CN107033226B

CN107033226B - Peste des petits ruminants virus F protein epitope peptide, determination method, preparation method and application thereof

Info

Publication number: CN107033226B
Application number: CN201710502539.0A
Authority: CN
Inventors: 窦永喜; 梁忠祥; 朱学亮; 蒙学莲; 张志东; 才学鹏
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2017-06-27
Filing date: 2017-06-27
Publication date: 2020-09-04
Anticipated expiration: 2037-06-27
Also published as: CN107033226A

Abstract

The invention relates to a Peste des petits ruminants virus F protein epitope peptide, the amino acid sequences of which are respectively as follows: f176:¹⁷⁶DYINNELVPSVHRMSCEL¹⁹³and/or F347:³⁴⁷MSPLLQECFRGSTKS³⁶¹and F391: or/and³⁹¹CKCYTTETVINQDPDKLL⁴⁰⁸and/or F417:⁴¹⁷TVINQDPDKGPVGSREYPD⁴³⁵and F430: or/and⁴³⁰SREYPDSVYLH⁴⁴⁰and/or F502:⁵⁰²VSLGLVTLICCCKGRCRNK⁵²⁰(ii) a The invention predicts the B cell epitope of the target protein, artificially synthesizes the predicted different epitopes respectively, and detects the reactogenicity of the antibody with the F protein, thereby identifying the B cell epitope of the PPRV F protein.

Description

Peste des petits ruminants virus F protein epitope peptide, determination method, preparation method and application thereof

Technical Field

The invention belongs to the technical field of bioinformatics and immunology, and particularly relates to a peste des petits ruminants virus F protein antigen epitope peptide, and a determination method, a preparation method and an application thereof.

Background

Peste des petits ruminants virus (PPRV) belongs to measles virus (Morblivirus) and is a pathogen causing acute infectious diseases of small ruminants such as goats and sheep, and the disease is characterized by high morbidity and mortality, is listed as a legal report animal infectious disease by the world animal health Organization (OIE), and is listed as a type I animal epidemic disease in China. The initial reports of peste des petits ruminants virus infection only in goats and sheep, but in recent years cases of interspecies viral transmission have been reported, and the disease is mainly distributed in africa and asia, and threatens europe.

At present, the main prevention means of the disease is attenuated vaccine immunity, but the vaccine has the problems of heat stability, strong toxicity and the like, and is not beneficial to the global elimination plan of the peste des petits ruminants, F is glycoprotein embedded on the capsule membrane of PPRV and forms fiber on the surface of virus particles, HN protein can interact with SLAM receptor on lymphocytes to mediate virus invasion, but the invasion process of the virus can be completed only by the help of F protein, the peste des petits ruminants virus can cause strong cellular immunity and humoral immunity response of organisms, and the F protein is a main protective antigen; in addition, the F protein is probably the main reason of having cross-protection property in morbillivirus, and the F protein is an important target point in the establishment of vaccine design and detection methods, so that the epitope mapping and antibody preparation of the F protein have great significance.

Disclosure of Invention

The invention aims to provide a peste des petits ruminants virus F protein epitope peptide.

The invention also aims to provide the application of the Peste des petits ruminants virus F protein epitope peptide in the preparation of Peste des petits ruminants virus epitope vaccine antigens and diagnostic reagent antigens.

The invention also aims to provide a method for determining and preparing the Peste des petits ruminants virus F protein epitope.

The technical scheme adopted by the invention is as follows: an epitope peptide of F protein of peste des petits ruminants virus, wherein the amino acid sequence of the epitope peptide is as follows: f176:¹⁷⁶DYINNELVPSVHRMSCEL¹⁹³and/or F347:³⁴⁷MSPLLQECFRGSTKS³⁶¹and/or F391:³⁹¹CKCYTTETVINQDPDKLL⁴⁰⁸and/or F417:⁴¹⁷TVINQDPDKGPVGSREYPD⁴³⁵and/or F430:⁴³⁰SREYPDSVYLH⁴⁴⁰and/or F502:⁵⁰²VSLGLVTLICCCKGRCRNK⁵²⁰。

an application of the antigen epitope peptide of F protein of peste des petits ruminants virus in preparing the antigen of epitope vaccine and the antigen of diagnostic reagent of peste des petits ruminants virus.

A method for determining and preparing Peste des petits ruminants virus F protein epitope comprises the following steps:

calculating and simulating:

step one, constructing a 3D structure of a virtual F protein by using molecular simulation software: performing homologous modeling on the PPRV-F protein of the vaccine strain by using SWISS-MODEL (GenBank accession number: X74443);

step two, searching the template: searching a PDB database (www.pdb.org), searching the structure of the homologous protein analyzed by experiments, selecting a protein structure with the sequence consistency of more than 20 percent and longer compared sequence as a template structure, and performing subsequent calculation;

step three, modeling: submitting the comparison result to a server, and inferring a structure according to the spatial coordinates and sequence similarity of the template protein, so as to calculate a target protein structure, namely generating 3 different target protein structures each time;

step four, evaluating the structure: evaluating the rationality of the protein structure by adopting a Laplace conformational diagram analysis method, and if the proportion of non-glycine/proline in an unlicensed region is not more than 5%, the simulated protein structure is reasonable and can be subjected to subsequent computational analysis;

prediction based on immune informatics:

step one, PPRV-HN protein B cell epitope prediction: analyzing the amino acid sequence of PPRV F protein by IEDB, Immunodicine Group and Bepided immune informatics analysis software, and comprehensively analyzing and predicting B cell epitope;

step two, synthesizing the epitope: sending the epitope amino acid sequence predicted by the software to GeneScript company, synthesizing by using a polypeptide synthesizer, and analyzing the purity of polypeptide synthesis and the correctness of amino acid by using High Performance Liquid Chromatography (HPLC) after synthesis;

preparation of antibody:

step one, immunizing animals: immunizing female Balb/c mice with 6-8 weeks old by using target antigen rPPRV-HN-F and peste des petits ruminants virus Glycoprotein (PPRV-Glycoprotein) with reactogenicity, injecting the mice at four subcutaneous points on the back of the neck, wherein the sample amount of the immune protein is 50 mu g/mouse, and the immune volume is 200 mu L/mouse; emulsifying the primary immunization with an equal volume of Freund complete adjuvant, and emulsifying the secondary immunization and the tertiary immunization with an equal volume of Freund incomplete adjuvant; immunizing once every 14 days, after immunizing for 14 days for three times, cutting the tail of the mouse, collecting blood, detecting the antibody in the serum, and when the titer of the antibody is 1: after 10000, four-way injection can be carried out without adjuvant;

step two, purifying the polyclonal antibody, namely putting absorbent cotton completely soaked by ether and a mouse into an anesthesia box at the same time, picking up eyeballs when the mouse is completely unconscious, taking the eyeball for collection, breaking the neck of the mouse to kill the mouse after collection is finished, incubating whole blood at 37 ℃ for 30 minutes, standing overnight at 4 ℃, centrifuging at 1500rpm for 15 minutes, and taking serum for later use at-20 ℃;

identification of an epitope;

the predicted epitope is biosynthesized, identified to meet the experimental requirements, and carried out by using an indirect ELISA method in which an aminated ELISA plate is used as a solid phase carrier;

diluting: diluting the synthesized and freeze-dried epitope peptide to 1 mu g/mu L by using high-pressure deionized water;

epitope peptide coating: coating the diluted epitope peptide on an amination enzyme label plate, wherein each hole is 50 mu L of coating solution, the content of antigen is 5 mu g, and the overnight incubation is carried out at 4 ℃;

washing the plate: throwing the liquid in the enzyme-labeled holes as far as possible, then adding about 250 mu LPBST into each hole, standing for 3 minutes, repeating for three times, and finally patting the enzyme-labeled plate on gauze;

and (3) sealing: adding 150 mu L of 1 multiplied sealing liquid into an enzyme labeling hole, incubating in an incubator at 37 ℃ for 1.5 hours, throwing the sealing liquid in the enzyme labeling hole as far as possible, and finally, patting an enzyme labeling plate on gauze for later use;

incubating the primary antibody: diluting the monoclonal antibody with a confining liquid according to a ratio of 1:500, adding 50 mu L of the monoclonal antibody into each hole of an enzyme label plate, and placing the enzyme label plate in a 37 ℃ incubator for incubation for 1 hour;

washing the plate: simultaneously washing the plate;

incubation of secondary antibody: diluting HRP-labeled anti-mouse IgG secondary antibody with a confining liquid according to a ratio of 1: 5000, adding 50 mu L of each hole of an enzyme label plate, and placing the enzyme label plate in a 37 ℃ incubator for incubation for 1 hour;

washing the plate: simultaneously washing the plate;

color development: adding 50 mu L of TMB color development liquid into each hole, and incubating for 15 minutes at 37 ℃ in a dark place;

and (4) terminating: adding 50 mu L of stop solution into each hole, and stopping color development;

reading: the ELISA plate was detected by end-point method at a wavelength of 450nm and the absorbance value of OD450nm was read. Multi-epitope antigen design:

the method comprises the following steps: designing multi-epitope gene and inducing expression, arranging epitopes in sequence in natural protein, adding GS joint molecules between epitopes, sending the epitopes to a synthesized gene fragment of Nanjing Kinshire company, connecting the synthesized gene fragment to a pET30a vector, transforming the vector into host bacteria, and inducing for 7 hours to induce expression according to the culture temperature of 37 ℃, the final concentration of an inducer IPTG being 1.0mM and an ampicillin LB culture medium;

step two: the purification of the multi-epitope antigen is carried out,

and (3) crushing thalli: repeatedly freezing and thawing the thallus for 3 times, and carrying out ultrasonic crushing on the thallus by using PBS buffer solution for heavy suspension;

centrifuging: centrifuging the crushed thallus at 4 ℃ and 10000 rpm for 20 minutes, and respectively collecting precipitate and supernatant;

sample preparation: resuspending the collected precipitate in buffer I (pH 7.4), mixing well, centrifuging at 8000 rpm and 4 ℃ for 20 min, and filtering the supernatant with 0.45 μm filter;

balancing Ni columns: adding a small amount of deionized water at the bottom of the column to remove air, then filling 2mL of Ni-NAT filler, washing the filled column with 5 times of deionized water to remove ethanol, and finally balancing the column material with 5 times of buffer solution I (pH is 7.4);

loading: combining the sample filtered by the filter with the column material in times, standing, collecting turbulent liquid after natural sedimentation, and storing at 4 ℃;

washing: buffer I (pH 7.4) was added to the column in portions, the total volume being greater than 5 times the volume of the above amount; and (3) elution: eluting proteins using elution buffers containing imidazole concentrations of 10mM, 20mM, 50mM, 100mM, 200mM, 300mM, and 400mM, each elution concentration being left for 15 minutes, collecting eluates at each concentration and placing on ice;

and (3) column preservation: after the elution step was completed, the column was washed with 5 column volumes of buffer I (pH 7.4), and then stored at 4 ℃ with an appropriate amount of 20% ethanol;

step three: the identification of the multi-epitope antigen is carried out,

protein electrophoresis sample preparation: putting 40 mu L of protein sample separated in the above step into a 1.5mL centrifuge tube, adding 10 mu L of 5 Xprotein loading buffer solution, boiling for 10 minutes, and placing in a refrigerator at 4 ℃ for rapid cooling; loading and electrophoresis: 10 μ L of the suspension was subjected to 100mV electrophoresis in 12% SDS-PAGE;

film transfer: cutting the NC membrane to be equal to the gel in size, placing the NC membrane in a transfer buffer solution for soaking for 5 minutes, placing thick filter paper, the gel, the NC membrane and the thick filter paper in sequence from the negative electrode to the positive electrode, and performing 200mA constant current wet rotation for 100 minutes while taking care to avoid bubbles;

and (3) sealing: taking out the transferred NC film, putting the transferred NC film in a proper amount of 5% skimmed milk powder, and sealing for two hours at room temperature;

adding a primary antibody: placing the sealed NC membrane in 5% BSA, adding a mouse anti-His monoclonal antibody (the dilution ratio is 1: 2500) and sheep-derived peste des petits ruminants positive serum (the dilution ratio is 1:250), and incubating overnight in a refrigerator at 4 ℃;

washing: after the primary antibody incubation was complete, wash 3 times with PBST shaking for 10 minutes each;

adding a secondary antibody: placing the NC membrane in a rabbit anti-mouse IgG-HRP (diluted by 1: 5000) and a donkey anti-sheep IgG-HRP (diluted by 1: 25000) secondary antibody, wherein the diluted solution is PBST containing 5% skimmed milk powder, and incubating for 1 hour at room temperature by shaking;

washing: washing in the same way;

color development: placing the washed NC membrane in a gel imaging system, mixing ECL (Ecl-super luminescent) liquid A and B, and dripping on the NC membrane to incubate for 3 minutes at room temperature;

and (4) observation: adjusting different exposure times until clear bands are seen;

step four: mice were immunized and mice were divided into 5 groups, group a: PBS + freund's incomplete adjuvant, group B: vaccine, group C: EHF1+ freund's incomplete adjuvant, group D: EHF2+ freund's incomplete adjuvant, group E: EHF3+ freund's incomplete adjuvant. Each group had 12 mice, 50. mu.L of each immune protein, and 200. mu.L of each immune volume. Injecting four points of the neck and back part subcutaneously, and immunizing again after 21 days;

step five: detecting the specific antibody of the mouse, collecting blood after tail breaking for 0 day, 7 days, 14 days, 28 days and 35 days, incubating for 30 minutes at 37 ℃, overnight at 4 ℃, centrifuging for 15 minutes at 3000rpm, collecting serum, and storing at-80 ℃ for later use. Detecting the antibody level by indirect enzyme-linked immunization coated with PPRV whole virus;

step six: detecting the level of mouse T lymphocytes, detecting the proliferation of immunized mouse CD3+ CD4+ T cells and CD3+ CD8+ T cells, detecting peripheral blood T cells at 0 day, 14 days and 26 days after immunization by adopting a flow cytometry technology, and detecting the T lymphocytes in the spleen at 35 day; the method comprises the following steps:

blood collection: anticoagulation of mice is adopted when the tail is broken, 5 mice are collected in each group, and each mouse is 100 mu L;

antibody incubation: dividing collected 100 mu L of anticoagulation blood into two parts on average, wherein 2.5 mu L of APC Hamsteranti-Mouse CD3e antibody and PE Rat Anti-Mouse CD4 antibody and 1 mu L of FITC Rat Anti-Mouse CD8a antibody are added into one part; adding 2.5 mu L of APC Hamster IgG1, kappa Isotype Control antibody, PE Rat IgG2a, kappa Isotype Control antibody, FITC Rat IgG2a and kappa Isotype Control antibody into the other part, mixing uniformly, and incubating for 15 minutes at 4 ℃ in a dark place;

and (3) cracking red blood cells: adding 1mL of 1X erythrocyte lysate, uniformly mixing, and incubating for 10 minutes at room temperature in a dark place;

washing: adding 1mL of Hank' S solution, mixing uniformly, centrifuging at 1000 g for 5 minutes, and repeating for 2 times;

and (3) filtering: filtering with a high-pressure 300-mesh nylon net before sample loading;

loading: the prepared sample is placed on a sample rack of the flow cytometer and the cells are prepared to be collected.

The invention has the beneficial effects that:

inoculating the VeroE6 cell with peste des petits ruminants virus, and detecting the reactogenicity of the polyclonal antibody and the peste des petits ruminants virus and the specificity of the antibody. The results show that rPPRV-HN-F polyclonal antibody (figure 1A) reacts with peste des petits ruminants virus in Vero cells, shows stronger green fluorescence, does not react with Vero cells, and does not fluoresce (figure 1C); mouse negative sera did not react with peste des petits ruminants virus in Vero cells (fig. 1B), with no fluorescence. The rPPRV-HN-F polyclonal antibody is a specific antibody of peste des petits ruminants virus; PPRV-Glycoprotein polyclonal antibody (figure 2) has good reactogenicity and specificity with Peste des petits ruminants virus.

The obtained rPPRV-HN-F (figure 3A) and PPRV-Glycoprotein (figure 3B) polyclonal antibodies are subjected to specificity detection based on prokaryotic expression and purification of rPPRV-HN-F protein by using a Western-Blotting method, an empty vector expression bacterium is used as a negative control, and the result shows that a single band appears in a rPPRV-HN-F protein lane, and no band exists in an empty vector expression bacterium lane, so that the two polyclonal antibodies have good reactogenicity with the rPPRV-HN-F protein and have good specificity without reacting with the empty vector expression bacterium.

The candidate epitopes are tested for the reactogenicity with rPPRV-HN-F polyclonal antibody (figure 4) and PPRV-Glycoprotein (figure 5) polyclonal antibody by using an aminated enzyme label plate, and the candidate epitopes F176, F347, F391, H417, F430 and F502 are determined.

The head structure of the F protein of the peste des petits ruminants virus Nigeria 75/1 strain was predicted using SWISS-MODEL, showing that F176, F347, F391, F417 and F430 are located on the surface of the F protein (fig. 6 blue panel).

The three synthesized multi-epitope genes are indirectly connected into a pET30a vector, and the double enzyme digestion identification (figure 7) shows that the target band is correctly inserted into the corresponding position of the vector. The result of plasmid sequencing by Cincisco TengZhi is compared with the synthesized sequence, and no mutation is found, which indicates that the correct sequence is inserted into the correct position of the vector.

The plasmid is induced to express and purified to obtain high-purity antigen protein (figure 8).

Western-Blotting specific detection is carried out on the expressed fusion protein by using a His tag antibody. The results show that a specific band appears in the lane of the positive clone, and no band exists in the lane of the empty vector host bacteria (FIG. 9), which indicates that the host bacteria successfully express the His fusion protein, namely the target protein.

The peripheral blood antibody level of the mice is detected, and the result based on the whole virus shows that each multi-epitope antigen can stimulate the antibody level of the mice, and the EHF1 proteome effect is good.

To understand the proliferation changes of T lymphocytes, proliferation of CD3+ CD4+ T lymphocytes and CD3+ CD8+ T lymphocytes in peripheral blood and spleen of mice was examined by flow cytometry. The levels of T lymphocytes at different time points on

days

0, 14, 26 and 35 post-immunization were examined and the results showed that the levels of CD3+ CD4+ T lymphocytes were different but not significantly changed in the different groups at the same time point (fig. 11). However, the levels of CD3+ CD8+ T lymphocytes did differ between groups at different time points (fig. 11). Therefore, the multi-epitope antigen can effectively stimulate the cellular immunity of the mice.

Drawings

FIG. 1 is a rPPRV-HN-F polyclonal antibody is a specific antibody of Peste des petits ruminants virus;

FIG. 2PPRV-Glycoprotein polyclonal antibody has good reactogenicity and specificity with Peste des petits ruminants virus as well;

FIG. 3Western Blotting detection shows that polyclonal antibody and rPPRV-HN-F protein have good reactogenicity and specificity;

FIG. 4rPPRV-HN-F polyclonal antibody screening F protein B cell epitope library results;

FIG. 5 PPRV-glycoprotin polyclonal antibody screening F protein B cell epitope library results;

FIG. 6 positions of the F176, F347, F391, F417 and F430 epitopes in the F protein structure;

FIG. 7 double restriction enzyme identification of recombinant plasmids pET30a-EHF1, pET30a-EHF2 and pET30a-EHF 3;

FIG. 8 SDS-PAGE analysis of purified polyepitope recombinant antigens EHF1, EHF2 and EHF 3;

FIG. 9Western-Blotting detection of His fusion protein expression;

FIG. 10 detection of mouse antibody levels based on whole virus indirect ELISA;

FIG. 11T lymphocyte proliferation changes in mice following immunization.

Detailed Description

The present invention will be described in further detail with reference to examples.

The antigen epitope peptide of the Peste des petits ruminants virus F protein has the amino acid sequence as follows:

F176：¹⁷⁶DYINNELVPSVHRMSCEL¹⁹³(SEQ ID NO.1)

and/or F347:³⁴⁷MSPLLQECFRGSTKS³⁶¹(SEQ ID NO.2)

and/or F391:³⁹¹CKCYTTETVINQDPDKLL⁴⁰⁸(SEQ ID NO.3)

or/and F417:⁴¹⁷TVINQDPDKGPVGSREYPD⁴³⁵(SEQ ID NO.4)

and/or F430:⁴³⁰SREYPDSVYLH⁴⁴⁰(SEQ ID NO.5)

or/and F502:⁵⁰²VSLGLVTLICCCKGRCRNK⁵²⁰(SEQ ID NO.6)。

the invention uses various kinds of immune informatics software to predict the B cell epitope of the target protein, then different predicted epitopes are artificially synthesized respectively, indirect ELISA method is used to verify the reactogenicity, different polypeptides coat the aminated enzyme label plate, and the reactogenicity of the antibody of the protein F is detected, thereby identifying the B cell epitope of the protein PPRV F.

The invention relates to a method for determining and preparing Peste des petits ruminants virus F protein epitope, which comprises the following steps:

calculating and simulating:

prediction based on immune informatics:

preparation of antibody:

identification of an epitope;

washing the plate: simultaneously washing the plate;

reading: detecting the ELISA plate at the wavelength of 450nm by using an end point method, and reading the absorbance value of OD450 nm;

multi-epitope antigen design:

the method comprises the following steps: designing multi-epitope gene and inducing expression, arranging epitopes in order in natural protein, adding GS joint molecules between epitopes, sending the mixture to Nanjing Kingsry company to synthesize gene fragment, and connecting the gene fragment to pET30a vector. The vector was transformed into a host cell, and induced for 7 hours for expression at a culture temperature of 37 ℃ in an ampicillin LB medium with a final inducer IPTG concentration of 1.0 mM.

Step two: the purification of the multi-epitope antigen is carried out,

washing: buffer I (pH 7.4) was added to the column in portions, the total volume being greater than 5 times the volume of the above amount;

and (3) elution: eluting proteins using elution buffers containing imidazole concentrations of 10mM, 20mM, 50mM, 100mM, 200mM, 300mM, and 400mM, each elution concentration being left for 15 minutes, collecting eluates at each concentration and placing on ice;

and (3) column preservation: after completion of the elution step, the column was washed with 5 column volumes of buffer I (pH 7.4), and stored at 4 ℃ with an appropriate amount of 20% ethanol.

Step three: the identification of the multi-epitope antigen is carried out,

protein electrophoresis sample preparation: putting 40 mu L of protein sample separated in the above step into a 1.5mL centrifuge tube, adding 10 mu L of 5 Xprotein loading buffer solution, boiling for 10 minutes, and placing in a refrigerator at 4 ℃ for rapid cooling;

loading and electrophoresis: 10 μ L of the suspension was subjected to 100mV electrophoresis in 12% SDS-PAGE;

washing: washing in the same way;

and (4) observation: the different exposure times were adjusted until clear bands were seen.

Step four: mice were immunized and mice were divided into 5 groups, group a: PBS + freund's incomplete adjuvant, group B: vaccine, group C: EHF1+ freund's incomplete adjuvant, group D: EHF2+ freund's incomplete adjuvant, group E: EHF3+ freund's incomplete adjuvant, 12 mice per group, 50 μ L of each immune protein, and 200 μ L of each immune volume. Injecting four points of the neck and back part subcutaneously, and immunizing again after 21 days;

step five: detecting a specific antibody of a mouse, collecting blood after tail breaking for 0 day, 7 days, 14 days, 28 days and 35 days, incubating for 30 minutes at 37 ℃, overnight at 4 ℃, centrifuging for 15 minutes at 3000rpm, collecting serum, storing at-80 ℃ for later use, and detecting the antibody level by an indirect enzyme-linked immunosorbent assay coated with PPRV whole virus;

step six: detecting the level of mouse T lymphocytes, detecting the proliferation of immunized mouse CD3+ CD4+ T cells and CD3+ CD8+ T cells, detecting peripheral blood T cells at 0 day, 14 days and 26 days after immunization by adopting a flow cytometry technology, and detecting the T lymphocytes in the spleen at 35 days, wherein the steps are as follows:

antibody incubation: dividing collected 100 mu L of anticoagulation blood into two parts on average, wherein 2.5 mu L of each of APCHAMSTER Anti-Mouse CD3e antibody and PE Rat Anti-Mouse CD4 antibody and 1 mu L of FITC Rat Anti-Mouse CD8a antibody are added into one part; adding 2.5 mu L of APC Hamster IgG1, kappa Isotype Control antibody, PE Rat IgG2a, kappa Isotype Control antibody, FITC Rat IgG2a and kappa Isotype Control antibody into the other part, mixing uniformly, and incubating for 15 minutes at 4 ℃ in a dark place;

As is apparent from FIGS. 1 and 2, the immunogenicity of polyclonal antibodies to Peste des petits ruminants virus and the specificity of the antibodies were examined by inoculating VeroE6 cells with Peste des petits ruminants virus. The results show that rPPRV-HN-F polyclonal antibody (figure 1A) reacts with peste des petits ruminants virus in Vero cells, shows stronger green fluorescence, does not react with Vero cells, and does not fluoresce (figure 1C); mouse negative sera did not react with peste des petits ruminants virus in Vero cells (fig. 1B), with no fluorescence. The rPPRV-HN-F polyclonal antibody is shown to be a specific antibody of peste des petits ruminants virus. PPRV-Glycoprotein polyclonal antibody (figure 2) has good reactogenicity and specificity with Peste des petits ruminants virus.

FIGS. 1 and 2 show that the polyclonal antibody has good reactogenicity and specificity with Peste des petits ruminants virus through indirect immunofluorescence detection.

VeroE6 cells showed syncytia, brighter fluorescence intensity and appeared on diseased cells, indicating that the antibodies had good reactogenicity.

Veroee 6 cells did not show fluorescence indicating that the control antibody did not react with the virus.

VeroE6 cells do not show fluorescence, indicating that the monoclonal antibody does not react with VeroE6 cells, and indicating that the monoclonal antibody has good specificity.

FIG. 3Western Blotting detection shows that the polyclonal antibody and rPPRV-HN-F protein have good reactogenicity and specificity.

FIG. 3A shows that a band is evident in lane 1 of rPPRV-HN-F protein, while a band is absent in lane 2 of empty vector-expressing bacteria, indicating that the polyclonal antibody has good reactogenicity and specificity with rPPRV-HN-F protein; the same result appears in fig. 3B.

FIG. 4rPPRV-HN-F polyclonal antibody screening F protein B cell epitope library results

And detecting the reactogenicity of the polyclonal antibody and the epitope peptide by using an indirect ELISA method taking an aminated enzyme label plate as a solid phase carrier. It is obvious that the OD450 value of the rPPRV-HN-F polyclonal antibody group in the F176, F347, F391, F417 and F502 epitope peptides is very significant compared with the OD450 value of the control group (p <0.00001), and the OD450 value of the rPPRV-HN-F polyclonal antibody group in the F430 epitope peptides is very significant compared with the OD450 value of the control group (p <0.001), which indicates that F176, F347, F391, F417, F430 and F502 have good reactogenicity with the rPPRV-HN-F polyclonal antibody, namely, the effective epitope.

FIG. 5PPRV-Glycoprotein polyclonal antibody screening F protein B cell epitope library results

And detecting the reactogenicity of the polyclonal antibody and the epitope peptide by using an indirect ELISA method taking an aminated enzyme label plate as a solid phase carrier. Obviously, the OD450 value of the PPRV-glycoprotin polyclonal antibody group in the F502 epitope peptide is extremely obvious to be compared with the OD450 value of a control group (p < 0.00001); in the F417 epitope peptide, the OD450 value of the PPRV-Glycoprotein polyclonal antibody group is very significant compared with the OD450 value of a control group (p is less than 0.001); in the F430 epitope peptide, the OD450 value of the PPRV-Glycoprotein polyclonal antibody group is obviously equal to the OD450 value of a control group (p is less than 0.05), which indicates that F417, F430 and F502 epitopes have good reactogenicity with the PPRV-Glycoprotein polyclonal antibody, namely the effective epitope.

FIG. 6 positions of the F176, F347, F391, F417 and F430 epitopes in the F protein structure

It can be clearly seen that the blue part in the figure is the epitope of the antigen, and it is obvious from the 3D structure that the epitope is located at a position where the surface of the antigen is more prominent, which is an important reason that the antibody corresponding to the epitope has good reactogenicity.

FIG. 7 double restriction enzyme identification of recombinant plasmids pET30a-EHF1, pET30a-EHF2 and pET30a-EHF3

M: DNA Marker; 1: pET30a vector double enzyme digestion product; 2: pET30a-EHF1 vector double enzyme digestion product; 3: pET30a-EHF2 vector double enzyme digestion product; 4: pET30a-EHF3 vector double enzyme digestion product

It is evident that both pET30a-EHF1, pET30a-EHF2 and pET30a-EHF3 are threaded with one strip aligned with pET30a and the other strip aligned with EHF1, EHF2 and EHF3, indicating that the target strip is correctly inserted into the corresponding position of the vector.

FIG. 8 SDS-PAGE analysis of multi-epitope recombinant antigens EHF1, EHF2 and EHF3 after purification

M: a protein Marker; 1-3: multi-epitope recombinant antigens EHF1, EHF2 and EHF3

The multi-epitope recombinant antigen is purified by a nickel sepharose column, target protein is eluted from the column by elution of different imidazole concentrations, and SDS-PAGE analysis results show that each lane of the multi-epitope recombinant antigen has no obvious impurity band, which indicates that relatively pure protein is obtained.

FIG. 9Western-Blotting detection of His fusion protein expression

1: precipitating the empty vector host bacteria; 2: pET30a-EHF3/BL21(DE3) precipitation; 3: pET30a-EHF2/BL21(DE3) precipitation; 4: pET30a-EHF1/BL21(DE3) precipitation

Specific bands appear in lanes precipitated by positive clones pET30a-EHF1/BL21(DE3), pET30a-EHF2/BL21(DE3) and pET30a-EHF3/BL21(DE3), wherein the specific bands are respectively around 25kDa, between 40-55kDa and between 55-70kDa, and no band exists in lanes of empty vector host bacteria, which indicates that the host bacteria successfully express His fusion protein, namely the target protein.

FIG. 10 detection of mouse antibody levels based on whole virus indirect ELISA

Mice in each group produced lower antibody levels on day 7, increased antibody levels in each group on

days

14 and 21, secondary immunizations on day 21, increased antibody levels in each group on

days

28 and 35, and no antibody in the PBS group at all times. However, the antibody level was lower in the test group compared to the vaccine group. However, the level of EHF1 protein was higher than that of the vaccine group at day 21. Compared with the three groups of test groups, the EHF1 protein group always maintains higher antibody level; the EHF2 proteome was significantly elevated after the second immunization, with comparable levels of EHF1 proteome antibodies at day 35; but EHF3 proteomic antibody levels were consistently the lowest.

FIG. 11 proliferation of mouse T lymphocytes after immunization

The percentage of CD3+ CD8+ T lymphocytes in the vaccine group, EHF1, EHF2, and EHF3 at day 14 post-immunization was significantly higher than in the PBS group; on day 26 after immunization, the percentage of CD3+ CD8+ T lymphocytes in different experimental groups was significantly different, the vaccine group, EHF1 group and EHF2 group were all significantly higher than the PBS group, and EHF3 was extremely significantly higher than the PBS group; compared with the EHF1 group, the EHF2 group and the EHF3 group, the EHF1 group and the EHF2 group are significantly higher than the vaccine group, and the EHF3 group is significantly higher than the vaccine group; on day 35 post-immunization, the CD3+ CD8+ T lymphocyte percentage was significantly higher for the vaccine group, EHF1 group, EHF2 group, and EHF3 group than for the PBS group, the CD3+ CD8+ T lymphocyte percentage was significantly higher for the EHF3 group than for the PBS group, and the CD3+ CD8+ T lymphocyte percentage was significantly higher for the EHF1 group than for the vaccine group.

The epitope peptide identified by the scheme provides a theoretical basis for the research and development of the preparation of the peste des petits ruminants virus epitope vaccine antigen and the diagnostic reagent antigen, and has great significance for the prevention and treatment of the peste des petits ruminants virus.

The present invention is not limited to the above-mentioned preferred embodiments, and any other products in various forms can be obtained by anyone in the light of the present invention, but any changes in the shape or structure thereof, which have the same or similar technical solutions as those of the present application, fall within the protection scope of the present invention.

SEQUENCE LISTING

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

<120> Peste des petits ruminants virus F protein epitope peptide, determination method, preparation method and application thereof

<130>2017

<160>6

<170>PatentIn version 3.3

<210>1

<211>18

<212>PRT

<213> Peste des petits ruminants virus

<400>1

DYINNELVPSVHRMSCEL

1 5 10 15

<210>2

<211>15

<212>PRT

<213> Peste des petits ruminants virus

<400>2

MSPLLQECFRGSTKS

1 5 10 15

<210>3

<211>18

<212>PRT

<213> Peste des petits ruminants virus

<400>3

CKCYTTETVINQDPDKLL

1 5 10 15

<210>4

<211>19

<212>PRT

<213> Peste des petits ruminants virus

<400>4

TVINQDPDKGPVGSREYPD

1 5 10 15

<210>5

<211>11

<212>PRT

<213> Peste des petits ruminants virus

<400>5

SREYPDSVYLH

1 5 10

<210>6

<211>19

<212>PRT

<213> Peste des petits ruminants virus

<400>6

VSLGLVTLICCCKGRCRNK

1 5 10 15

Claims

1. A Peste des petits ruminants virus F protein epitope peptide is characterized in that: the epitope peptide is F502, or a combination of F502 and one or more of F176, F347, F391, F417 and F430;

wherein the amino acid sequence of F502 is: 502VSLGLVTLICCCKGRCRNK 520;

the amino acid sequence of F176 is: 176DYINNELVPSVHRMSCEL 193;

the amino acid sequence of F347 is: 347MSPLLQECFRGSTKS 361;

the amino acid sequence of F391 is: 391CKCYTTETVINQDPDKLL 408;

the amino acid sequence of F417 is: 417TVINQDPDKGPVGSREYPD 435;

the amino acid sequence of F430 is: 430SREYPDSVYLH 440.

2. The use of the Peste des petits ruminants virus F protein epitope peptide according to claim 1 in the preparation of a Peste des petits ruminants virus diagnostic reagent.

3. The use of the Peste des petits ruminants virus F protein epitope peptide according to claim 1 in the preparation of Peste des petits ruminants virus vaccines, characterized in that: the epitope peptide has the amino acid sequence as follows: f176, F347, F391, F417, F430 and F502 are arranged in sequence, and the peptide fragments are connected with each other through a GS linker molecule.