Preparation method and application of polyclonal antibody of long oyster insulin-like peptide receptor

Abstract

The application belongs to the field of marine biotechnology, and specifically discloses a preparation method of a long oyster insulin-like peptide receptor (ILPR) polyclonal antibody and application thereof. The method is characterized by the following steps: specific antigen epitopes of the long oyster ILPR protein are screened through bioinformatics analysis, a pET32a-ILPR recombinant plasmid is constructed and induced to express in Escherichia coli; the purified recombinant protein is used as an immunogen to immunize New Zealand white rabbits, and rabbit antisera are collected after four times of booster immunization; specific polyclonal antibodies are purified by using antigen affinity chromatography technology, and can specifically recognize the ILPR protein in long oyster tissues. The antibody prepared by the application has high specificity and sensitivity, and provides an important biological tool for studying the insulin signal transduction pathway, growth regulation and energy metabolism mechanism of long oysters.

Description

Technical Field

[0001] This invention belongs to the field of marine biotechnology, specifically relating to a polyclonal antibody against the insulin-like peptide receptor (ILPR) of the oyster, as well as the preparation method and uses of the polyclonal antibody. Background Technology

[0002] In vertebrates, growth is centrally regulated by the hypothalamic-pituitary growth axis. Under the dual influence of hypothalamic growth hormone-releasing factor (GHRH) and somatostatin (SS), growth hormone (GH) is secreted by pituitary growth hormone-secreting cells (Somatotropes). GH travels through the circulatory system to the liver, inducing the expression of insulin and insulin-like growth factor (IGF). Insulin / IGF bind to their respective target cell surface receptors, mediating GH's growth regulation. However, in invertebrates such as oysters, which lack brain and pituitary structures, how is growth regulated? To date, no GH or receptor genes or GH-related regulatory factors homologous to vertebrates have been found in invertebrates. However, insulin-like peptides, insulin-like peptide receptors, and their binding proteins have been discovered, playing important roles in growth regulation and energy homeostasis in invertebrates.

[0003] Vertebrates possess two receptor genes, IR and IGFR. Both IR and IGFR are heterodimers composed of α and β subunits. The α subunit contains a ligand-binding site that binds to free insulin, while the β subunit contains a tyrosine kinase domain for ligand signal transduction. Insulin / IGF binds to IR / IGFR on the cell membrane, triggering a signaling cascade network. Free insulin in plasma binds to the extracellular α subunit, activating the intracellular β subunit, which undergoes autophosphorylation and exerts tyrosine kinase activity. This transmits the insulin signal to the IRS, inducing IRS phosphorylation and activating downstream PI3K-AKT and RAS-MAPK signaling pathways, amplifying the insulin signal cascade and regulating cell proliferation and energy metabolism. Unlike vertebrates, which possess two highly homologous insulin signaling receptors (IR and IGFR), most invertebrates have only one insulin-like peptide receptor identified.

[0004] In our previous research, through extensive multi-omics data mining, we identified an insulin-like peptide receptor gene, ILPR, in the genome of the Pacific oyster. We found that ILPR is significantly influenced by artificial selection, and its expression level in the rapidly growing Pacific oyster "Haida No. 1" is significantly higher than in wild oysters. Our studies also revealed higher expression levels in neurally rich tissues, suggesting that the insulin-like peptide receptor may play an important role in the growth of the Pacific oyster. Furthermore, although the insulin receptor is a cell membrane receptor, there has long been evidence that it exists in the cell nucleus, leading to the hypothesis that the insulin receptor itself can directly participate in gene transcriptional regulation (Goldfine and Smith 1976; Podlecki et al. 1987). Recently, in mammals such as humans and mice, it has been confirmed that the insulin receptor can respond to glucose stimulation, migrate to the cell nucleus, interact with RNA polymerase II (Pol II), bind to the promoter regions of numerous genes, activate the expression of these genes, and thus participate in lipid metabolism, protein synthesis, and transcriptional regulation. However, is this insulin receptor-mediated transcriptional regulation mechanism evolutionarily conserved in invertebrates such as oysters? Does the oyster insulin-like peptide receptor (ILPR) regulate the growth process through a similar transcriptional regulatory mechanism? To answer these questions, the precise localization of the oyster insulin-like peptide receptor protein is first required.

[0005] However, currently available commercially available antibodies are specific to the insulin receptor in higher animals. Due to the significant evolutionary differences between mollusks and vertebrates, the homology of receptor protein sequences is low. These cross-species antibodies struggle to specifically bind to the insulin-like peptide receptor in the Pacific oyster, failing to accurately identify the target protein. This limits their application in protein expression detection, tissue localization, and related physiological studies. Therefore, developing a Pacific oyster-specific ILPR polyclonal antibody to overcome these technological limitations and use it as a tool to further elucidate the growth regulatory network it mediates is of significant scientific value and practical application. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a polyclonal antibody against insulin-like peptide receptor from oyster. Another purpose of this invention is to provide a specific application of the oyster insulin-like peptide receptor-specific polyclonal antibody (anti-ILPR).

[0007] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows: A polyclonal antibody against the insulin-like peptide receptor of *Crassostrea gigas* is obtained by immunizing rabbits with the antigen protein expressed by the pET32a-ILPR recombinant plasmid. The antigenic epitopes used to prepare the *Crassostrea gigas* insulin-like peptide receptor antibody are anti-ILPR, and their corresponding antigenic epitopes are shown in SEQ ID NO. 1. After expressing the above amino acid sequences in a plasmid, the antigen protein is obtained through induction expression and purification.

[0008] Biological material containing the above-mentioned antigenic polypeptide encoding gene, wherein the biological material is an expression plasmid and a host bacterium, wherein the expression plasmid is pET32a-ILPR, and the host bacterium is a Rosetta(DE3) expression strain containing the above-mentioned recombinant plasmid.

[0009] The method for preparing the insulin-like peptide receptor polyclonal antibody includes the following steps: predicting the signal peptide of the insulin-like peptide receptor, removing the signal peptide sequence, and then analyzing the antigenicity of the remaining amino acid sequence of the insulin-like peptide receptor; constructing a recombinant plasmid: designing a pair of specific primers for the insulin-like peptide receptor to amplify the antigen fragment, ligating the amplified antigen fragment to the prokaryotic expression vector pET32a (the vector contains a 6×His tag that can be used for subsequent verification of the recombinant protein), and constructing the pET32a-ILPR recombinant plasmid; introducing the above recombinant plasmid into Rosetta(DE3) for induced expression, and purifying the recombinant protein; emulsifying the recombinant protein and administering it to rabbits via multiple subcutaneous injections, followed by five immunizations; capturing the required immunized rabbits, anesthetizing them by intravenous injection of 1 ml of 3% sodium pentobarbital per kilogram of blood, collecting and separating the rabbit serum containing the antibody using cardiac blood collection, and purifying the serum using Protein A / G affinity chromatography to obtain the insulin-like peptide receptor polyclonal antibody.

[0010] The aforementioned polyclonal antibody against insulin-like peptide receptor protein can be used for tissue expression and localization of insulin-like peptide receptor in oyster, and for constructing a gene expression regulatory network mediated by insulin-like peptide receptor in oyster.

[0011] Advantages and beneficial effects of this invention: This invention constructs a recombinant expression plasmid, induces the expression of a recombinant protein, and uses the recombinant protein as an antigen to immunize rabbits, thereby obtaining a specific polyclonal antibody against the insulin-like peptide receptor of the oyster *Crassostrea gigas*. The polyclonal antibody against the insulin-like peptide receptor of the oyster *Crassostrea gigas* provided by this invention has the advantages of high specificity and high sensitivity, and can meet the experimental requirements of immunohistochemistry, Western blotting, and laser confocal microscopy.

[0012] This invention provides a foundation for establishing in vitro immunoassays and studying the physiological function of the insulin-like peptide receptor (IRP) in the Pacific oyster. It holds significant promise for applications in the expression and localization of the IRP protein in the Pacific oyster, and lays the groundwork for revealing the interacting proteins and target genes regulated by the IRP after it enters the cell nucleus, as well as for constructing a gene expression regulatory network mediated by the Pacific oyster's IRP receptor. Attached Figure Description

[0013] Figure 1 This is a graph showing the predicted signal peptide of the insulin-like peptide receptor protein in the oyster.

[0014] Figure 2 Figure showing the predicted hydrophobicity of insulin-like peptide receptor protein in oyster.

[0015] Figure 3 This is a diagram showing the predicted transmembrane region of the insulin-like peptide receptor protein in the oyster.

[0016] Figure 4 Figure showing the predicted phosphorylation sites of insulin-like peptide receptor protein in oyster.

[0017] Figure 5 This is a diagram showing the predicted results of the insulin-like peptide receptor protein antigenic epitope in the oyster.

[0018] Figure 6 This is an SDS-PAGE image showing the results of induction of recombinant insulin-like peptide receptor protein from oyster. M represents the molecular weight of the protein; IPTG concentration: 0.6 mol / L; temperature and time: 37℃ for 6 h and 16℃ for 16 h. Lane 1 represents the sample before induction; lane 2 represents the sample after induction at 37℃; lane 3 represents the total protein after lysis at 37℃; lane 4 represents the precipitated protein after lysis at 37℃; lane 5 represents the supernatant protein after lysis at 37℃; lane 6 represents the sample after induction at 16℃; lane 7 represents the total protein after lysis at 16℃; lane 8 represents the precipitated protein after lysis at 16℃; and lane 9 represents the supernatant protein after lysis at 16℃.

[0019] Figure 7 The figure shows the purification results of recombinant insulin-like peptide receptor protein from oyster; where M represents the molecular weight standard of the protein, lane 1 represents the sample before induction, lane 2 represents the sample after induction, lane 3 represents the total protein after disruption, lane 4 represents the supernatant protein after disruption, lane 5 represents the precipitated protein after disruption, lanes 6-7 represent the washing buffer, and lane 8 represents the elution buffer.

[0020] Figure 8 The results validated the His monoclonal antibody against the recombinant insulin-like peptide receptor protein from the long oyster.

[0021] Figure 9 This is the result of Western blot specific detection of insulin-like peptide receptor antibody in oyster.

[0022] Figure 10 The results of immunofluorescence analysis of insulin-like peptide receptor in visceral ganglion tissue from Ostrea gigas are shown. Detailed Implementation

[0023] 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. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1: Preparation of polyclonal antibodies against insulin-like peptide receptor protein Specific polyclonal antibodies were designed targeting the ILPR protein of the Pacific oyster. First, the nucleotide sequence of the ILPR gene was deduced into an amino acid sequence using ORF software. Then, bioinformatics software was used to analyze the function and structure of the Pacific oyster ILPR protein. Specific software and websites are listed in Table 1. The physicochemical properties of the ILPR protein are as follows: its relative molecular mass is 178.28 kDa, and its isoelectric point is 5.92, classifying it as an unstable protein. Signal peptide prediction using Signal 6.0 software showed that the protein contains amino acids 1-21 of the signal peptide (…). Figure 1 The hydrophilicity / hydrophobicity results of the ILPR protein showed that the hydrophilic region was significantly larger than the hydrophobic region, classifying it as a hydrophilic protein. Figure 2 Transmembrane structure prediction shows that the ILPR protein has one transmembrane region located at amino acids 1034-1056. Figure 3 Protein phosphorylation site prediction results showed that ILPR has 29 tyrosine phosphorylation sites, 69 serine phosphorylation sites, and 50 threonine phosphorylation sites. Figure 4 ); Antigenic epitope prediction results found ( Figure 5 The ILPR protein contains 65 B-cell antigenic epitopes.

[0025] Based on the above analysis results, and considering factors such as antigenicity, structural stability, and expression feasibility, this embodiment selects a fragment located at amino acid positions 1098-1529 of the ILPR protein (amino acid sequence as shown in SEQ ID NO.1) as the immunogenic antigen. The segment selected in this embodiment is only a preferred option; other immunogenic fragments derived from the ILPR protein can also be used for the preparation of the polyclonal antibody of this invention.

[0026] Table 1 Bioinformatics Analysis Software Construction of recombinant plasmid: Specific primers anti-ILPR-F (SEQ ID NO.2) and anti-ILPR-R (SEQ ID NO.3) were designed for the insulin-like peptide receptor to amplify the corresponding antigen fragment. The amplified antigen fragment was then ligated to the prokaryotic expression vector pET32a to construct the pET32a-ILPR recombinant plasmid.

[0027] Recombinant protein expression and purification: The recombinant plasmid was introduced into *E. coli* Rosetta (DE3) for expression. The recombinant protein was purified according to the His Trap™ HP (cytiva) instructions. The results of induced expression are as follows: Figure 6 As shown. Figure 7 The results show the purification of the ILPR recombinant protein. The results indicate that the purified oyster insulin-like peptide receptor recombinant protein exhibits a single band around 70 kDa.

[0028] After emulsifying the recombinant protein, New Zealand white rabbits were immunized subcutaneously four times. One mg of immunogen was dissolved in 500 µL of 0.01 mol / L PBS and mixed with an equal volume of 500 µL of Freund's complete adjuvant (incomplete Freund's adjuvant was used for subsequent booster immunizations). The mixture was emulsified (a drop of emulsion forming a spherical shape on the water surface without spreading indicates sufficient emulsification). The emulsified recombinant protein was used as the antigen to immunize rabbits four times via subcutaneous injection. Approximately one week after the last immunization, blood was collected from the tail, and rabbit antiserum was collected. After standing at 4 ℃ for 1 h, the serum was centrifuged at 3500 g, and the supernatant was collected. The antibody titer in the serum was detected using ELISA. After the titer reached the target, blood was collected from the retro-orbital venous plexus to collect immune serum, which was stored at -80 ℃ for later use.

[0029] Rabbit serum containing antibodies was collected and separated, and the serum was purified by antigen affinity chromatography to obtain the antibodies.

[0030] Example 2: Validation of a specific His monoclonal antibody against recombinant insulin-like peptide receptor from oyster shell. To verify the specificity of the purified recombinant protein, the purified protein was subjected to SDS-PAGE electrophoresis (separating gel 80 V 40 min, stacking gel 120 V 120 min). The target protein was then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk at 37°C for 2 h. The membrane was washed twice (5 min each time) with TBST buffer on a shaker. His monoclonal antibody (1:1000 dilution) was incubated overnight at 4°C on a shaker. The membrane was then washed three times (15 min each time) with TBST buffer on a shaker. Subsequently, the membrane was incubated with secondary antibody at 37°C for 1 h (1:2000 dilution), and washed three times (15 min each time) with TBST buffer on a shaker. ECL chemiluminescence solution was poured onto the membrane, and exposure and color development were performed using a chemiluminescent gel imaging system. Antibody specificity was then detected. The analytical results are as follows: Figure 8 As shown, the purified ILPR proteins all exhibited a single band at 70 kDa, indicating that the recombinant proteins obtained after prokaryotic expression and induction purification were of the correct size and had good specificity, and could be used as antigens for subsequent animal immunization.

[0031] Example 3: Determination of the titer of polyclonal antibodies To determine the titer of the obtained polyclonal antibodies, rabbit serum collected before the first immunization in the polyclonal antibody preparation steps of Example 1 was used as a negative control. Serum collected after the last immunization was diluted between 1:250 and 1:1024000 to serve as the primary antibody. HRP-labeled goat anti-rabbit IgG was used as the secondary antibody, and TMB was used as the chromogenic solution. The OD of the post-immunization serum samples was determined. 450 A titer ≥0.6 was used as the judgment criterion. The test results are shown in Table 2. The results show that the antibodies obtained in the rabbit serum all have good specificity, and the antibody titer of anti-ILPR is 64 K.

[0032] Table 2. Potency determination of rabbit antiserum against insulin-like peptide receptor Example 4: Specificity verification of polyclonal antibody against insulin-like peptide receptor from oyster shell Because the folding state of recombinant proteins may differ from that of natural proteins, to verify whether the antibody prepared in this invention can effectively recognize the natural ILPR protein in Pacific oysters, total protein was extracted from Pacific oyster tissue samples and subjected to SDS-PAGE electrophoresis (separating gel 80V 40 min, stacking gel 120V 120 min). The target protein was transferred to a PVDF membrane; PVDF was blocked with 5% skim milk powder at 37 ℃ for 2 h or overnight at 4 ℃; the membrane was washed three times with TBST buffer on a shaker for 5 minutes each time; rabbit antiserum (1:5000 dilution) was incubated overnight on a shaker at 4 ℃; the membrane was washed three times with TBST buffer on a shaker for 15 minutes each time; subsequently, secondary antibody incubation (1:2000 dilution) was performed, and the membrane was washed three times with TBST buffer on a shaker; ECL chemiluminescent solution was poured onto the membrane, and exposure and color development were performed using a chemiluminescent gel imaging system. Antibody specificity was detected. The analytical results are as follows. Figure 9 As shown, the prepared polyclonal antibody against insulin-like peptide receptor in oysters identified a single band (approximately 95 kDa) in oyster tissue, demonstrating that the prepared polyclonal antibody has good specificity and can be used for subsequent quantification and localization of insulin-like peptide receptor in oysters.

[0033] Example 5: Immunofluorescence analysis of insulin-like peptide receptor protein in oyster. The visceral ganglion tissue of *Oyster amurensis* was embedded in a suitable sample holder using cryo-tissue embedding (OCT) medium and sectioned to a thickness of 17 μm. The sections were fixed with 4% paraformaldehyde at room temperature for 15 min and washed three times with PBS. They were then permeabilized with PBS containing 0.1% Triton X-100 for 10 min and washed three times with PBS (5 min each time). The sections were blocked with PBS containing 5% bovine serum for 30 min at room temperature. A 1:500 dilution of Insulin Receptor Rabbit pAb primary antibody was added, and the sections were incubated overnight at 4°C; the sections were washed three times with PBS (15 min each time). Then, a 1:1000 dilution of CY3-labeled rabbit antibody was added, and the sections were incubated at room temperature in the dark for 60 min; the sections were washed three more times with PBS (15 min each time). The sections were stained with DAPI for 30 min, washed six times with PBS (5 min each time), and mounted with anti-fluorescence quenching mounting medium. Finally, the sections were observed and photographed under a confocal microscope. The results are as follows: Figure 5 As shown in the figure, immunohistochemical analysis revealed a specific positive signal for the insulin-like peptide receptor (ILPR) in the visceral ganglion of the Pacific oyster. This result indicates that the Pacific oyster ILPR polyclonal antibody prepared in this invention can specifically bind to the natural antigen in tissue sections, exhibiting extremely high sensitivity and spatial localization accuracy. This antibody can not only be used for qualitative analysis of ILPR proteins but also serve as a powerful tool for studying the insulin signaling pathway and neuroendocrine regulatory mechanisms in Pacific oysters, demonstrating promising scientific research applications and commercial value.

[0034] The above results demonstrate that this invention has the following advantages compared to existing technologies: This invention employs molecular biology and genetic engineering methods to construct a recombinant expression vector for the insulin-like peptide receptor in *Crassostrea gigas*, inducing expression and obtaining purified recombinant protein via affinity chromatography. The polyclonal antibody against the insulin-like peptide receptor in *Crassostrea gigas* prepared by this invention exhibits strong specificity and can be used for experiments requiring immunohistochemistry, immunoblotting, and laser confocal microscopy, establishing in vitro immunoassays and providing an important tool for in-depth research on the physiological function of insulin-like peptides in the growth of *Crassostrea gigas*. The recombinant protein against the insulin-like peptide receptor in *Crassostrea gigas* provided by this invention can act as an active factor, enabling a series of related in vitro studies. The polyclonal antibody against the insulin-like peptide receptor in *Crassostrea gigas* prepared by this invention can be widely applied to the biological immunoassay of *Crassostrea gigas*. The recombinant protein against the insulin-like peptide receptor in *Crassostrea gigas* provided by this invention can serve as an active factor, enabling a series of related in vitro studies. This invention solves the problem of the lack of commercially available specific antibodies against the insulin-like peptide receptor in *Crassostrea gigas*, providing important technical support for further exploring the key role of insulin-like peptides in the growth of *Crassostrea gigas*.

[0035] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A polyclonal antibody against the insulin-like peptide receptor (ILPR) of the long oyster, characterized in that, The insulin-like peptide receptor antibody is anti-ILPR, and its corresponding antigenic epitope is shown in SEQ ID NO.

1.

2. A biomaterial containing the antigenic epitope as described in claim 1, characterized in that, The biological materials are an expression plasmid and a host bacterium. The expression plasmid is pET32a-ILPR, and the host bacterium is a Rosetta (DE3) expression strain containing the above recombinant plasmid.

3. The method for preparing the insulin-like peptide receptor protein polyclonal antibody includes the following steps: S1. Predict the signal peptide of the insulin-like peptide receptor, remove the signal peptide sequence, and then analyze the antigenicity of the remaining amino acid sequence of the insulin-like peptide receptor. S2. Construction of recombinant plasmid: A pair of specific primers targeting the insulin-like peptide receptor were designed to amplify the antigen fragment. The amplified antigen fragment was then ligated to the prokaryotic expression vector pET32a to construct the pET32a-ILPR recombinant plasmid. S3. The above recombinant plasmid was introduced into Escherichia coli Rosetta (DE3) for expression, and the recombinant protein was purified; S4. After emulsifying the recombinant protein, New Zealand white rabbits were injected subcutaneously and immunized four times. S5. Collect and separate rabbit serum containing antibodies, and purify the serum using antigen affinity chromatography to obtain polyclonal antibodies against insulin-like peptide receptor protein.

4. The application of the polyclonal antibody against insulin-like peptide receptor in oyster as described in claim 1 in detecting ILPR protein expression in oyster.

5. The application as described in claim 3, characterized in that, The polyclonal antibody was used as the primary antibody to specifically bind to the ILPR protein in the oyster sample to be tested. The self-expression of ILPR protein in oyster was detected and analyzed by secondary antibody color development or signal detection.

6. The application as described in claim 3, used for tissue expression and localization of insulin-like peptide receptor in oyster, and for the construction of a gene expression regulatory network mediated by insulin-like peptide receptor in oyster.

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