CN106749557B

CN106749557B - Specific antigen epitope polypeptide of bluetongue virus VP7 protein group and application thereof

Info

Publication number: CN106749557B
Application number: CN201611112486.3A
Authority: CN
Inventors: 唐丽杰; 李一经; 徐义刚; 乔薪瑗; 王丽; 臧明鑫; 武啸; 李佳璇
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2016-12-06
Filing date: 2016-12-06
Publication date: 2020-02-11
Anticipated expiration: 2036-12-06
Also published as: CN106749557A

Abstract

The invention discloses a specific epitope polypeptide of a bluetongue virus VP7 protein group and application thereof. The invention uses the monoclonal antibody 3H7 cell strain of the VP7 protein of the bluetongue virus prepared in the early stage and utilizes the phage display peptide technology to screen the epitope recognized by the 3H7 cell strain. Sequencing after 3 rounds of panning shows that the epitope recognized by the 3H7 cell strain is QYHAM. Further synthesizing short peptides, and identifying the 3H7 cell strain by using indirect ELISA, wherein an experimental result shows that the epitope recognized by the 3H7 cell strain is QYHAM; amino acid sequence alignment shows that the epitope is conserved among different sources of type 25 bluetongue virus strains. The invention identifies the specific epitope polypeptide of the VP7 protein group of the bluetongue virus, lays a foundation for establishing a competitive ELISA method aiming at the bluetongue virus, and the identified epitope can be applied to the design of vaccines in the future and has important significance for preventing and treating the bluetongue in China.

Description

Specific antigen epitope polypeptide of bluetongue virus VP7 protein group and application thereof

Technical Field

The invention relates to an epitope polypeptide, in particular to a specific epitope polypeptide of a bluetongue virus VP7 protein group identified by a bluetongue virus 25 type VP7 protein monoclonal antibody 3H7 cell strain and application thereof in detecting bluetongue viruses, belonging to the technical field of biology.

Background

Bluetongue virus (BTV) has been isolated in many countries of the world and is distributed globally as the global climate warms. Up to now, there have been found 27 serotypes of bluetongue virus, with no cross protection between the types ^[1-2]. The world animal health Organization (OIE) lists the bluetongue as a necessary epidemic disease to be notified, and China also lists the bluetongue as a type of epidemic disease. The disease is mainly transmitted by Culicoides culans, Aedes aegypti, etc., and can also be vertically transmitted by placenta infected fetus ^[3]. BTV mainly infects ruminants such as sheep and the like, and has the main symptoms of body temperature rise of sick livestock, listlessness, anorexia and salivation, and edema of various degrees at various parts of affected livestock. Congestion, erosion and cyanosis of oral mucosa are typical symptoms of bluetongue. BTV25 type was isolated from a sample of goat blood from switzerland in 2008. VP2 of BTV25 type has only 23% -79% homology with other serotype viruses VP 2. The livestock infected with BTV25 has no typical symptoms, is often overlooked, and causes extremely serious economic loss once epidemic disease occurs ^[4]。

Since the first discovery of bluetongue in 1876, new serotypes were continuously discovered, and the range of outbreaks was gradually expanded. The bluetongue virus type 27 is discovered and separated for the first time in the island of Coxijia France in 2014 ^[5]It has been reported that bluetongue virus type 28 and type 29 are now found and isolated sequentially in the middle east and south Africa ^6-7]. Seven serotypes BTV-1, BTV-2, BTV-3, BTV-4, BTV-12, BTV-15 and BTV-16 have been detected in China, wherein the types BTV1 and 16 are main pathogenic serotypes in China ^[8]。

The BTV genome consists of 10 linear double-stranded RNAs with a total molecular weight of 1.3X 107 Da. The genome consists of a large fragment (L1-L3), a middle fragment (M4-M6) and a small fragment (S7-S10), and the genome totally encodes 7 structuresProtein (VP1-VP7) and 4 non-structural proteins (NS1, NS2, NS3/NS3a and NS4) ^[9-10]. The bluetongue virus has a double-coat structure, and the VP2 and VP5 encoded by the L2 and M5 genes constitute the major components of the BTV coat, which is about 40% of the total viral protein. The viral inner shell, the core capsid, is composed primarily of VP3 and VP7 encoded by L3 and S7. VP7 exists primarily as a trimer within the nucleocapsid, VP3 is the major protein of the sub-core particle, and both are BTV group-specific antigens.

The virus genome and three structural proteins of VP1, VP4 and VP6 are wrapped inside the core capsid ^[11]. The VP7 protein is the best choice for establishing a BTV group-specific serological detection method, since its sequence and antigenicity are highly conserved among all serotypes. After BTV infection of cells, VP7 was the first antigen detected, and after BTV infection of animals, anti-VP 7 antibody was one of the major anti-BTV antibodies produced by the body. VP7 can regulate adsorption and invasion of BTV to insect cells in the absence of VP2 or VP5 ^[12]。

Therefore, the identification of the group-specific epitope polypeptide of the bluetongue virus VP7 protein has important significance for the design of BT vaccines and the development of high-throughput bluetongue virus detection kits.

Reference documents:

[1] miao Zhi Qiang, Zheng Ming school, etc., epidemic situation and prevention and control measure [ J ]. Heilongjiang animal husbandry veterinarian, 2010 (5): 28-30.

[2]Legisa.，et al.Bluetongue virus in South America，Central Americaand the Caribbean[J].Virus research，2014，182：87-94.

[3] Preparation of monoclonal antibody of VP2 protein type 17 of bluetongue virus and identification of epitope thereof [ J ]. Chinese veterinary medical report of prevention 2011, 33 (6): 465-470.

[4]Hofmann M A，Renzullo S，Mader M，et al.Genetic characterization oftoggenburg orbivirus，a new bluetongue virus，from Goats，Switzerland[J].EmergInfect Dis，2008，14(12)：1855-1861.

[5]Zientara S，Sailleau C，Viarouge C，et al.Novel bluetongue virus inGoats，Corsica，France，2014[J].Emerg Infect Dis，2014，20(12)：2123-2125.

[6]Maan S，Maan N S，Nomikou K，et al.Novel bluetongue virus serotypefrom Kuwait[J].Emerg Infect Dis，2011，17(5)：886-889.

[7]Maan S，Maan N S，Belaganahalli M N，et al.Full-genome sequencing asa basis for molecular epidemiology studies of bluetongue virus in India[J].PLoS One，2015，10(6)：e0131257.

[8] Yunnan bluetongue virus type 1 strain M6 gene sequence analysis [ J ]. Chinese veterinary medicine, 2016, 43 (2): 340-347

[9]Umeshappa CS，Singh KP，Pandey A B，et al.Cell-mediated immuneresponse and cross-protective efficacy of binary ethylenimine-inactivatedbluetongue virus serotype-1vaccine in sheep[J].Vaccine，2010，28(13)：2522-2531.

[10] Yellow embroidery, Pushuying, Gansu province animal bluetongue epidemiological investigation and pathogen separation, identification, the first treatise on the discussion of bluetongue academy of Pacific region in southeast Asia [ C ]. Yunnan Kunming, 1995.

[11]Hassan S.H，Wirblich C，Forzan M，et al.Expression and functionalcharacterization of bluetongue virus VP5protein：role in cellularpermeabilization[J].Virol，2001，75(18)：8356-8367.

[12]Mertens，P.，Burroughs，J.，Walton，A.et al.，Enhanced Infectivity ofModified Bluetongue Virus Particles for Two Insect Cell Lines and forTwoCulicoidesVector Species[J].Virology.1996，217：582-593.

Disclosure of Invention

The invention aims to solve the technical problem of providing a specific antigen epitope polypeptide of a bluetongue virus VP7 protein group, application thereof and application thereof in detecting bluetongue viruses.

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

the invention uses the early-stage prepared hybridoma cell strain 3H7 which stably secretes anti-25-type BTV VP7, and utilizes phage display peptide technology to screen the epitope recognized by the 3H7 cell strain. Sequencing after 3 rounds of panning revealed that the epitope recognized by the 3H7 cell line was QYHAM (shown in SEQ ID NO. 1). Further synthesis of short peptides 3H7 cell line was identified by indirect ELISA. The experimental result shows that the epitope recognized by the 3H7 cell strain is QYHAM; through comparison with amino acid sequences of 25 bluetongue virus VP7 proteins in NCBI, analysis shows that the linear epitope QYHAM recognized by the 3H7 cell strain and the bluetongue virus VP7 protein have two amino acid mutations, namely that the 239 th amino acid is mutated from arginine to tyrosine, the 240 th amino acid is mutated from asparagine to histidine, and other amino acids are highly conserved in the 25 serotypes of the bluetongue virus. Therefore, the polypeptide can be used as specific epitope polypeptide of the blue tongue virus VP7 protein group and used for detecting the blue tongue virus.

On the basis of the research, the invention provides a specific antigen epitope polypeptide of a bluetongue virus VP7 protein group, and the amino acid sequence of the antigen epitope polypeptide is shown as SEQ ID NO. 1.

The nucleotide sequence for encoding the epitope polypeptide, the expression vector containing the nucleotide sequence and the host cell containing the expression vector are also within the protection scope of the invention.

Furthermore, the invention also provides application of the antigen epitope polypeptide in preparation of a reagent for detecting the bluetongue virus.

The invention provides technical support for detecting the bluetongue virus by adopting an indirect ELISA method, and has important significance for early detection of the bluetongue virus.

Drawings

FIG. 1 shows the results of indirect immunofluorescence reactions of 3H7 with BHK cells;

FIG. 2 shows the Western blot identification results of recombinant protein VP7 and 3H7 cell supernatant;

m, protein Marker; 1. purifying the protein by pET-28a-VP7/BL 21; 2. before induction, pET-28a-VP7/BL 21;

FIG. 3 shows the sequencing results of 12 peptides displayed by 18 phage clones;

FIG. 4 shows the synthetic short peptide and the indirect ELISA result of the monoclonal antibody supernatant and ascites;

FIG. 5 shows the indirect ELISA identification results of synthetic short peptides and different types of BTV positive sera;

FIG. 6 shows the BLAST results of the short peptide sequence and the gene sequence BTV1-BTV 12;

FIG. 7 shows the BLAST results of the short peptide sequence and the gene sequences BTV13-BTV 26.

Detailed Description

The invention will be further described with reference to specific embodiments and drawings, the advantages and features of which will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 13 identification of epitope Polypeptides recognized by H7 cell line

1 materials and methods

1.1 cells, viruses and antibodies

Hybridoma cell line 3H7 (which is described in the following non-patent documents: prokaryotic expression of proteins of BTV VP7 and VP2 of type 25 and preparation of monoclonal antibodies thereof, Wutsu, Master academic thesis of northeast university of agriculture, 2015) which stably secretes anti-25 BTV VP7, blue tongue virus positive serum, BHK21 cells stored in the laboratory, horseradish peroxidase-labeled goat anti-mouse IgG, FITC fluorescence-labeled goat anti-mouse, mouse anti-His-labeled monoclonal antibodies purchased from China fir gold bridge Limited, and standard positive blood and negative serum of blue tongue disease purchased from blue tongue disease reference laboratory Pirbrigentitite.

1.2 Primary reagents

PRMI 1640 medium was purchased from Gibicol; HAT (50X), HT (100X), dimethyl sulfoxide (DMSO), and antibody subclass chain specificity identification kit were purchased from SIGMA; protein Marker was purchased from precious bioengineering (Dalian) Inc.

1.3 cell recovery and identification

The hybridoma cell strain 3H7 which is stored in the laboratory and stably secretes anti-25 BTV VP7 is recovered. And carrying out continuous passage, and reserving cell supernatant. And (3) identifying the monoclonal antibody supernatant by using a competitive ELISA method, taking the VP7 protein which is subjected to prokaryotic expression and purification as an antigen, taking the monoclonal antibody supernatant as a primary antibody, and taking the goat anti-mouse IgG marked by the HRP as a secondary antibody.

1.4 Indirect immunofluorescence assay

BHK21 cells were transferred to a 24-well plate, and the plates were placed in the wells and inoculated with 8-type BTV after the cells grew a monolayer, while normal BHK21 cells were set as negative controls. Culturing at 37 ℃ and 5% CO2 for 48h, then discarding the culture supernatant, gently washing with PBS 3 times, fixing with 4% paraformaldehyde fixing solution at room temperature for 30min, adding 3% BSA to each well at 37 ℃ and sealing for 1h, adding 1ml hybridoma cell culture supernatant to each well as a control SP2/0 cell culture supernatant, acting at 37 ℃ for 1h, adding FITC-labeled goat anti-mouse IgG diluted with 0.3% BSA as a secondary antibody at 200. mu.L/well, acting at 37 ℃ in a dark place for 1h, observing the result under a fluorescence microscope, and taking pictures to record.

1.5 Western-Blot identification

The laminated gel part is cut off orderly by carrying out SDS-PAGE electrophoresis on the protein which is expressed and purified by pronucleus, the laminated gel part is electrically transferred to a nitrocellulose membrane, 50mA is transferred for 50min, then the NC membrane is put into 5 percent skim milk to act for 2.5H at 37 ℃, the NC membrane is taken out and washed by PBST for three times, 10 min/time, then 3H7 cell culture supernatant is used as a primary antibody for incubation, and the mixture is sealed at 4 ℃ for overnight. Taking out the NC membrane, washing the NC membrane for three times by PBST for 10 min/time, then putting the NC membrane into HRP-labeled goat anti-mouse IgG diluted by 5% skim milk according to a ratio of 1: 2000, carrying out secondary antibody incubation, acting at room temperature for 2.5H, taking out the NC membrane, washing the NC membrane for three times by PBST for 10 min/time, developing at room temperature by DAB-H2O2 developing solution, and observing the result.

1.6 epitope identification

The phage peptide library was panned 3 times with purified monoclonal antibody IgG, according to the ph.d. -12TM13 phage peptide library instructions. The purified monoclonal antibody ascites IgG was diluted to a concentration of 100ng/mL with a coating diluent and added to a microplate in a coating amount of 150. mu.L/well. Removing the coating liquid, and filling sealing liquid into each hole. mu.L of the original phage library was diluted with 100. mu.L of LPBST and added to the plate and gently shaken at room temperature. Add 100. mu.L of elution buffer to the microplate and shake gently at room temperature. And taking out the eluate and adding a neutralizing solution to obtain the phage after the first round of panning. 100 mu L of panned phage is added into ER2738 bacterial liquid for phage amplification, and shake culture is carried out at 37 ℃. The supernatant was taken, 2.67mL of PEG/NaCl solution was added, and the amplified phage was precipitated overnight at 4 ℃. The cell-spraying solution precipitated overnight was centrifuged at 4 ℃ and the supernatant was discarded, centrifuged briefly and the residual solution was aspirated. And taking the supernatant, adding PEG/NaCl, and acting on ice for 1h. The phage was precipitated by centrifugation at 4 ℃ and the supernatant was discarded, centrifuged briefly and the residual solution was aspirated. The precipitate was redissolved with 200. mu. LTBS, centrifuged at 4 ℃ for 1min, and the supernatant was transferred to a new centrifuge tube, which was the amplified phage. Taking 10 mu L of amplified phage, using LB liquid culture medium to dilute 10 times, respectively taking 10 mu L of each diluted solution to inoculate 200mL of host bacteria ER2738, after 5min at room temperature, adding the diluted solution into 3mL of top-layer agar preheated at 45 ℃, quickly mixing and pouring the mixture on an LB/IPTG/X-gal solid agar hoof plate preheated at 37 ℃, culturing overnight at 37 ℃ in a dark place to check the plate, counting the number of spots on the plate with about 100 blue plaques, and multiplying the number by the corresponding dilution multiple, namely the titer (pfu) of each 10 mu L of amplified phage. Panning was repeated three times. The products after three panning were sequenced. And synthesizing short peptide and monoclonal antibody supernatant to carry out indirect ELISA identification.

1.7 short peptide ELISA identification

Coating: diluting the short peptide by using a coating diluent to 100 mu l/hole coating an enzyme label plate, and standing overnight at 4 ℃; and (3) sealing: discarding the coating liquid in the plate, adding 200 mul/hole PBST solution, shaking to wash each hole, adding 200 mul of confining liquid (5% skim milk), diluting positive serum with 5% skim milk, adding 100 mul to each hole, making 2 parallel holes for each sample, simultaneously setting blank control holes, acting for 1h at 37 ℃, using 5% skim milk for the second antibody to label the goat anti-mouse enzyme-labeled second antibody, adding 100 mul/hole to each reaction hole, and acting for 1h at 37 ℃. Color development: adding OPD-H2O2 substrate color development solution prepared at present, developing 100 μ l/hole at 37 deg.C in dark for 15min, adding stop solution(2M/H ₂SO ₄) The color development was stopped at 50. mu.l/well.

1.8 conservative analysis

Epitope conservation of 3H7 cell line was analyzed by Blast in NCBI database.

2 results

2.1 specific identification of monoclonal antibodies

The results of indirect immunofluorescence reaction of 3H7 with BHK cells are shown in FIG. 1, wherein, FIG. A shows the results of reaction of 3H7 cell supernatant with 21 cells of BHK cells inoculated with BTV type 25. FIG. B shows the reaction of 3H7 supernatant with BHK21 cells that were not inoculated with BTV type 25. The result shows that 3H7 cells are obviously positive reaction and negative reaction with the control.

2.2 Western-Blot identification of monoclonal antibodies

The purified VP7 protein of prokaryotic expression is detected by 3H7 supernatant, and a blank thallus control is arranged at the same time. The result of DAB color development shows that the antibody secreted by 3H7 can be specifically combined with the protein expressed by pronucleus, and can not be specifically combined with blank thallus contrast. The purified VP7 protein of prokaryotic expression is detected by 3H7 supernatant, and a blank thallus control is arranged at the same time. The results after DAB color development show that the antibody secreted by 3H7 can be specifically combined with the protein expressed by the pronucleus, and can not be specifically combined with the blank thallus control (figure 2).

2.3 DNA sequencing and epitope analysis

DNA sequencing is carried out on 18 phage positive clones, the displayed short peptides are subjected to sequence alignment, the result is shown in figure 3, and the result shows that 18 phage clones display a common sequence QYHAM (shown in SEQ ID NO. 1). And the sequence alignment shows that the sequence is relatively conserved in each BTV serotype. We speculate that the QYHAM sequence may be a linear B cell epitope.

2.4 short peptide ELISA identification results

The synthetic short peptide is respectively reacted with the monoclonal antibody supernatant and the ascites, the obtained result is the average value of 20 times of ELISA reactions, and the result shows that the value of the reaction with the ascites is obviously higher than that of the supernatant, which is probably because the nonspecific antibody combined with the short peptide in the ascites is more. The S/N ratios of the short peptide and the monoclonal antibody supernatant are both larger than 2, which shows that the short peptide can be specifically combined with the antibody in the supernatant (figure 4).

The results of indirect ELISA using synthetic short peptides to identify BTV positive sera of different serotypes are shown in FIG. 5, and show that synthetic peptide QYHAM positively reacts with BTV2 types, 3 types, 5 types, 7 types, 8 types, 11 types, 12 types, 13 types, 14 types, 15 types, 16 types, 17 types, 18 types, 19 types, 20 types, 21 types, 22 types, 23 types and 24 types. The reaction was weakly positive for BTV1 type, BTV4 type, BTV9 type, and BTV10 type. The synthetic peptide can have positive reaction with most BTV serotype positive serums, and the QYHAM sequence is a key epitope of the bluetongue virus group specific antigen.

2.5 results of conservative analysis

The short peptide sequence obtained by sequencing result of phage clone display technology is compared with amino acid sequence of 25 serotype bluetongue virus VP7 proteins in Genbank, and the result is shown in FIGS. 6 and 7, and the result shows that the mutation from arginine to tyrosine is found at the 239 th amino acid sequence and the mutation from asparagine to histidine is found at the 240 th amino acid sequence. Alanine to tyrosine at the 239 th amino acid sequence of BTV 7, BTV15, BTV 19.

The indirect ELISA identification is carried out by using the synthesized short peptide to identify different serotype BTV positive serums, and the results show that the synthesized peptide has positive reactions with BTV2 types, 3 types, 5 types, 7 types, 8 types, 11 types, 12 types, 13 types, 14 types, 15 types, 16 types, 17 types, 18 types, 19 types, 20 types, 21 types, 22 types, 23 types and 24 types. The reaction was weakly positive for BTV1 type, BTV4 type, BTV9 type, and BTV10 type. The synthetic peptide can have positive reaction with most BTV serotype positive serums, and the QYHAM sequence is a key epitope of the bluetongue virus group specific antigen.

3. Discussion of the related Art

To date, 27 serotypes of bluetongue have been found, and there is no cross-protection between the individual types. There is no effective treatment for bluetongue, so the disease is mainly prevented. In the research, the monoclonal antibody 3H7 strain preserved in a laboratory is used as an experimental basis, and the epitope of the monoclonal antibody is detected by using a phage display technology. One linear epitope QYHAM of the monoclonal antibody is detected by three times of panning. The synthesized short peptide reacts with 24 positive serums of the bluetongue virus by a competitive ELISA method, the result shows that the synthesized short peptide can positively react with most of the positive serums, and the analysis reason is that the synthesized short peptide can weakly positively react with a few serums due to the fact that the positive serums of the bluetongue virus are stored at the temperature of minus 80 ℃ for a long time and the serum titer is reduced. Through comparison with amino acid sequences of 25 bluetongue virus VP7 proteins in NCBI, analysis shows that the linear epitope QYHAM recognized by the 3H7 cell strain and the bluetongue virus VP7 protein have two amino acid mutations, namely that the 239 th amino acid is mutated from arginine to tyrosine, the 240 th amino acid is mutated from asparagine to histidine, and other amino acids are highly conserved in the 25 serotypes of the bluetongue virus. The invention identifies the specific epitope polypeptide of the VP7 protein group of the bluetongue virus, lays a foundation for establishing a competitive ELISA method aiming at the bluetongue virus, and the identified epitope can be applied to the design of vaccines in the future and has important significance for preventing and treating the bluetongue in China.

Sequence listing

<110> northeast university of agriculture

<120> bluetongue virus VP7 protein group specific antigen epitope polypeptide and application thereof

<130>KLPI161343

<160>1

<170>PatentIn 3.5

<210>1

<211>5

<212>prt

<213> epitope Polypeptides

<400>1

Gln Tyr His Ala Met

Claims

1. The bluetongue virus VP7 protein group specific antigen epitope polypeptide is characterized in that the amino acid sequence of the antigen epitope polypeptide is shown as SEQ ID NO. 1.

2. A polynucleotide encoding the epitope polypeptide of claim 1.

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

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

5. Use of the epitope polypeptide of claim 1 in the preparation of a reagent for detecting bluetongue virus.