Carp edema virus P4a recombinant protein, polyclonal antibody as well as preparation method and application of polyclonal antibody

Abstract

The invention discloses a carp edema virus P4a recombinant protein, a polyclonal antibody as well as a preparation method and application of the polyclonal antibody, a recombinant vector comprises a gene for expressing the carp edema virus P4a recombinant protein, and the gene sequence of the gene for expressing the carp edema virus P4a recombinant protein is shown as Seq. ID No. 1 or Seq. ID No. 2. According to the invention, prokaryotic expression is carried out on the CEV P4a protein and the polyclonal antibody is prepared for the first time, and the method has important significance in further development of a novel CEV detection technology, research on biological functions of the P4a protein, research on antibody molecular preparations, vaccine research and the like.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a carp edema virus P4a recombinant protein, a polyclonal antibody and a preparation method and application thereof. Background Art

[0002] Carp edema disease is a viral infectious disease caused by infection with carp edema virus, with a mortality rate of up to 80%-100%. It was first reported in my country in 2016 and was included in the "List of Aquatic Animal Diseases Surveillance in the Asia-Pacific Region" in 2017 (Lei 2017; Lv et al. 2022). Carp edema virus (CEV) can cause carp and koi with lethargy, sunken eyes, rotten gills, edema and other clinical symptoms. It is a highly pathogenic and highly lethal viral infectious disease, causing huge economic losses to carp farming and koi export trade worldwide (Jung-Schroers et al. 2015; Lv et al. 2022; Zrn i et al. 2020). CEV is a double-stranded linear DNA virus with a size of 250-400nm in the Poxviridae family and the Poxvirinae subfamily of the Chordata phylum. It replicates and proliferates in the cytoplasm and is oval and round after maturity and moves outside the cell (Lewisch et al. 2015; Machat et al. 2021; Rud et al. 2023). The CEV genome sequence is 456821bp long, with an A+T content of 71.2%. However, the current phylogenetic research on the CEV genome is still relatively limited, mainly relying on the virus core protein P4a (Matras et al. 2017). The gene sequence of the P4a protein is 2028bp long. It is a highly conserved gene in the middle region of the poxvirus genome and plays an important role in CEV infection and pathogenesis (Gubser et al. 2004; Sahoo et al. 2022). However, the infection mechanism, epidemiology, sensitive cell lines, etc. of CEV are still unknown, and there is no effective treatment for carp edema caused by CEV infection.

[0003] At present, the main detection methods of CEV include electron microscopy, histopathological observation, and nucleic acid detection. Electron microscopy can directly observe virus particles to determine whether it is CEV virus. Although this method is intuitive and effective, the instrument is expensive, the sample preparation is complicated, the detection personnel are required to have high skills, and the detection success rate varies greatly. It is not suitable for rapid and clinical detection of CEV (Miyazaki et al. 2005; Way et al. 2017); histopathological diagnosis is relatively simple and fast, with high accuracy, but when pathogens with similar clinical symptoms are mixed, such as CEV and KHV, the diagnostic difficulty and misjudgment rate are greatly increased (Davis et al. 2008; Witeska et al. 2005); nucleic acid detection is currently the most commonly used identification method for CEV (Lv Xiaonan et al. 2018), with strong sensitivity and good specificity, but existing nucleic acid detection methods such as PCR, qPCR, LAMP, RPA-LFD, etc. have their own advantages and disadvantages, and missed detection, false positive, and false negative results often exist. Due to the mismatch between different infection stages and viral loads of carp edema disease, asymptomatic infection and persistent reactivation of asymptomatic infection, the existing CEV detection methods cannot meet current detection needs. Summary of the invention

[0004] The purpose of the present invention is to provide a recombinant vector in view of the fact that the existing CEV detection method cannot meet the current detection needs.

[0005] Another object of the present invention is to provide a recombinant Escherichia coli based on the recombinant vector.

[0006] Another object of the present invention is to provide a recombinant protein of carp edema virus P4a produced by the recombinant Escherichia coli.

[0007] Another object of the present invention is to provide a polyclonal antibody based on the carp edema virus P4a recombinant protein.

[0008] Another object of the present invention is to provide an application of the polyclonal antibody.

[0009] The technical solution adopted to achieve the purpose of the present invention is:

[0010] A recombinant vector comprises a gene for expressing a recombinant protein of carp edema virus P4a, wherein the gene sequence of the gene for expressing the recombinant protein of carp edema virus P4a is as shown in Seq.ID No.1 or Seq.ID No.2.

[0011] In the above technical solution, the recombinant vector is the prokaryotic cloning vector PMD18-T-P4a or the prokaryotic expression vector pET-32a-P4a of the carp edema virus P4a protein.

[0012] In the above technical solution, the prokaryotic cloning vector PMD18-T-P4a is constructed by the following method:

[0013] Step A1, connecting the gene expressing the carp edema virus P4a recombinant protein with the pMD18-T cloning vector to obtain a connection product;

[0014] Step A2, transferring the ligation product obtained in step A1 into DH5α competent cells, spreading it on LB solid culture medium containing ampicillin, culturing it upright until the liquid is completely absorbed, and then culturing it inverted;

[0015] Step A3, pick white positive colonies and add them to LB liquid culture medium containing ampicillin, mix well and culture on a shaking platform;

[0016] Step A4, extracting plasmid DNA from the positive bacterial solution and performing PCR amplification verification to obtain the prokaryotic cloning vector PMD18-T-P4a.

[0017] In the above technical solution, the prokaryotic expression vector pET-32a-P4a is constructed by the following method:

[0018] Step B1, connecting the gene expressing the carp edema virus P4a recombinant protein with the pET-32a expression vector to obtain a connection product;

[0019] Step B2, transferring the ligation product obtained in step B1 into BL21 competent cells, spreading it on LB solid culture medium containing ampicillin, culturing it upright until the liquid is completely absorbed, and then culturing it inverted;

[0020] Step B3, pick white positive colonies and add them to LB liquid culture medium containing ampicillin, mix and culture on a shaking table;

[0021] Step B4, extracting plasmid DNA from the positive bacterial solution and performing PCR amplification verification to obtain the prokaryotic expression vector pET-32a-P4a.

[0022] Another aspect of the present invention provides a recombinant Escherichia coli, which includes the recombinant vector or the gene expressing the recombinant protein of carp edema virus P4a is introduced into the genome of the recombinant Escherichia coli through the recombinant vector.

[0023] In the above technical solution, the recombinant Escherichia coli is recombined by the following method:

[0024] Step 1, constructing a recombinant vector;

[0025] Step 2, introducing the recombinant vector into Escherichia coli;

[0026] Step 3, screening and obtaining recombinant Escherichia coli expressing the recombinant protein of carp edema virus P4a.

[0027] Another aspect of the present invention provides a recombinant protein of carp edema virus P4a, which is produced by the recombinant Escherichia coli. The amino acid sequence of the recombinant protein of carp edema virus P4a is shown in Seq.ID No.3.

[0028] Another aspect of the present invention provides a polyclonal antibody against the P4a protein of carp edema virus, which is prepared by the recombinant protein of the P4a protein of carp edema virus.

[0029] In the above technical solution, the polyclonal antibody against the P4a protein of carp edema virus is an inclusion body protein.

[0030] Another aspect of the present invention provides a use of the polyclonal antibody against the P4a protein of carp edema virus in the preparation of immunological detection products or vaccines.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The present invention is the first to express CEV P4a protein in prokaryotes and prepare polyclonal antibodies, which is of great significance for further developing new CEV detection technologies, studying the biological functions of P4a protein, antibody molecular preparations and vaccine research.

[0033] 2. The present invention constructs a CEV P4a protein prokaryotic cloning vector PMD18-T-P4a and a prokaryotic expression vector pET-32a-P4a through an Escherichia coli prokaryotic expression system. Both sequences have a homology of 99% with the theoretical sequence of the P4a protein. NCBI Blast comparison analysis shows that the vector sequence has a homology of 99% with the theoretical sequence of the P4a protein. The P4a polyclonal antibody still has high immunogenicity when diluted 1:4000 times. The BCA method measures the concentration of the P4a polyclonal antibody to be 2.05 mg / mL. SDS-PAGE and Western Blot identification show that the antibody is an inclusion body protein with good immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Shown is the electrophoresis result of the PCR amplification product of the P4a gene.

[0035] Figure 2 shows the result of cloning the recombinant plasmid PMD18-T-P4a, where: Figure 2A This is the electrophoresis result of PCR amplification product of cloned recombinant plasmid DNA. Figure 2B This is the sequencing result of the target gene in PMD18-T-P4a.

[0036] FIG3 shows the expression result of the recombinant plasmid pET-32a-P4a, wherein: Figure 3A This is the electrophoresis result of PCR amplification product expressing recombinant plasmid DNA. Figure 3B This is the sequencing result of the target gene in pET-32a-P4a.

[0037] Figure 4 The figure shows the SDS-PAGE analysis of P4a recombinant protein. Figure 4 In A: M: Rainbow pre-stained protein marker, 1: bacterial solution before induction; 2: bacterial solution after induction; 3: bacterial solution after sonication; Figure 4 In B: M: Rainbow pre-stained protein marker; 1: precipitate after ultrasound; 2: supernatant after ultrasound; Figure 4 In C: M: Rainbow pre-stained protein marker; 1: purified His fusion protein.

[0038] Figure 5 Shown is the BCA standard curve.

[0039] Figure 6 The figure shows the Western Blot analysis of polyclonal antibodies, in which: M: rainbow pre-stained protein marker; 1: 500-fold dilution of rabbit antiserum; 2: 1000-fold dilution of rabbit antiserum; 3: 2000-fold dilution of rabbit antiserum; 4: negative serum control; 5: 4000-fold dilution of rabbit antiserum.

[0040] Figure 7 Shown is the plasmid map of pET-32a-P4a.

[0041] Figure 8 Shown is the plasmid map of PMD18-T-P4a.

[0042] Fig. 9 Shown is the amino acid sequence alignment of P4a recombinant protein and P4a protein. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0044] In the specific implementation manner of the present invention, the carp edema virus (CEV) positive tissue sample is stored and provided by Tianjin Animal Disease Prevention and Control Center; the healthy New Zealand test rabbit (weighing about 1.5Kg) is provided by Tianjin Experimental Animal Center.

[0045] PrimeScript in the specific implementation mode of the present invention TM RT Master Mix, pMD TM18-T Vector Cloning Kit and T4 DNA Ligase were purchased from Bioray Biotechnology Co., Ltd. (Beijing); IPTG, X-Gal, pET-32a, ampicillin, enhanced DAB colorimetric kit, goat anti-rabbit IgG-HRP, HRP-labeled antibody diluent, BCA protein concentration determination kit, Rainbow 180 broad-spectrum protein Marker (11-180KD), and dry dialysis bag were purchased from Solebow Technology Co., Ltd. (Beijing); SalI and XhoI were purchased from Thermo Fisher Scientific; TRNzol, 2×Taq PCR MasterMixⅡ, DH5α, BL21, agarose gel DNA recovery kit, and rapid plasmid extraction kit were purchased from TIANGEN; nitrocellulose membrane (0.45μm), skimmed milk powder, and His-tag protein purification kit were purchased from Beyotime Biotechnology Co., Ltd.; Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma-Aldrich.

[0046] Example 1 Preparation of recombinant vector

[0047] Example 1.1 Extraction of target gene

[0048] The complete genome sequence of CEV P4a protein (GenBank No: MH645915.1) was searched in GenBank, and primers P4A-F / R were designed using Primer 5.0 software. The amplified target gene fragment was 2028 bp in length. The primer sequences are shown in Table 1. The primer sequences were synthesized and purified by Shanghai Sangon Biotechnology Co., Ltd., and the enhanced polyacrylamide gel electrophoresis (ULTRAPAGE) purification method was selected.

[0049] Table 1Sequence of Cloned Primers

[0050]

[0051] Note: The black underlined sequences are the recognition sequences of restriction endonucleases SalI and XhoI (the restriction sites are determined by the complete genome sequence of P4a protein and the pET-32a plasmid sequence)

[0052] The total RNA of CEV-positive tissue samples was extracted by Trizol method, and the RNA concentration and purity were tested by ultra-micro nucleic acid UV spectrophotometer; refer to PrimeScript TMAccording to the RT Master Mix manual, RNA was reverse transcribed into cDNA. The system was as follows: 5× PrimeScript RT Master Mix 2μl, RNA 1μl (<500ng), ddH2O 7μl; the reaction procedure was as follows: 37℃15min, 85℃5s, 4℃ temporary storage. The target gene was amplified according to the 2×Taq PCR MasterMixⅡ manual. The system was as follows: cDNA 1μl, P4A-F / R (10μM) 0.5μl, 2×Taq PCR MasterMixⅡ10μl, ddH2O 8μl; the reaction procedure was as follows: 94℃3min; 94℃30s, 62℃30s, 72℃1min, 35 cycles; 72℃5min, 4℃ temporary storage, and the amplified product was detected by 1% agarose gel electrophoresis. According to the TIANGEN agarose gel DNA recovery kit manual, the target band was recovered to obtain the purified target DNA solution.

[0053] Total RNA was extracted from CEV-positive tissue samples and detected by ultra-micro nucleic acid UV spectrophotometer. The A260 / A280 value of this RNA was between 1.8 and 2.0, indicating that this RNA can be used as a template for reverse transcription. This RNA was used as a template for reverse transcription to obtain cDNA. PCR amplification was performed using full-length primers P4A-F / R. The results of 1% agarose gel electrophoresis were as follows: Figure 1 The results show that the electrophoresis bands are consistent with the expected band sizes.

[0054] Example 1.2 Construction of prokaryotic cloning vector of CEV P4a protein

[0055] The target DNA solution obtained in Example 1.1 was connected to the pMD18-T cloning vector at 16°C overnight. The connection system is as follows: pMD18-T 1μl, DNA 1μl, ddH2O 3μl. The connection product was transferred into DH5α competent cells, spread on LB solid culture medium containing ampicillin, and cultured upright at 37°C for 1h until the liquid was completely absorbed, and then cultured inverted for 12h. Pick white positive colonies to LB liquid culture medium containing ampicillin, mix well, and culture at 37°C shaker at 150rpm for 4-6h. Extract plasmid DNA from the positive bacterial solution according to the instructions of the TIANGEN rapid plasmid extraction kit, and perform PCR amplification verification. The plasmid solution with the target band was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. After correct alignment by NCBI Blast, the cloned recombinant plasmid was named PMD18-T-P4a. The plasmid map of PMD18-T-P4a is shown below. Figure 8 shown.

[0056] The extracted cloned recombinant plasmid DNA was amplified by PCR using primers P4A-F / R, and the results were detected by 1% agarose gel electrophoresis ( Figure 2A ) showed that the electrophoresis band was consistent with the expected target band size, indicating that the plasmid DNA was CEV DNA.

[0057] The sequencing results of the cloned recombinant plasmid PMD18-T-P4a were analyzed by Blast analysis on NCBI. The results showed that the sequence had a homology of 99% ( Figure 2B ), proving that the prokaryotic cloning vector of CEV P4a protein was successfully constructed.

[0058] Example 1.3 Construction of prokaryotic expression vector of CEV P4a protein

[0059] The target band verified correctly in Example 1.2 was cut and recovered by gel cutting, and the gel recovery product and the pET-32a expression vector were double-digested at the same time. The digestion reaction system was as follows: DNA / pET-32a 2μl, SalI / XhoI 1μl, 10X FastDigestGreen Buffer 2μl, ddH2O 14μl, 37℃ reaction for 2h, and then heated at 80℃ for 5min to terminate the reaction. The double digestion product was detected by 1% agarose gel electrophoresis and then the target band was cut and recovered by gel cutting. The double digestion product after gel recovery was connected. The connection reaction system was as follows: DNA double digestion product 2μl, pET-32a double digestion product 1μl, 10X T4 DNA Ligase Buffer 1μl, T4 DNA Ligase 1μl, ddH2O 15μl, 4℃ overnight connection. The ligation product was transferred into BL21 competent cells, spread onto LB solid culture medium containing ampicillin, cultured upright at 37°C for 1 hour until the liquid was completely absorbed, and then cultured inverted for 12 hours. Pick white positive colonies to LB liquid culture medium containing ampicillin, mix well and culture at 37°C shaker at 150rpm for 4-6 hours. Extract plasmid DNA from positive bacterial solution according to the instructions of TIANGEN Rapid Plasmid Extraction Kit, and perform PCR amplification verification. The plasmid solution with the target band was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. After correct alignment by NCBI Blast, the expression recombinant plasmid was named pET-32a-P4a. The plasmid map of pET-32a-P4a is shown as follows. Figure 7 The prokaryotic expression system based on the pET-32a plasmid contains two highly regulated promoters and a His tag, which can improve the purity of the recombinant protein during protein purification and also facilitate subsequent Western Blot identification.

[0060] The extracted recombinant plasmid DNA was amplified by PCR using primers P4A-F / R, and the results were detected by 1% agarose gel electrophoresis ( Figure 3A ) showed that the electrophoresis band was consistent with the expected target band size, indicating that the plasmid DNA was CEV DNA.

[0061] The sequencing results of the expression recombinant plasmid pET-32a-P4a were analyzed by Blast analysis on NCBI. The results showed that the sequence had a homology of 99% ( Figure 3B ), proving that the prokaryotic expression vector of CEV P4a protein was successfully constructed.

[0062] Example 2 Prokaryotic expression, purification and renaturation of recombinant P4a protein

[0063] The positive bacterial solution verified in Example 1.3 was cultured in a 37°C shaker at 200 rpm until OD 600 When the concentration is about 0.6-0.8, IPTG is added to a final concentration of 1mmol / L, and the culture is continued at 37℃ and 200rpm on a shaker for 6h. The induced bacterial solution is centrifuged at 4℃ to separate the supernatant and bacterial precipitate, and the bacterial precipitate is washed twice with 0.01MPBS (pH7.2), and then 25ml PBS is added to resuspend the bacteria for low-temperature ultrasonic disruption (ultrasonication for 5s, intermittent for 5s, 90 times), and the supernatant and bacterial precipitate are separated by centrifugation at 4℃ after ultrasonication. The bacterial solution before induction, the bacterial solution after induction, the bacterial solution after ultrasonication, the supernatant after ultrasonication, and the precipitate after ultrasonication are taken for 12% SDS-PAGE to detect the protein expression form.

[0064] Take the supernatant of the recombinant protein after ultrasound, and purify the target protein in the supernatant using a 6×His tag protein purification kit. Take out the 6×His tag protein affinity pre-packed column, first rinse the Ni column with 5 volumes of deionized water, and then add 5 volumes of BufferA to balance the Ni column; take 5ml of the above supernatant and filter it through a 0.45μm filter membrane, repeat the Ni column 5 times at a flow rate of 1ml / min and collect each flow-through; after the injection, use Buffer B solution for stage elution, the volume of the eluent is 5 column volumes, the elution concentration is 10%B, 20%B, 50%B, 100%B, the elution flow rate is 2ml / min, and the eluent is collected. The eluate was added to a dialysis bag, transferred to an 8M urea solution for overnight dialysis, and then transferred to a 6M, 4M, 2M, and 0M urea solution for 6 hours. The whole process was maintained on a magnetic stirrer for slow and uniform dialysis at low temperature. The dialyzed bacterial solution was centrifuged at 4°C to separate the supernatant and precipitate, and stored at -20°C. The His-tagged P4a fusion protein, i.e., the carp edema virus P4a recombinant protein, was identified by 12% SDS-PAGE. The amino acid sequence of the carp edema virus P4a recombinant protein is shown in Seq.ID No.3. The amino acid sequence of the carp edema virus P4a recombinant protein and the carp edema virus P4a protein is compared as shown in Fig. 9 shown.

[0065] The pET-32a-P4a recombinant expression bacterial solution verified by sequencing was induced to express protein by IPTG, ultrasonically broken, and renatured with gradient urea. In the experiment, the bacterial solution before induction, the bacterial solution after induction, the bacterial solution after ultrasonication, the precipitation after ultrasonication, the supernatant after ultrasonication, and the purified His-tagged PAa fusion protein were collected for 12% SDS-PAGE detection. Figure 4 As shown, the results showed that there was a target band of expected size near 76kDa, indicating that the P4a recombinant protein was successfully expressed; after the recombinant expression bacteria were induced by IPTG, the expression level of the target protein increased, indicating that the induction effect was good; the recombinant protein after induction was subjected to low-temperature ultrasonic fragmentation, and the target protein band became more obvious, indicating that the protein was an endosomal protein.

[0066] Example 3 Preparation of polyclonal antibodies

[0067] Example 3.1 Preparation and concentration determination of polyclonal antibodies

[0068] The concentration of the supernatant after dialysis, i.e., the His-tagged P4a fusion protein, was determined. The supernatant was mixed with Freund's complete adjuvant in a 1:1 ratio and emulsified to an oil-in-water state. The experimental rabbits were immunized subcutaneously at multiple points on the back at a dose of 1 mg / kg. Subsequently, the His-tagged PAa fusion protein and Freund's incomplete adjuvant were immunized 2, 3, and 4 times every other week in the same manner. After the fourth immunization, blood was collected from the heart and serum was separated and stored at -80°C. The concentration of the rabbit antiserum was determined according to the instructions of the BCA protein concentration determination kit, and this concentration was the concentration of the polyclonal antibody.

[0069] The absorbance value of the standard protein was determined by the BCA method, and a standard curve was drawn as follows: Figure 5 As shown, the standard curve y = 1.0322x-0.1446, R 2 =0.9994>0.995, the standard curve is established and the results are reliable. The calculated polyclonal antibody concentration is 2.05 mg / mL.

[0070] Example 3.2 Western blot identification of polyclonal antibodies

[0071] The His-tagged P4a fusion protein was subjected to 12% SDS-PAGE electrophoresis, and then wet-transferred to a nitrocellulose membrane at low temperature, rinsed with PBST three times, blocked with 5% skim milk powder at 4°C overnight, and washed with PBST three times. The rabbit antiserum diluted 500, 1000, 2000, and 4000 times was used as the primary antibody, and the nitrocellulose membrane was cut into strips and placed in the diluted primary antibody solution respectively. After marking, it was incubated at room temperature for 2 hours and washed with PBST three times. The goat anti-rabbit IgG-HRP solution diluted 5000 times was used as the secondary antibody and incubated at room temperature for 1 hour and washed with PBST three times. The DAB color development working solution (the ratio of reagents A, B, and C was 1:1:18) was prepared according to the instructions of the enhanced DAB color development kit (20×), and the nitrocellulose membrane was immersed in the DAB color development working solution, incubated at room temperature in the dark until the background was light and the target band was clear, and the color development reaction was terminated by rinsing with deionized water twice. Take out the nitrocellulose membrane and place it on a gel imager to take pictures and save them.

[0072] Western Blot identification was performed using rabbit antiserum diluted 1:500, 1:1000, 1:2000, and 1:4000 as primary antibodies and goat anti-rabbit IgG-HRP solution diluted 1:5000 as secondary antibodies. Figure 6 As shown, it indicates that the target protein appears in the 76kDa attachment at all four dilutions and the immunogenicity of the antibody is high.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant vector, characterized in that: The recombinant vector includes a gene for expressing the recombinant protein of carp edema virus P4a, and the gene sequence of the gene for expressing the recombinant protein of carp edema virus P4a is shown in Seq.ID No.1 or Seq.ID No.

2.

2. The recombinant vector according to claim 1, characterized in that The recombinant vector is a prokaryotic cloning vector PMD18-T-P4a or a prokaryotic expression vector pET-32a-P4a of the carp edema virus P4a protein.

3. The recombinant vector according to claim 2, characterized in that The prokaryotic cloning vector PMD18-T-P4a was constructed by the following method: Step A1, connecting the gene expressing the carp edema virus P4a recombinant protein with the pMD18-T cloning vector to obtain a connection product; Step A2, transferring the ligation product obtained in step A1 into DH5α competent cells, spreading it on LB solid culture medium containing ampicillin, culturing it upright until the liquid is completely absorbed, and then culturing it inverted; Step A3, pick white positive colonies and add them to LB liquid culture medium containing ampicillin, mix well and culture on a shaking platform; Step A4, extracting plasmid DNA from the positive bacterial solution and performing PCR amplification verification to obtain the prokaryotic cloning vector PMD18-T-P4a.

4. The recombinant vector according to claim 2, characterized in that The prokaryotic expression vector pET-32a-P4a was constructed by the following method: Step B1, connecting the gene expressing the carp edema virus P4a recombinant protein with the pET-32a expression vector to obtain a connection product; Step B2, transferring the ligation product obtained in step B1 into BL21 competent cells, spreading it on LB solid culture medium containing ampicillin, culturing it upright until the liquid is completely absorbed, and then culturing it inverted; Step B3, pick white positive colonies and add them to LB liquid culture medium containing ampicillin, mix and culture on a shaking table; Step B4, extracting plasmid DNA from the positive bacterial solution and performing PCR amplification verification to obtain the prokaryotic expression vector pET-32a-P4a.

5. A recombinant Escherichia coli, characterized in that The recombinant Escherichia coli comprises the recombinant vector according to any one of claims 1-4, or the gene expressing the recombinant protein of carp edema virus P4a is introduced into the genome of the recombinant Escherichia coli through the recombinant vector according to any one of claims 1-4.

6. The recombinant Escherichia coli according to claim 5, characterized in that The recombinant Escherichia coli is recombined by the following method: Step 1, constructing a recombinant vector; Step 2, introducing the recombinant vector into Escherichia coli; Step 3, screening and obtaining recombinant Escherichia coli expressing the recombinant protein of carp edema virus P4a.

7. A recombinant protein of carp edema virus P4a, characterized in that: The recombinant protein of carp edema virus P4a is produced and prepared by the recombinant Escherichia coli as described in claim 5, and the amino acid sequence of the recombinant protein is shown in Seq.IDNo.

3.

8. A polyclonal antibody against carp edema virus P4a protein, characterized in that: The method is prepared by using the carp edema virus P4a recombinant protein according to claim 7.

9. The polyclonal antibody to the P4a protein of carp edema virus according to claim 8, characterized in that: The carp edema virus P4a protein polyclonal antibody is an inclusion body protein.

10. Use of the polyclonal antibody against carp edema virus P4a protein as claimed in any one of claims 8 to 9 in the preparation of immunological detection products or vaccines.

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