Preparation of Zika virus non-structural protein NS2A polypeptide and its antibody

Abstract

Provided is the preparation of a Zika virus non-structural protein NS2A polypeptide and its antibody. A polypeptide is provided, which comprises the amino acid sequence shown in SEQ ID NO.1 or a variant thereof, and the variant has 90% sequence identity with the amino acid sequence shown in SEQ ID NO.1. The present invention for the first time provides a ZIKV NS2A polypeptide and a polypeptide antibody. After the polypeptide is coupled with KLH, a ZIKV NS2A antibody is prepared, and the antibody can be used in Western blot, immunofluorescence experiments and other antigen-antibody recognition experiments.

Description

Technical Field

[0001] The present invention relates to polyclonal antibodies, and particularly to the preparation of Zika virus non-structural protein NS2A polypeptides and antibodies thereof. Background Art

[0002] Zika virus (ZIKV) is an enveloped, single-stranded positive-sense RNA virus mainly transmitted by mosquitoes, and belongs to the genus Flavivirus of the family Flaviviridae, together with Dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), etc. As of July 2019, mosquito-borne infections of ZIKV have been reported in approximately 87 countries or territories around the world. Infection of pregnant women with Zika virus can lead to microcephaly or abnormal development of nerve cells in infants, and infection of adults can lead to neurological damage diseases such as Guillain-Barré syndrome, neuritis or meningitis. Existing studies have shown that ZIKV infection can directly or indirectly cause disorders in neurogenesis, development and maturation.

[0003] The ZIKV genome has an open reading frame, and the length of its coding region is approximately 10.8 kb, encoding three structural proteins in sequence: capsid protein (C), membrane protein and its precursor (M / prM), and envelope protein (E protein), and seven non-structural proteins, namely NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5. Among them, NS2A is involved in ZIKV replication, assembly and budding and helps the virus escape from host immunity, and also disrupts neurogenesis in the cerebral cortex by degrading connexins and cadherins, thereby leading to microcephaly. NS2A contains 226 amino acids, including seven transmembrane segments (pTMS), the N-terminal region (pTMS1-pTMS2) is close to the ER lumen, pTMS3 spans the ER membrane, and the C-terminal region (pTMS4-pTMS7) is close to the cytoplasm. The multiple transmembrane structure of NS2A has led to the absence of a commercial antibody against NS2A protein on the market, and thus relatively few scientific studies have been carried out on NS2A.

[0004] There is a need in the art for immunogenic fragments that can elicit anti-NS2A antibodies and anti-NS2A antibodies. Summary of the Invention

[0005] Currently, there is no commercially available ZIKV NS2A antibody on the market. The multiple transmembrane structure of the NS2A antibody makes it difficult to express its protein in vitro, while polypeptides can be obtained by synthesis. In the present invention, the antigen region of the NS2A protein is predicted by antigen software, and polypeptides are obtained through extensive screening work. Subsequently, the polypeptide is synthesized, conjugated with KLH, and used to immunize New Zealand white rabbits. After obtaining the immune serum, the NS2A antibody is finally obtained through antibody purification. It has been detected that this NS2A antibody can detect the ZIKV NS2A protein.

[0006] In one aspect, the present invention provides a polypeptide comprising the amino acid sequence shown in SEQ ID NO.1 or a variant thereof, which variant has 90% sequence identity with the amino acid sequence shown in SEQ ID NO.1.

[0007] In one aspect, the present invention provides a conjugate comprising a carrier protein and the polypeptide described herein. In one embodiment, the carrier protein is KLH protein.

[0008] In one aspect, the present invention provides a polyclonal antibody that is directed against the Zika virus NS2A protein. In one embodiment, the polyclonal antibody of the present invention can be obtained by immunizing an animal with the conjugate described herein and then purifying it.

[0009] In one aspect, the present invention provides nucleic acid. In one embodiment, the nucleic acid of the present invention may comprise a nucleotide sequence encoding the polypeptide described herein.

[0010] In one embodiment, the nucleic acid further comprises a nucleotide sequence encoding a purification tag, such as a His tag, GST tag, MBP tag, SUMO tag or NusA tag. In one embodiment, the nucleic acid further comprises a nucleotide sequence encoding a leader sequence.

[0011] In one aspect, the present invention provides a vector comprising the nucleic acid described herein. In one embodiment, the vector comprises expression control elements operably linked to the nucleic acid, such as a promoter, terminator and / or enhancer.

[0012] In one aspect, the present invention provides a host cell comprising the nucleic acid described herein or the vector described herein. In one embodiment, the host cell is a eukaryotic cell or a prokaryotic cell. In one embodiment, the eukaryotic cell is a yeast cell, an animal cell and / or an insect cell. In one embodiment, the prokaryotic cell is an Escherichia coli cell.

[0013] In one aspect, the present invention provides a composition. The composition of the present invention may comprise the polypeptides described herein. In one embodiment, the composition of the present invention may comprise the conjugates described herein. In one embodiment, the composition of the present invention may further comprise a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier is an adjuvant. In one embodiment, the composition further comprises another peptide derived from the Zika virus NS2A protein.

[0014] In one aspect, the present invention provides a method for preparing polyclonal antibodies, which comprises immunizing a mammal with the polypeptides described herein, the conjugates described herein, or the composition comprising the polypeptides or conjugates described herein, collecting blood from the mammal, and collecting polyclonal antibodies from the blood. In one embodiment, the mammal is a rabbit.

[0015] In one aspect, the present invention provides a method for preparing the polypeptides described herein, which comprises culturing the host cells described herein and collecting the polypeptides.

[0016] In one aspect, the present invention provides the use of the polyclonal antibodies described herein in the preparation of a kit for treating and / or diagnosing Zika virus infection in a subject or for detecting the NS2A protein of Zika virus.

[0017] In another aspect, the present invention provides a method for detecting Zika virus in vitro or ex vivo, which comprises the step of contacting the polyclonal antibodies described herein with an object suspected of containing Zika virus. The object may be a cell or a substance containing cells.

[0018] In another aspect, the present invention provides a method for detecting the NS2A protein, which comprises using the polyclonal antibodies described herein to detect the NS2A protein in cells infected with Zika virus by Western blot or immunofluorescence detection methods.

[0019] The advantages of the present invention include:

[0020] 1. The present invention for the first time provides a ZIKV NS2A polypeptide and a polypeptide antibody, the amino acid sequence of which is GSTDHMDHFSLGVLC. After the polypeptide is conjugated with KLH, a ZIKV NS2A antibody is prepared.

[0021] 2. The present invention provides a polypeptide sequence that can be used to prepare a ZIKV NS2A antibody, including its conjugation with the enhanced antigen protein KLH, including its use of the antibody in WB and IF experiments, and other similar antigen-antibody recognition experiments.

[0022] 3. Compared with other peptides derived from the natural NS2A protein, the polypeptide of the present invention can detect the NS2A protein in cells infected with Zika virus by Western blot and immunofluorescence detection methods.

[0023] 4. The polypeptide of the present invention is only 14 amino acids in length, is easy to synthesize, and can generate high-titer anti-NS2A antibodies in rabbits.

[0024] 5. Compared with the currently available commercial antibodies for detecting Zika virus infection, the polyclonal antibodies of the present invention can better detect Zika virus infection (at earlier infection time points and with higher sensitivity). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Western blot detection of purified antibody polypeptide 4 antibody. Detection results of different antibodies over time.

[0026] Figure 2 : Western blot detection of purified antibody polypeptide 1 antibody. The left figure is polypeptide 1 antibody used at a concentration of 0.65 μg / ml; the right figure is polypeptide 1 antibody used at a concentration of 1 μg / ml.

[0027] Figure 3 : ELISA detection of purified antibody polypeptide 4 antibody.

[0028] Figure 4 : Immunofluorescence (IF) detection of purified antibody polypeptide 4 antibody. The first two rows are the results of polypeptide 4 antibody detection; the middle two rows are the results of E antibody detection; the last two rows are the results of NS1 antibody detection. The E antibody is a murine monoclonal antibody prepared from 4G2 hybridoma cells (produced by the hybridoma 4G2 presented by Professor Jiang Lifang of Sun Yat-sen University).

[0029] Figure 5 : For Figure 4 the experimental results were statistically analyzed. Three fields of view were randomly selected from the Figure 4 photos for statistical analysis. The statistical software used was SPSS 13.0, and the statistical method was a two-tailed T-test, *, P < 0.5; ***, P < 0.001.

[0030] Figure 6 : Polypeptide 4 antibody specifically recognizes NS2A of Zika virus. A) Immunofluorescence (IF) detection of polypeptide 4 antibody against different viruses. B) WB detection of polypeptide 4 antibody against different viruses. C) Polypeptide sequence alignment of different virus strains. DETAILED DESCRIPTION OF THE INVENTION

[0031] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0032] As used herein, "polypeptide" refers to a ZIKV NS2A-derived polypeptide having immunogenicity or antigenicity. The length of the polypeptide can be 10-15 amino acid residues, such as 11, 12, 13, and 14 amino acid residues. In this article, the polypeptide comprises the amino acid sequence shown in SEQ ID NO.1 (GSTDHMDHFSLGVLC) or a variant thereof, which has 90% sequence identity with the amino acid sequence shown in SEQ ID NO.1, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0033] As used herein, "carrier protein" refers to any immunologically acceptable protein for forming a complete antigen. Carrier proteins include, but are not limited to, bovine serum albumin, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin (HSA), and synthetic polylysine (PLL), etc. In this article, the carrier protein can be keyhole limpet hemocyanin (KLH). The carrier protein can be crosslinked to haptens and other antigens, such as polypeptides, to enhance their immunogenicity.

[0034] As used herein, "conjugate" refers to a substance formed by conjugating a polypeptide with a carrier protein, which can produce an antibody immune response in the body. The method of producing the conjugate is well known in the art. For example, chemical reagents such as iodoacetamide, maleimide, or alkyl halide can be used to conjugate the carrier protein with the antigen peptide. This can be carried out through the amino group on the carrier protein and the sulfhydryl group on the antigen peptide.

[0035] As used herein, "adjuvant" refers to a substance that can enhance an immune response or alter the type of immune response, and can be injected into an organism simultaneously with or prior to an antigen during application. An adjuvant can itself be immunogenic or non-immunogenic. Adjuvants can include inorganic adjuvants (such as aluminum hydroxide, alum, etc.); biological adjuvants (such as Mycobacterium tuberculosis, Bacillus Calmette-Guérin, Corynebacterium parvum, Bordetella pertussis, Gram-negative bacterial endotoxin, cholera toxin B subunit, muramyl dipeptide, and cytokines, etc.); synthetic adjuvants (such as double-stranded polyinosinic acid: cytidylic acid (Poly I:C), double-stranded polyadenylic acid: uridylic acid); Freund's complete adjuvant, peanut oil emulsion; nano adjuvants, etc.

[0036] As used herein, "variant" refers to a polypeptide having one or more amino acid residue mutations (such as additions, deletions, substitutions, insertions) compared to a reference polypeptide. For example, compared to the amino acid sequence shown in SEQ ID NO.1, the variant can have one or more amino acid residue mutations (such as additions, deletions, substitutions, insertions). Preferably, the mutations are conservative amino acid substitutions.

[0037] As used herein, "nucleic acid" refers to multiple nucleotides linked by internucleotide linkages. The internucleotide linkages can be, for example, phosphodiester bonds. The nucleic acids herein can include polynucleotides encoding the polypeptides of the present invention.

[0038] As used herein, "polyclonal antibody" refers to a mixture of immunoglobulins produced against a specific antigen, and each immunoglobulin can recognize an epitope on the antigen molecule. In this document, polyclonal antibodies are produced by immunizing rabbits with the conjugates described herein. The polyclonal antibodies herein can be used to detect ZIKV NS2A or to detect Zika virus in vitro or ex vivo.

[0039] For the convenience of subsequent polypeptide processing, the nucleic acids of the present invention can also include nucleotides encoding purification tags, such as His tag, GST tag, MBP tag, SUMO tag, or NusA tag, and nucleotides encoding a leader sequence when needed.

[0040] As used herein, the term "vector" is a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, enabling the genetic material elements it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses, etc. A vector can contain various elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector can also contain an origin of replication. A vector can contain the nucleic acid of the present invention to facilitate its introduction into a cell for expression. A vector can contain expression control elements operably linked to the nucleic acid, such as promoters, terminators, and / or enhancers.

[0041] As used herein, the term "host cell" is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfecting with viral vectors, transforming with plasmid vectors, and introducing naked DNA by electroporation, lipofection, and particle gun acceleration. A host cell can be a eukaryotic cell or a prokaryotic cell. For example, eukaryotic cells are yeast cells, animal cells, and / or insect cells. Prokaryotic cells can be Escherichia coli cells.

[0042] Polypeptides, carrier proteins, and conjugates

[0043] The present invention provides polypeptides, carrier proteins, and conjugates. The polypeptide can be the amino acid sequence shown in SEQ ID NO.1 (GSTDHMDHFSLGVLC) or a variant thereof. The polypeptide can be used as an immunogenic polypeptide or an antigenic polypeptide to immunize an animal to produce polyclonal antibodies. Herein, a carrier protein can be conjugated to the polypeptide to obtain a conjugate for immunizing an animal to increase the production of polyclonal antibodies. The conjugation method is known in the art. For example, chemical reagents such as iodoacetamide, maleimide, or alkyl halide can be used to conjugate the carrier protein to the antigenic peptide. This can be carried out through the amino group on the carrier protein and the sulfhydryl group on the antigenic peptide. The method of immunizing an animal with the polypeptide or conjugate to obtain polyclonal antibodies is known in the art. The animal can be a mammal, such as a rabbit, a mouse, a guinea pig, etc.

[0044] Kits

[0045] The various materials of the present invention, including polypeptides, carrier proteins, conjugates, antibodies (such as polyclonal antibodies), adjuvants, etc., can be prepared into kits. The kits also contain any one or more of the various materials (such as polypeptides, carrier proteins, conjugates, antibodies) described herein. The kits can be used for various purposes, such as for treating and / or diagnosing Zika virus infection in a subject, for detecting Zika virus in vitro or ex vivo, or for detecting the NS2A protein of Zika virus.

[0046] Application

[0047] The various materials of the present invention, including polypeptides, carrier proteins, conjugates, antibodies (such as polyclonal antibodies), can be applied in multiple aspects. For example, the polyclonal antibodies of the present invention can be used for treating and / or diagnosing Zika virus infection in a subject or for methods of detecting the NS2A protein of Zika virus. The method can include the step of detecting the NS2A protein using the polyclonal antibody. For example, the presence of the NS2A protein in cells infected with Zika virus can be detected by Western blot or immunofluorescence detection methods. The polyclonal antibodies of the present invention can also be used for methods of detecting Zika virus in vitro or ex vivo, which include the step of contacting the polyclonal antibody with an object suspected of containing Zika virus, such as a cell.

[0048] Examples

[0049] The following examples are provided to illustrate the present invention. Those skilled in the art should understand that the examples are merely illustrative and not restrictive. The present invention is defined only by the scope of the appended claims.

[0050] Example 1: Screening of antigenic regions

[0051] To screen for the antigenic regions of NS2A, first, the GenScript OptimumAntigen TM design software was used to predict and analyze the antigenicity, hydrophilicity / hydrophobicity, transmembrane regions, homology, helical regions, signal peptides, etc. of NS2A. The results of the prediction and analysis are shown in Table 1. Table 1 ranked the extracellular polypeptides 1 - 8 according to the level of the predicted values of polypeptide antigenicity, and polypeptide 1 had the highest antigenicity.

[0052] Table 1: Results of screening for NS2A antigen polypeptides

[0053]

[0054] Example 2: Preparation of polypeptide antibodies

[0055] The inventors selected polypeptides 1 - 8 and commissioned GenScript Biotech Corporation to synthesize the polypeptides and prepare antibodies. Specifically, GenScript Biotech Corporation was commissioned to commercially synthesize polypeptides 1 - 8 and conjugate them with KLH protein. The polypeptides conjugated with KLH were mixed with GenScript Biotech Corporation's proprietary adjuvant and used to immunize New Zealand white rabbits at multiple points on the back for multiple times. After the final immunization, the antiserum of the rabbits was collected, approximately 40 ml per rabbit. The antibodies were purified by antigen affinity, and the OD was detected 280 The antibody concentration was detected and antibodies prepared from each of polypeptides 1 - 8 were produced (the above steps were commissioned to GenScript for completion, using the internal immunization process of GenScript Biotech Corporation).

[0056] Example 3: WB Detection of Polypeptide Antibodies

[0057] In this example, the pure products of the antibodies prepared from polypeptides 1 and 4 respectively (polypeptide 1 antibody and polypeptide 4 antibody) were provided by GenScript Biotech Corporation. The inventors performed Western Blot (WB) immunoblotting using the polypeptide 1 antibody and the polypeptide 4 antibody respectively. It was found that the polypeptide 4 antibody had a better detection effect in Western Blot (WB) immunoblotting.

[0058] 1. Western Blot (WB) immunoblotting is a method in which proteins are denatured and transferred to a membrane, and then detected using antibodies. It is commonly used to detect the expression level of target proteins; in this example, this method was used to detect the recognition ability of the rabbit antibody prepared from polypeptide 4 for the linear epitope of the target protein NS2A. The specific principle is as follows: First, protein samples are collected. After separation by SDS-PAGE gel, the proteins are transferred to a solid-phase carrier, nitrocellulose membrane. The solid-phase carrier adsorbs proteins in a non-covalent bond form. Since the proteins were denatured before loading, the protein epitopes exposed in the sample at this time are mainly linear epitopes. Using the proteins on the solid-phase carrier as antigens, they react with the corresponding antibodies, and then react with the horseradish peroxidase-labeled secondary antibody. After substrate color development, the specific target proteins separated by electrophoresis are detected.

[0059] HMC3 cells in good growth state were seeded in 6-well plates, 5X10 5 / Wells, and used after culturing at 37°C and 5% CO2 for 24 h. Inoculate the ZIKV / SZ01 virus strain (GenBank accession number: KU866423) (0.1 MOI) into HMC3 cells, and collect cell lysates for WB after culturing at 37°C and 5% CO2 for 0, 12, 24, 36, 48, and 60 h. Add the samples to an SDS-PAG gel with a 5% stacking gel (PG113, YaEn Biotech) and a 12% separating gel (PG113, YaEn Biotech), and run at 80 V and 120 V for 20 and 80 min, respectively. Subsequently, transfer the protein samples to a PVDF membrane at a constant current of 300 mA for 60 min. After blocking with 5% non-fat milk powder (prepared with TBS solution) for 60 min, remove the blocking solution, cut the membrane at the expected position of the band to be observed, and then add the NS2A antibody polypeptide 4 antibody, NS1 antibody (GTX634158, GeneTex), or E antibody (B1845, Beijing Bioclone) (1 μg / ml) and anti-GAPDH antibody. The antibodies are diluted with TBS and incubated overnight at 4°C. Wash three times with TBST, 10 min each time. Add HRP-labeled anti-rabbit secondary antibody (SA00001-2, Proteintech) or anti-mouse secondary antibody (SA00001-1, Proteintech), incubate at room temperature for 1 h, and wash with TBST 10 min × 3 times. Use a chromogenic solution (WBKLS0500, Millipore) and a fully automatic chemiluminescence imaging analysis system (5200, Tanon) for color development.

[0060] Figure 1 The results of this experiment are shown in

[0061] As can be seen from Figure 1 It can be seen that the antibody prepared from polypeptide 4 specifically recognizes a band between 15 - 25 kDa after ZIKV infects cells (consistent with the expected NS2A protein), and its expression increases with the prolongation of ZIKV infection time. Since this expression trend is consistent with the expression trend of its NS1 protein after ZIKV infection, and the expression of the ZIKV structural protein E tends to be stable at 24 h, it is considered that the antibody can specifically recognize the ZIKV NS2A linear epitope. In this detection project, the NS1 antibody (GTX634158) purchased from GeneTex and the E antibody (B1845) purchased from Beijing Bioclone were used as antibody controls for detecting protein samples after virus-infected cells.

[0062] 2. Seed well-grown HMC3 cells in a 6-well plate, 5X10 5 / Wells were used after culturing at 37°C and 5% CO2 for 24 h. The ZIKV / SZ01 virus strain (0.1 MOI) was inoculated into HMC3 cells. Cell lysates were collected after culturing at 37°C and 5% CO2 for 12, 24, and 48 h for WB. The non-infected group (Mock, without adding virus) was used as a control. Samples were added to the SDS-PAG gel with 5% stacking gel and 12% separating gel (PG113, Yaenzyme Biotechnology) and run at 80 V and 120 V for 20 and 80 min respectively. Subsequently, protein samples were transferred to PVDF membranes at a constant current of 300 mA for 60 min. After blocking with 5% non-fat milk powder for 60 min, the blocking solution was removed, and NS2A antibody polypeptide 1 antibody (0.65 or 1 μg / ml) was added and incubated overnight at 4°C. It was washed 3 times with TBST, 10 min each time. HRP-labeled anti-rabbit secondary antibody was added and incubated at room temperature for 1 h, and then washed with TBST for 10 min × 3 times. A chromogenic solution (WBKLS0500, Millipore) and a chemiluminescence imaging analysis system (5200, Tanon) were used for color development.

[0063] Figure 2 The results of this experiment are shown in

[0064] From Figure 2 It can be seen that although bands were seen on the PVDF membrane for the antibody prepared with polypeptide 1, corresponding bands were present at the control group positions, with many non-specific bands and poor specificity, and non-specific bands were detected at different antibody concentrations.

[0065] The experiment in Example 3 demonstrated that the antibody induced by polypeptide 1 was verified to be unable to effectively recognize NS2A in WB. Compared with the antibody prepared with polypeptide 1 (polypeptide 1 antibody), the antibody prepared with polypeptide 4 (polypeptide 4 antibody) could specifically detect the NS2A protein.

[0066] Example 4: ELISA Detection of Polypeptide Antibody (Polypeptide 4 Antibody)

[0067] Enzyme-linked immunosorbent assay (ELISA) is an enzyme-labeled solid-phase immunoassay technique used to detect the ability of an antibody to recognize an antigen. The detection principle of this example is: First, the antigen (polypeptide 4) is bound to a solid-phase carrier, namely the ELISA plate; subsequently, the antibody prepared from rabbits is added to the plate to recognize the antigen in the plate, and then the unreacted part is removed by washing; subsequently, an anti-rabbit-horseradish peroxidase-labeled secondary antibody is used to recognize the remaining antibody in the plate, and the unreacted part is removed again by washing; a substrate is added, and the substrate catalyzed by the enzyme bound to the solid-phase carrier produces a colored substance. By qualitatively or quantitatively detecting the amount of the colored product, the content of the antibody in the sample can be determined. The higher the measured value, the more antibodies bound to the antigen, that is, the more antibodies that can recognize polypeptide 4.

[0068] Coat a 96-well ELISA plate with 4 μg / ml of the polypeptide using PBS coating buffer, 100 μL per well, and incubate overnight at 4°C. The next day, wash 3 times with PBST, add 100 μl of 3% BSA to each well, and block at 37°C for 1 h. After washing 3 times with PBST, dilute the 1 mg / ml polypeptide 4 antibody and pre-immune rabbit serum from 1:1000 by two-fold serial dilution to 1:512000. Incubate at 37°C for 1 h. After washing 5 times with PBST, add HRP-conjugated anti-rabbit secondary antibody and incubate at 37°C for 1 h. After washing 5 times with PBST, add the chromogenic agent and develop for 5 - 20 min, then terminate with the stop solution, and read the OD 450 absorbance value. Figure 3 The results of the detection are shown in

[0069] It can be seen from Figure 3 that the ELISA titer of the polypeptide 4 antibody recognizing polypeptide 4 is higher than 1:521000, indicating that the antibody can recognize polypeptide 4 well.

[0070] Example 5: Immunofluorescence (IF) assay of polypeptide 4 antibody

[0071] The immunofluorescence assay is a method for detecting the in-situ expression of antigens based on the antigen-antibody reaction. The main principle in this example is as follows: First, fix the cells with a fixative to protect the cell morphology and the antigenicity of proteins. Since the samples are fixed in the experiment, the epitopes exposed by the proteins are mainly conformational epitopes at this time. Then, use a perforating agent to make holes so that the antibody can enter the cells to recognize the antigen. Subsequently, use a fluorescently labeled secondary antibody to recognize the primary antibody, thereby achieving the in-situ recognition of the antigen. This example is used to detect the recognition of the conformational epitope of the target protein NS2A by the rabbit antibody prepared from polypeptide 4.

[0072] Seed well-grown Vero cells in a 24-well plate containing Vero cell coverslips, 5X10 4 / Wells, and used after culturing at 37°C and 5% CO2 for 24 h. Inoculate 0.1 MOI of ZIKV / SZ01 into Vero cells, and use after culturing at 37°C and 5% CO2 for 48 h. Discard the supernatant, and fix the cells with 250 μL of 4% paraformaldehyde for 10 min. Subsequently, treat the cells with 250 μL of 0.2% TritonX-100 for 10 min, and wash the cells with PBS for 2 min × 3 times. After blocking non-specific antigens with 3% BSA for 30 min, remove the blocking solution, and add the primary antibodies NS2A antibody polypeptide 4 antibody, ZIKV E structural epitope 4G2 antibody diluted with 1% BSA or NS1 antibody GTX634158 (5 μg / ml) purchased from GeneTex Inc. and incubate at room temperature for 1 h. The uninfected virus control group was treated in the same way. Subsequently, wash with PBS for 3 min × 3 times, discard the supernatant, add the secondary antibody Alexa Fluor 488 Donkey Anti-rabbit IgG (thermo fisher Scientific) (diluted 1:1000) diluted with 1% BSA, incubate at room temperature for 1 h, and wash with PBS for 3 min × 5 times. Mount the slides with an anti-quenching mounting medium containing DAPI. After the slides are dried, observe the experimental results under a laser confocal microscope. To detect whether the antibody can recognize the structural epitope of ZIKV NS2A, Vero cells infected with ZIKV were detected, and the results are as Figure 4 , Figure 5 shown. Specific expression of NS2A was observed in ZIKV-infected cells, and it was mainly expressed in the cytoplasm. To evaluate the recognition efficiency of the NS2A antibody, the inventors used the widely used ZIKV E structural epitope 4G2 antibody and NS1 antibody GTX634158 purchased from GeneTex Inc. as controls. As Figure 4 , 5 seen, the infection rates of ZIKV on Vero cells detected by the ZIKV NS2A antibody and the ZIKV E protein antibody were 34.29% and 47.29% respectively, while the infection rate detected by the NS1 antibody was only 7.09%. The recognition efficiency of the NS2A antibody was significantly higher than that of the NS1 antibody for NS1. It shows that the antibody prepared from polypeptide 4 can effectively recognize the expression of NS2A protein after ZIKV infection.

[0073] Example 6: Specific recognition of Zika virus NS2A by polypeptide 4 antibody

[0074] To detect whether the polypeptide 4 antibody specifically recognizes ZIKV NS2A, spread well-grown BHK21 cells on a 24-well plate with cell culture slides, 5X10 4 / Wells, and used after culturing at 37°C and 5% CO2 for 24 h. Inoculate 0.1 MOI of ZIKV / SZ01, ZIKV / FLR, ZIKV / MR766, DENV-2 or YFV-17D virus strains into BHK21 cells, and use after culturing at 37°C and 5% CO2 for 30 h. Discard the supernatant, and fix the cells with 250 μL of 4% paraformaldehyde for 10 min. Subsequently, treat the cells with 250 μL of 0.2% TritonX-100 for 10 min, and wash the cells with PBS for 2 min × 3 times. After blocking non-specific antigens with 3% BSA for 30 min, remove the blocking solution, and add the primary antibody NS2A antibody polypeptide 4 antibody (5 μg / ml) diluted with 1% BSA and incubate at room temperature for 1 h. Wash with PBS for 3 min × 3 times, discard the supernatant, add the secondary antibody Alexa Fluor488 Donkey Anti-rabbit IgG (thermofisher Scientific) (diluted 1:1000) diluted with 1% BSA, incubate at room temperature for 1 h, and wash with PBS for 3 min × 5 times. Mount the slides with an anti-quenching mounting medium containing DAPI. After the slides are dried, observe the experimental results under a laser confocal microscope. As can be seen from Figure 6 Figure A of Figure 6 , the polypeptide 4 antibody can effectively recognize cells infected with multiple ZIKV strains, including ZIKV / SZ01, ZIKV / FLR, ZIKV / MR766, but does not recognize cells infected with DENV-2 and YFV-17D, indicating that the polypeptide 4 antibody specifically recognizes NS2A of Zika virus.

[0075] To further detect whether the polypeptide 4 antibody specifically recognizes ZIKV NS2A, Vero cells in good growth state were seeded in 6-well plates, 5X10 5 / Wells were used after culturing at 37°C and 5% CO₂ for 24 h. The Vero cells were inoculated with 0.1 MOI of ZIKV / SZ01, ZIKV / FLR, ZIKV / MR766, DENV-2 or YFV-17D virus strains (see Yu, Y., Deng Y.Q., Zou P., et al., A peptide-based viral inactivator inhibits Zika virus infection in pregnant mice and fetuses. Nat Commun, 2017.8:15672) (0.1 MOI), and the cell lysates were collected after culturing at 37°C and 5% CO₂ for 36 h for WB. The samples were added to the SDS-PAG gel with 5% stacking gel (PG113, Yaenzyme Biotechnology) and 12% separating gel (PG113, Yaenzyme Biotechnology), and run at 80 V and 120 V for 20 and 80 min respectively. Subsequently, the protein samples were transferred to the PVDF membrane at a constant current of 300 mA for 60 min. After blocking with 5% skim milk powder (prepared with TBS solution) for 60 min, the blocking solution was removed, the membrane was cut according to the expected position of the target band, and then the NS2A antibody polypeptide 4 antibody (1 μg / ml) or anti-GAPDH antibody was added respectively. The antibodies were diluted with TBS and incubated overnight at 4°C. Washed 3 times with TBST, 10 min each time. Add HRP-labeled anti-rabbit secondary antibody (SA00001-2, Proteintech) or anti-mouse secondary antibody (SA00001-1, Proteintech), incubated at room temperature for 1 h, and washed with TBST 10 min x 3 times. Developed with chromogenic solution (WBKLS0500, Millipore) and fully automatic chemiluminescence imaging analysis system (5200, Tanon). As shown by Figure 6 Figure B of Figure 6 , specific bands were detected in the cell samples infected with ZIKV / SZ01, ZIKV / FLR, and ZIKV / MR766 by polypeptide 4 antibody. The sizes of the bands were all between 15 - 25 Kda, while no specific bands were seen in the cells infected with DENV-2 and YFV-17D ( Figure 6 Figure B of Figure 6 ). Further indicated that polypeptide 4 antibody specifically recognizes NS2A of Zika virus.

[0076] Subsequently, the inventors used MAGE5.1 software to align the NS2A proteins of ZIKV / SZ01, ZIKV / FLR, ZIKV / MR766, DENV-2 and YFV-17D viruses with polypeptide 4, and found that the corresponding amino acid sequences of NS2A of different strains were 100% identical to the amino acid sequence of polypeptide 4; while the homologies of DENV-2 and YFV-17D were 57.1% and 14.3% respectively ( Figure 6(Figure C). Differences in amino acid sequence homology may lead to differences in the specific recognition of polypeptide 4 for different flavivirus NS2A proteins.

[0077] The inventors found that peptides with relatively high antigenicity predicted by antigen analysis software may not induce specific antibodies when immunizing animals. Although the software takes into account possible secondary structures, it mainly analyzes the amino acid sequence of proteins and may lose some information. In the absence of crystal structures, the analysis software cannot replace considering the overall conformation of proteins. Therefore, antigen prediction, animal immunization, and antibody specificity detection are indispensable for preparing antibodies with polypeptides.

Claims

1. A polypeptide consisting of the amino acid sequence shown in SEQ ID NO.

1.

2. A conjugate comprising a carrier protein and the polypeptide according to claim 1, wherein the carrier protein is KLH protein.

3. A polyclonal antibody obtained by purifying after immunizing an animal with the conjugate according to claim 2.

4. A nucleic acid comprising a nucleotide sequence encoding the polypeptide according to claim 1.

5. The nucleic acid according to claim 4, further comprising a nucleotide sequence encoding a purification tag.

6. The nucleic acid according to claim 5, wherein the purification tag is His tag, GST tag, MBP tag, SUMO tag or NusA tag.

7. The nucleic acid according to any one of claims 4 - 6, further comprising a nucleotide sequence encoding a leader sequence.

8. A vector comprising the nucleic acid according to any one of claims 4 - 7.

9. The vector according to claim 8, wherein the vector comprises an expression control element operably linked to the nucleic acid.

10. The vector according to claim 9, wherein the expression control element includes a promoter, a terminator and / or an enhancer.

11. A host cell comprising the nucleic acid according to any one of claims 4 - 7 or the vector according to any one of claims 8 - 10; wherein the host cell is a eukaryotic cell or a prokaryotic cell; the eukaryotic cell is a yeast cell, an animal cell or an insect cell.

12. The host cell according to claim 11, wherein the prokaryotic cell is an Escherichia coli cell.

13. A composition comprising the polypeptide according to claim 1 and / or the conjugate according to claim 2 and a pharmaceutically acceptable carrier.

14. The composition according to claim 13, further comprising an adjuvant.

15. A method for preparing a polyclonal antibody, comprising immunizing a mammal with the polypeptide according to claim 1, the conjugate according to claim 2, or the composition according to claim 13 or 14, collecting blood from the mammal, and collecting the polyclonal antibody from the blood.

16. The method according to claim 15, wherein the mammal is a rabbit.

17. A method for preparing the polypeptide according to claim 1, comprising culturing the host cell according to claim 11 or 12, and collecting the polypeptide.

18. Use of the polyclonal antibody according to claim 3 in the preparation of a medicament for treating Zika virus infection in a subject or a reagent for diagnosing Zika virus infection in a subject.

19. Use of the polyclonal antibody according to claim 3 in the preparation of a kit for detecting the NS2A protein of Zika virus.

20. A method for detecting Zika virus in vitro or ex vivo, comprising the step of contacting the polyclonal antibody according to claim 3 with an object suspected of containing Zika virus; the method is for non - diagnostic purposes.

21. The method according to claim 20, wherein the object is a cell.

22. A method for non-therapeutic or non-diagnostic purposes of detecting the NS2A protein of Zika virus, which comprises using the polyclonal antibody of claim 3 to detect the NS2A protein in cells infected with Zika virus by Western blot or immunofluorescence detection method.

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