CN107082802B - Neutralizing B cell epitope polypeptide of type 4 bluetongue virus VP2 protein and application thereof - Google Patents

Neutralizing B cell epitope polypeptide of type 4 bluetongue virus VP2 protein and application thereof Download PDF

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CN107082802B
CN107082802B CN201710116956.1A CN201710116956A CN107082802B CN 107082802 B CN107082802 B CN 107082802B CN 201710116956 A CN201710116956 A CN 201710116956A CN 107082802 B CN107082802 B CN 107082802B
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唐丽杰
李一经
徐义刚
王丽
乔薪瑗
姜艳平
崔文
李佳璇
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Northeast Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12111—Orbivirus, e.g. bluetongue virus
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    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12111—Orbivirus, e.g. bluetongue virus
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    • C—CHEMISTRY; METALLURGY
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    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12111—Orbivirus, e.g. bluetongue virus
    • C12N2720/12134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The invention discloses a neutralizing B cell epitope polypeptide of type 4 bluetongue virus VP2 protein and application thereof. The invention adopts the phage display technology to screen the BTV VP2 protein epitope amino acid. After 3 rounds of panning, sequencing is carried out, and the sequencing results are analyzed and compared to obtain a common short peptide sequence of 160NH 161. The experiment proves that the method has the advantages that, 160NH 161reacts specifically with both supernatant and ascites of 4A-1G7 160NH 161The reaction specificity with BTV positive standard serum of 1-24 types is good. The invention provides a new technical means for clinical diagnosis of the type 4BTV and lays a material foundation for the subsequent establishment of a BTV type specificity differential diagnosis method and the preparation of a type 4BTV related epitope vaccine.

Description

Neutralizing B cell epitope polypeptide of type 4 bluetongue virus VP2 protein and application thereof
Technical Field
The invention relates to an epitope polypeptide and application thereof, in particular to a neutralizing B cell epitope polypeptide of type 4 bluetongue virus VP2 protein and application thereof in diagnosis or treatment of bluetongue, and belongs to the technical field of medicines.
Background
Bluetongue (BT) is an insect-borne viral infectious disease common in tropical and subtropical countries that infects ruminants. BT is an infectious disease caused by Bluetongue Virus (BTV) and can infect ruminants such as sheep, cattle and camels. BTV infects animals, plants, insects and is transmitted by arthropods such as mosquitoes, culicodes, ticks, and the like. BTV belongs to the genus circovirus (Orbivirus) which is one of twelve genera of the Reoviridae (Reoviridae). BTV was first discovered in 1876 and is distributed worldwide. BTV has been isolated from several tropical, subtropical and temperate countries in africa, europe, asia, north america, south america and oceans and its distribution is expanding and the hazard is increasing. Ten serotypes BTV-1, BTV-2, BTV-3, BTV-4, BTV-5, BTV-7, BTV-12, BTV-15, BTV-16 and BTV-24 have been detected in China, wherein BTV-1 and BTV-16 are main pathogenic serotypes in China.
The VP2 protein is the main structural protein constituting the BTV envelope, and most of the neutralizing epitopes exist on the VP2 protein, so that the VP2 protein can induce the body to generate a neutralizing response to BTV. The VP2 protein is encoded by the L2 gene. Data show that the L2 gene is greatly different among serotypes, and the variation of the L2 gene is closely related to BTV serotypes, so that the VP2 protein is a type-specific protein and can be used for distinguishing the difference among the BTV serotypes, and therefore, a main determinant of the VP2 protein serotype.
BTV has a tendency to spread and new epidemics have continuously appeared in recent years. BTV is heterogeneous and it is subject to variation, and 27 serotypes have been isolated without cross-immunity between serotypes. Meanwhile, the 4-type BTV can infect ruminants such as sheep and camel, the morbidity is urgent, the mortality rate is high, and the damage to the breeding industry is huge. Therefore, it is important to establish a type-specific differential diagnosis method to prevent and control BTV.
Disclosure of Invention
The invention aims to solve the technical problem of providing a neutralizing B cell epitope polypeptide of type 4 bluetongue virus VP2 protein and application thereof in detecting bluetongue viruses.
In order to achieve the purpose, the invention adopts the following technical means:
the invention uses phage random peptide library to carry out 3 rounds of biological panning on the 4A-1G7 monoclonal antibody which is successfully prepared by self, and the result after sequencing is as follows: the 12 phage sequences contained NH consistently, which has a certain similarity to amino acids 160-161 of the BTV VP2 amino acid type 4. To further identify the sequence, the short peptide sequence was synthesized 160NH 161The short peptide is found by adopting an indirect ELISA method to react with BTV positive serum of 24 serotypes 160NH 161Can show obvious negative reaction with most positive serums and positive reaction with a few serums. And is 158AMNSY 162， 158AMNHNSY 162Reacting with BTV-4 standard positive serum respectively, containing 160NH 161The fragments are more reactive. The results show that the epitope has good specificity. In addition, the spatial conformation of the screened phage polypeptide is similar to the natural epitope by the phage competitive inhibition test and the ELISA method, thereby suggesting that the short peptide can be used for preparing the type 4BTV related epitope vaccine.
Therefore, on the basis of the research, the invention provides a neutralizing B cell epitope polypeptide of the type 4 bluetongue virus VP2 protein, and the epitope polypeptide contains an amino acid sequence shown in SEQ ID NO. 1.
In the invention, preferably, the amino acid sequence of the epitope polypeptide is shown as SEQ ID NO.1 or SEQ ID NO. 2.
The nucleotide sequence for coding the neutralizing B cell epitope polypeptide and an expression vector containing the nucleotide sequence are also within the protection scope of the invention.
Furthermore, the invention also provides application of the neutralizing B cell epitope polypeptide in preparation of a reagent for detecting or diagnosing bluetongue virus infection, and application of the neutralizing B cell epitope polypeptide in preparation of a medicine for preventing or treating bluetongue virus.
Among them, preferably, the bluetongue virus includes BTV-4, BTV-8, BTV-9, BTV-18, BTV-20 and BTV-9.
The invention provides a new technical means for clinical diagnosis of the type 4BTV and lays a material foundation for the subsequent establishment of a BTV type specificity differential diagnosis method and the preparation of a type 4BTV related epitope vaccine.
Drawings
FIG. 1 shows the 161. sup. st and 162. sup. th bit sequences of VP2 in BTV1-24 serotype;
FIG. 2 shows the indirect ELISA assay results of short peptides and MAb;
FIG. 3 shows the indirect ELISA identification results of short peptides and BTV positive sera of different serotypes;
FIG. 4 shows a short peptide 158AMNSY 162、 158AMNHNSY 162And indirectly ELISA identifying results with BTV-4 positive serum.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example identification of neutralizing B cell epitopes of the type 14 bluetongue Virus VP2 protein
1 materials and methods
1.1 cells with Primary reagents
The MAb 1G7 cell line of the anti-VP 2 protein is preserved in the laboratory. Goat anti-mouse IgG labeled with HRP was purchased from sequoia jessamine limited; BTV Standard Positive and negative sera types 1-24 were purchased from the bluetongue reference laboratory Pirbright institute; the Ph.D. -12TM13 phage peptide library kit was purchased from New England Biolabs (NEB) Inc., USA.
1.2 Gene sequences
The GenBank accession numbers for BTV types 1-24 used for the alignment are shown in table 1.
TABLE 1 GenBank accession numbers for BTV types 1-24
Figure BDA0001235834060000041
1.3 epitope screening
And (3) adding purified MAb ascites fluid serving as an antigen into a microplate according to the specification of the Ph.D. -12TM13 phage peptide library, performing 3 times of panning on the phage peptide library, and sequencing products obtained after the three times of panning.
1.4 location and Authenticity analysis of epitope recognized by monoclonal antibody
Comparing and analyzing 20 random 12 peptide sequences displayed on the phage through sequence determination, determining a consensus amino acid sequence, and determining the position of the consensus sequence on the amino acid sequence of the encoding serotype 4BTV VP2 protein, wherein the position region is the region of the epitope recognized by the monoclonal antibody. The obtained epitope sequence was synthesized into short peptides by Shanghai Brilliant Biotech Co., Ltd.
Meanwhile, in order to prove the authenticity of the epitope recognized by the monoclonal antibody, the short peptide is diluted by a coating diluent in a multiple ratio, an enzyme label plate is coated by 100 mu l/hole, the supernatant and the purified ascites diluent of 4A-1G7 are respectively used as the antibody, and the goat marked by the HRP is usedAnti-mouse IgG as a secondary antibody, the OPD-H prepared at present was added 2O 2And (3) adding a terminating solution after the substrate is developed for 15min, and identifying the reaction activity of the short peptide. And synthesized and removed by Shanghai Brilliant Biotech Ltd 160NH 1611Is/are as follows 158AMNSY 162With the original BTV-4 fragment 158AMNHNSY 162. Are respectively provided with 158AMNSY 162、 158AMNHNSY 162For coating antigen, the 4 type BTV standard positive serum and the negative serum are used as antibodies to carry out indirect ELISA verification so as to prove the authenticity of the epitope recognized by the monoclonal antibody.
1.5 conservative analysis and specificity analysis of epitope recognized by monoclonal antibody
Epitope sequence recognized by MAb4A-1G7 using Blast software in NCBI database 160NH 161Conservative analysis and specificity analysis are carried out. And performing indirect ELISA verification by using short peptide as a coating antigen and using 1-24 type BTV standard positive serum and negative serum as antibodies to prove the specificity of the epitope recognized by the monoclonal antibody.
2 results
2.1 DNA sequencing and epitope analysis of epitopes recognized by monoclonal antibodies
The epitope of the obtained 4A-1G7 monoclonal antibody is subjected to sequence alignment display by using a phage display technology, and is displayed by phage genome sequencing: 6 of the 20 phage clones panned by monoclonal antibody 4A-1G7 showed the NH sequence at amino acid positions 161-162 of the 4BTV VP2 protein used in this study (Table 2). The sequence is analyzed by sequence alignment, and the amino acids 161-162 of VP2 in the serotype of BTV1-24 are not conserved and have stronger specificity (figure 1). So presume that 160NH 161The sequence (shown in SEQ ID NO. 1) may be a linear B cell epitope.
TABLE 2 determination of the sequence of the short peptides
2.2 Indirect ELISA method for identifying reactivity of short peptide and 4A-1G7
Aiming at the identified amino acid region, based on the principle that hydrophobic amino acid mutates to hydrophilic amino acid and hydrophilic uncharged amino acid mutates to hydrophilic amino acid, and hydrophilic amino acid with positive and negative charges, the inventor artificially synthesizes a short peptide (shown as SEQ ID N O.1) only containing NH sequence, and utilizes indirect ELISA method to detect whether the short peptide sequence reacts with 4A-1G7 monoclonal antibody supernatant and ascites. The results are shown in FIG. 2, where the monoclonal antibody supernatant and the ascites both reacted specifically with the short peptide, and the short peptide could be specifically bound to the antibody in the monoclonal antibody supernatant and the ascites. And the value of the reaction with ascites was significantly higher than that of the supernatant, probably due to the higher amount of nonspecific antibodies bound to the short peptide in the ascites.
2.3 Indirect ELISA method for identifying specificity of short peptides
The indirect ELISA method detects the reaction of the synthesized short peptide and the 1-24 type BTV positive serum, the result is shown in figure 3, and the result shows that: it can be seen that the reaction of the short peptide with BTV-4, BTV-8, BTV-9, BTV-18 and BTV-20 positive serum is positive, and the reaction of the short peptide with BTV-9 positive serum is weak positive.
Detection of synthetic knockouts by indirect ELISA 160NH 161BTV-4 fragment of (A) 158AMNSY 162With the original BTV-4 fragment 158AMNHNSY 162(SEQ ID NO. 2) and the results are shown in FIG. 4. Equal amount of 158AMNSY 162， 158AMNHNSY 162The short peptide AMNHNSY is more reactive with BTV-4 standard positive serum.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> northeast university of agriculture
<120> type 4 bluetongue virus VP2 protein neutralizing B cell epitope polypeptide and application thereof
<130>KLPI170141
<160>2
<170>PatentIn 3.5
<210>1
<211>2
<212>PRT
<213> short peptide
<400>1
Asn His
<210>2
<211>7
<212>PRT
<213> short peptide
<400>2
Ala MET Asn His Asn Ser Tyr

Claims (4)

1.4 Bluetongue Virus (BTV) VP2 protein neutralizing B cell epitope polypeptide, which is characterized in that the amino acid sequence of the epitope polypeptide is shown as SEQ ID NO. 2.
2. A polynucleotide encoding the neutralizing B cell epitope polypeptide of claim 1.
3. An expression vector comprising the polynucleotide of claim 2.
4. Use of the neutralizing B cell epitope polypeptide of claim 1 in the preparation of a reagent for detecting or diagnosing type 4 bluetongue virus infection.
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Citations (1)

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CN106190986A (en) * 2016-07-07 2016-12-07 东北农业大学 The monoclonal antibody of resistant to bluetongue virus serum 4 type VP2 albumen, secretes hybridoma cell strain and the purposes of this monoclonal antibody

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106190986A (en) * 2016-07-07 2016-12-07 东北农业大学 The monoclonal antibody of resistant to bluetongue virus serum 4 type VP2 albumen, secretes hybridoma cell strain and the purposes of this monoclonal antibody

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Identification of a novel bluetongue virus 1-specific B-cell epitope using a monoclonal antibody against the VP2 protein;P Wei等;《Archives of Virology》;20130105;第158卷(第5期);第1099-1104页 *
应用噬菌体展示技术(12肽文库)进行蓝舌病1型病毒VP2蛋白型特异性表位分析;宋建领等;《中国畜牧医学会2014年学术年会论文集》;20141108;材料与方法及结果与讨论部分 *
蓝舌病病毒VP7和VP2蛋白的原核表达及其单克隆抗体的制备;刘琦;《中国优秀硕士学位论文全文数据库 农业科技辑》;20160415;摘要 *

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