A duck Tembusu virus polyclonal antibody and preparation method thereof

Abstract

The present invention discloses a duck Tembusu virus polyclonal antibody and a preparation method thereof, the preparation method comprising: S100, connecting the Capsid gene of the duck Tembusu virus with an empty vector to form a recombinant plasmid; S200, transforming the recombinant plasmid obtained in step S100 into competent cells, culturing and purifying to obtain a purified recombinant Capsid protein; S300, immunizing with the recombinant Capsid protein obtained in step S200 as an antigen to obtain a duck Tembusu virus polyclonal antibody; wherein the duck Tembusu virus has a deposit number of CCTCC No: V202262, and is deposited in the China Center for Type Culture Collection on September 15, 2022. The polyclonal antibody prepared by the present invention provides experimental materials for establishing a detection method for DTMUV and subsequent in-depth research, and lays an experimental foundation.

Description

Technical Field

[0001] The present invention relates to the field of antibody preparation, and in particular to a duck Tembusu virus polyclonal antibody and a preparation method thereof. Background Art

[0002] Duck Tembusu virus disease is an acute infectious disease caused by Duck Tembusu virus (DTMUV) infecting laying ducks, meat ducks, geese, sparrows, etc. The main clinical symptoms of infected ducks are a rapid decline in egg production, ataxia, ovarian hemorrhage, inflammation, and even death.

[0003] At present, most of the research on duck Tembusu virus focuses on E protein, NS1 protein or NS3 protein, and there is basically no relevant research on the interaction between Capsid protein and host protein.

[0004] At present, the detection of DTMUV mainly relies on multiplex PCR detection methods, while serological detection methods are relatively rare. The only reported method is the Epitope-ELISA method established based on the DTMUV E protein antigenic epitope (Li Chenxi, Bai Xiaofei, Shao Zhou, Wulin, et al. Identification of specific antigenic epitopes of duck Tembusu virus E protein and establishment of Epitope-ELISA serological diagnostic method [J]. Acta Animal Husbandry and Veterinary Medicine, 2017, 48(10): 1958-1968.).

[0005] Therefore, providing a mouse polyclonal antibody against Capsid protein and establishing a new DTMUV detection method are the problems that the present invention urgently needs to solve. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to address the problem that there are certain limitations in the research on the Capsid protein of duck Tembusu virus in the prior art, thereby providing a mouse polyclonal antibody against Capsid protein, which can provide experimental materials and lay the foundation for subsequent in-depth research on the biological function of Capsid protein and the pathogenic mechanism of DTMUV, and a method for preparing the duck Tembusu virus polyclonal antibody.

[0007] In order to achieve the above object, the present invention provides a method for preparing duck Tembusu virus polyclonal antibodies, the preparation method comprising:

[0008] S100, the Capsid gene of duck Tembusu virus was connected to an empty vector to construct a recombinant plasmid;

[0009] S200, transforming the recombinant plasmid obtained in step S100 into competent cells, and culturing and purifying to obtain purified recombinant Capsid protein;

[0010] S300, using the recombinant Capsid protein obtained in step S200 as an antigen for immunization to obtain duck Tembusu virus polyclonal antibodies; wherein,

[0011] The deposit number of the duck Tembusu virus is CCTCC No: V202262, and it was deposited in the China Center for Type Culture Collection on September 15, 2022.

[0012] Preferably, step S100 specifically includes:

[0013] S101, inoculating DEF cells with duck Tembusu virus for culture, harvesting the cells, extracting RNA and performing reverse transcription to obtain cDNA;

[0014] S102, using the cDNA obtained in step S101 as a template, amplifying the Capsid gene of duck Tembusu virus by PCR;

[0015] S103, connecting the Capsid gene obtained in step S102 to an empty vector to obtain a connection product;

[0016] S104, transforming the ligation product obtained in step S103 into DH5α competent cells, culturing the cells, performing restriction digestion and sequencing, and constructing a recombinant plasmid, which is recorded as pET-30a-Capsid.

[0017] Preferably, the amplification primers in step S102 are shown as SEQ ID No: 1 and SEQ ID No: 2.

[0018] Preferably, in step S102, the reaction conditions of the amplification process are: 94°C, 2 min; 98°C, 10 s; 68°C, 1 min, 30 cycles; 68°C, 5 min.

[0019] Preferably, step S200 specifically includes:

[0020] S201, transforming the empty vector and pET-30a-Capsid into BL21 (DE3) competent cells at the same time for culture, picking a single colony after culture and inoculating it into kanamycin-resistant LB medium to form a bacterial solution for culture;

[0021] S202, detecting the OD600 value of the bacterial solution in step S201, and when the OD600 value is 0.6-0.9, adding IPTG for induction culture, collecting the bacterial cells by centrifugation, washing them, resuspending them, and ultrasonically disrupting them until the solution is clear, separating the supernatant and the precipitate, collecting the precipitate for purification, and obtaining the purified recombinant Capsid protein.

[0022] Preferably, step S300 specifically includes:

[0023] S301, using the purified recombinant Capsid protein and ISA206 adjuvant mixture as antigen, the mice were immunized for the first time;

[0024] S302. After the mice are immunized for the second time after the first immunization, the mouse serum is collected to obtain corresponding polyclonal antibodies against duck Tembusu virus.

[0025] Preferably, the sequence of the Capsid gene of the duck Tembusu virus is as shown in SEQ ID No:3.

[0026] Preferably, in step S300, the amino acid sequence of the recombinant Capsid protein is as shown in SEQ ID No:4.

[0027] The present invention also provides a duck Tembusu virus polyclonal antibody prepared according to the above-mentioned preparation method.

[0028] Through the above technical scheme, the present invention clones and expresses the Capsid gene of a specific duck Tembusu virus in a prokaryotic manner, thereby successfully preparing a polyclonal antibody against the Capsid protein, providing experimental materials and laying a foundation for establishing a detection method for DTMUV, as well as subsequent in-depth research on the biological function of the Capsid protein and the pathogenic mechanism of DTMUV. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention, but do not constitute a limitation of the present invention. In the accompanying drawings:

[0030] Figure 1 is a graph showing the agarose gel electrophoresis identification result in detection example 1;

[0031] Figure 2 is a diagram showing the results of SDS-PAGE analysis of the unpurified recombinant Capsid protein in Detection Example 2;

[0032] Figure 3 is a diagram showing the results of SDS-PAGE analysis of the purified recombinant Capsid protein in Detection Example 2;

[0033] Figure 4 is a Western-blot detection result diagram of the purified recombinant Capsid protein in Detection Example 2;

[0034] Figure 5 is a graph showing the titer determination results of the mouse polyclonal antibody obtained in Detection Example 3;

[0035] Figure 6This is a graph showing the protein expression results of HEK293T cells transfected with Flag-tagged Capsid detected by Western-blot in Detection Example 4;

[0036] Figure 7 This is the result of Western-blot detection of DEFs Capsid protein expression in DTMUV infection in Detection Example 4;

[0037] Figure 8 This is the result of detecting the expression of Capsid protein in DEFs infected with DTMUV by IFA in Detection Example 5;

[0038] Fig. 9 is a graph showing the titer determination results of the rabbit polyclonal antibody obtained in Detection Example 3;

[0039] Fig.10 This is a graph showing the protein expression results of detection in Detection Example 5 using Western-blot. DETAILED DESCRIPTION

[0040] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0041] Specifically, the present invention provides a duck Tembusu virus polyclonal antibody and a preparation method thereof, which will be further described below in conjunction with specific examples.

[0042] Duck Tembusu virus (CCTCC NO: V202262) was deposited in the China Center for Type Culture Collection. pET-30a(+) and pCS2-FLAG-Capsid plasmids were preserved by Jiangsu Provincial Key Laboratory of Veterinary Biopharmaceutical High-Tech Research. Escherichia coli DH5α and BL21 (DE3) competent cells were purchased from Suzhou Xinsaimei Biotechnology Co., Ltd. SPF-grade 4-6 week-old BALB / c pure female mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. Among them, the pCS2-FLAG-Capsid plasmid was specifically constructed by the following method:

[0043] DEF cells infected with DTMUV (duck Tembusu virus strain with a deposit number of CCTCC No: V202262) were collected, and the total RNA in the cells was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit V2. The RNA concentration, purity, and integrity were detected using a Nano-100C micro-spectrophotometer and 1% agarose gel electrophoresis. 1 μg of total RNA was taken from each sample and III RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit was used for reverse transcription to obtain cDNA. The cDNA was used as a template for PCR reaction to amplify the DTMUV Capsid gene. PCR reaction system 50μL: PrimeSTAR Max Premix (2×) 25μL, pCS2-FLAG-Capsid forward and reverse primers (10μM) 1μL each, genome 1μL, ddH2O 22μL. PCR reaction program: 98℃ pre-denaturation for 5min; 98℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 30s, a total of 30 cycles; 72℃ extension for 5min. The FLAG-pCS2 vector was double-digested with EcoR I and Xba I, and the PCR product and enzyme digestion product were quickly recovered. Ligation reaction system 20μL: 2×Hieff The amount of Enzyme Premix is ​​10 μL, the linearized vector is 1 μL, the DTMUV Capsid gene fragment obtained by the above amplification is 3 μL, and the amount of ddH2O is 6 μL. Place it at 50°C for reaction for 20 minutes, take 10 μL to transform Escherichia coli DH5α competent cells, pick a single clone for colony PCR identification after overnight culture, and extract the endotoxin-free plasmid after expanding the positive bacteria. It is named pCS2-FLAG-Capsid plasmid, and is stored after EcoRI and Xba I double enzyme digestion identification. Among them, the pCS2-FLAG-Capsid forward and reverse primers used in PCR amplification are shown in SEQ ID No: 5 and SEQ ID No: 6.

[0044] High-fidelity KOD enzyme was purchased from Takara; restriction endonucleases EcoR I and Xho I were purchased from New England Biolabs; plasmid extraction kit and gel recovery kit were purchased from Omega BioTek; homologous recombination kit, peroxidase-labeled goat anti-mouse IgG (H+L) and 488-labeled rabbit anti-mouse IgG (H+L) were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; IPTG was purchased from Kangwei Century Biotechnology Co., Ltd. Protein purification nickel column with His tag was purchased from GE, USA. Amplification primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0045] Preparation Example 1: Amplification of Capsid Gene and Construction of Recombinant Plasmid

[0046] DEF cells were inoculated with duck Tembusu virus with a deposit number of CCTCC No: V202262. After 48 hours, the cells were harvested and RNA was extracted. The cDNA obtained by reverse transcription was used as a template, and the Capsid gene was amplified by PCR using the upstream and downstream primers shown in SEQ ID No: 1 and SEQ ID No: 2. 50 μL of PCR amplification system: 5 μL of 10×PCR Buffer for KOD-Plus-Neo, 5 μL of 2 mmol / L dNTPs, 3 μL of 25 mmol / L MgSO4, 1.5 μL of upstream and downstream primers (10 μmol / L), 200 ng of cDNA, 1 μL of KOD-Plus-Neo (1 U / μl), and 50 μL of ddH2O. PCR reaction conditions: 94℃, 2min; 98℃, 10s, 68℃, 1min, 30 cycles, 68℃, 5min; at the same time, the pET-30a(+) empty vector was double-digested with EcoRI and XhoI. The Capsid gene fragment and pET-30a(+) empty vector were recovered after gel running and then connected using homologous recombinase. The connection product was transformed into DH5α and coated. After inverted overnight culture at 37℃ for 12h, the plasmid with correct enzyme digestion was selected for gene sequencing. The plasmid with successful sequencing was pET-30a-Capsid.

[0047] Preparation Example 2: Prokaryotic expression and purification of recombinant Capsid protein

[0048] The vector plasmid pET-30a(+) and the correctly identified prokaryotic expression plasmid pET-30a-Capsid were transformed into BL21(DE3) competent cells and plated, and a single colony was picked and inoculated into 10mL of kanamycin-resistant LB medium, and then transferred to kanamycin-resistant LB medium at a ratio of 1:100 the next day; when the OD600 of the bacterial solution was about 0.6-0.9, IPTG with a final concentration of 1.0mmol / L was added, and the culture was induced at 37℃ for 4h, and the bacteria were collected by centrifugation, washed with PBS 2-3 times and resuspended, ultrasonically broken under ice bath conditions until the solution was clear, and the supernatant and precipitate were separated. The solubility of the recombinant Capsid protein was analyzed by SDS-PAGE, and the reactivity of the recombinant Capsid protein was analyzed by Western-blot. Subsequently, the correctly expressed pET-30a-Capsid plasmid was induced in large quantities, and the recombinant Capsid protein was purified using the His tag nickel column affinity chromatography method. The purified protein was identified by SDS-PAGE, and its concentration was determined by BCA method and stored at -80°C for future use.

[0049] Preparation Example 3: Preparation of mouse polyclonal antiserum against recombinant Capsid protein

[0050] Using purified recombinant Capsid protein as antigen, four 4-6 week-old BALB / c mice were used, and each mouse was subcutaneously immunized with 50 μg of antigen mixed with an equal volume of ISA206 adjuvant. Two weeks later, the second immunization was performed, and the second immunization method was the same as the first immunization. Two weeks after the second immunization, blood was collected from the eye sockets and serum was collected.

[0051] Preparation Example 4: Preparation of rabbit polyclonal antiserum against recombinant Capsid protein

[0052] Using the purified recombinant Capsid protein as the antigen, two 6-week-old New Zealand white rabbits were used, and 500 μL of pre-immune serum was taken from each rabbit for preservation. 400 μg of antigen was used for each rabbit, and it was thoroughly mixed with an equal volume of complete Freund's adjuvant (CFA). On the 14th day, 100 μg of antigen was used for each rabbit, and it was thoroughly mixed with an equal volume of CFA for the second immunization. On the 28th, 35th, 42nd, 49th, 56th and 63rd days, 100 μg of antigen was used for each rabbit, and it was thoroughly mixed with an equal volume of incomplete Freund's adjuvant (IFA) for booster immunization. Serum was collected for ELISA testing on the 42nd day, and the rabbits were finally bled on the 70th day to collect serum.

[0053] Detection Example 1: Verification of Capsid Gene and Recombinant Plasmid

[0054] The gene fragment, empty vector, recombinant plasmid and recombinant plasmid after double enzyme digestion in Preparation Example 1 were identified by agarose gel electrophoresis. The results were as follows: Figure 1 Lane M is a molecular weight marker, lane 1 is the Capsid gene fragment after PCR amplification, lane 2 is the pET-30a(+) empty vector after double digestion with EcoRI and XhoI, lane 3 is the recombinant plasmid pET-30a-Capsid without enzyme digestion, and lane 4 is the recombinant plasmid pET-30a-Capsid after double digestion with EcoRI and XhoI.

[0055] From the above agarose gel electrophoresis, it can be seen that a single band consistent with the expected result can be seen between 250 bp and 500 bp, and its size is about 360 bp. After double digestion of EcoRI and XhoI, the recombinant plasmid pET-30a-Capsid obtained a fragment of about 360 bp in size, which is consistent with the expected result. The sequencing results showed that the Capsid gene sequence was consistent with the original strain sequence, indicating that the Capsid gene has been successfully cloned into the vector pET-30a(+).

[0056] Detection Example 2: Identification of Recombinant Capsid Protein

[0057] The recombinant Capsid protein in Preparation Example 2 was subjected to SDS-PAGE and Western-blot detection to identify it accordingly.

[0058] Among them, the SDS-PAGE analysis results of the recombinant Capsid protein are as follows Figure 2 and Figure 3 shown.

[0059] according to Figure 2 It can be seen that the target band appears at 18 ku. The DNAstar Lasergene 7.1 software predicts that the size of the Capsid protein is about 13 ku. The pET-30a(+) vector contains a 6×His tag with a size of about 5 ku, which is consistent with the expected size, indicating that the recombinant Capsid protein was successfully induced to express. The recombinant Capsid protein was mainly found in the precipitate, indicating that the recombinant Capsid protein mainly exists in the form of inclusion bodies. Figure 2 In the figure, lane M is a molecular weight marker, lane 1 is induced pET-30a(+), lane 2 is the bacterial solution after uninduced culture, lane 3 is the bacteria collected after induced culture, lane 4 is the supernatant separated after the bacteria collected after induced culture were crushed, and lane 5 is the precipitate separated after the bacteria collected after induced culture were crushed.

[0060] The recombinant Capsid protein successfully expressed in the inclusion body was purified by nickel column affinity chromatography to obtain a recombinant Capsid protein of high purity. The results of SDS-PAGE analysis are as follows: Figure 3 Among them, lane M is a molecular weight marker, and lane 1 is the purified recombinant Capsid protein.

[0061] The purified recombinant Capsid protein was detected by Western-blot using anti-His tag mouse monoclonal antibody as the primary antibody. The results are shown in Figure 4 As shown, according to Figure 4 It can be seen that the target band appears at 18 ku, indicating that the recombinant protein has good reactivity. Lane M is a molecular weight marker, lane 1 is induced pET-30a (+), and lane 2 is the purified recombinant Capsid protein.

[0062] Test Example 3: Indirect enzyme-linked immunosorbent assay (ELISA)

[0063] Dilute the purified recombinant Capsid protein obtained in Preparation Example 2 to 0.5 μg / mL, and coat 100 μL per well at 4°C overnight. Add 200 μL of 5% skim milk to each well and block at 37°C for 1 hour. Dilute the polyclonal antibody (i.e., the serum collected in Preparation Example 3) in 2-fold ratios from 1:1000 to 1:512000, add 100 μL to each well, and incubate at 37°C for 1 hour. Then dilute HRP-labeled goat anti-mouse IgG at 1:5000 as a secondary antibody, add 100 μL to each well, and incubate at 37°C for 1 hour. Add 100 μL of TMB to each well for color development, incubate at room temperature away from light, and add 100 μL of stop solution to each well after 30 minutes to stop color development, and read the OD with an enzyme reader. 450 nm value.

[0064] The above method uses purified recombinant Capsid protein to coat the ELISA plate, and the titer of the mouse-derived and rabbit-derived Capsid protein polyclonal antibodies in Preparation Example 3 and Preparation Example 4 (i.e., the corresponding sera obtained in Preparation Example 3 and Preparation Example 4, respectively) is determined by the indirect ELISA method, and the pre-immune serum is used as a negative control. Among them, the detection results of the mouse-derived Capsid protein polyclonal antibody are as follows Figure 5 As shown in the figure, the titer of the prepared mouse Capsid protein polyclonal antibody can reach 1:256000. The test results of the rabbit Capsid protein polyclonal antibody are shown in the figure. Fig. 9 As shown, the results showed that the titer of the prepared rabbit Capsid protein polyclonal antibody was >1:128000.

[0065] Therefore, in comparison, the Capsid protein polyclonal antibody prepared according to the method of the present invention has a higher titer when obtained from mouse sources.

[0066] Detection Example 4: Western-blot detection of mouse capsid protein polyclonal antibody (i.e., serum obtained in Preparation Example 3)

[0067] The pCS2-FLAG-Capsid recombinant plasmid was transfected into HEK293T cells, and the cells were collected after 48 hours. At the same time, DEFs were infected with 0.1MOIDTMUV. After 48 hours, the cells were lysed on ice with RIPA lysis buffer, the supernatant was collected, the proteins were separated by 15% SDS-PAGE, and then transferred to the PVDF membrane by wet transfer. Blocked with 5% skim milk at room temperature for 2 hours, the anti-Capsid protein serum prepared in Preparation Example 3 was diluted (1:800) as the primary antibody and incubated at 4°C overnight; HRP-labeled goat anti-mouse IgG (1:10000) was diluted as the secondary antibody, incubated at room temperature for 4 hours, and the color was developed using the ECL luminescence imaging system. The results obtained are as follows. Figure 6 and Figure 7 shown.

[0068] in, Figure 6 This is the result of Western-blot detection of protein expression in HEK293T cells transfected with pCS2-FLAG-Capsid. Figure 7 The figure shows the results of Western-blot detection of Capsid protein expression in DEFs infected with DTMUV. Figure 6 and Figure 7 As shown, the Capsid proteins expressed after DEFs were infected with pCS2-FLAG-Capsid and DTMUV could be specifically recognized by mouse anti-Capsid protein polyclonal antibody, indicating that the prepared mouse anti-Capsid protein polyclonal antibody can be used for Western-blot detection of Capsid protein.

[0069] Detection Example 5: Western-blot detection of rabbit-derived capsid protein polyclonal antibody (i.e., serum obtained in Preparation Example 4)

[0070] According to the method of Detection Example 4, Western-blot was used to detect the protein expression of the recombinant Capsid protein in Preparation Example 2 or the Capsid protein in DEFs infected with DTMUV. Fig.10 As shown, lane M is a molecular weight marker, and lane 1 is the recombinant Capsid protein in Preparation Example 2. It can be seen that the recombinant Capsid protein in Preparation Example 2 can be recognized by the serum obtained in Preparation Example 4, but the Capsid protein expressed in DEFs infected with DTMUV cannot be recognized by the serum obtained in Preparation Example 4. This shows that the rabbit anti-Capsid protein polyclonal antibody (serum obtained in Preparation Example 4) can be used for Western-blot detection of recombinant Capsid protein, but cannot be used for Western-blot detection of Capsid protein expressed in DEFs infected with DTMUV.

[0071] It can be seen from Detection Examples 4 and 5 that the mouse Capsid protein polyclonal antibody can simultaneously detect the Capsid protein expressed after pCS2-FLAG-Capsid and DTMUV infected DEFs based on Western-blot detection, while the rabbit Capsid protein polyclonal antibody can only detect the recombinant Capsid protein in Preparation Example 2, and cannot detect the Capsid protein expressed after virus infected DEFs.

[0072] Test Example 6: Indirect Immunofluorescence (IFA) Identification

[0073] DEFs were infected with 0.1 MOI DTMUV, fixed and permeabilized with formaldehyde after 48 hours, blocked with 5% skim milk for 1 hour, and anti-capsid protein polyclonal antibody (i.e., serum obtained in Preparation Example 3 and Preparation Example 4) was diluted 1:100 with 5% skim milk and incubated at room temperature for 4 hours; 488-labeled rabbit anti-mouse IgG (H+L) was diluted 1:250 and incubated at room temperature in the dark for 1 hour. After DAPI staining at room temperature, the cells were observed under a fluorescence microscope. The results obtained in Preparation Example 3 are shown in Figure 2. Figure 8 shown.

[0074] Depend on Figure 8 It can be seen that specific green fluorescence can be detected 48 hours after DEF cells were infected with DTMUV, while the uninfected control group had no fluorescence. The IFA results showed that the mouse capsid protein polyclonal antibody prepared in this study can be used to detect the capsid protein of DTMUV with good specificity.

[0075] The results corresponding to Preparation Example 4 are: no specific green fluorescence was detected in DEF cells infected with DTMUV for 48 hours, and similarly, no fluorescence was detected in the uninfected control group. The IFA results showed that the rabbit anti-capsid protein polyclonal antibody (serum obtained in Preparation Example 4) could not be used to detect the capsid protein expressed in DEFs infected with DTMUV.

[0076] Through the above technical scheme, the present invention is based on duck Tembusu virus with a preservation number of CCTCC No: V202262, and uses a one-step cloning method to clone the DTMUV Capsid gene into the expression vector pET-30a(+). Compared with the traditional method, the one-step cloning technology does not require enzyme digestion of the PCR fragment, which can effectively improve the cloning efficiency. And the Capsid protein expressed by the Escherichia coli expression system exists in the expression product in the form of inclusion body precipitation.

[0077] Furthermore, the polyclonal antibody prepared by immunizing mice with ISA206 adjuvant emulsified protein can specifically recognize the Capsid protein encoded by DTMUV after DEF cells are infected. Compared with the traditional Freund's adjuvant, its oil content is lower, and only one step of emulsification is required to prepare a water-in-oil-in-water vaccine, which is not only less toxic, but also more convenient to use.

[0078] Based on this, the technical scheme of the present invention obtains DTMUV Capsid protein with good reactogenicity and immunogenicity, and successfully prepares mouse anti-Capsid protein polyclonal antibodies with good specificity, laying a foundation for the development of DTMUV diagnostic preparations and further clarifying the DTMUV replication mechanism, and also provides experimental materials for in-depth research on the structure and function of DTMUV Capsid protein.

[0079] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0080] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0081] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for preparing duck Tembusu virus polyclonal antibodies, characterized in that: The preparation method comprises: S100, the Capsid gene of duck Tembusu virus was connected to an empty vector to construct a recombinant plasmid; S200, transforming the recombinant plasmid obtained in step S100 into competent cells, and culturing and purifying to obtain purified recombinant Capsid protein; S300, using the recombinant Capsid protein obtained in step S200 as an antigen for immunization to obtain duck Tembusu virus polyclonal antibodies; wherein, The sequence of the Capsid gene of the duck Tembusu virus is shown in SEQ ID No:

3.

2. The method for preparing a duck Tembusu virus polyclonal antibody according to claim 1, characterized in that: Step S100 specifically includes: S101, inoculating DEF cells with duck Tembusu virus for culture, harvesting the cells, extracting RNA and performing reverse transcription to obtain cDNA; S102, using the cDNA obtained in step S101 as a template, amplifying the Capsid gene of duck Tembusu virus by PCR; S103, connecting the Capsid gene obtained in step S102 to an empty vector to obtain a connection product; S104, transforming the ligation product obtained in step S103 into DH5α competent cells, culturing the cells, performing restriction digestion and sequencing, and constructing a recombinant plasmid, which is recorded as pET-30a-Capsid.

3. The method for preparing a duck Tembusu virus polyclonal antibody according to claim 2, characterized in that: The amplification primers in step S102 are shown as SEQ ID No: 1 and SEQ ID No:

2.

4. A method for preparing a duck Tembusu virus polyclonal antibody according to claim 2 or 3, characterized in that: In step S102, the reaction conditions of the amplification process are: 94°C, 2 min; 98°C, 10 s, 68°C, 1 min, 30 cycles; 68°C, 5 min.

5. The method for preparing a duck Tembusu virus polyclonal antibody according to claim 2, characterized in that: Step S200 specifically includes: S201, transforming the empty vector and pET-30a-Capsid into BL21 (DE3) competent cells at the same time, picking a single colony after culture and inoculating it into kanamycin-resistant LB medium to form a bacterial solution for culture; S202, detecting the OD600 value of the bacterial solution in step S201, and when the OD600 value is 0.6-0.9, adding IPTG for induction culture, collecting the bacterial cells by centrifugation, washing them, resuspending them, and ultrasonically disrupting them until the solution is clear, separating the supernatant and the precipitate, collecting the precipitate for purification, and obtaining the purified recombinant Capsid protein.

6. A method for preparing a duck Tembusu virus polyclonal antibody according to claim 1 or 2, characterized in that: Step S300 specifically includes: S301, using the purified recombinant Capsid protein and ISA206 adjuvant mixture as antigen, the mice were immunized for the first time; S302. After the mice are immunized for the second time after the first immunization, the mouse serum is collected to obtain corresponding polyclonal antibodies against duck Tembusu virus.

7. A method for preparing a duck Tembusu virus polyclonal antibody according to any one of claims 1 to 3, characterized in that: In step S300, the amino acid sequence of the recombinant Capsid protein is shown in SEQ ID No:

4.

8. A duck Tembusu virus polyclonal antibody prepared according to the preparation method according to any one of claims 1 to 7.

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