Preparation method and application of duck hepatitis a virus type 3 VP0 polypeptide and polyclonal antibody thereof

Abstract

The application discloses a duck hepatitis A virus type 3 VP0 polypeptide and a preparation method and application of a polyclonal antibody thereof, and belongs to the technical field of biochemistry and molecular immunology. The duck hepatitis A virus type 3 VP0 polypeptide has an amino acid sequence as shown in SEQ ID NO. 2. A polyclonal antibody is prepared by immunizing Kunming mice with the polypeptide. The polyclonal antibody of the duck hepatitis A virus type 3 VP0 polypeptide is obtained by immunizing an antiserum with a VP0 polypeptide coupled with KLH as an antigen. The polyclonal antibody preparation method is simple, low in cost, high in antibody titer, and capable of specifically recognizing the duck hepatitis A virus type 3 VP0 protein in eukaryotic cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biochemistry and molecular immunology, and particularly relates to a duck hepatitis A virus type 3 VP0 polypeptide and a preparation method and application of a polyclonal antibody thereof. BACKGROUND

[0002] Duck viral hepatitis (DVH) is an acute and highly contagious disease caused by duck hepatitis virus (DHV) infection in ducklings. The disease mainly affects ducklings under 4 weeks of age, and is characterized by local epidemic, rapid transmission, short course and high mortality. It is one of the main diseases that endanger the duck industry. The dead ducklings have their heads tilted backward, showing "angular bowstring" shape, and the liver is enlarged and has a large number of hemorrhagic spots. DHV has been divided into three serotypes, namely type I, type II and type III. DHV-I has been renamed as duck hepatitis A virus (DHAV). DHAV is divided into three serotypes, namely type 1, type 2 and type 3, and DHAV-3 is widely prevalent in China, causing great harm to the duck industry.

[0003] As a small RNA virus, the life cycle of DHAV-3 is first adsorbed to the host cell through the receptor, and its RNA genome is delivered into the cytoplasm, and the virus is expressed and assembled using the cell's own metabolism to form new virus particles. In this process, the structural protein plays a huge function, such as binding to cell surface receptors, maintaining the stability of the virus form. In addition, the structural protein plays an important role in stimulating the body to produce humoral immunity and cellular immunity.

[0004] Ducks are an important waterfowl in China, and the study of DHAV can effectively alleviate the loss of the duck industry due to the epidemic. VP0 is an important structural protein on the surface of DHAV-3, which has good immunogenicity and host protection sites. The study of VP0 can fill the gap in the mechanism of DHAV-3 infection of cells. Because the preparation of DHAV-3 VP0 monoclonal antibody is complex, high cost and time-consuming, it seriously restricts the detection of DHAV-3, and there is a lack of detection of DHAV-3 VP0 antibody on the market. Therefore, the preparation of polyclonal antibody against DHAV-3 VP0 protein is of great significance, which can be used for detecting the infection of DHAV-3, and can also meet the market demand for this antibody. SUMMARY

[0005] To solve the above technical problems, the application provides a preparation method and application of a duck hepatitis A virus type 3 VP0 polypeptide and a polyclonal antibody of the duck hepatitis A virus type 3 VP0 polypeptide.

[0006] To achieve the above object, the application provides a duck hepatitis A virus type 3 VP0 polypeptide, wherein the amino acid sequence of the duck hepatitis A virus type 3 VP0 polypeptide is shown as SEQ ID NO. 2.

[0007] The application further provides a polyclonal antibody of the duck hepatitis A virus type 3 VP0 polypeptide, wherein the polyclonal antibody of the duck hepatitis A virus type 3 VP0 polypeptide is obtained by immunization of the duck hepatitis A virus type 3 VP0 polypeptide as an antigen, and the amino acid sequence of the duck hepatitis A virus type 3 VP0 polypeptide is shown as SEQ ID NO. 2.

[0008] Preferably, the duck hepatitis A virus type 3 VP0 polypeptide is coupled with the hemocyanin by a coupling agent MBS.

[0009] The application further provides a preparation method of the polyclonal antibody of the duck hepatitis A virus type 3 VP0 polypeptide, comprising the following steps.

[0010] The duck hepatitis A virus type 3 VP0 polypeptide is coupled with the hemocyanin (KLH) by the coupling agent MBS to form a complete antigen; the complete antigen is mixed with a Bio-o-Lon rapid immunization water adjuvant, and Kunming mice are immunized; serum containing the duck hepatitis A virus type 3 VP0 polypeptide antibody is collected, and the duck hepatitis A virus type 3 VP0 polyclonal antibody is obtained.

[0011] Preferably, the complete antigen is used to immunize the Kunming mice, and the method comprises the following steps.

[0012] The antigen is diluted and mixed with an equal amount of the Bio-o-Lon rapid immunization water adjuvant, and then the leg muscle injection is performed, the Kunming mice are immunized for the first time, the booster immunization is performed after three weeks, and the booster immunization is performed once.

[0013] Preferably, the collection of the serum containing the mouse anti-duck hepatitis A virus type 3 VP0 polypeptide antibody comprises the following steps.

[0014] The capillary eye orbit blood collection method is used for the Kunming mice, the blood is collected, and then the blood is placed at an inclination at 37 DEG C for 1 h, and then the blood is placed at 4 DEG C for 12 h, and the antiserum is separated and obtained.

[0015] Preferably, after the antiserum is obtained, the titer of the antiserum is detected by using the indirect ELISA method.

[0016] Preferably, the polyclonal antibody is used for immunoblotting detection of the duck hepatitis A virus type 3 VP0 protein in eukaryotic cells.

[0017] The application also provides the use of the polyclonal antibody or the polyclonal antibody prepared by the preparation method in detecting the duck hepatitis A virus type 3 VP0 protein in eukaryotic cells.

[0018] Compared with the prior art, the application has the following advantages and technical effects:

[0019] 1. Based on the duck hepatitis A virus type 3 VP0 gene sequence, the antigenicity, hydrophilicity and homology of the duck hepatitis A virus type 3 VP0 protein sequence are analyzed, and a suitable peptide segment sequence is predicted as a target sequence for artificial synthesis.

[0020] 2. The peptide segment sequence after prediction and synthesis is coupled with a carrier protein hemocyanin to obtain duck hepatitis A virus type 3 VP0 polypeptide-KLH coupling protein.

[0021] 3. Kunming mice are immunized with the prepared duck hepatitis A virus type 3 VP0 polypeptide-KLH coupling protein, and the titer of the antiserum is detected by an indirect ELISA method, and the ELISA detection result shows that the titer of the prepared antiserum is above 1:51200.

[0022] The prepared mouse anti-duck hepatitis A virus type 3 VP0 polypeptide antibody can specifically recognize the duck hepatitis A virus type 3 VP0 protein in eukaryotic cells, fills the blank in the field of detection of the duck hepatitis A virus type 3 VP0 protein, and lays a material foundation for pathogenicity research of duck hepatitis A virus type 3 infection. DETAILED DESCRIPTION

[0023] The various illustrative embodiments of the application are now described in detail. The following detailed description is merely intended to teach a person skilled in the art further details about certain aspects of the application and is not intended to limit the scope of the application. Only the claims define the scope of the application.

[0024] It should be understood that the terms used in the present application are merely used to describe particular embodiments and are not intended to limit the present application. In addition, for the numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range and any other stated value or stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0025] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains unless otherwise specifically defined herein. Although preferred methods and materials are described herein, any method and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described herein. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0026] Many modifications and variations of the present application described in the specific embodiments of the application can be made by those skilled in the art without departing from the spirit or scope of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0027] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed materials and methods.

[0028] The source of the drug used in the embodiments of the present application:

[0029] Female Kunming mice were purchased from Chengdu Dashuo Experimental Animal Co., Ltd., and fast immune water adjuvant was purchased from Boaolong Biological Technology Co., Ltd. Embodiments

[0030] 1.3 type duck hepatitis A virus VP0 protein sequence analysis and design and synthesis of 3 type duck hepatitis A virus VP0 polypeptide

[0031] Analysis of the DHAV-3 VP0 gene sequence shows that: DHAV-3 VP0 is 768 bp, encoding 256 aa.

[0032] The full-length amino acid sequence of DHAV-3 VP0 protein SEQ ID NO. 1: MDTLTKNIEDETVKIIGSCAEKAQEAISGLGAVESVASTNSVVATANATTTQTIPDPTDGSTDDFYSCSYEVGAQGDNISRLVHLHTGQWSTQHGVTTCLRWLATPGCFYTVNTQPAYGQTRYFRFIRCGYHFRLLVNAPSGAAGGLMMVWMPYPYCRVLTGSYNVDASVDRRSLLNLPYAILDLRTNTEIDLVIPYVNFRNYVEITATDSVGGAICVFVLGAFTHGSGTSNTVDYTLFGEMLETDLQCPRPFNDQ.

[0033] The antigenicity of the VP0 protein sequence of type 3 duck hepatitis A virus was analyzed by using different bioinformatics websites (ABCPred, BcePred, and BepiPred-3.0), and a segment of amino acid sequence was selected as the target sequence for antigen synthesis, SEQ ID NO. 2: CRVLTGSYNVDASVDRRSL (160-178 aa).

[0034] The purity of the DHAV-3 VP0 polypeptide was detected by high performance liquid chromatography (HPLC), and the results are shown in Table 1. Figure 4 The molecular weight of the DHAV-3 VP0 polypeptide was detected by mass spectrometry, and the results are shown in Table 2. Figure 5 The molecular weight of the DHAV-3 VP0 polypeptide was detected by mass spectrometry, and the results are shown in Table 2.

[0035] 2. Coupling of DHAV-3 VP0 polypeptide and carrier protein keyhole limpet hemocyanin (KLH)

[0036] 5.0 mg of DHAV-3 VP0 polypeptide was coupled with 5.0 mg of carrier protein KLH by using a coupling agent MBS, and the coupling was completed by Abtek Biological Technology Co., Ltd. to obtain DHAV-3 VP0 polypeptide-KLH coupled protein, i.e., a complete antigen.

[0037] 3. Immunization of experimental animals and preparation of antisera

[0038] Female Kunming mice were selected as immunized animals, and blood was collected from the tail tips of the Kunming mice before the first immunization (all blood collection was performed in the morning before feeding) as control sera for subsequent ELISA detection.

[0039] The antigen peptide is sent in dry powder form and the total amount is 5 mg. The dry powder is diluted with sterile ddH2O to a final concentration of 1 mg / mL and can be stored at -20°C. The adjuvant is stored at 4°C and the amount is 50 μL per mouse. From the day of immunization, it is recorded as day 1. On day 1, the antigen is diluted to 2 times the final concentration (prepared according to the amount of 50 μL per injection). The amount of antigen for each mouse is 20 μg, so based on the antigen peptide diluted with ddH2O, 0.3 mg of antigen is needed for 15 mice. 0.3 mg of antigen is diluted with normal saline, that is, 300 μL of antigen diluted with ddH2O is supplemented with 750 μL of normal saline, and the final concentration of the antigen is 0.4 mg / mL. After the adjuvant is thoroughly mixed, the adjuvant and the diluted antigen are mixed in a volume ratio of 1:1 to prepare for immunization. At this time, 750 μL of adjuvant is prepared for 15 mice, and after the antigen is mixed, the total volume is 1500 μL, and the final concentration of the antigen is 0.2 mg / mL, that is, 20 μg of antigen is contained in every 100 μL. The mice are immunized by intramuscular injection in the hind leg, and each mouse is immunized with 100 μL. On day 21, the same method is used for a booster immunization. On day 35, blood is collected for ELISA titer determination. According to the results of the detected antibody titer, it is determined whether further booster immunization is needed.

[0040] After the second immunization, the antiserum is collected, and the capillary is used for the orbital blood collection of Kunming mice. After a large amount of blood is collected, the blood is collected at 37°C and inclined for 1 h, and then transferred to a 4°C refrigerator and inclined for 12 h, so that it is fully analyzed for antiserum. Centrifugation at 4°C, 2500 r / min for 20 min, separation of antiserum, aliquot storage in a -80°C refrigerator, and standby.

[0041] 4. Indirect ELISA method for detecting the titer of antiserum

[0042] Coating. The purified VP0 protein was diluted to 50 μg / mL with coating solution (0.05 mol / L carbonate buffer, pH = 9.6, 0.75 g of sodium carbonate, 1.46 g of sodium bicarbonate, and deionized water were weighed, dissolved, and made up to 500 mL) to coat the enzyme-labeled plate, 100 μL / well, and placed at 4°C overnight; washing. PBST was used to wash 4 times, and the liquid was discarded after each shaking; blocking. 5% skim milk powder, 300 μL / well, was added, and blocked at 37°C for 1 h; washing. PBST was used to wash 4 times, and the liquid was discarded after each shaking; incubation of the primary antibody. The separated VP0 group serum and negative serum were added to the enzyme-labeled plate at different dilutions (1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:51200, 1:102400, 1:204800), 100 μL / well, at 37°C for 2 h (4 replicates were set for each group); washing. PBST was used to wash 4 times, and the liquid was discarded after each shaking; incubation of the secondary antibody. HRP-goat anti-mouse IgG was diluted with PBS, 100 μL / well, and reacted at 37°C for 1 h (the antibody dilution was 1 / 10 of the blocking solution); washing. PBST was used to wash 4 times, and the liquid was discarded after each shaking; color development. TMB color developing liquid, 100 μL / well, was added, and color developed at 37°C for 20 min (or at room temperature for 30 min) in the dark; determination of OD450. The OD450 value was determined after 2 M H2SO4, 50 μL / well, was added to terminate the reaction; when the ratio to the negative control serum was greater than 2.1, the antibody titer was calculated.

[0043] The detection results are shown in Table 1. Figure 6 The antibody titer of the DHAV-3 VP0 antiserum was 1:51200.

[0044] 5. Western Blot detection of DHAV-3 VP0 protein in eukaryotic cells

[0045] The gel reagent kit was prepared according to the SDS-PAGE preparation gel reagent kit of Solibao. The eukaryotic expressed DHAV-3 VP0 protein was sampled with 5xloading buffer, added to the loading hole of the vertical electrophoresis tank, and subjected to SDS-PAGE gel electrophoresis at 90 V for 30 min, and then the voltage was adjusted to 120 V for 2 h until the bromophenol blue ran to 1.5 cm from the bottom of the gel, and then the electrophoresis was terminated.

[0046] After SDS-PAGE electrophoresis, wet transfer was used to transfer the protein to PVDF membrane. Blocking was performed with 5% skim milk powder for 3h at room temperature, and the membrane was washed with TBST for 3 times, 5min each time. Primary antibody (prepared mouse anti-DHAV-3 VP0 polypeptide polyclonal antibody, different dilution degrees were used for dilution, dilution degrees were 1:200, 1:400, 1:800, 1:1600, 1:3200 respectively) was added, and incubation was performed at 4°C overnight, and the membrane was washed with TBST for 6 times; HRP-labeled goat anti-mouse IgG was used as secondary antibody for incubation, and incubation was performed at room temperature for 1h, and the membrane was washed with TBST for 3 times; after mixing, ECL luminescent solution reacted with PVDF membrane in dark, and a fluorescent luminescence image was collected. After color development, the same operation was performed using tag antibody as primary antibody to ensure that the protein size was consistent, so that it was determined that the DHAV-3 VP0 polypeptide antiserum could recognize DHAV-3 VP0 protein.

[0047] The results are shown in Figure 7 The bands are clear, and the DHAV-3 VP0 antiserum has good specificity.

[0048] The results are shown in Figure 8 The bands are clear, and the DHAV-3 VP0 antiserum can specifically recognize VP0 protein in the process of virus infection.

[0049] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

[0050] Figure 1 The results of analysis of the antigen index of the VP0 amino acid sequence of duck hepatitis A virus type 3 by using the bioinformatics website ABCPred

[0051] Figure 2 The results of analysis of the antigen index of the VP0 amino acid sequence of duck hepatitis A virus type 3 by using the bioinformatics website BcePred

[0052] Figure 3 The results of analysis of the antigen index of the VP0 amino acid sequence of duck hepatitis A virus type 3 by using the bioinformatics website BepiPred-3.0

[0053] Figure 4 The results of HPLC purity detection of the VP0 polypeptide of duck hepatitis A virus type 3

[0054] Figure 5 The results of mass spectrometry detection of the VP0 polypeptide of duck hepatitis A virus type 3

[0055] Figure 6 The mouse anti-DHAV-3 VP0 serum titer detected by the indirect ELISA method

[0056] Figure 7 The Western Blot result of the polyclonal antibody of the mouse anti-3 duck hepatitis A virus VP0 polypeptide prepared by using the application for detecting the eukaryotic expression VP0 protein (the VP0 protein is fused with the HA tag, and the HA tag antibody is the control group)

[0057] Figure 8 The Western Blot result of the polyclonal antibody of the mouse anti-3 duck hepatitis A virus VP0 polypeptide prepared by using the application for detecting the VP0 protein in the DHAV-3 infected cell.

Claims

1. A duck hepatitis A virus type 3 VP0 polypeptide, characterized in that: The amino acid sequence of the type 3 duck hepatitis A virus VP0 polypeptide is shown in SEQ ID NO.

2.

2. A polyclonal antibody against duck hepatitis A virus type 3 VP0 polypeptide, characterized in that: The amino acid sequence of the type 3 duck hepatitis A virus VP0 polypeptide is shown in SEQ ID NO.

2. The polyclonal antibody against the type 3 duck hepatitis A virus VP0 polypeptide is coupled with the type 3 duck hepatitis A virus VP0 polypeptide and hemocyanin (KLH) via a coupling agent MBS to obtain a complete antigen; the complete antigen is then mixed with Biolong rapid immune water adjuvant, Kunming mice are immunized, and serum containing antibodies against the type 3 duck hepatitis A virus VP0 polypeptide is collected.

3. A method for preparing a polyclonal antibody against the duck hepatitis A virus type 3 VP0 polypeptide according to claim 2, characterized in that: The following steps are involved: The type 3 duck hepatitis A virus VP0 polypeptide is coupled to hemocyanin (KLH) to obtain a complete antigen; the complete antigen is mixed with Biolong rapid immune water adjuvant and used to immunize Kunming mice; serum containing antibodies to the type 3 duck hepatitis A virus VP0 polypeptide is collected to obtain antiserum, and the amino acid sequence of the type 3 duck hepatitis A virus VP0 polypeptide is shown in SEQ ID NO.

2.

4. The preparation method according to claim 3, wherein: The complete antigen and Biolong rapid immune water adjuvant are mixed in equal volumes to immunize Kunming mice, comprising the following steps: The amount of immunizing antigen for each mouse was 20 μg. After the antigen was diluted, it was mixed with an equal volume of Biolong rapid immunization water adjuvant and then injected into the leg muscle. This was the first immunization of Kunming mice. A booster immunization was performed 3 weeks after the first immunization, and the number of booster immunizations was 1.

5. The preparation method according to claim 3, wherein: The method of collecting serum containing type 3 duck hepatitis A virus VP0 polypeptide antibodies comprises the following steps: The blood was collected from Kunming mice using a capillary orbital blood collection method. After the blood was collected, it was tilted and allowed to stand at 37°C for 1 hour, and then allowed to stand at 4°C for 12 hours to separate and obtain the antiserum.

6. The preparation method according to claim 3, wherein: It also includes immunoblotting detection of eukaryotic expressed VP0 protein using polyclonal antibodies against duck hepatitis A virus type 3 VP0 polypeptide.

7. The preparation method according to claim 3, wherein: It also includes immunoblotting detection of VP0 protein in virus-infected cells using polyclonal antibodies against duck hepatitis A virus type 3 VP0 polypeptide.

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