Anti-grouper PSMB8 protein polyclonal antibody as well as preparation method and application thereof

Abstract

The invention provides an anti-grouper PSMB8 protein polyclonal antibody as well as a preparation method and application thereof. The anti-grouper PSMB8 protein polyclonal antibody is obtained by taking recombinant protein EcPSMB8 as an antigen immune animal, and the amino acid sequence of the recombinant protein EcPSMB8 is as shown in SEQ ID NO: 1. The polyclonal antibody for resisting the grouper PSMB8 protein, provided by the invention, has high specificity on the grouper PSMB8 protein, the problem that a high-specificity antibody for the grouper PSMB8 protein is lacked in the prior art is solved, and the grouper PSMB8 protein can be better and more effectively researched.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a polyclonal antibody against grouper PSMB8 protein, a preparation method thereof, and an application thereof. Background Art

[0002] The PSMB8 gene (Proteasome Subunit Beta Type-8), also known as LMP7 (Large Multifunctional Protease 7), encodes the PSMB8 protein (also known as beta5i protein), which is one of the key subunits of the immunoproteasome. The PSMB8 protein is a beta-type proteasome subunit that mainly participates in protein degradation, especially for misfolded or damaged proteins within cells. This process is crucial for maintaining intracellular homeostasis. It has been found that the PSMB8 protein can replace certain subunits in the proteasome to form an immunoproteasome. The immunoproteasome is induced to express under the stimulation of inflammatory cytokines such as interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), enhancing the generation of antigenic peptides, thereby promoting MHC-I class molecule-mediated antigen presentation and participating in the regulation of inflammatory signaling pathways, such as rheumatoid arthritis and psoriasis. In these diseases, the expression level of PSMB8 may regulate the severity of the pathological inflammatory response. Mutations in the PSMB8 gene may cause proteasome dysfunction, resulting in abnormal protein degradation and abnormal activation of inflammatory factors. Such mutations will disrupt the protein metabolic balance of cells and induce severe inflammatory or immune diseases. Therefore, the detection of the expression level and mutations of PSMB8 can be used as a biomarker for certain immune diseases. The PSMB8 gene plays an important role in protein metabolism, immune regulation, and inflammatory responses, and its abnormal function is closely related to various diseases.

[0003] Although there are antibodies against human PSMB8 protein on the market, due to species differences, such antibodies do not have specificity for the PSMB8 protein of orange-spotted grouper and cannot accurately recognize the PSMB8 protein of orange-spotted grouper. Moreover, there is currently no antibody with high specificity for the PSMB8 protein of orange-spotted grouper. Therefore, there is an urgent need to develop an antibody against grouper PSMB8 protein with high specificity for grouper PSMB8 protein. Summary of the Invention

[0004] In order to solve the problem that the existing antibodies against human PSMB8 protein do not have specificity for the PSMB8 protein of orange-spotted grouper, the present invention provides a polyclonal antibody against grouper PSMB8 protein, a preparation method thereof, and an application thereof.

[0005] According to the first aspect of the present invention, a polyclonal antibody against grouper PSMB8 protein is provided. This polyclonal antibody is obtained by immunizing an animal with the recombinant protein EcPSMB8 as an antigen, and the amino acid sequence of the recombinant protein EcPSMB8 is as shown in SEQ ID NO: 1.

[0006] The inventors of the present application extracted grouper tissue RNA from grouper tissue, obtained grouper tissue cDNA after reverse transcription, used this cDNA as a template to amplify the gene encoding grouper PSMB8 protein (EcPSMB8 gene), constructed the EcPSMB8 gene onto the PET-32a plasmid to obtain the recombinant plasmid PET-32a-EcPSMB8, then transformed the recombinant plasmid PET-32a-EcPSMB8 into an engineering bacterium and induced the engineering bacterium to express the recombinant protein EcPSMB8 with the nucleotide sequence shown in SEQ ID NO: 1, and immunized an animal with this recombinant protein EcPSMB8 as an antigen to obtain a polyclonal antibody against grouper PSMB8 protein.

[0007] The polyclonal antibody against grouper PSMB8 protein provided by the present invention has high specificity for grouper PSMB8 protein, solves the problem of the lack of a highly specific antibody against grouper PSMB8 protein in the prior art, and can conduct better and more effective research on grouper PSMB8 protein.

[0008] Preferably, the recombinant protein EcPSMB8 is obtained by transforming the recombinant plasmid PET-32a-EcPSMB8 into an engineering bacterium and inducing expression. The recombinant plasmid PET-32a-EcPSMB8 contains the gene encoding grouper PSMB8 protein (EcPSMB8 gene), and the nucleotide sequence of the gene encoding grouper PSMB8 protein is as shown in SEQ ID NO: 2.

[0009] Preferably, the above-mentioned engineering bacterium is Escherichia coli BL21.

[0010] According to the second aspect of the present invention, a method for preparing a polyclonal antibody against grouper PSMB8 protein is provided, including the following steps:

[0011] S1. Construct a recombinant plasmid PET-32a-EcPSMB8 containing the gene encoding grouper PSMB8 protein, and the nucleotide sequence of the gene encoding grouper PSMB8 protein is as shown in SEQ ID NO: 2;

[0012] S2. After transforming the engineering bacteria with the recombinant plasmid pET-32a-EcPSMB8, induce the engineering bacteria to express the recombinant protein EcPSMB8 of grouper PSMB8. After purification, the recombinant protein EcPSMB8 with the amino acid sequence shown in SEQ ID NO: 1 is obtained;

[0013] S3. Immunize an animal with the recombinant protein EcPSMB8 to obtain immune serum, and purify the immune serum to prepare a polyclonal antibody against grouper PSMB8 protein.

[0014] The polyclonal antibody against grouper PSMB8 protein prepared by the above method has high specificity for grouper PSMB8 protein, making the binding force between the polyclonal antibody and grouper PSMB8 protein stronger.

[0015] The His tag (histidine tag) consists of 6-10 histidine residues, with a molecular weight of less than 0.84 KD, and is usually inserted at the C-terminus or N-terminus of the target protein. The His tag can bind to a metal ion column (such as a nickel column), and utilize the affinity between the histidine chelating metal and the metal ions on the column, thereby achieving efficient purification of the protein containing the His tag. The His tag is the most commonly used tag for recombinant protein expression, and can be purified by immobilized metal ion affinity chromatography (IMAC) whether the expressed protein is soluble or inclusion body. Therefore, during the process of transforming the engineering bacteria with the recombinant plasmid pET-32a-EcPSMB8 and inducing the engineering bacteria to express the recombinant protein EcPSMB8 of grouper PSMB8 and purifying it, a His tag can be inserted at the C-terminus or N-terminus of the recombinant protein, which is beneficial to the efficient purification of the recombinant protein EcPSMB8.

[0016] Preferably, in S1, the coding gene of grouper PSMB8 is amplified by the following steps: extract grouper tissue RNA from grouper tissue and then perform reverse transcription to obtain grouper tissue cDNA. Using the grouper tissue cDNA as a template, amplify the coding gene of grouper PSMB8 with the first upstream primer and the first downstream primer, wherein the nucleotide sequence of the first upstream primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the first downstream primer is shown in SEQ ID NO: 4.

[0017] Preferably, in S1, the recombinant plasmid pET-32a-EcPSMB8 is prepared by the following steps:

[0018] S1-1. Using the RT-PCR method, clone and amplify the grouper tissue RNA with the second upstream primer and the second downstream primer. After recovery and purification, the grouper tissue RNA is obtained. Among them, the nucleotide sequence of the second upstream primer is as shown in SEQ ID NO: 5, and the nucleotide sequence of the second downstream primer is as shown in SEQ ID NO: 6;

[0019] S1-2. Reverse transcribe the grouper tissue RNA to obtain grouper tissue cDNA. Cut the grouper tissue cDNA with Bam HI and HindIII. After recovery and purification, the first target fragment is obtained;

[0020] S1-3. Cut the PET-32a(+) plasmid with Bam HI and Hind III. After recovery and purification, the second target fragment is obtained;

[0021] S1-4. Mix the first target fragment and the second target fragment at a mass ratio of 3-5:1, add T4 ligase, and react at 15-17 °C for 10-14 hours to obtain a ligation product. Transform the ligation product into E. coli DH5α competent cells and culture them. Extract the plasmid to obtain the recombinant plasmid PET-32a-EcPSMB8.

[0022] Preferably, in S2, the method for expressing and purifying the recombinant protein EcPSMB8 includes the following steps: Expand the culture of the engineering bacteria until the OD 600 value reaches 0.4-0.6, add IPTG to make the IPTG concentration in the culture system 0.1-0.3 mM, induce and culture at 15-17 °C for 18-22 hours, collect the bacterial cells, purify through a column to obtain inclusion bodies, and denature and renature the inclusion bodies to obtain the recombinant protein EcPSMB8.

[0023] Preferably, S3 includes the following operations: Measure the protein concentration of the recombinant protein EcPSMB8, and immunize New Zealand rabbits subcutaneously once every 2 weeks. The immunization dose of the recombinant protein EcPSMB8 used for each immunization is 500 μg / rabbit. Immunize 4 times, extract the blood of the New Zealand rabbits to prepare antiserum, and obtain the anti-grouper PSMB8 protein polyclonal antibody.

[0024] According to the third aspect of the present invention, there is provided an anti-grouper PSMB8 protein polyclonal antibody, which is prepared by the method for preparing the anti-grouper PSMB8 protein polyclonal antibody described above.

[0025] According to the fourth aspect of the present invention, there is provided an application of the polyclonal antibody against grouper PSMB8 protein or the polyclonal antibody against grouper PSMB8 protein prepared by the preparation method of the polyclonal antibody against grouper PSMB8 protein in the preparation of a product for detecting grouper PSMB8 protein.

[0026] The polyclonal antibody against grouper PSMB8 protein provided by the present invention has high specificity for grouper PSMB8 protein. When the polyclonal antibody against grouper PSMB8 protein provided by the present invention is applied to the preparation of a product for detecting grouper PSMB8 protein, the product can have a good detection effect on grouper PSMB8 protein.

[0027] According to the fifth aspect of the present invention, there is provided a specific detection method for the polyclonal antibody against grouper PSMB8 protein, which is characterized by including the following steps:

[0028] Step 1, coating an ELISA plate with recombinant protein EcPSMB8 and PBS buffer solution with pH = 7.4 to obtain an ELISA plate coated with recombinant protein EcPSMB8;

[0029] Step 2, adding the polyclonal antibody against grouper PSMB8 protein to the ELISA plate coated with the recombinant protein EcPSMB8 and culturing at 36 - 38 °C for 2 - 4 h. After washing with PBS buffer solution, adding goat anti-rabbit-HRP and incubating at 36 - 38 °C for 0.5 - 2 h. After washing the incubated product with PBS buffer solution, performing color development with a color developing solution, and observing the position and size of the band to evaluate the specificity of the polyclonal antibody against grouper PSMB8 protein.

[0030] The specific detection method for the polyclonal antibody against grouper PSMB8 protein provided by the present invention coats recombinant protein EcPSMB8 on an ELISA plate, incubates with the polyclonal antibody against grouper PSMB8 protein as the primary antibody and goat anti-rabbit-HRP as the secondary antibody, and then performs a color development reaction. The specificity of the polyclonal antibody against grouper PSMB8 protein is evaluated according to the position and size of the band in the color development result, and the detection result is accurate. Description of the Drawings

[0031] Figure 1 It is a result diagram of identifying the PCR product of the antigen binding site of Epinephelus coioides PSMB8 by 1% agarose gel electrophoresis in Example 1.

[0032] Figure 2 It is a result diagram of identifying recombinant plasmid PET-32a-EcPSMB8 by 1% agarose gel electrophoresis in Example 1.

[0033] Figure 3Figure showing the identification results of the prokaryotic expression of the recombinant plasmid pET-32a-EcPSMB8 by 10% SDS-PAGE electrophoresis in Example 1.

[0034] Figure 4 Figure showing the identification results of the product after ultrasonic disruption of the prokaryotic expression of the recombinant plasmid pET-32a-EcPSMB8 by SDS-PAGE electrophoresis in Example 1.

[0035] Figure 5 Figure showing the identification results of the product after ultrasonic disruption of the prokaryotic expression of the recombinant plasmid pET-32a-EcPSMB8 by 10% SDS-PAGE electrophoresis and elution of the precipitate with 4M urea in Example 1.

[0036] Figure 6 Figure showing the verification results of incubating the product (1) after ultrasonic disruption of the prokaryotic expression of the recombinant plasmid pET-32a-EcPSMB8 and the unsonicated induced product (2) with grouper PSMB8 in Example 3.

[0037] Figure 7 Figure showing the verification results of incubating the product after prokaryotic expression of the recombinant plasmid pET-32a-EcPSMB8 with a polyclonal antibody against grouper PSMB8 protein in Example 3. Detailed implementation manners

[0038] The technical features in the technical solutions provided by the present invention are further clearly and completely described below in conjunction with the detailed implementation manners. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0039] Example 1

[0040] A polyclonal antibody against grouper PSMB8 protein, which is prepared by the following steps:

[0041] 1. Construction of the recombinant plasmid pET-32a-EcPSMB8

[0042] (1) Preparation of cDNA with grouper PSMB8 gene

[0043] Table 1 Primers for amplifying grouper tissue RNA

[0044] Primer NameSequence Number Specific Nucleotide Sequence Second Upstream Primer SEQ ID NO: 5 ATGGCTCTTTTCGAAGTAACTGGT Second Downstream Primer SEQ ID NO: 6 TCAGAACATTCCCTTCCTGTAGCGGT

[0045] Table 2 EcPSMB8 primers for amplifying the full-length open reading frame of EcPSMB8

[0046] EcPSMB8 Primer Sequence Number Specific Nucleotide Sequence First Upstream Primer SEQ ID NO: 3 CGGGATCCATGGCTCTTTTCGAAGTAACTGGT First Downstream Primer SEQ ID NO: 4 CCCAAGCTTGGTCAGAACATTCCCTTCCTGTAGCGGT

[0047] Anatomize healthy Epinephelus coioides, and take tissues such as its head kidney, body kidney, liver, spleen, gill, heart, intestine, stomach, brain, skin, and muscle. Extract Epinephelus coioides tissue RNA from the above tissues. Using the RT-PCR method, clone and amplify the Epinephelus coioides tissue RNA with the primers shown in Table 1 (including the second upstream primer with the nucleotide sequence shown in SEQ ID NO: 5 and the second downstream primer with the nucleotide sequence shown in SEQ ID NO: 6). After recovery, obtain Epinephelus coioides tissue RNA, and reverse transcribe the Epinephelus coioides tissue RNA to obtain Epinephelus coioides tissue cDNA. Among them, the reverse transcription system (total volume of the system is 20 μL) is as follows: Add Primer Mix, Enzyme Mix, and 5×RT Buffer in sequence according to the volume ratio of 1:1:4. Add 2000 ng of RNA sample, and make up to 20 μL with ddH 2 O. The reverse transcription program: React at 37 °C for 25 min, react at 98 °C for 5 min, and keep warm at 4 °C; Then, based on the Epinephelus coioides transcriptome expression sequence, design the EcPSMB8 primers shown in Table 2 (including the first upstream primer with the nucleotide sequence shown in SEQ ID NO: 3 and the first downstream primer with the nucleotide sequence shown in SEQ ID NO: 4). Using the Epinephelus coioides tissue cDNA as a template, amplify the full-length open reading frame (ORF) of EcPSMB8 with the EcPSMB8 primers, so as to obtain the Epinephelus coioides PSMB8 protein-coding gene (i.e., the EcPSMB8 gene), and its nucleotide sequence is shown in SEQ ID NO: 2.

[0048] SEQ ID NO: 2

[0049] ATGGCTCTTTTCGAAGTAACTGGTTTTAAGCCTTATGCTGAACTCCGTGGGC

[0050] AGATTCTTCCAGCAGGACAGACGCACCTCGTCGACCGAACCAACCACTAC

[0051] AACTTCGGGGCCAAAACTAACGAATTTGCTGTCCCTCTGGGTGTAGATCCC

[0052] TCCGGGTTTATCCAATCCTGCAGCCGTGACGGAGGTGTATGTATAGACCTGA

[0053] ACCATGGTACGACCACTCTGGCCTTCAAGTTCAGACATGGAGTCATCGTGG

[0054] CTGTGGACTCCAGAGCCTCAGCTGGTCGTTACTTAGCATCCAACGACGTCA

[0055] ACAAGGTCATAGAGATCAATCCCTACCTGCTGGGCACCATGTCAGGGAGTG

[0056] CTGCAGACTGCCAGTACTGGGAGAGACTCCTGGCCAAAGAATGCAGGCTG

[0057] TACCGGCTGAGGAACAACCACAGGATCTCTGTGGCTGCTGCCTCCAAGCT

[0058] GCTGTCTAACATGATGCTAGGTTACAGAGGCATGGGCCTCTCCATGGGAAG

[0059] CATGATCTGTGGATGGGACAAAGAGGGCCCTGGTTTGTACTATGTGGATGAT

[0060] AATGGGACACGTCTCTCGGGCCGTATGTTCTCGACCGGCTGTGGTAACAGT

[0061] TATGCCTATGGTGTGGTGGACAGCGGTTACCGGGAAGACATGACAGTGGAG

[0062] GAGGCATATGAACTGGGCCGTCGGGGCATCGCTCACGCCACACACAGAGA

[0063] TGCCTACTCTGGAGGGGTGGTCAACATGTACCACATGCAGGAGGACGGCT

[0064] GGATAAAGGTGTGTAAGGAAGATGTATCTGAGTTGATCCACCGCTACAGGA

[0065] AGGGAATGTTCTGA

[0066] (2) Preparation of recombinant plasmid pET-32a-EcPSMB8

[0067] A. Using the healthy Epinephelus coioides cDNA as a template, specific primers (including the first upstream primer with the nucleotide sequence shown in SEQ ID NO: 3 and the first downstream primer with the nucleotide sequence shown in SEQ ID NO: 4) were used to perform PCR amplification on the template to obtain a PCR product. Among them, the PCR reaction system was: 2×PCR Buffer 25 μL, dNTP 10 μL, the second upstream primer 1.5 μL, the second downstream primer 1.5 μL, DNA polymerase (KOD high-fidelity enzyme) 1 μL, cDNA 1 μL, ddH 2 O 10 μL, and the total PCR reaction system was 50 μL; the PCR amplification conditions were: 98 °C for 2 min; 95 °C for 10 s, 56 °C for 10 s, 72 °C for 1 min, for 36 cycles; 72 °C for 10 min.

[0068] After performing PCR amplification on the Epinephelus coioides cDNA, the obtained PCR product was detected by 1% agarose gel electrophoresis, and the results were as Figure 1 shown. It can be Figure 1 seen that the target gene fragment was significantly amplified.

[0069] B. The OMEGA Gel Extraction Kit was used to purify the PCR amplification product to obtain the target gene fragment, and the recovered product (i.e., Epinephelus coioides tissue cDNA) was obtained. At 37 °C, the recovered product and the empty PET-32a plasmid were double-digested with Bam HI and Hind III respectively, electrophoresed, and the target fragment was recovered and purified. The two were reacted with T4 ligase at a ratio of 4:1 at 16 °C for 12 hours to obtain a ligation product. All the ligation product was used to transform E. coli DH5α competent cells and then spread on an LB plate containing 100 mg / mL ampicillin and cultured overnight at 37 °C in an inverted position. Positive clones were selected for sequencing, and the positive clones with correct sequencing were expanded and cultured, and then the plasmid was extracted using the OMEGA Mini Plasmid Extraction Kit to obtain the recombinant prokaryotic expression plasmid PET-32a-EcPSMB8 (i.e., the recombinant plasmid PET-32a-EcPSMB8).

[0070] The recombinant plasmid PET-32a-EcPSMB8 was identified by electrophoresis, and the results were as Figure 2 shown. It can be Figure 2 seen that the Epinephelus coioides tissue cDNA was successfully ligated to the PET-32a plasmid, that is, the recombinant plasmid PET-32a-EcPSMB8 was successfully constructed.

[0071] 2. Prokaryotic expression and purification of the recombinant plasmid

[0072] A. Prokaryotic expression of the recombinant plasmid

[0073] The recombinant plasmid pET-32a-EcPSMB8 was transformed into E. coli BL21 competent cells (E. coli BL21 was purchased from Guangzhou Xinkailai Biotechnology Co., Ltd.), and the cells were spread on an LB plate containing 100 mg / mL ampicillin and cultured overnight at 37 °C in an inverted position. The positive clones screened were the engineered bacteria containing the recombinant plasmid pET-32a-EcPSMB8. Then, the engineered bacteria containing the recombinant plasmid pET-32a-EcPSMB8 were expanded in an LB liquid medium containing 100 mg / mL ampicillin, and the engineered bacteria strain containing the recombinant plasmid pET-32a-EcPSMB8 was preserved with glycerol. The preserved engineered bacteria containing the recombinant plasmid pET-32a-EcPSMB8 were inoculated into 1 mL of LA liquid medium (LA medium is an LB medium with + resistance, that is, the antibiotic ampicillin [Ampicillin] was added to the LB medium) at a volume ratio of 1:1000 and cultured overnight at 37 °C for activation. The activated engineered bacteria containing the recombinant plasmid pET-32a-EcPSMB8 were inoculated into fresh LB liquid medium (containing 100 mg / L ampicillin) at a volume ratio of 1:100 and cultured at 37 °C for about 4 hours until the OD 600 value was about 0.6. Isopropyl-β-D-thiogalactoside (IPTG) was added to the culture system to make the concentration of IPTG in the culture system 0.2 mM, and the engineered bacteria containing the recombinant plasmid pET-32a-EcPSMB8 were induced to express at 16 °C. After 20 h of induction, the bacterial liquid was collected.

[0074] The bacterial protein samples in the bacterial liquid after IPTG induction treatment were detected by 10% SDS-PAGE electrophoresis, and the detection results are as Figure 3 shown, where 0, 1:100, and 1:1000 represent the volume ratios of IPTG to the culture system. It can be Figure 3 seen that adding IPTG to the culture system can successfully induce the engineered bacteria containing the recombinant plasmid pET-32a-EcPSMB8 to express the recombinant protein HIS-EcPSMB8, and its amino acid sequence is as shown in SEQ ID NO: 1.

[0075] SEQ ID NO: 1

[0076] MALFEVTGFKPYAELRGQILPAGQTHLVDRTNHYNFGTKTKEFAVPLGVDPSG

[0077] FIQSCNRDGGVCIDLNHGTTTLAFKFRHGVIVAVDSRASAGRYLASNDVNKVI

[0078] EINPYLLGTMSGSAADCQYWERLLAKECRLYQLRNNHRISVAAASKLLSNMM

[0079] LGYRGMGLSMGSMICGWDKEGPGLYYVDDNGTRLSGHMFSTGCGNSYAYG

[0080] VVDSGYREDMTVEEAYELGRRGIAHATHRDAYSGGVVNMYHMQEDGWIKVCKEDVSELIHRYRKGMF*

[0081] In SEQ ID NO: 1, * represents a stop codon.

[0082] B. Purification and extraction of recombinant protein HIS-EcPSMB8

[0083] Take the collected bacterial solution and induce it at 16 °C for 20 h. After centrifugation at 5000 g, collect the bacterial cell precipitate. Use PBS buffer (8.0 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 、0.24 g KH 2 PO 4 ) to resuspend the bacterial cell precipitate, and use ultrasonic waves to lyse the cells. Use 10% SDS-PAGE electrophoresis to detect the bacterial protein samples in the IPTG-induced bacterial solution (whole bacteria), the supernatant after centrifugation of the bacterial solution, and the precipitate. The results are as Figure 4 shown; at the same time, centrifuge the obtained bacterial solution, take the precipitate collected after centrifugation and resuspend it with 4 M urea, repeat the operation 3 times, then resuspend it with 1% TritonX-100 (pH 8.0), centrifuge to collect the precipitate, resuspend it with PBS and then centrifuge to remove the supernatant, repeat three times. Use 10% SDS-PAGE electrophoresis to detect the bacterial protein samples in the IPTG-induced bacterial solution. The results are as Figure 5 shown.

[0084] From Figure 4 and Figure 5 electrophoresis results, it can be seen that more than 80% of the recombinant protein HIS-EcPSMB8 exists in the form of inclusion bodies in the bacterial cell precipitate, and the precipitated protein obtained by extraction is the recombinant protein HIS-EcPSMB8.

[0085] 3. Preparation of polyclonal antibody against antigen-binding site of grouper EcPSMB8

[0086] The recombinant protein HIS-EcPSMB8 was detected by 10% SDS-PAGE electrophoresis, and the purity of the recombinant protein HIS-EcPSMB8 was analyzed using the professional analysis software Grab-it 2.5. The protein concentration of the recombinant protein HIS-EcPSMB8 was determined by the BCA method. Two New Zealand rabbits weighing 2 - 2.5 kg were immunized subcutaneously, once every two weeks. The immunization dose of the recombinant protein GST-TvbS1 was 500 μg per rabbit each time, and a total of 4 immunizations were carried out. After the immunization, the blood of the New Zealand rabbits was collected to prepare antiserum, and a polyclonal antibody against grouper PSMB8 protein was obtained.

[0087] Example 2

[0088] A specific detection method for a polyclonal antibody against grouper PSMB8 protein, comprising the following steps:

[0089] Step 1: The engineering bacteria containing the recombinant plasmid PET-32a-EcPSMB8 were induced to express at a temperature of 16 °C and an IPTG concentration of 0.2 mM for 20 hours. Subsequently, centrifugation was carried out at 3000 g for 10 min, the supernatant was discarded, and the precipitate was resuspended with PBS buffer.

[0090] Step 2: The product obtained in Step 1 was subjected to Western Blot experiment. The polyclonal antibody against grouper PSMB8 protein prepared in Example 1 was used as the primary antibody, divided into 4 groups. 5% skim milk was added to each group, and each group was diluted to 250, 500, 1000, and 2000 times respectively, and incubated overnight at 4 °C. Goat anti-rabbit-HRP was used as the secondary antibody, diluted to 5000 times, incubated at 37 °C for 45 min. After that, the incubated product was washed with PBS buffer, and then developed using a chromogenic solution (ELISA ELC chromogenic solution). The position and size of the bands were observed to evaluate the specificity of the polyclonal antibody against grouper PSMB8 protein.

[0091] Example 3

[0092] The specificity of the polyclonal antibody against grouper PSMB8 protein provided in Example 1 was detected using the specific detection method for the polyclonal antibody against grouper PSMB8 protein provided in Example 2. The results are as Figure 6 、 Figure 7 shown. Among them, the polyclonal antibody against grouper PSMB8 protein used was diluted to 2000 times, and at Figure 6In it, M represents Marker. Lanes 1 - 2 show the results of Western blot detection of the expression product obtained by inducing the engineering bacteria containing the recombinant plasmid pET - 32a - EcPSMB8 to express and collecting the expression product, and directly incubating with the polyclonal antibody against grouper PSMB8 protein after ultrasonic disruption. Two replicates were made for the same sample to be tested. Lane 3 shows the results of Western blot detection of the expression product obtained by inducing the engineering bacteria containing the recombinant plasmid pET - 32a - EcPSMB8 to express and collecting the expression product, and directly incubating with the polyclonal antibody against grouper PSMB8 protein; in Figure 7 In it, from left to right, the three lanes are as follows: Lane 1 shows the results of Western blot detection of directly incubating with the polyclonal antibody against grouper PSMB8 protein after collecting the protein sample of GS (grouper spleen) cells. Lanes 2 - 3 show the results of Western blot detection of directly incubating with the polyclonal antibody against grouper PSMB8 protein after overexpressing the EcPSMB8 gene in GS cells and collecting the expression product. Two replicates were made for the same sample to be tested.

[0093] From Figure 6 and 7 it can be seen that after the polyclonal antibody against grouper PSMB8 protein prepared in Example 1 was diluted 2000 - fold, the electrophoresis bands were still relatively obvious, indicating that the polyclonal antibody against grouper PSMB8 protein prepared in Example 1 has a high specific recognition ability for the recombinant protein EcPSMB8.

[0094] In summary, in the embodiments of the present invention, through the amplification, cloning and prokaryotic expression of the grouper PSMB8 gene, a large amount of recombinant protein EcPSMB8 was rapidly and effectively prepared. Using the recombinant protein EcPSMB8 as an antigen, a polyclonal antibody against grouper PSMB8 protein was prepared, making the obtained polyclonal antibody against grouper PSMB8 protein highly specific for the recombinant protein EcPSMB8, and solving the problem that there is a lack of a highly specific antibody against grouper PSMB8 protein in existing antibodies. The polyclonal antibody against grouper PSMB8 protein provided by the present invention can better and more effectively study the grouper PSMB8 protein due to its high specificity.

[0095] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. A polyclonal antibody against grouper PSMB8 protein, characterized in that: The polyclonal antibody is obtained by immunizing an animal with the recombinant protein EcPSMB8 as an antigen. The amino acid sequence of the recombinant protein EcPSMB8 is shown in SEQ ID NO:

1.

2. The anti-grouper PSMB8 protein polyclonal antibody according to claim 1, characterized in that: The recombinant protein EcPSMB8 is obtained by transforming an engineering bacterium with the recombinant plasmid PET-32a-EcPSMB8 and inducing expression. The recombinant plasmid PET-32a-EcPSMB8 contains a grouper PSMB8 protein encoding gene, and the nucleotide sequence of the grouper PSMB8 protein encoding gene is shown in SEQ ID NO:

2.

3. The anti-grouper PSMB8 protein polyclonal antibody according to claim 2, characterized in that: The engineered bacteria is Escherichia coli BL21.

4. A method for preparing a polyclonal antibody against grouper PSMB8 protein, characterized in that: The following steps are involved: S1. constructing a recombinant plasmid PET-32a-EcPSMB8 containing a gene encoding a grouper PSMB8 protein, wherein the nucleotide sequence of the gene encoding a grouper PSMB8 protein is shown in SEQ ID NO: 2; S2. After transforming the engineering bacteria with the recombinant plasmid PET-32a-EcPSMB8, the engineering bacteria are induced to express the recombinant protein EcPSMB8 of grouper PSMB8, and after purification, the recombinant protein EcPSMB8 having an amino acid sequence as shown in SEQ ID NO: 1 is obtained; S3. Immunize animals with the recombinant protein EcPSMB8 to obtain immune serum, purify the immune serum, and prepare the anti-grouper PSMB8 protein polyclonal antibody.

5. The method for preparing the anti-grouper PSMB8 protein polyclonal antibody according to claim 4, characterized in that: In S1, the grouper PSMB8 protein encoding gene is amplified by the following steps: extracting grouper tissue RNA from grouper tissue and then performing reverse transcription to obtain grouper tissue cDNA, using the grouper tissue cDNA as a template, and amplifying the grouper PSMB8 protein encoding gene using a first upstream primer and a first downstream primer, wherein the nucleotide sequence of the first upstream primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the first downstream primer is shown in SEQ ID NO:

4.

6. The method for preparing the anti-grouper PSMB8 protein polyclonal antibody according to claim 5, characterized in that: In S1, the recombinant plasmid PET-32a-EcPSMB8 is prepared by the following steps: S1-1. Using the RT-PCR method, the grouper tissue RNA is cloned and amplified using the second upstream primer and the second downstream primer, and the grouper tissue RNA is obtained after recovery and purification, wherein the nucleotide sequence of the second upstream primer is shown in SEQ ID NO: 5, and the nucleotide sequence of the second downstream primer is shown in SEQ ID NO: 6; S1-2. Reverse transcription of the grouper tissue RNA was performed to obtain grouper tissue cDNA, and the grouper tissue cDNA was cut using Bam HI and Hind III, and the first target fragment was obtained after recovery and purification; S1-3. The PET-32a(+) plasmid was cut with Bam HI and Hind III, and the second target fragment was obtained after recovery and purification; S1-4. The first target fragment and the second target fragment are mixed in a mass ratio of 3 to 5:1, T4 ligase is added, and the mixture is reacted at 15 to 17°C for 10 to 14 hours to obtain a ligation product. The ligation product is transformed into Ecoli.DH5α competent cells and cultured, and the plasmid is extracted to obtain the recombinant plasmid PET-32a-EcPSMB8.

7. The method for preparing the anti-grouper PSMB8 protein polyclonal antibody according to claim 4, characterized in that: In S2, the expression and purification method of the recombinant protein EcPSMB8 comprises the following steps: expanding the engineering bacteria to an OD value of 600 When the value reaches 0.4 to 0.6, IPTG is added to make the IPTG concentration in the culture system 0.1 to 0.3 mM, and the culture is induced at 15 to 17° C. for 18 to 22 hours, the bacteria are collected, and the inclusion bodies are purified by column. The inclusion bodies are denatured and renatured to obtain the recombinant protein EcPSMB8. and / or, The S3 includes the following operations: measuring the protein concentration of the recombinant protein EcPSMB8, and subcutaneously immunizing New Zealand rabbits once every 2 weeks, with the immunization dose of the recombinant protein EcPSMB8 used for each immunization being 500 μg / rabbit, immunizing 4 times, extracting New Zealand rabbit blood to prepare antiserum, and obtaining the anti-grouper PSMB8 protein polyclonal antibody.

8. A polyclonal antibody against grouper PSMB8 protein, characterized in that: The polyclonal antibody is prepared by the method for preparing an anti-grouper PSMB8 protein polyclonal antibody according to any one of claims 4 to 7.

9. Use of the anti-grouper PSMB8 protein polyclonal antibody according to any one of claims 1 to 3 or the anti-grouper PSMB8 protein polyclonal antibody prepared by the method for preparing the anti-grouper PSMB8 protein polyclonal antibody according to any one of claims 4 to 7 in the preparation of a product for detecting grouper PSMB8 protein.

10. The specific detection method of anti-grouper PSMB8 protein polyclonal antibody according to claim 1, characterized in that: The following steps are involved: Step 1, coating an ELISA plate with the recombinant protein EcPSMB8 and a PBS buffer solution with a pH value of 7.4 to obtain an ELISA plate coated with the recombinant protein EcPSMB8; Step 2: Add the anti-grouper PSMB8 protein polyclonal antibody to the ELISA plate coated with the recombinant protein EcPSMB8 and culture at 36-38° C. for 2-4 hours. After washing with PBS buffer, add goat anti-rabbit-HRP and incubate at 36-38° C. for 0.5-2 hours. After washing the incubated product with PBS buffer, develop the color with a color developing solution, and observe the position and size of the band to evaluate the specificity of the anti-grouper PSMB8 protein polyclonal antibody.

Images