Pseudosciaena crocea IRE1 alpha protein polyclonal antibody as well as preparation method and application thereof

Abstract

The invention relates to the technical field of genetic engineering, in particular to a small yellow croaker IRE1 alpha protein polyclonal antibody as well as a preparation method and application thereof. The invention provides a preparation method of a small yellow croaker IRE1alpha protein polyclonal antibody. The preparation method comprises the following steps: cloning an open reading frame of a small yellow croaker IRE1alpha gene, and constructing a small yellow croaker IRE1alpha gene recombinant expression vector pET-30a (+)-IRE1alpha. Performing inducible expression and purification on the recombinant protein of the Larimichthys polyactis IRE1 alpha, and performing specific analysis on Western Blot; and preparing the polyclonal antibody of the small yellow croaker IRE1 alpha. According to the invention, a recombinant expression vector of the small yellow croaker IRE1alpha gene is constructed by adopting molecular biology and genetic engineering methods, purified recombinant protein is obtained through induced expression and affinity chromatography, the prepared polyclonal antibody can be used for expression detection of the small yellow croaker IRE1alpha protein, and an important material foundation is laid for research on functions and action mechanisms of the small yellow croaker IRE1alpha protein.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a polyclonal antibody against IRE1α protein in small yellow croaker, its preparation method, and its application. Background Technology

[0002] Environmental stress can induce endoplasmic reticulum stress (ERS) in the body or cells, thereby activating the unfolded protein response (UPR) to alleviate ERS. Inositol-requiring enzyme 1α (IRE1α) is an important transmembrane protein of the endoplasmic reticulum in the UPR signaling pathway, and the signaling pathway it mediates is the most evolutionarily conserved and important pathway in the UPR pathway. Excessive ERS prevents the intracellular environment from restoring stability in time, leading to apoptosis. ERS regulates apoptosis through three pathways: activation of transcription factor CHOP, activation of the JNK kinase pathway, and activation of Caspase-12. IRE1α is the only signaling molecule that can simultaneously activate all three pathways. Therefore, the IRE1α gene is a key signaling molecule in the induction of apoptosis and plays an important role in ERS-mediated apoptosis.

[0003] Small yellow croaker ( Larimichthys polyactis As one of my country's traditional "four major marine products," it has significant economic value. Existing research indicates that the IRE1α gene responds to high-temperature stress and Pseudomonas proteus (…). Pseudomonas plecoglossicida Infection with pathogens induces apoptosis in the liver cells of small yellow croaker, leading to severe liver damage. Therefore, in-depth research into the regulatory mechanisms of IRE1α in response to high-temperature stress and pathogen infection is beneficial for exploring the endogenous protective potential of the liver and providing a scientific basis for the prevention and treatment of liver-damaging diseases. However, the lack of commercially available antibodies that specifically recognize the IRE1α protein in small yellow croaker limits further in-depth research. Summary of the Invention

[0004] In view of the problems existing in the prior art, the purpose of this invention is to design a method for preparing a polyclonal antibody against IRE1α protein in small yellow croaker and a technical solution for its application.

[0005] The present invention is implemented using the following technical solutions: The first aspect of this invention provides a method for preparing a polyclonal antibody against IRE1α protein in small yellow croaker, comprising: (1) Cloning of the open reading frame of the IRE1α gene of small yellow croaker: Total RNA was extracted from the liver tissue of small yellow croaker. The cDNA obtained by reverse transcription of total RNA was used as a template. Based on the IRE1α gene sequence reported in GenBank database, the first specific primers IRE1α-ORF-F and IRE1α-ORF-R were designed and PCR amplification was performed to obtain the open reading frame fragment of the IRE1α gene of small yellow croaker. (2) Construction of the recombinant expression vector of IRE1α gene in small yellow croaker: IRE1α-PE-F and IRE1α-PE-R, which contain suitable restriction enzyme sites NdeI and XhoI, were designed. The cDNA obtained in step (1) was used as a template for PCR amplification. The recovered target DNA fragment was double-digested with pET-30a (+) expression vector, ligated with T4 ligase, and transformed into competent cells. Positive clones were screened to obtain the recombinant expression vector pET-30a-IRE1α. (3) Induction, purification and Western Blot specificity analysis of IRE1α recombinant protein in small yellow croaker: The recombinant expression vector obtained in step (2) was transformed into the expression strain BL21 (DE3). After expression was induced by IPTG, the bacterial cells were collected, the bacterial cells were lysed and the recombinant protein was purified by nickel affinity chromatography. The specificity of the purified protein was then verified by Western Blot. (4) Preparation of polyclonal antibody against IRE1α in small yellow croaker: The IRE1α protein purified in step (3) was used as the antigen to subcutaneously immunize New Zealand white rabbits at 400 μg / kg, and immunized once every 2-3 weeks; when the titer was greater than 1:50,000, blood was collected to prepare antiserum and purified to obtain polyclonal antibody against IRE1α protein in small yellow croaker.

[0006] Furthermore, the first specific primer includes: IRE1α-ORF-F: 5'-CATGAAAACAAGAGTTTCGGG-3', as shown in SEQ ID NO.1; IRE1α-ORF-R: 5'-CCTGGTTGTTGGCAGAGGAA-3', as shown in SEQ ID NO.2.

[0007] Furthermore, the PCR amplification system includes 2.0 μL of cDNA from the liver of small yellow croaker, 2.0 μL each of upstream and downstream primers, 25.0 μL of Mix, and ddH2O added to a volume of 50.0 μL; the PCR amplification reaction conditions include 94 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s, 60 ℃ annealing for 30 s, 72 ℃ extension for 45 s, for 35 cycles; and 72 ℃ extension for 10 min.

[0008] Furthermore, the second specific primer includes: IRE1α-PE-F: 5'-GGACATATGCATGAAAACAAGAGTTTCGGG-3', as shown in SEQ ID NO.3; IRE1α-PE-R: 5'-CTCG CTCGAGCCTGGTTGTTGGCAGAGGAA-3', as shown in SEQ ID NO.4.

[0009] Furthermore, the competent cells are Escherichia coli Trans5α, and positive clones are screened by bacterial culture PCR.

[0010] Furthermore, the PCR amplification system includes 1.0 μL of cDNA from the liver of small yellow croaker, 0.5 μL each of upstream and downstream primers, 10.0 μL of Mix, and 8.0 μL of ddH2O; the PCR amplification reaction conditions include: 98 ℃ pre-denaturation for 3 min; 98 ℃ denaturation for 10 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 35 cycles; and 72 ℃ extension for 10 min.

[0011] Furthermore, the conditions for inducing expression include bacterial culture OD... 600 When the concentration reaches 0.6-0.8, IPTG is added, with a final IPTG concentration of 0.2 mM, and the mixture is induced overnight; the molecular weight of the recombinant IRE1α protein from the small yellow croaker is 107.16 kDa.

[0012] The second aspect of the present invention provides a polyclonal antibody against IRE1α protein of small yellow croaker obtained by the preparation method described above.

[0013] The third aspect of this invention provides applications of the polyclonal antibody against IRE1α protein in small yellow croaker, including: applications in detecting the expression pattern of IRE1α protein in the liver of small yellow croaker under high temperature stress; applications in detecting the effect of high temperature stress on the expression of IRE1α protein in primary hepatocytes of small yellow croaker; and applications in detecting the expression characteristics of IRE1α protein in small yellow croaker after Pseudomonas proteus infection.

[0014] The present invention has the following beneficial effects: The polyclonal antibody against IRE1α in small yellow croaker prepared in this invention can be used for the detection of IRE1α protein expression in small yellow croaker, laying an important material foundation for the study of IRE1α protein function and mechanism of action. In-depth research into the regulatory mechanism of IRE1α response to high-temperature stress and pathogen infection is beneficial for exploring the endogenous protective potential of the liver and providing a scientific basis for the prevention and treatment of liver-damaging diseases. Attached Figure Description

[0015] Figure 1 Restriction enzyme digestion map (M: DNA marker, 1: plasmid, 2: plasmid digested with NdeI-XhoI); Figure 2 Protein expression identification by SDS-PAGE analysis (M: protein molecular weight standard, 1: pET30a induction (empty vector), 2: no induction, 3: after induction, 4: supernatant after induction and lysis, 5: precipitate after induction and lysis). Figure 3 Protein purification SDS-PAGE analysis (M: protein molecular weight standard, 1: post-disruption sample, 2: eluent, 3: elution). Figure 4 Protein analysis (M: molecular weight standard of protein, 1: 0.5 mg / mL BSA, 2: purified sample); Figure 5 Western blotting analysis of proteins (M: molecular weight standard of protein, 1: purified sample). Figure 6 SDS-PAGE analysis of purified IRE1α antibody (M is the protein molecular weight standard; 1 is the result of antibody purification analysis). Figure 7 Analysis of IRE1α protein expression characteristics in the liver of small yellow croaker under high temperature stress (green fluorescence represents IRE1α signal, blue light represents DAPI-stained cell nuclei. Scale bar is 100 μm). Figure 8 Analysis of the expression characteristics of IRE1α protein in primary hepatocytes of small yellow croaker under high temperature stress; Figure 9 Characterization of IRE1α protein expression levels in small yellow croaker 0-96 h after Pseudomonas proteus infection. Detailed Implementation

[0016] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0017] Example 1: Cloning of the open reading frame of the IRE1α gene in small yellow croaker Healthy 8-month-old yellow croakers were randomly selected, anesthetized, and rapidly dissected. Liver tissue was collected, flash-frozen in liquid nitrogen, and stored at -80°C. TRIzol was used. TM RNA was extracted using the Thermo Fisher Scientific (Thermo Fisher, Waltham, MA, USA) method, and its concentration and purity were determined using a NanoDrop 2000 (Thermo Scientific). RNA band integrity was verified by 1% agarose gel electrophoresis. cDNA was synthesized using the Hifair® Ⅲ 1st Strand cDNASynthesis SuperMix for qPCR reverse transcription kit from Hifair Biotechnology (Shanghai) Co., Ltd., and stored at -80℃ for later use.

[0018] According to reports in the GenBank database IRE1α Gene sequence-specific primers IRE1α-ORF-F (5'-CATGAAAACAAGAGTTTCGGG-3', as shown in SEQ ID NO.1) and IRE1α-ORF-R (5'-CCTGGTTGTTGGCAGAGGAA-3', as shown in SEQ ID NO.2) were designed for PCR amplification to obtain the open reading frame fragment of the IRE1α gene from the small yellow croaker. The PCR amplification system was as follows: 2.0 μL of small yellow croaker liver cDNA, 2.0 μL each of forward and reverse primers, 25.0 μL of mix, and ddH2O added to a volume of 50.0 μL. The amplification program was as follows: 94 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s, 60 ℃ annealing for 30 s, 72 ℃ extension for 45 s, 35 cycles; 72 ℃ extension for 10 min.

[0019] After the amplified product was checked for integrity by 1% agarose gel electrophoresis, it was extracted using a SanPrep column DNA gel extraction kit (Sangon Biotech, Shanghai). After confirming that the concentration was qualified, it was ligated into the pESI-T vector and then transformed into DH5α competent cells. The cells were placed in a 4 ℃ refrigerator for 30 min, heat-shocked in a 42 ℃ metal bath for 90 s, placed in a -20 ℃ refrigerator for 5 min, and then 940 μL of ampicillin-free LB medium was added. The cells were shaken at 37 ℃ and 180 rpm for 10 min and centrifuged at 5000 rpm for 1 min.

[0020] 200 μL of bacterial culture was plated and incubated at 37 ℃ for 24 h. Single colonies were then picked and placed in centrifuge tubes, and 1000 μL of LB medium containing ampicillin was added. The culture was incubated at 37 ℃ and 220 rpm for at least 4 h until the culture became turbid. Positive clones were identified by PCR using the bacterial culture as a template. The clones were then sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. After successful sequencing, the gene sequence was assembled using BioEdit software. The open reading frame sequence of the *IRE1α* gene in *Siniperca spp.* is 2640 bp, encoding 879 amino acids, with a relative molecular weight of 109.01 kDa. The open reading frame sequence of the *IRE1α* gene in *Siniperca spp.* is shown in SEQ ID NO. 5, and the amino acid sequence encoded by the *IRE1α* gene in *Siniperca spp.* is shown in SEQ ID NO. 6.

[0021] Example 2: Construction of a recombinant expression vector for the IRE1α gene in small yellow croaker Obtaining the target gene fragment Select appropriate restriction enzyme sites NdeI (CATATG) and XhoI (CTCGAG), and design specific primers containing these restriction enzyme site sequences: IRE1α-PE-F: 5'-GGACATATGCATGAAAACAAGAGTTTCGGG-3', as shown in SEQ ID NO.3; IRE1α-PE-R: 5'-CTCGCTCGAGCCTGGTTGTTGGCAGAGGAA-3', as shown in SEQ ID NO.4.

[0022] Using cDNA as a template, the target gene fragment was amplified by PCR. The PCR amplification system was as follows: 1.0 μL of cDNA from the liver of small yellow croaker, 0.5 μL each of forward and reverse primers, 10.0 μL of Mix, and 8.0 μL of ddH2O. The PCR amplification reaction conditions included: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, and 72℃ extension for 30 s, for 35 cycles; and 72℃ extension for 10 min. After agarose gel electrophoresis, the target band was selected and the DNA was extracted using a SanPrep column-based DNA gel extraction kit (Sangon Biotech, Shanghai). The recovered DNA was stored at -20℃ for later use.

[0023] Construction of pET-30a-IRE1α recombinant plasmid The recovered DNA and pET-30a (+) expression vector were double-digested with enzymes. The double-digestion reaction system consisted of: 2.0 μL 10 × K buffer, 1.0 μL each of NdeI and XhoI, and 16.0 μL DNA / pET-30a (+). After overnight digestion at 30 °C, the cells were digested again at 37 °C for 1.5 h. The products were then subjected to agarose gel electrophoresis, and the linear DNA and pET-30a (+) plasmid were excised and recovered. The recovered linear DNA and plasmid were ligated using T4 ligase. The T4 ligase system consisted of: 2.0 μL linear DNA, 3.5 μL ddH2O, 2.5 μL linear pET-30a (+), 1.0 μL T4 Ligase, and 1.0 μL Ligase buffer. The mixture was vortexed, centrifuged, and ligated at 16 °C for 1–5 h.

[0024] Next, 10 μL of the ligation product was gently added to 25 μL of competent Trans5α cells. The cells were incubated on ice for 0.5 h, then incubated in a water bath at 42 ℃ for 90 s, followed by an ice bath for 2–5 min. 700 μL of antibiotic-free LB medium (1 g tryptone, 0.5 g yeast extract, 1 g NaCl dissolved in 100 mL of pure water) was added for propagation. The cells were incubated at 220 rpm and 37 ℃ for 1–2 h. The plates were then plated (+Kana) and incubated overnight at 37 ℃. Once colonies reached an appropriate size, positive colonies were selected and propagated on TB medium (+Kana) for 2 h. Bacterial culture was identified using specific primers (IRE1α-PE-F: 5'-GGACATATGCATGAAAACAAGAGTTTCGGG-3', as shown in SEQ ID NO.1; IRE1α-PE-R: 5'-CTCGCTCGAGCCTGGTTGTTGGCAGAGGAA-3', as shown in SEQ ID NO.2). Cultures containing the target band were sent for testing. Cultures with correct sequencing were then subjected to plasmid mini-prep. Restriction enzyme digestion analysis showed two clear bands after agarose gel electrophoresis: one approximately 5.4 kb, consistent with the theoretical molecular weight of the double-digested pET-30a empty vector; and the other approximately 2.6 kb, consistent with the theoretical length (2640 bp) of the ORF region of the IRE1α gene in the small yellow croaker. This indicates that the pET-30a-IRE1α recombinant plasmid was successfully constructed. Figure 1 ).

[0025] Example 3: Induction, purification, and Western Blot specificity analysis of recombinant IRE1α protein from small yellow croaker protein expression The constructed pET-30a-IRE1α plasmid was transformed into the BL21(DE3) expression strain. After transformation, amplification, sequencing, and plasmid extraction, bacterial cultures containing the target fragment were selected. 100 μL of this bacterial culture was inoculated into 5 mL of TB medium (+Kana) and incubated overnight at 220 rpm and 37 °C. 2 mL of the overnight culture was added to 200 mL of TB medium (+Kana) and incubated at 220 rpm and 37 °C for 4 h until the OD value at 600 nm was 0.6–0.8. IPTG was added to 200 mL of the bacterial culture to a final concentration of 0.2 mM, and after induction overnight, 1 mL of the bacterial culture was centrifuged at 5000 rpm for 5 min. The supernatant was discarded, and 100 μL of 1×PBS solution was added to the precipitate. The mixture was then thoroughly mixed by pipetting, followed by 25 μL of SDS-PAGE protein loading buffer (5X). After mixing, denature in a boiling water bath for 5-10 min, cool in an ice bath, and then perform SDS-PAGE gel electrophoresis. After electrophoresis, Coomassie brilliant blue staining is used to identify the induction effect.

[0026] Protein purification The 200 mL induced bacterial culture was centrifuged multiple times in 50 mL centrifuge tubes, discarding the supernatant and retaining the precipitate in the tubes. Each centrifuge tube contained less than 100 mL of bacterial culture to prevent incomplete sonication and facilitate subsequent washing of contaminating proteins. The centrifuged bacterial culture was resuspended in 10 mL of inclusion body washing buffer (prepared in 1 L: 120.12 g urea, 20 mL 1 M Tris-HCl, 8.77 g NaCl, 0.29 g EDTA, 5 mL Triton X-100, pH 7.9), and 100 μl of 100 mg / mL lysozyme was added. The mixture was then incubated on ice for 30 min. The 50 mL centrifuge tubes were vertically inserted into a 500 mL beaker filled with crushed ice, secured with a sponge, and sonicated at 300 W for 20 min until mostly clear. The mixture was then centrifuged at 12000 rpm for 10 min at 4 ℃, and the supernatant was discarded. The precipitate was resuspended in 10 mL of inclusion body lysis buffer (prepared in 1 L: 480.48 g urea, 20 mL 1 M Tris-HCl, 8.77 g NaCl, 1 mL β-mercaptoethanol, 2 mL Triton X-100, 2.04 g imidazole, pH 7.9) and sonicated at 300 W for 15 min. It was then centrifuged at 12000 rpm for 20 min at 4 °C, and the supernatant was collected.

[0027] Before protein purification, the nickel column needs to be pretreated: New nickel columns are washed twice with 4 mL of sterile water and equilibrated with inclusion body lysis buffer for 5-10 min. Used nickel columns are washed four times with 4 mL of sterile water and equilibrated with inclusion body lysis buffer for 5-10 min. The resulting supernatant is added to the nickel column and incubated at 4°C for 2 h. 100 mL of inclusion body lysis supernatant is added to every 1 mL of nickel medium. The effluent from the nickel column is collected in a 15 mL sterile centrifuge tube. This effluent is repeated twice, and finally 1 mL of effluent is collected. The nickel column is washed with 10 mL of protein washing buffer (prepared in 1 L: 360.36 g urea, 20 mL 1 M Tris-HCl, 8.77 g NaCl, 1 mL β-mercaptoethanol, 1 mL Triton X-100, 1.36 g imidazole, pH 7.9) ten times consecutively. Add 0.6 mL of denatured protein elution buffer (prepared in 100 mL solutions: 36.36 g urea, 1 mL 1 M Tris-HCl, 0.29 g NaCl, 3.4 g imidazole, pH 4.5), and discard immediately. Then add 1 mL of denatured protein elution buffer, soak for 10 min, and collect the eluent (repeat 5 times). After use, wash the nickel column 2-5 times with sterile water, add 4 mL of 20% ethanol, and store at 4 °C.

[0028] SDS-PAGE Rubber Tapping Recycling Purification and denaturation of proteins Add 1 / 4 of the SDS-PAGE protein loading buffer (5×) to the five elution buffers mentioned above, mix well, boil in boiling water for 5-10 min, then immediately cool in an ice bath and store at -20 ℃ for later use.

[0029] SDS-PAGE validation of purified protein First, clean and dry the electrophoresis mold and glass plates, and install the gel casting plate. Pour the prepared 12% separating gel and 5% stacking gel sequentially into the casting plate (after preparing the lower separating gel, wait 30 minutes before preparing the upper stacking gel). After the stacking gel covers the entire casting plate, insert a 1 mm comb, let it stand for 1 hour, remove the comb, and store at 4°C for later use. Add 1× protein electrophoresis buffer (Tris 3.03 g, glycine 18.77 g, SDS 1 g, adjusted to 1000 mL) to the electrophoresis tank until it covers the wells. Then, add 10 μl of denatured protein sample, 1 mg / mL BSA, and 5 μl of protein marker to the wells sequentially. Electrophoresis is performed at a constant voltage of 80 V. After the bromophenol blue enters the separating gel, electrophoresis is performed at a constant voltage of 120 V until the bromophenol blue reaches the bottom of the protein gel (during electrophoresis, the electrophoresis tank is placed in a foam box containing crushed ice for cooling).

[0030] Protein purification by SDS-PAGE gel extraction and recovery Prepare the protein gel (when adding the separating gel, do not insert the comb; leave a distance of approximately 1 cm between the separating gel and the top edge of the gel casting plate). Add 1 mL of denatured protein to the gel casting plate and perform electrophoresis according to step (3b). After electrophoresis, remove the protein gel, cut off the stacking gel and the separating gel containing bromophenol blue, and develop the remaining gel in pre-cooled 0.25 M KCl solution and cut the gel, collecting it into a 10 mL EP tube. Add an appropriate amount of 1× PBS to the recovered protein gel and homogenize it using a homogenizer or mortar until it does not clog the needle when aspirating with a 1 mL syringe.

[0031] Western Blot Specificity Analysis Protein sample impurity removal The purified IRE1α recombinant protein was added to 5×SDS loading buffer at a volume ratio of 4:1. The mixture was heated in a boiling water bath for 10 min to denature the protein and separate it from impurities. After centrifugation at 12000×g for 5 min, the supernatant was used as the Western blot sample to avoid precipitation interfering with the bands.

[0032] Protein quality testing Prepare SDS-PAGE gels with 12% separating gel and 5% stacking gel. Take 10 μL of the treated sample and add 0.5 mg / mL BSA. Electrophore at a constant voltage of 80 V until bromophenol blue enters the separating gel. Adjust the voltage to 120 V and electrophore until bromophenol blue reaches the bottom of the gel. Stain with Coomassie Brilliant Blue for 2 h, and destain with destaining solution until the bands are clear. Observe whether the target band is single and free of impurities or tails to ensure that the sample purity meets the requirements for Western blotting.

[0033] Western Blot Detection After electrophoresis, proteins from the gel were transferred to a PVDF membrane using a wet transfer method. After transfer, the membrane was washed three times with TBST buffer for 5 min each time. 5% skim milk powder was added, and the membrane was blocked on a shaker at room temperature for 2 h. The blocking buffer was discarded, and the membrane was washed three times with TBST. The anti-His tag monoclonal antibody was diluted 1:1,000, and the PVDF membrane was immersed in the antibody dilution solution and incubated overnight on a shaker at 4°C. The secondary antibody was diluted 1:5,000 and incubated on a shaker at room temperature for 1 h. The location and specificity of the target bands were analyzed by chemiluminescence immunoassay.

[0034] The results showed no band in the negative control, but the band appeared in the precipitate after induction, indicating successful expression of the IRE1α recombinant protein. The molecular weight of the recombinant protein was 107.16 kDa. Figure 2 The results showed that the target band was clear after fragmentation, with no obvious impurities, indicating that the IRE1α protein was successfully purified. Figure 3The results showed that the purified sample had a single band with a high protein concentration, and the band could be specifically recognized by the anti-His tag antibody, indicating that the purified IRE1α protein was successfully obtained. Figure 4 , Figure 5 ).

[0035] Example 4: Preparation of IRE1α polyclonal antibody in small yellow croaker After determining the BCA concentration of the obtained IRE1α protein, two New Zealand white rabbits (2-2.5 kg) were immunized, with a repeat immunization every 2-3 weeks. Freund's adjuvant was administered subcutaneously at a ratio of 1:1 (antigen solution volume: adjuvant volume), with an injection of 400 μg / kg antigen. Blood samples were collected for testing. The titer of the antiserum against IRE1α protein was determined using an indirect ELISA method. Once the titer was greater than 1:50,000, final blood samples were collected to prepare the antiserum for purification. An antigen affinity purification chromatography column was prepared by coupling IRE1α protein with agarose medium. The obtained antiserum was mixed with an equal volume of PBS and slowly loaded onto the column. After antibody binding, elution with glycine elution buffer yielded the desired purified antibody. The antibody was immediately dialyzed overnight at 4°C in PBS. Purity, concentration, and titer were determined the following day. The titer of the purified antibody was detected by ELISA, and the concentration of the obtained antibody was determined using a BCA concentration assay kit. The purity of the purified antibody was observed by SDS-PAGE electrophoresis and Coomassie brilliant blue staining.

[0036] The purified IRE1α antibody concentration was 1.32 mg / mL. SDS-PAGE electrophoresis and Coomassie brilliant blue staining confirmed that the purity of the purified IRE1α antibody was above 85%. Figure 6 The titer of IREIα antibody was found to be above 3280.5 K by ELISA detection (Table 1).

[0037] Table 1. Results of indirect ELISA titer assay for IRE1α purified antibody.

[0038] Note: The initial dilution of antibody samples is 1:500; titer: the highest dilution where the sample OD / blank OD is ≥ 2.1.

[0039] Example 5: Expression patterns of IRE1α protein levels in the liver in response to high temperature stress (1) High temperature stress experiment One hundred and eighty healthy, one-year-old small yellow croakers (weight: 45.20 ± 12.42 g) of relatively uniform size, with no external injuries, were selected and cultured in six 0.5 m... 3Six circular culture tanks, each containing 30 experimental fish, were temporarily housed for one week. At the start of the experiment, the fish were randomly divided into two groups: the high-temperature group (HT) and the control group (CT), with three replicates per group. The culture water temperature was measured at 20 ± 0.5 ℃. The CT group was continuously cultured at this temperature. The HT group had its culture water temperature raised from 20 ℃ to 32 ℃ at a rate of 2 ℃ / h using a 1000 W heater and maintained at this temperature. After reaching the target temperature, small yellow croakers from the HT group (HT-0 h, 6 h, 12 h, 24 h) were collected at 0 h, 6 h, 12 h, and 24 h, with three fish per replicate. The liver tissue was quickly dissected, immersed in 4% paraformaldehyde, and stored at 4 ℃ for later use. Samples from the CT group were also collected at the same time.

[0040] (2) Immunofluorescence staining Remove the liver tissue of small yellow croaker that has been fixed for more than 24 hours in a fume hood, trim it smooth, and place it in an embedding cassette lined with gauze. Place the embedding cassette in a dehydrator and dehydrate it with graded alcohols, then soak it in paraffin. Transfer the paraffin-soaked liver tissue to the embedding machine for embedding, and trim it after the paraffin block solidifies. Place the trimmed paraffin block in a microtome for sectioning. After spreading and baking the sections, store them at room temperature for later use. After dewaxing and rehydrating the paraffin sections, antigens were added according to the antibody loading order for retrieval. After washing, circles were drawn along the tissue with a histochemical pen, and 3% BSA was added inside the circles, followed by blocking for 30 min. After blocking, primary antibody (IRE1α antibody, 1:3,000) was added, and the slides were incubated overnight at 4 °C in a humidified chamber. The slides were removed, washed with PBS, and the corresponding secondary antibody was added, followed by incubation at room temperature in the dark for 50 min. After incubation, the slides were washed with PBS, and DAPI staining solution was added to cover the tissue, followed by incubation at room temperature in the dark for 10 min. The slides were removed, washed again, and autofluorescence quencher solution B was added to the tissue. After 5 min, the slides were rinsed with running water for 10 min. Antifluorescence quenching mounting medium was added for mounting. The slides were then transferred to a fluorescence microscope for observation, image acquisition, and analysis.

[0041] The results are as follows Figure 7 As shown, no obvious IRE1α fluorescence signal was observed in the normal temperature control group, indicating that IRE1α protein was essentially not expressed. At 0 h of high-temperature treatment, IRE1α fluorescence signal began to appear in the liver. With increasing high-temperature treatment time, the fluorescence signal reached its peak at 6 h, decreased significantly at 12 h, and then significantly increased again at 24 h. This indicates that the expression pattern of IRE1α protein in the liver after high-temperature stress is characterized by an initial increase, a decrease, and a rebound.

[0042] Example 6: Effects of high temperature stress on IRE1α protein expression in primary hepatocytes of small yellow croaker Cell transfection Primary liver cells from small yellow croaker were seeded in 6-well plates (6 × 10⁶ cells per well).5 After culturing at 28 °C and 5% CO2 for 24 h, the synthesized GRP94 overexpression plasmid and siRNA were transfected using Lipofectamine™ 3000 transfection reagent (Thermo, USA).

[0043] (2) Immunoblotting Cells regulated with GRP94 were cultured at 28 °C and 32 °C for 48 h, respectively. Cells were then digested with trypsin and collected. After cell lysis, total protein was extracted using RIPA buffer, quantified by the BCA method, and adjusted to the target concentration before denaturation treatment (98 °C for 5 min). SDS-PAGE electrophoresis (100 V, 1 h) was performed, followed by transfer to a PVDF membrane (100 V, 1 h), and incubation overnight at 4 °C with primary antibodies (IRE1α antibody, 1:1,500; GAPDH antibody, 1:1,000), then incubated with secondary antibodies for 2 h. IRE1α protein expression was finally detected by chemiluminescence. The results showed that the expression level of IRE1α protein was relatively low at 28 °C. After treatment at 32 °C, its expression level significantly increased. Figure 8 ).

[0044] Example 7: Characterization of IRE1α protein expression in small yellow croaker after Pseudomonas proteus infection Small yellow croaker artificially infected with Pseudomonas proteus One hundred and eighty healthy 8-month-old yellow croakers were randomly selected and cultured in six 0.5 m deep pools. 3 Thirty experimental fish were placed in each of the circular culture tanks. They were temporarily housed in filtered seawater (18 ℃) for one week, fed twice daily (morning and evening) with formulated feed. About half an hour after feeding, feces and uneaten food were removed from the bottom of the tank, and two-thirds of the water was replaced to maintain water cleanliness. The fish were fasted for 24 hours before the challenge experiment. The six tanks of fish were randomly divided into two groups: the challenge group and the control group, with three replicates per group. *Pseudomonas proteus* strains were activated in TSA solid medium and incubated overnight in TSB liquid medium at 28 ℃ with shaking. The concentration of the microcrystal solution was calculated using colony-forming units (CFU / mL). The challenge group received an intraperitoneal injection of 0.5 mL of a 1×10⁻⁵ CFU / mL solution. 3 The control group received an intraperitoneal injection of 0.5 mL of bacteria-free TSB solution containing CFU / mL of *Pseudomonas proteus*. Liver tissue was collected from fish in the TL and CL groups at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h post-injection, with six fish in each parallel group. The liver samples were rapidly dissected, flash-frozen in liquid nitrogen, and stored at -80 °C.

[0045] Immunoblotting A certain amount of liver tissue was weighed, and total protein was extracted using RIPA buffer. Quantification was performed using the BCA method, and the concentration was adjusted to the target level before denaturation treatment (98 °C for 5 min). SDS-PAGE electrophoresis was performed (100 V, 1 h), and the sample was transferred to a PVDF membrane (100 V, 1 h). The membrane was incubated overnight at 4 °C with primary antibodies (IRE1α antibody, 1:1,500; Actin antibody, 1:1,000), followed by incubation with secondary antibodies for 2 h. Finally, IRE1α protein expression was detected by chemiluminescence immunoassay. Results are as follows: Figure 9 As shown, the expression level of IRE1α protein in the liver of small yellow croaker showed an overall upward trend after infection with *Pseudomonas proteus*. Compared with 0 h after infection, the protein expression level was significantly upregulated at 72 and 96 h after infection (P<0.05).

Claims

1. A method for preparing polyclonal antibodies of IRE1α protein of Pseudosciaena heterolava, characterized in that, Comprising: S.1 Cloning of the open reading frame of the IRE1α gene of P. olivaceus: total RNA was extracted from the liver tissue of P. olivaceus, and cDNA obtained by reverse transcription of the total RNA was used as a template, first specific primers IRE1α-ORF-F and IRE1α-ORF-R were designed according to the IRE1α gene sequence reported in the GenBank database, and the open reading frame fragment of the IRE1α gene of P. olivaceus was obtained by PCR amplification; S.2 Construction of the recombinant expression vector of the IRE1α gene of P. olivaceus: second specific primers IRE1α-PE-F and IRE1α-PE-R containing suitable enzyme digestion sites NdeI and XhoI sequences were designed, and cDNA obtained in step S.1 was used as a template for PCR amplification, the recovered target DNA fragment was double-digested with pET-30a (+) expression vector, and after ligation by T4 ligase, it was transformed into competent cells, and positive clones were screened to obtain the recombinant expression vector pET-30a-IRE1α; S.3 Induced expression, purification and Western Blot specificity analysis of the IRE1α recombinant protein of P. olivaceus: the recombinant expression vector obtained in step S.2 was transformed into the expression strain BL21 (DE3), and after IPTG induced expression, the bacterial body was collected, the bacterial body was lysed and the recombinant protein was purified by nickel affinity chromatography, and the specificity of the purified protein was verified by Western Blot; S.4 Preparation of the polyclonal antibody of the IRE1α protein of P. olivaceus: the IRE1α protein purified in step S.3 was used as an antigen, and New Zealand white rabbits were immunized subcutaneously at a dose of 400 μg / kg, and immunized once every 2-3 weeks; when the titer was greater than 1:50,000, the final blood collection was performed to prepare the antiserum, and the polyclonal antibody of the IRE1α protein of P. olivaceus was purified.

2. The method for preparing polyclonal antibody of IRE1α protein of Pseudosciaena heterolava according to claim 1, characterized in that, The first specific primers in step S.1 include: IRE1α-ORF-F: 5'-CATGAAAACAAGAGTTTCGGG-3', as shown in SEQ ID NO. 1; IRE1α-ORF-R: 5'-CCTGGTTGTTGGCAGAGGAA-3', as shown in SEQ ID NO.

2.

3. The method for preparing polyclonal antibody of IRE1α protein of Pseudosciaena heterolava according to claim 1, characterized in that, The PCR amplification system in the PCR amplification in step S.1 includes 2.0 μL of cDNA of P. olivaceus liver, 2.0 μL of each of the upper and lower primers, 25.0 μL of Mix, and ddH2O is added to a volume of 50.0 μL; the PCR amplification reaction conditions in the PCR amplification include 94 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s, 60 ℃ annealing for 30 s, 72 ℃ extension for 45 s, 35 cycles; 72 ℃ extension for 10 min.

4. The method for preparing polyclonal antibody of IRE1α protein of Pseudosciaena heterolava according to claim 1, characterized in that, The second specific primers in step S.2 include: IRE1α-PE-F: 5'-GGACATATGCATGAAAACAAGAGTTTCGGG-3', as shown in SEQ ID NO. 3; IRE1a-PE-R: 5'-CTCGCTCGAGCCTGGTTGTTGGCAGAGGAA-3', as shown in SEQ ID NO.

4.

5. The method for preparing polyclonal antibody of IRE1α protein of Pseudosciaena heterolava according to claim 1, characterized in that, The competent cell in step S.2 is E. coli Trans5a, and the positive clones are screened by broth PCR.

6. The method for preparing polyclonal antibody of IRE1α protein of Pseudosciaena heterolava according to claim 1, characterized in that, The PCR amplification system in the PCR amplification in step S.2 comprises 1.0 μL of P. olivaceus liver cDNA, 0.5 μL of each of the upper and lower primers, 10.0 μL of Mix, and 8.0 μL of ddH2O; and the PCR amplification reaction conditions comprise: 98 ℃ pre-denaturation for 3 min; 98 ℃ denaturation for 10 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 35 cycles; and 72 ℃ extension for 10 min.

7. The method for preparing polyclonal antibody of IRE1α protein of Pseudosciaena heterolava according to claim 1, characterized in that, The conditions for inducing expression described in step S.3 include bacteria liquid OD 600 The conditions for inducing expression described in step S.3 include bacteria liquid OD 600 The conditions for inducing expression described in step S.3 include bacteria liquid OD 600 The conditions for inducing expression described in step S.3 include bacteria liquid OD 600 The conditions for inducing expression described in step S.3 include bacteria liquid OD 600 The conditions for inducing expression described in step S.3 include bacteria liquid OD 600 The conditions for inducing expression described in step S.3 include bacteria liquid OD 600 The conditions for inducing expression described in step S.3 include bacteria liquid OD 600 8. The polyclonal antibody of the P. olivaceus IRE1a protein obtained by the preparation method of any one of claims 1-7.

9. The use of the polyclonal antibody of the IRE1α protein of the Pseudosciaena heteroloba according to claim 8, characterized in that, The applications include the application in detecting the expression regularity of the IRE1a protein in the P. olivaceus liver under high-temperature stress; the application in detecting the influence of high-temperature stress on the expression of the IRE1a protein in the primary hepatocytes of the P. olivaceus; and the application in detecting the expression characteristics of the IRE1a protein of the P. olivaceus after the infection of Pseudomonas plecoglossicida.

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