 The dynamic changes of the number of B cells in peripheral blood;

[0026] Figure 4 The dynamic changes of IgA levels in OPF;

[0027] Figure 5 Figure 3 is the ROC curve of the two indicators at 35 dpc, and the area under the curve (AUC) represents the diagnostic performance. DETAILED DESCRIPTION

[0028] The present invention is further described below. The implementation cases introduced in this description are only exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that without departing from the principles and methods of the present invention, the details and forms of the technical solution of the present invention may be partially modified or replaced, but such modification or replacement is within the scope of protection of the present invention.

[0029] Example 1

[0030] method:

[0031] 1. Isolation of peripheral blood mononuclear cells (PBMCs): PBMCs were isolated from fresh anticoagulated blood by standard density gradient centrifugation using Ficoll Plus 1.083 solution. Briefly, each blood sample was diluted with an equal volume of 1×PBS (pH 7.4), and an equal volume of diluted whole blood was gently layered on top of the Ficoll Plus 1.083 solution, ensuring that the two layers did not mix, and centrifuged at 800×g for 30 minutes at room temperature. After centrifugation, cells at the interface were carefully aspirated and transferred to a new 15 ml centrifuge tube, washed twice with 1×PBS (pH 7.4) containing 2% v / v fetal bovine serum, and then PBMCs were resuspended in RIPM-1640 containing 10% v / v fetal bovine serum and 1% v / v dual antibody, and the cell number and viability of the cells were detected using the Countess II automated cell counter. PBMCs were then resuspended in freezing medium containing 80% v / v fetal bovine serum, 10% v / v DMSO and 10% v / v RIPM-1640 and frozen in a -80°C freezer for 24 hours using a NALGENETM Cryo 1°C Gradient Freezing Box and then transferred to liquid nitrogen for storage until use.

[0032] 2.scRNA-seq: Frozen PBMCs were thawed and washed with PBS containing 0.04% BSA. Dead cells and cell debris were removed using the MACS Dead Cell Removal Kit (Miltenyi Biotec). Cell capture and library construction were performed using the ChromiumTM Controller and ChromiumTM Single Cell 3'Reagent Kits v3 (10×Genomics), and the quality of the library was then tested using the Agilent Bioanalyzer. Finally, the library was sequenced on the IlluminaNovaSeq 6000 platform (paired-end, 2×150bp), with a minimum depth of 20,000 reads per cell.

[0033] 3. Bioinformatics analysis: Sequencing results were converted to FASTQ format using Illumina bcl2fastq software. CellRanger was used to sort samples, barcode and count single-cell 3' genes, and single-cell RNA-seq data were aligned with the Ensembl genome ARS-USD1.2 cattle reference genome using the STAR aligner to generate a matrix of gene counts and cells. The CellRanger output was loaded into Seurat, and the gene barcode matrix of the 9 samples was integrated to calculate the mitochondrial gene content in each sample. Unqualified cells were filtered using the following parameters: (1) Number of genes expressed in each cell: 500 < number of genes < 5000; (2) All genes expressed in less than 3 cells were removed; (3) Mitochondrial gene counts < 20%. Doublet cells were then identified and removed using the R package DoubletFinder. Next, the data were normalized and corrected using the "NormolizedData" algorithm in the Seurat software, and the 2,000 characteristic genes that showed high inter-cell differences in the data set were calculated using the "FindVariableFeatures" algorithm. The expression levels of these genes were scaled using the "ScaleData" function, and the principal component analysis was performed in the variable gene space using the "RunPCA" function. Harmony was then used to correct the batch effect. After removing the batch effect, the principal component was reduced in dimension using the "FindNeighbors" algorithm, and the data was reduced in dimension and clustered using the "FindClusters" algorithm. The data was then nonlinearly reduced in dimension using the "RunUMAP" algorithm and plotted using the "DimPlot" function. Finally, the cell types were annotated using recognized cell surface markers and highly expressed characteristic genes, and the cell type proportions and molecular characteristics of the persistent infection group and the recovery group were compared.

[0034] 4. Flow cytometry: thaw the frozen PBMCs and wash them with RPIM-1640 medium, then resuspend them in RPIM-1640 medium with 10% fetal bovine serum and place them at 37°C for at least one hour. Centrifuge to remove cell debris and count the cells. 6 The cells were seeded into 96-well U-bottom plates (Costar) and incubated with 5% bovine serum for 20 min at 4°C to inhibit Fc receptors. TM The cells were stained at 780, washed twice with staining solution, and stained with anti-bovine CD21 monoclonal antibody containing fluorescent labeling for 30 minutes on ice. Finally, the cells were washed twice with staining solution and resuspended in staining solution, and the data were collected by flow cytometer CytExpert and analyzed using FlowJo software. The staining solution was DPBS or PBS buffer containing 2% v / v fetal bovine serum and 0.09% w / v sodium azide, or the staining solution was DPBS or PBS buffer containing 2% w / v BSA and 0.09% w / v sodium azide.

[0035] 5. Sucrose Gradient Purification of 146s FMDV / O / BY / 2010 Particles

[0036] FMDV / O / BY / 2010 propagated with BHK-21 (the virus has been recorded in the patent application with publication number CN112852745A, the invention name is "BHK-21 cell line with HDAC3 gene knockout and its construction method and application", and is preserved and provided by Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences.) The virus was repeatedly frozen and thawed three times at -80, first centrifuged at 4,000g at 4℃ for 30min, and then centrifuged at 10,000g at 4℃ for 30min to remove cell debris; then the virus solution was concentrated 50 times, and then centrifuged at 10,000g at 4℃ for 30min, and the precipitate was discarded. Ultracentrifuged at 36,000rpm at 4℃ for 2.5h, the precipitated virus was soaked in NET (0.1MNaCl-0.004M EDTA-0.05M Tris, pH 8.0) buffer, placed at 4°C overnight; resuspend the precipitate, grind on ice for 5-6h; centrifuge at 10,000g, 30min, 4°C to remove impurities, and discard the precipitate; prepare 15% w / w and 45% w / w sucrose solutions with NET buffer, filter with 0.22um filter membrane, add 15% w / w sucrose solution to the sucrose tube first, then inject 45% w / w sucrose solution into the bottom of the tube, prepare sucrose pad with sucrose density gradient preparation instrument, and use The sucrose density gradient is: 15% w / w-25% w / w-35% w / w-45% w / w, add the virus solution to the sucrose pad, centrifuge at 36,000rpm, 4℃ for 2.5h; use a 1ml pipette to continuously take 500ul equal portions of sucrose solution into continuous EP tubes, measure OD260, and draw a curve based on the absorbance measurement to determine and calculate the distribution and content of virus 146S. Virus 146S is located near the highest peak. Virus content calculation: The absorbance value of the blank sucrose solution is X1. According to the curve, the highest peak tube and the two tubes before and after it are combined, and the absorbance value is X2. According to the formula, the 146S content is 132×(X2-X1).

[0037] 6. IgA ELISA: 96-well plates were directly coated with 2ug / mL sucrose gradient purified 146s FMDV / O / BY / 2010 particles in carbonate / bicarbonate buffer (pH: 9.6, 100μL per well). The coated plates were first blocked with PBS containing 1% w / v BSA, washed 5 times with PBST and drained, and incubated with OPF (1:4) at 37°C for 1 hour, washed 5 times with PBST and drained, and then incubated with monoclonal antibodies against IgA-HRP. After washing 5 times with PBST, TMB substrate was added to each well for 10 to 30 minutes, 0.2M H2SO4 was added to stop the reaction, and read in a BioRad450 ELISA plate reader.

[0038] result:

[0039] 1. Differences in cell clustering and cell type proportions

[0040] First, we performed 10×Genomics scRNA-seq on PBMCs samples from three unimmunized healthy controls (HC), three FMDV persistently infected cows (CR), and three recovered cows (NCR). After quality control and filtering, we used UMAP unsupervised clustering and identified eight major immune cell lineages based on typical lineage marker genes and genes specifically upregulated in each cell cluster, namely B cells, CD8+T cells, CD4+T cells, γδT cells, NKT-like cells, NK cells, proliferating T cells, and myeloid cells ( Figure 1 A). By comparing the relative proportions of various immune cells among the three groups, we found that the proportion of B cells in CRs was the lowest, and the proportions of other cell types had no significant differences among the three tissues ( Figure 1 B).

[0041] 2. Molecular characteristics of persistent FMDV infection in bovine B cells

[0042] Next, we analyzed the differences in molecular characteristics of B cells between FMDV persistently infected cattle and recovered cattle to explain the reason for the reduction of B cells in CR. The KEGG analysis of differentially expressed genes in B cells of FMDV persistently infected cattle relative to recovered cattle found that upregulated genes were significantly enriched in pathways related to B cell development and activation, such as C-type lectin receptor signaling pathway, BCR signaling pathway, MAPK pathway, NF-κB signaling pathway, cell adhesion molecules, leukocyte transendothelial migration, chemokine signaling pathway, etc. ( Figure 2 A). The expression of representative genes related to B cell development and activation, PRDM1, XBP1 and IRF4, and chemokine receptor CXCR4, was higher in FMDV persistently infected cattle than in recovered cattle ( Figure 2 B). This indicates that B cells in cattle with persistent FMDV infection are still active and that more B cells may migrate to secondary lymphoid organs. We also observed high levels of IgG secreting cells in the lymph nodes and tonsils around the nasopharynx ( Figure 2 C) These results suggest that the low number of circulating B cells in cattle persistently infected with FMDV may be closely related to the specific tissue localization of FMDV under persistent infection.

[0043] 3. Dynamic changes in the number of B cells in peripheral blood

[0044] Using flow cytometry, we detected the dynamic changes in the number of B cells in cattle with persistent FMDV infection and recovered cattle at different time points after infection. Figure 3The results show that although the trend of change over time is basically the same, from 7dpc to 35dpc, the CD21 in the peripheral blood of cattle persistently infected with FMDV + The increasing trend of B cell proportion in cattle was significantly slower than that in recovered cattle. At 35 dpc, CD21 in peripheral blood of cattle with persistent FMDV infection + The proportion of B cells was significantly lower (p<0.01) than that of CD21 in the peripheral blood of recovered cattle. + B cell ratio.

[0045] 4. Dynamic changes of IgA levels in OPF

[0046] Subsequently, we detected the dynamic changes of IgA levels in oropharyngeal fluid (OPF). Figure 4 As shown in the figure, the level of IgA in OPF of recovered cattle began to increase from 7 dpc, reached the highest level at 21 dpc, and then decreased, while the level of IgA in OPF of cattle with persistent FMDV infection did not change significantly before the establishment of persistent infection. At 35 dpc, the level of IgA in OPF of cattle with persistent FMDV infection was significantly higher (p<0.05) than that in OPF of recovered cattle, and this result was consistent with the expression of CD21 in peripheral blood. + The decrease in the number of B cells corresponds to the increase in the number of B cells in the persistent infection site. This indicates that at 35 dpc, cattle with persistent FMDV infection are still in a state of viral infection, thus recruiting a large number of B cells at the infection site to secrete antibodies to eliminate the virus. Therefore, simultaneous detection of the number of B cells in peripheral blood and the level of IgA in OPF 28 days after FMDV infection can more effectively screen out cattle with persistent FMDV infection.

[0047] 5. Diagnostic Performance Evaluation

[0048] Next, we will analyze these two markers - CD21 in peripheral blood + The number of B cells and IgA levels in OPF were analyzed by ROC analysis, and the area under the curve (AUC) and its 95% confidence interval (95% CI), as well as the sensitivity and specificity of diagnosis were calculated. Figure 5 As shown in the figure, the ROC curve of the combination of these two indicators showed a higher diagnostic accuracy. At 35 dpc, the AUC was 1.000 (95% confidence interval, 0.782-1.000), and the sensitivity and specificity of diagnosis reached 100%. This result shows that the simultaneous detection of CD21 in peripheral blood + The number of B cells and the level of IgA in OPF can more accurately screen cattle for persistent FMDV infection.

Claims

1. A marker for screening for persistent infection of cattle by foot-and-mouth disease virus (FMDV), characterized in that: The marker is CD21 in bovine peripheral blood + B cell and IgA composition in bovine oropharyngeal fluid.

2. The marker according to claim 1, characterized in that Simultaneous detection of CD21 in bovine peripheral blood 28 days after foot-and-mouth disease virus infection + B cell numbers and IgA levels in oropharyngeal fluid, and CD21 in peripheral blood of cattle persistently infected with FMDV + The proportion of B cells was significantly lower than that of CD21 in the peripheral blood of recovered cattle. + The proportion of B cells and the level of IgA in OPF of cattle with persistent FMDV infection were significantly higher than those in OPF of recovered cattle, which can more effectively screen out cattle with persistent FMDV infection.

3. Use of the marker according to claim 1 or 2 in the preparation of a reagent for screening cattle persistently infected with foot-and-mouth disease virus.

4. Used to detect CD21 in bovine peripheral blood + Application of reagents for B cell number and IgA expression level in bovine oropharyngeal fluid in the preparation of a kit for screening cattle persistently infected with foot-and-mouth disease virus.

5. A kit for screening cattle persistently infected with foot-and-mouth disease virus, characterized in that: The kit contains a protein for detecting CD21 in bovine peripheral blood. + B cell number reagent and a reagent for detecting the expression level of IgA in bovine oropharyngeal fluid, wherein, preferably, the reagent for detecting CD21 in bovine peripheral blood + B cell count was determined by flow cytometry to detect CD21 + The reagent for B cells, the reagent for detecting the expression level of IgA in bovine oropharyngeal fluid is a reagent for detecting the expression level of IgA by ELISA method.

6. The kit according to claim 5, characterized in that Detection of CD21 by flow cytometry + B cell reagents include anti-bovine CD21 monoclonal antibody, active dye eFluor TM 780 and a staining solution, wherein the staining solution is DPBS or PBS buffer containing 2% v / v fetal bovine serum and 0.09% w / v sodium azide, or the staining solution is DPBS or PBS buffer containing 2% w / v BSA and 0.09% w / v sodium azide; the reagent for detecting the expression level of IgA by ELISA method includes 146s FMDV / O / BY / 2010 particles, PBS blocking solution containing 1% w / v BSA, PBST washing solution, IgA-HRP monoclonal antibody, TMB color developing solution and 0.2M H2SO4.

7. The kit according to claim 5, characterized in that Detection of CD21 by flow cytometry + When B cells were isolated, the following steps were followed: 1) thawed frozen PBMCs and washed with RPIM-1640 medium, then resuspended in RPIM-1640 medium containing 10% v / v fetal bovine serum and placed at 37°C for at least one hour, centrifuged to remove cell debris, and counted the cells; the staining step was performed after the freshly isolated PBMCs were counted; 2) Staining: 1×10 6 PBMCs were seeded into 96-well U-bottom plates and incubated with 5% v / v bovine serum for 20 min to inhibit Fc receptors, washed twice with PBS, and then incubated with the vital dye eFluor 40 diluted in PBS. TM The cells were stained with 780 for 30 min, washed twice with staining solution, and stained with anti-bovine CD21 monoclonal antibody containing fluorescent label for 30 min. All staining steps were performed on ice. Finally, the cells were washed twice with staining solution and resuspended in staining solution. The data were collected by flow cytometer CytExpert and analyzed by FlowJo software.

8. The kit according to claim 5, characterized in that When detecting the IgA expression level by ELISA method, the following steps were followed: 96-well plates were directly coated with 2ug / mL sucrose gradient purified 146sFMDV / O / BY / 2010 particles in carbonate / bicarbonate buffer at pH 9.

6. The coated plates were first blocked with PBS containing 1% w / v BSA, washed 5 times with PBST and drained, and incubated with bovine oropharyngeal fluid at 37°C for 1 hour, washed 5 times with PBST and drained, and then incubated with monoclonal antibody against IgA-HRP. After washing 5 times with PBST, TMB colorimetric solution was added to each well for reaction for 10 to 30 minutes, 0.2MH2SO4 was added to stop the reaction, and the plate was read in a BioRad450 ELISA plate reader.

9. The kit according to claim 5, characterized in that The sucrose gradient purified 146sFMDV / O / BY / 2010 particles were prepared by the following method: the FMDV / O / BY / 2010 virus propagated by BHK-21 was repeatedly frozen and thawed three times at -80, first centrifuged at 4,000g at 4°C for 30min, and then centrifuged at 10,000g at 4°C for 30min to remove cell debris; The virus solution was then concentrated 50 times, centrifuged at 10,000 g for 30 min at 4°C, and the precipitate was discarded; ultracentrifuged at 36,000 rpm at 4°C for 2.5 h, and the precipitated virus was soaked in NET buffer and placed at 4°C overnight. The NET buffer was 0.05 M Tris, pH 5.0, containing 0.1 M NaCl and 0.004 M EDTA. 8.0; resuspend the precipitate, grind on ice for 5-6h; 10,000g, 30min, 4℃ centrifuge to remove impurities, discard the precipitate; prepare 15% w / w and 45% w / w sucrose solutions with NET buffer, filter with 0.22um filter membrane, add 15% w / w sucrose solution to the sucrose tube first, then inject 45% w / w sucrose solution to the bottom of the tube, prepare sucrose pad with sucrose density gradient preparation instrument, so that the sucrose density gradient is: 15% w / w-25% w / w-35% w / w-45% w / w, add virus solution to the sucrose pad, 36,000rpm, 4℃ centrifuge for 2.5h; use 1ml pipette to continuously take 500ul equal portions of sucrose solution into continuous EP tubes, measure OD260, and draw a curve chart according to the measured absorbance value to determine and calculate the distribution and content of virus 146S, and virus 146S is located near the highest peak.

10. Use of the kit according to claims 5-9 in preparing a reagent for screening cattle persistently infected with foot-and-mouth disease virus.

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