Greater wax moth taste receptor GR43a antigen polypeptide, polyclonal antibody as well as preparation method and application of greater wax moth taste receptor GR43a antigen polypeptide and polyclonal antibody

Abstract

The invention discloses a greater wax moth taste receptor GR43a antigen polypeptide, a polyclonal antibody as well as a preparation method and application thereof. In order to solve the problems of non-specific binding, high background value and high cost of the existing greater wax moth taste receptor GR43a polyclonal antibody, a model insect greater wax moth is used as a material, the greater wax moth taste receptor GR43a is selected as a research object, bioinformatics analysis and antigen epitope prediction are performed on GR43a protein, and the polyclonal antibody is obtained. A polypeptide segment as shown in SEQ ID No.1 is selected for preparing a polyclonal antibody, the titer of the prepared greater wax moth taste receptor GR43a polyclonal antibody is 1: 128000, the greater wax moth taste receptor GR43a polyclonal antibody can be specifically combined with greater wax moth taste receptor GR43a protein, and the greater wax moth taste receptor GR43a protein can be accurately recognized or positioned by adopting methods such as immunohistochemical positioning and immunoblotting; the method has the advantages of simple preparation route, strong specificity, high titer, low cost and the like.

Description

Technical Field

[0001] The present invention relates to insect taste receptor polyclonal antibodies, in particular to a greater wax moth (Galleria mellonella) taste receptor GR43a antigen polypeptide, polyclonal antibodies and preparation methods and applications thereof, belonging to the field of greater wax moth taste receptor GR43a polyclonal antibodies and preparation and application thereof. Background Art

[0002] Insects possess an extremely sensitive taste sensory system that plays a vital role in numerous life processes, including feeding, mating, and reproduction. Specific taste receptors within the insect taste sensory system sense and recognize food chemical cues, thereby determining whether to feed continuously and in large quantities. Taste receptors play a key role in insect food selection. Studying the mechanism of action of insect taste receptors is crucial for elucidating insect food selection mechanisms and developing agents to modulate insect feeding behavior.

[0003] The Greater Wax Moth (Gallus wax moth) is a worldwide pest of honey bees and one of the most significant enemies of the Chinese honey bee. It not only disrupts the normal activities of Chinese honey bees and can even cause bee colonies to abandon their nests, directly impacting the conservation of Chinese honey bee resources. This reduces pollination in mountain forests, diminishes plant diversity, and leads to declines in the number of bird and insect species associated with plants, causing widespread outbreaks of pests and diseases, and causing adverse ecological impacts. Current research on the GGM moth focuses on two key areas: First, from the perspective of bee colony pest management, researchers have conducted extensive research on the pathology of the moth, using biological control methods such as parasitic wasps, pathogenic nematodes, and pathogenic bacteria, chemical control methods such as biopesticides, and physical control methods such as temperature manipulation. This has provided technical support for comprehensive GGM control. However, in production, beekeepers still often use chemical fumigation to store honeycombs to prevent GGM moth infestation, resulting in drug residues on the honeycombs, causing a series of problems including food safety and environmental pollution. Therefore, there is an urgent need to develop and implement environmentally friendly bee pest control technologies, such as repellents and antifeedants that modulate their behavior. Secondly, from the perspective of insect resource utilization, researchers have found that the larvae of the greater wax moth are rich in protein and can be used as bait for some small animals. Because of its advantages such as short growth cycle and easy breeding, the greater wax moth has been widely used as a model insect for scientific research. In 2017, Spanish researchers discovered that the larvae of the greater wax moth can also gnaw on plastic (polyethylene) and mineralize and degrade the plastic, providing another important idea for the control of "white pollution". Therefore, whether as a bee pest or a resource insect, the greater wax moth has very important research value. Studying the feeding behavior of the greater wax moth based on taste perception is also of great practical significance for controlling its harm and resource utilization.

[0004] During its long evolutionary process, the wax moth has developed a unique diet, whereby its larvae grow and develop by feeding on honeycombs rich in honey (primarily composed of fructose and glucose). Currently, the mechanism of action of the wax moth's taste receptors remains unclear, hindering our understanding of the wax moth's taste perception and diet selection mechanisms, the co-evolutionary mechanisms of the wax moth and bees, and hindering efforts to control pests by regulating feeding behavior through taste perception. In 2017, the whole-genome sequencing data of the wax moth (Accession No. PRJNA401315) was published; in 2019, the whole-genome sequence of the wax moth was further optimized and improved based on the novel Pacbio sequencing technology (Accession No. PRJNA386430). These genomic data lay the foundation for studying the perception mechanism of the wax moth's taste receptors.

[0005] Insect taste receptors can be divided into four major categories: sugar receptors, bitter receptors, CO2 receptors, and GR43a-like receptors. GR43a taste receptors function as fine-tuning fructose receptors in insect gustatory neurons. As an important nutrient sensor in insects, the GR43a membrane protein may play a key role in the feeding selection process of the greater wax moth larvae. By identifying the fructose taste receptor gene gr43a in the greater wax moth, and using techniques such as qPCR and immunohistochemistry to localize the odorant receptor GR43a membrane protein, we can investigate its biological function. Therefore, the development of specific antibodies is of great significance for the recognition of taste receptor ligands, the signal transduction mechanism, and the study of receptor localization.

[0006] The molecular biology method based on PCR technology has complicated operation steps. Generally, DNA recombination technology is used to add a specific tag to the target protein. After fusion expression of the commercial His tag and the target protein, affinity purification is performed through nickel columns (Ni++affinityresins), which usually does not affect the biological activity and intracellular localization of the target protein. Although His-tagged antibodies are versatile, there is potential non-specific binding, resulting in high background. Secondly, there is also the issue of cost. High-quality His-tagged antibodies, especially imported products, are relatively expensive and have their limitations. His-tagged antibodies are only suitable for detecting proteins with His tags, and the genetic manipulation of tagging requires a more complex technical system to support them. Summary of the Invention

[0007] One of the purposes of the present invention is to provide a G. mellonella taste receptor GR43a antigen polypeptide;

[0008] The second object of the present invention is to provide a fusion protein obtained by coupling the G. mellonella taste receptor GR43a antigen polypeptide and a tag protein;

[0009] The third object of the present invention is to provide a gene encoding a G. mellonella taste receptor GR43a antigen polypeptide or a gene encoding a fusion protein obtained by coupling the G. mellonella taste receptor GR43a antigen polypeptide and a tag protein;

[0010] A fourth object of the present invention is to provide a recombinant vector containing a gene encoding the G. mellonella taste receptor GR43a antigen polypeptide or a gene encoding a fusion protein obtained by coupling the G. mellonella taste receptor GR43a antigen polypeptide and a tag protein;

[0011] The fifth object of the present invention is to provide a recombinant host bacteria containing the recombinant vector;

[0012] A sixth object of the present invention is to provide a polyclonal antibody to the wax moth taste receptor GR43a prepared using the wax moth taste receptor GR43a antigen polypeptide, a fusion protein obtained by coupling the wax moth taste receptor GR43a antigen polypeptide and a tag protein, an encoding gene, a recombinant vector or a recombinant host bacterium;

[0013] The seventh object of the present invention is to apply the polyclonal antibody against the G. mellonella taste receptor GR43a to the recognition or localization of the G. mellonella taste receptor GR43a membrane protein.

[0014] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0015] One aspect of the present invention is to provide a G. mellonella taste receptor GR43a antigen polypeptide, the amino acid sequence of which is shown as IPVAAAKPRRSRST (SEQ ID No. 1).

[0016] The invention analyzes the immunobiological characteristics of the greater wax moth taste receptor GR43a membrane protein antigen epitope, screens out the polypeptide segment IPVAAAKPRRSRST, adds specific amino acid residues to the end of the polypeptide segment, and synthesizes the antigen polypeptide for preparing polyclonal antibodies in vitro.

[0017] The second aspect of the present invention is to provide a fusion protein obtained by coupling a G. mellonella taste receptor GR43a antigen polypeptide and a tag protein; the fusion protein comprises a fusion protein obtained by connecting the antigen polypeptide described in SEQ ID No. 1 to a linker and coupling it to a tag protein, wherein the tag protein is selected from any one of conventional tag proteins such as KLH (keyhole limpet hemocyanin), 6×His, GST, FLAG or MAP, and is preferably KLH.

[0018] The third aspect of the present invention is to provide a gene encoding a G. mellonella taste receptor GR43a antigen polypeptide or a gene encoding a fusion protein obtained by coupling the G. mellonella taste receptor GR43a antigen polypeptide and a tag protein.

[0019] Preferably, the polynucleotide sequence of the gene encoding the G. mellonella taste receptor GR43a antigen polypeptide is as shown in (a), (b) or (c):

[0020] (a) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1;

[0021] (b) a polynucleotide sequence that can hybridize to the complementary sequence of the polynucleotide sequence shown in (a) under stringent hybridization conditions;

[0022] (c) a polynucleotide sequence having at least 80% homology with the polynucleotide sequence shown in (a); preferably, a polynucleotide sequence having at least 90% homology with the polynucleotide sequence shown in (a); more preferably, a polynucleotide sequence having at least 95% homology with the polynucleotide sequence shown in (a).

[0023] The fourth aspect of the present invention is to provide a recombinant vector containing the coding gene of the G. mellonella taste receptor GR43a antigen polypeptide or the coding gene of the fusion protein obtained by coupling the G. mellonella taste receptor GR43a antigen polypeptide and a tag protein;

[0024] The fifth aspect of the present invention is to provide a recombinant host bacteria containing the recombinant vector;

[0025] A sixth aspect of the present invention is to provide a polyclonal antibody to the greater wax moth taste receptor GR43a, the preparation method of which comprises: linking the antigen polypeptide described in SEQ ID No. 1 to a linker and coupling it to a tag protein to obtain a fusion protein; immunizing an animal with the fusion protein as an immunogen, and collecting the serum of the immunized animal to obtain the obtained antibody; preferably, linking the antigen polypeptide described in SEQ ID No. 1 to a cysteine linker and then coupling it to the tag protein to obtain the fusion protein (i.e., the antigen polypeptide); immunizing New Zealand rabbits with the antigen polypeptide by subcutaneous multi-point injection on days 0, 7, 14, and 21, respectively, wherein the first time the coupled polypeptide is emulsified with Freund's complete adjuvant and co-injected with 200 μg, and from the second time onwards, the coupled polypeptide is mixed with Freund's incomplete adjuvant and co-injected with 200 μg; 4 days before immunization, 1 mL of rabbit serum is collected from the ear vein as a negative control; on the 7th day after each immunization, the serum of the immunized animal is tested by enzyme-linked immunosorbent assay (ELISA) to determine the level of immune response; after the ELISA test results are ideal, all the serum of the immunized New Zealand rabbits is collected and the antigen affinity polyclonal antibody is purified.

[0026] The seventh aspect of the present invention is to use the polyclonal antibody against the G. mellonella taste receptor GR43a to prepare a reagent or drug for identifying or locating the G. mellonella taste receptor GR43a.

[0027] For reference, the present invention provides an immunohistochemical detection kit for identifying or locating the wax moth taste receptor GR43a, comprising: a primary antibody, a secondary antibody and a color developer; wherein the primary antibody is a polyclonal antibody to the wax moth taste receptor GR43a; and the color developer is DAB.

[0028] The present invention aims to solve the problems of non-specific binding, high background value and high cost of the existing polyclonal antibodies against the taste receptor GR43a of the wax moth. The model insect wax moth is used as the material, the wax moth taste receptor GR43a is selected as the research object, and the RT-qPCR technology is used to study its tissue differential expression characteristics; primers are designed and gene cloning and sequencing are performed; bioinformatics analysis and antigen epitope prediction of GR43a protein are performed, and SEQ ID NO: 1 is selected. The polypeptide segment shown in No. 1 was used to prepare a polyclonal antibody, and the titer of the prepared polyclonal antibody to the wax moth taste receptor GR43a was 1:128,000. The polyclonal antibody to the wax moth taste receptor GR43a prepared by the present invention was used as a primary antibody to localize the wax moth taste receptor GR43a using an immunohistochemical localization method, and the results showed that clear immunohistochemical localization results of the wax moth taste receptor GR43a protein could be obtained. The polyclonal antibody to the wax moth taste receptor GR43a prepared by the present invention can specifically bind to the wax moth taste receptor GR43a protein, and can be used to accurately identify or localize the wax moth taste receptor GR43a protein using methods such as immunohistochemical localization and Western blot. The antibody has the advantages of a simple preparation route, strong specificity, high titer, and low cost.

[0029] Definitions of terms used in this invention

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

[0031] The term "homology" refers to sequence similarity to a natural nucleic acid sequence. "Homology" includes nucleotide sequences that are preferably 85% or higher, more preferably 90% or higher, and most preferably 95% or higher identical to the nucleotide sequence of the regulatory fragment of the present invention. Homology can be evaluated visually or with computer software. Using computer software, the homology between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the homology between related sequences.

[0032] The term "complementary" herein refers to two nucleotide sequences comprising antiparallel nucleotide sequences that can pair with each other after hydrogen bonding between the complementary base residues of the antiparallel nucleotide sequences. It is known in the art that the nucleotide sequences of two complementary strands are reverse complementary to each other when both sequences are viewed in a 5' to 3' direction. It is also known in the art that two sequences that can hybridize to each other under a given set of conditions are not necessarily 100% completely complementary.

[0033] The term "stringent hybridization conditions" refers to conditions of low ionic strength and high temperature as known in the art. Generally, under stringent conditions, a probe hybridizes to its target sequence to a greater extent than to other sequences (e.g., at least 2-fold above background). Stringent hybridization conditions are sequence-dependent and will vary under different environmental conditions, with longer sequences specifically hybridizing at higher temperatures. By controlling the stringency of hybridization or washing conditions, a target sequence that is 100% complementary to the probe can be identified. For detailed guidance on nucleic acid hybridization, reference can be made to the relevant literature (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes," Overview of principles of hybridization and the strategy of nucleic acid assays. 1993). More specifically, the stringent conditions are usually selected to be about 5-10°C lower than the thermal melting point (Tm) of the specific sequence at a specified ionic strength and pH. Tm is the temperature (under specified ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (because the target sequence is present in excess, 50% of the probes are occupied at equilibrium at Tm). Stringent conditions may be those wherein the pH is The salt concentration is less than about 1.0 M sodium ion concentration at 7.0 to 8.3, typically about 0.01 to 1.0 M sodium ion concentration (or other salts), and the temperature is at least about 30°C for short probes (including but not limited to, 10 to 50 nucleotides) and at least about 60°C for long probes (including but not limited to, greater than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least twice the background hybridization, optionally 10 times the background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5× SSC, and 1% SDS, incubation at 42°C; or 5× SSC, 1% SDS, incubation at 65°C, wash in 0.2× SSC, and wash in 0.1% SDS at 65°C. Washes can be performed for 5, 15, 30, 60, 120 minutes, or longer.

[0034] The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector, such as a plasmid, or may be integrated into the host genome.

[0035] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.). Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. In particular, degenerate codon substitutions can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The polyclonal antibody prepared by the present invention is used to perform tissue localization on the G. mellonella taste receptor GR43a protein. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0038] Example 1 Preparation of polyclonal antibodies against the greater wax moth taste receptor GR43a

[0039] Total RNA was extracted from G. mellonella and reverse transcribed to obtain cDNA. The primers for cloning the G. mellonella fructose taste receptor gene gr43a were as follows:

[0040] F:ATGTTGATAAAGTCGAGTGTTTACTGCGTG;

[0041] R: CTAAGTGTCGTATCTCTGGAACTGTATC. Using cDNA as a template, the full-length glycanthus mellonella fructose taste receptor gene gr43a was cloned. The cloned gene was sequenced and identified. The full-length gene sequence was 1404 bp. The specific sequence is as follows:

[0042] ATGGGCGTGGATTCTACGAAAGTGGAAGGAGCGAGCGACATTCC

[0043] GGTTGCGGCCGCGAAACCTCGGCGCTCCCGGTCCACACGCTGCGTCGT

[0044] TGGAGGAGCTCATGCACTAGTTCTGAGGGTATCGAGTTTGTTCGGACT

[0045] GGCGCCGCTGAAGTTTGAGGCGTGTGATGGCGGGTATTACGTCACAAT

[0046] ATCCAGTGCTATGTGTATATATAGTTATATACTGGTCACAGTACTAGTA

[0047] ATATTAACAATATCCGGCCTGGTGGCGGAGATCAAGGTGGGCGTGGA

[0048] GTTGGCAGTGCGTATGTCCTCGACCACGTCACAGATCGTGTCCATGTG

[0049] CGACATATTAGTGGTGGTGCTGACCGCCGGCGCGGGTGTCTACGGCGC

[0050] GCCGAAACGTATGCGAAGCATGCTCAAGTTTATGGACAGCGTTGCTTC

[0051] GGTTGATACCAGCATAGGTGGTAAATATTCAGAGACGACAGAGCGCA

[0052] AAATATGCGCGATACTCATAGCAATTCTCGTGTTCTTCTCCGGCTTGAT

[0053] TATTGATGACATCTGCTTCTATGTGATACATGCTAAAAAAGTGGGAAG

[0054] ACAATGGGACGTGCTGCTCAACTACATAGGTTTCTACTTGCTATGGTA

[0055] CATAGTCATGATATTGGAACTGCAGTTTGCTTTTACTGCTCTGTCGGTG

[0056] CGCGTGCGCTTCCAAGCCATCAACGACACGCTCGTGCTTACCGCCAAG

[0057] AAAGCATCAATGCCAATGTCCGCAGTGGAGAAGACTAGGAGTCCAGC

[0058] GTCGCTGAACATCTTCGCGATCAGCGTGGCGTCGTCGGATATGCAGCG

[0059] TGCGGGCAACGTCAACCTCCTGGTGGACTCGCACGACGACTCCGACCA

[0060] TGATGTCATCGTGTCCAAATCTGTGGGAGGTGAGCCCCGGCTGCTGGT

[0061] GTCCGCCGGCAGGGCGGCCGAGCAGCTGTCGAAGCTGCACGGCGCGC

[0062] TATGCTGCGCGGTGCGGCGCGTGGAGCACAGCTACGGGCTGCCGCTCG

[0063] TGGTCATACTCATCTCCACGCTGCTCCACCTCATCGTCACGCCGTACTT

[0064] CCTCATCATGGAGATAATCGTGTCTACGAATCGTGTACACTTCCTGGT

[0065] ACTGCAGTTCCTGTGGTGCGTCACGCATTTACTGCGGTTAGTCATCGTC

[0066] GTGGAACCCTCCCATTACACAATAATTGAAGGTAAAAGAACCCGTAGC

[0067] CTGGTGTGTCGATTAATGTCATTGACACCGACAACTGGGGAGTTCCCT

[0068] GCACGTCTCGAAGTGTTCTGCCGACAACTGATGCTGCAGACCACCTCC

[0069] TATATGCCGATGGGCATGTGTACTTTGGACCGGCCACTTGTTGCTTCGA

[0070] TACTCGGAGCAGTCACTACGTACTTAGTCATACTGATACAGTTCCAGA

[0071] GATACGACACTTAG (SEQ ID No. 2).

[0072] The corresponding amino acid sequence of the greater wax moth fructose taste receptor GR43a protein is 467 amino acid residues, and its amino acid sequence is as follows:

[0073] MGVDSTKVEGASDIPVAAAKPRRSRSTRCVVGGAHALVLRVSSLFG

[0074] LAPLKFEACDGGYYVTISSAMCIYSYILVTVLVILTISGLVAEIKVGVELAV

[0075] RMSSTTSQIVSMCDILVVVLTAGAGVYGAPKRMRSMLKFMDSVASVDTS

[0076] IGGKYSETTERKICAILIAILVFFSGLIIDDICFYVIHAKKVGRQWDVLLNYI

[0077] GFYLLWYIVMILELQFAFTALSVRVRFQAINDTLVLTAKKASMPMSAVEK

[0078] TRSPASLNIFAISVASSDMQRAGNVNLLVDSHDDSDHDVIVSKSVGGEPRL

[0079] LVSAGRAAEQLSKLHGALCCAVRRVEHSYGLPLVVILISTLLHLIVTPYFLI

[0080] MEIIVSTNRVHFLVLQFLWCVTHLLRLVIVVEPSHYTIIEGKRTRSLVCRL

[0081] MSLTPTTGEFPARLEVFCRQLMLQTTSYMPMGMCTLDRPLVASILGAVTT

[0082] YLVILIQFQRYDT*(SEQ ID No. 3).

[0083] Bioinformatics analysis of the membrane protein sequence of the greater wax moth fructose taste receptor GR43a revealed a molecular formula of C 2317 H 3762 N 604 O 641 S 29 The molecular weight is 51233.76 Da, the theoretical isoelectric point is 8.93, and it is a slightly alkaline and unstable protein. The aliphatic amino acid index is 120.36. The G. mellonella taste receptor GR43a membrane protein has a half-life of approximately 30 hours in vitro, greater than 20 hours in yeast, and greater than 10 hours in Escherichia coli. The G. mellonella taste receptor GR43a membrane protein is a transmembrane protein with seven transmembrane domains and an intracellular N-terminus, consistent with the basic structural properties of taste receptor proteins. It lacks a signal peptide site, indicating that it is a non-secreted protein.

[0084] The immunobiological properties of epitopes on the membrane protein of the G. mellonella taste receptor GR43a were analyzed. Bioinformatics software was used to analyze the hydrophilicity, hydrophobicity, surface accessibility, and flexibility of the GR43a protein, and potential epitopes were comprehensively predicted. The results revealed that regions of high surface accessibility in the G. mellonella taste receptor GR43a protein were primarily located in peptide segments 19-25 and 248-254. Karplus-Schulz analysis of the plastic regions of the GR43a protein revealed that peptide segments 20-28 and 249-255 exhibited high flexibility and were readily accessible to antibodies, suggesting a high likelihood of forming epitopes. The Jameson-Wolf analysis of cellular epitopes on the GR43a protein revealed that these were primarily located in peptide segments 18-30 and 247-257. Immunobioinformatics tools were used to analyze the immunobiological characteristics of the antigenic epitopes of these peptide segments by amino acid residue step-by-step analysis. Based on antigenicity, surface probability, hydrophilicity parameters, and homology comparison parameters with Mus musculus, Rattus norvegicus, and Rabbit, the peptide segment IPVAAAKPRRSRST with the best immunogenicity and conservation was screened as the antigenic peptide. After adding cysteine (Cys) at the end, it was synthesized in vitro and used as the antigen for synthesizing polyclonal antibodies.

[0085] A synthetic peptide with a cysteine (Cys) linker was conjugated to hemocyanin (KLH) to produce an antigenic polysaccharide. This hemocyanin-KLH-conjugated antigenic peptide was then immunized in New Zealand rabbits using multiple subcutaneous injections on days 0, 7, 14, and 21. The first injection consisted of 200 μg of the antigenic peptide emulsified with Freund's complete adjuvant. From the second and subsequent injections, 200 μg of the antigenic peptide was mixed with Freund's incomplete adjuvant and injected. Four days before immunization, 1 mL of rabbit serum was collected from the ear vein as a negative control. Seven days after each immunization, sera from the immunized animals were analyzed using an enzyme-linked immunosorbent assay (ELISA) to determine the level of immune response. After satisfactory ELISA results, the entire serum from the immunized New Zealand rabbits was collected and purified for antigen-affinity polyclonal antibodies. Antibody concentration and purity were determined, and polyclonal antibodies of acceptable quality were used.

[0086] Test Example 1: Test for the potency determination of polyclonal antibodies against the greater wax moth taste receptor GR43a

[0087] The polyclonal antibody prepared and purified in Example 1 (ie, serum from immune New Zealand rabbits) and pre-immune serum were detected by enzyme-linked immunosorbent assay (ELISA). The ELISA results of the pre-immune serum and the purified polyclonal antibody are shown in Table 1.

[0088] Table 1 ELISA results of pre-immune serum and purified antibodies

[0089]

[0090] According to the measurement results in Table 1, the titer of the polyclonal antibody against the G. mellonella taste receptor GR43a prepared in Example 1 of the present invention is 1:128,000.

[0091] Test Example 2 Application of polyclonal antibodies against the greater wax moth taste receptor GR43a

[0092] Using immunohistochemistry (IHC) technology, antibodies and antigens are highly specifically bound to each other, and DAB is then used to display the antigen-antibody complex in a specific color to indicate the expression site of the receptor, thereby locating the tissue expressing the taste receptor and clarifying the specific distribution of the taste receptor.

[0093] The tissue localization of the wax moth taste receptor GR43a protein was performed using the gradient dilution of the polyclonal antibody to the wax moth taste receptor GR43a prepared and purified in Example 1. The insect tissue was incubated at 4°C overnight using the primary antibody at different dilutions. After 13 hours, the tissue was rewarmed at room temperature. After incubation with the secondary antibody and DAB color development, it was found that when the dilution was 1500 times, clear immunohistochemical localization results of the wax moth taste receptor GR43a protein could be obtained ( Figure 1), the red arrow indicates the expression site of GR43a protein in the body tissue of G. mellonella to which the polyclonal antibody specifically binds.

Claims

1. A greater wax moth taste receptor GR43a antigen polypeptide, characterized in that Its amino acid sequence is shown in SEQ ID No.

1.

2. A fusion protein, characterized in that The peptide is obtained by coupling the G. mellonella taste receptor GR43a antigen polypeptide according to claim 1 with a tag protein; preferably, the tag protein is selected from any one of keyhole limpet hemocyanin, His, GST, FLAG or MAP, and most preferably keyhole limpet hemocyanin.

3. The gene encoding the G. mellonella taste receptor GR43a antigen polypeptide according to claim 1; preferably, the polynucleotide sequence of the encoding gene is as shown in (a), (b) or (c): (a) A polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No.1; (b) a polynucleotide sequence that is capable of hybridizing to a complementary sequence of the polynucleotide sequence shown in (a) under stringent hybridization conditions; (c) A polynucleotide sequence having at least 80% homology with the polynucleotide sequence shown in (a); preferably, a polynucleotide sequence having at least 90% homology with the polynucleotide sequence shown in (a); more preferably, a polynucleotide sequence having at least 95% homology with the polynucleotide sequence shown in (a). The gene encoding the fusion protein of claim 2 .

5. A recombinant vector containing the coding gene according to claim 3 or 4.

6. A recombinant host bacteria containing the recombinant vector according to claim 5.

7. A polyclonal antibody against the wax moth taste receptor GR43a, characterized in that: include: The method is prepared by immunizing an animal with the greater wax moth taste receptor GR43a antigen polypeptide according to claim 1 or the fusion protein according to claim 2.

8. The polyclonal antibody against the greater wax moth taste receptor GR43a according to claim 7, characterized in that include: Connecting the antigen polypeptide described in SEQ ID No. 1 to a linker and coupling it with a tag protein to obtain a fusion protein; The fusion protein is used as an immunogen to immunize an animal, and the serum of the immunized animal is collected to obtain the antigen polypeptide; more preferably, the antigen polypeptide described in SEQ ID No. 1 is connected to cysteine as a linker and then coupled to a tag protein to obtain the antigen polypeptide; the antigen polypeptide is immunized in New Zealand rabbits by subcutaneous multi-point injection on days 0, 7, 14, and 21, and the first time the coupled polypeptide is emulsified with Freund's complete adjuvant and co-injected with 200 μg, and from the second time onwards, the coupled polypeptide is mixed with Freund's incomplete adjuvant and co-injected with 200 μg; 4 days before immunization, 1 mL of rabbit serum is collected from the ear vein as a negative control; on the 7th day after each immunization, the serum of the immunized animal is tested by enzyme-linked immunosorbent assay (ELISA) to determine the level of immune response; after the ELISA test results are ideal, all the serum of the immunized New Zealand rabbits is collected and purified.

9. Use of the polyclonal antibody against the G. mellonella taste receptor GR43a according to claim 7 or 8 in the preparation of an agent or drug for recognizing or localizing the G. mellonella taste receptor GR43a.

10. A detection kit for identifying or locating the greater wax moth taste receptor GR43a, comprising: A primary antibody, a secondary antibody and a color developing agent; characterized in that the primary antibody is the polyclonal antibody against the greater wax moth taste receptor GR43a according to claim 7 or 8; and the color developing agent is DAB.

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