Phosphoglycerate kinase 1 gene and recombinant protein of lucilia cepat

Abstract

This invention belongs to the field of biotechnology and proposes the *Gnaphalium gravidarum* phosphoglycerate kinase PGK1 gene and its recombinant protein. The sequence of the *Gnaphalium gravidarum* phosphoglycerate kinase PGK1 gene is shown in SEQ ID NO.3, and the amino acid sequence of the recombinant protein is shown in SEQ ID NO.4. This invention also proposes a kit for detecting antibodies against *Gnaphalium gravidarum* infection, which can react with antibodies in the serum of horses infected with *Gnaphalium gravidarum*. The kit established using the recombinant *Gnaphalium gravidarum* phosphoglycerate kinase PGK1 protein as the coating antigen exhibits good ability to distinguish between positive and negative sera, low cross-reactivity, and good reproducibility.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a PGK1 gene, a recombinant PGK1 protein, and an in vitro detection kit containing the recombinant protein. Background Technology

[0002] Equine stomach fly myiasis is a parasitic disease caused by the larvae of the genus *Equine stomach fly* that infest the digestive tract of equines such as horses and donkeys. *Equine stomach fly* ( Gasterophilus spp. The horse stomach fly goes through four developmental stages: egg, larva, pupa, and adult. The larval stage involves parasitizing the host and causing primary damage. Current methods for identifying live horse stomach flies primarily rely on observing eggs on the body surface, expelling worms after deworming, endoscopy, or necropsy. Early or mild infections often present with atypical clinical symptoms. Endoscopic examination requires equipment and individual horse-by-horse operation, making it unsuitable for rapid screening of large herds on ranches. The presence of worms in feces and eggs on the body surface is also affected by season and developmental stage.

[0003] Previous studies have established serological detection methods using antigens excreted / secreted by second-stage larvae of the horse gastropod fly or natural proteins from the larvae. Sánchez-Andrade et al. used *Gastropoda equina* (…). Gasterophilus intestinalis ) and nasal stomach flies ( Gasterophilus nasalis Establishment of ELISA method for excretion / secretion antigens in second-stage larvae (Sánchez-Andrade R, Cortiñas FJ, Francisco I, et al. A novel second instar Gasterophilus Excretory / secretory antigen-based ELISA for the diagnosis of gasterophilosis in grazing horses[J]. Veterinary parasitology, 2010, 171(3-4): 314-320. However, crude antigens extracted from insect bodies, larval tissue antigens, and excretory / secretory antigens all contain multiple proteins, and their composition and relative content are easily affected by insect species, developmental stage, collection season, storage conditions, and extraction processes, making it difficult to guarantee batch-to-batch consistency. Complex natural antigens may also contain conserved components shared with other organisms, increasing the risk of non-specific reactions. Currently, there is a lack of recombinant antigen detection systems with well-defined sequences, batch-to-batch stability, scalable preparation, and validated by equine serum systems. Population detection still requires recombinant antigens with clearly defined sources, definitive sequences, scalable preparation, and reproducible performance.

[0004] Current serological detection methods for *Gnaphalium equineumoniae* rely on natural antigens from larvae, such as crude antigens extracted from the larvae, larval tissue antigens, or antigens excreted / secreted by second-stage larvae. Currently, publicly available data does not include chromosome-level genome sequencing data or transcriptomic data for different developmental stages. Furthermore, there is a lack of molecularly defined antigens that have been screened from *Gnaphalium equineumoniae* omics data, expressed in soluble recombinant form, and validated using equine serum systems. Obtaining antigens that span the entire life cycle, are expressible during the parasitic larval stage, are suitable for prokaryotic soluble preparation, and possess infection-related immunoreactivity is a key technical challenge that needs to be addressed in developing standardized in vitro diagnostic kits. Summary of the Invention

[0005] To address the deficiency in existing technologies that lack molecularly defined antigens for *Gnaphalium gravidarum*, the first objective of this invention is to propose a *Gnaphalium gravidarum* phosphoglycerate kinase PGK1 gene.

[0006] The second objective of this invention is to provide a recombinant protein from *Gastropoda melanogaster* with a well-defined sequence and origin, capable of stably yielding soluble protein, and recognizable by positive horse serum infected with *Gastropoda melanogaster*.

[0007] The third objective of this invention is to propose a kit based on this recombinant protein that can achieve high positive and negative differentiation, low cross-reactivity within the detection range, and good batch-to-batch reproducibility in equine serum, thereby improving the problems of complex natural parasite antigen composition, insufficient batch consistency, and difficulty in standardization in the detection of equine gastric fly myiasis.

[0008] The technical solution for achieving the above-mentioned objective of this invention is as follows: A *Gnaphalium gravidarum* phosphoglycerate kinase PGK1 gene, the sequence of which is shown in SEQ ID NO.3.

[0009] The inventors first constructed a chromosome-level reference genome of *Gnaphalium affine* and obtained reference transcriptome expression data for four developmental stages: egg, larva, pupa, and adult. Based on this, initial screening was conducted according to the sequence reliability, cross-stage expression, parasitic larval stage expression, and functional information of candidate genes. The detection of expression in all four stages was used as the initial screening criterion to exclude candidates with transient expression only in a single stage, prioritizing proteins that are expressed throughout the life cycle and have a basis for expression in the parasitic larval stage. Subsequently, multidimensional screening was conducted combining larval stage expression, protein structure and potential antigenicity, feasibility of prokaryotic soluble expression, and possible cross-reactivity risks. This determined that PGK1 encoded by SEQ ID NO.3 could be used as a candidate antigen for detecting *Gnaphalium affine* infection-related antibodies. The material used in the experiment was *Gnaphalium affine* (*Gnaphalium affine*). Gasterophilus pecorum This is a species of horse stomach fly. The PGK1 gene that was screened is present in all horse stomach fly species.

[0010] This project identified PGK1 as a candidate antigen through omics and bioinformatics screening. Then, the recombinant PGK1 protein was cloned, expressed in prokaryotes, and purified. Western blot confirmed that the recombinant protein could be recognized by positive horse serum infected with Gastropoda melanogaster. iELISA (indirect enzyme-linked immunosorbent assay) verified that it had good ability to distinguish between positive and negative sera and low cross-reactivity. Therefore, it was determined to be a recombinant antigen that can be used for in vitro detection of Gastropoda melanogaster infection-related antibodies.

[0011] A recombinant protein of phosphoglycerate kinase PGK1 from the black-bellied gastric fly, the amino acid sequence of which is shown in SEQ ID NO.4, is capable of generating an immune response with antibodies in the serum of black-bellied gastric flies infected with positive horses.

[0012] A preferred embodiment of the present invention is that the recombinant protein of *Gnaphalium melanogaster* phosphoglycerate kinase PGK1 is prepared by the following steps: S1: Total RNA was extracted from second-stage larvae of the black-bellied gastric fly and the first strand of cDNA was synthesized using a reverse transcription kit. S2: Primers PGK1 F ​​and PGK1 R were designed targeting the PGK1 gene, with sequences SEQ ID NO. 1 and SEQ ID NO. 2, respectively. The primer sequences were then modified by adding... Eco RI and Xho I restriction site, synthesize PGK1 primer; S3: Using cDNA from *Gnaphalium affine*, *Gnaphalium affine*, *Gnaphalium affine*, *Parascaridium equine*, and *Strongyloides equine* as templates, PCR amplification and specificity verification were performed using the PGK1 primers, confirming that the primers could only amplify the target fragment from *Gnaphalium affine* cDNA. S4: Ligate the PCR product obtained in step S3 with the cloning vector pMD19-T, transform the ligation product into TOP10 competent cells for culture, and use the cultured bacteria for PCR identification. Extract plasmids from bacteria that are positive for PCR identification and perform double enzyme digestion identification. Sequencing is performed on recombinant clones that are preliminarily identified as positive by PCR and double enzyme digestion products. S5: The recombinant cloning bacterial strain and the pET-30a(+) expression vector strain were cultured separately. The recombinant cloning plasmid and the pET-30a(+) expression plasmid were extracted and subjected to double enzyme digestion. The target gene fragment and the linearized pET-30a(+) vector fragment were recovered and ligated. The ligation product was transformed into competent cells, cultured, and screened to identify the PGK1 recombinant expression strain. S6: When the PGK1 recombinant expression bacteria reach an OD600 value of 0.4-0.6, IPTG is added for induction. The induced bacterial cells are collected, resuspended in buffer, treated with lysozyme, sonicated, and centrifuged. The soluble supernatant containing the recombinant protein is collected and processed using Ni... 2+Affinity chromatography column purification.

[0013] Preferably, in step S6, the final concentration of IPTG (isopropyl-β-D-thiogalactoside) added to the system for culturing the PGK1 recombinant expression bacteria is 0.1 mmol / L.

[0014] More preferably, in step S6, the induction temperature is 16°C and the induction time is 12 h.

[0015] In step S6, the bacterial cells are resuspended in TBS buffer, lysozyme solution is added at 1 / 10 of the bacterial resuspension volume, the mixture is sonicated, centrifuged, and the soluble supernatant is filtered through a 0.45 μm pore size filter before being processed with Ni. 2+ Affinity column chromatography.

[0016] The ultrasonic disruption time in step S6 can be 10~20 min, and the centrifugation conditions can be: 4℃, 12000×g centrifugation for 30 min.

[0017] A kit for detecting antibodies against *Gastropoda melanogaster* infection in isolated horse serum includes an enzyme-linked immunosorbent assay (ELISA) plate coated with the recombinant protein of *Gastropoda melanogaster* phosphoglycerate kinase PGK1. The kit shows no cross-reactivity with positive horse serum from *Strongyloides equine*, *Parascaris equine*, and *Gastropoda equinea*.

[0018] More preferably, the coating concentration of the recombinant protein of the black-bellied gastric fly phosphoglycerate kinase PGK1 is 1.5 μg / mL.

[0019] More preferably, the kit further comprises a blocking solution and an enzyme-labeled secondary antibody; the blocking solution is 5% BSA, and the enzyme-labeled secondary antibody is HRP-labeled goat anti-horse IgG diluted 1:2000. The kit has a cutoff value of 0.492.

[0020] The beneficial effects of this invention are as follows: Compared with existing natural larval antigens, this invention uses a single recombinant protein with a defined amino acid sequence as the antigen, which facilitates standardized production and quality control. The recombinant protein PGK1 of *Gnaphalium affine* proposed in this invention is mainly expressed in a soluble form when induced by 0.1 mmol / L IPTG for 12 h at 16°C, reducing the impact of inclusion body refolding on protein conformation and batch stability.

[0021] Western blot results showed that the recombinant PGK1 protein could be recognized by serum from horses infected with *Gastropoda melanogaster*. In iELISA assays of 120 known positive sera and 30 known negative sera containing the recombinant PGK1 protein, the area under the ROC curve was 0.999 (95% confidence interval 0.996–1.000), and the cutoff value was 0.492; the positive concordance rate was 97.50% (117 / 120), and the negative concordance rate was 100.00% (30 / 30). No cross-reactivity was detected in 43 positive serum samples from *Strongyloides equine*, *Parascaris equine*, and *Taenia equine*; the intra-assay coefficient of variation was 1.07%–5.19%, and the inter-assay coefficient of variation was 2.47%–9.52%. These results indicate that the recombinant protein and the kit have good ability to distinguish between positive and negative sera, low cross-reactivity, and reproducibility.

[0022] An in vitro detection kit established using recombinant protein PGK1 phosphoglycerate kinase from the black-bellied gastric fly as the coating antigen exhibits good ability to distinguish between positive and negative sera, low cross-reactivity, and good reproducibility. Attached Figure Description

[0023] Figure 1 The images show the amplification results of the gene coding region. The left image shows the PCR amplification results of the PGK1 gene; M: DL 2000 DNA Marker; lanes 1-3: PGK1 amplification products. The right image shows the specificity verification results of the PGK1 gene primer amplification; M: DL 2000 DNA Marker; lane 1: *Gnaphalium affine*; lane 2: *Gnaphalium affine*; lane 3: *Massock beetle*; lane 4: *Parascaridium equine*; lane 5: *Strongyloides equine*.

[0024] Figure 2 The images show the identification results of the cultured bacterial culture. The left image shows the PCR amplification results of the bacterial culture (M: DL 2000 DNA Marker; 1-3: PGK1), and the right image shows the double enzyme digestion results of the recombinant cloning plasmid PGK1 gene (M: DL 2000 DNA Marker; 1-2: PGK1).

[0025] Figure 3 Electrophoresis results of PGK1 gene gel recovery products; M: DL 2000 DNA Marker; Lanes 1-3: PGK1 gel recovery products.

[0026] Figure 4 The results of double enzyme digestion identification of the pET-PGK1 recombinant expression plasmid are shown; M1: DL 2000 DNA Marker; M2: DL10000 DNA Marker; Lanes 1-2: double enzyme digestion products of the pET-PGK1 recombinant expression plasmid.

[0027] Figure 5 The results of the optimized induction temperature for the recombinant expression strain of BL21(pET-PGK1); M: protein molecular weight standard; Lane 1: empty BL21(DE3) strain; Lane 2: empty vector strain of BL21(pET-30a(+)) before induction; Lane 3: empty vector strain of BL21(pET-30a(+)) after induction; Lane 4: BL21(pET-PGK1) before induction; Lanes 5-7: BL21(pET-PGK1) after induction at 16℃, 25℃ and 37℃, respectively.

[0028] Figure 6 The results of optimized induction time for BL21(pET-PGK1) recombinant expression bacteria; M: protein molecular weight standard; lane 1: BL21(DE3) empty bacteria; lane 2: BL21(pET-30a(+)) empty vector bacteria before induction; lane 3: BL21(pET-30a(+)) empty vector bacteria after induction; lane 4: BL21(pET-PGK1) before induction; lanes 5-9: BL21(pET-PGK1) after induction for 2 h, 4 h, 6 h, 8 h and 12 h, respectively.

[0029] Figure 7 The results show the optimized IPTG induction concentration for BL21(pET-PGK1) recombinant expression bacteria; M: protein marker; Lane 1: empty BL21(DE3) bacteria; Lane 2: empty vector BL21(pET-30a(+)) bacteria before induction; Lane 3: empty vector BL21(pET-30a(+)) bacteria after induction; Lanes 4-9: induction samples of BL21(pET-PGK1) recombinant expression bacteria with final IPTG concentrations of 0.05, 0.1, 0.2, 0.6, 1.0, and 2.0 mmol / L, respectively.

[0030] Figure 8 The results show the expression form of the PGK1 recombinant protein; M: protein molecular weight standard; lanes 1-2: supernatant of cell lysis; lanes 3-4: cell lysis precipitate.

[0031] Figure 9 The results show the purification of PGK1 recombinant protein; M: protein molecular weight standard; lanes 1-3: purified PGK1 protein.

[0032] Figure 10 Western blot identification results of the His tag for recombinant PGK1 protein; M: protein molecular weight standard; lanes 1-2: recombinant PGK1 protein.

[0033] Figure 11Western blot results for serum immunoreactivity of recombinant PGK1 protein; M: protein molecular weight standard; lanes 1-2: positive horse serum group infected with Gastropoda melanogaster; lanes 3-4: horse serum group not infected with Gastropoda melanogaster; lanes 5-6: observation group of sheep serum infected with other parasites.

[0034] Figure 12 Results of optimized coating temperature and time for PGK1 recombinant protein iELISA antigen.

[0035] Figure 13 The results show the optimization of blocking solution and blocking time for PGK1 recombinant protein iELISA. The left figure shows the optimization of the blocking solution, and the right figure shows the optimization of the blocking time.

[0036] Figure 14 The results show the optimized incubation time of the primary antibody (serum) for iELISA of recombinant PGK1 protein and the optimized TMB color development time. The left figure shows the optimized primary antibody (serum) incubation time, and the right figure shows the optimized TMB color development time.

[0037] Figure 15 The distribution and judgment values ​​of OD450 values ​​in negative and positive sera for PGK1 recombinant protein iELISA are shown. Blue scatter plots represent known negative sera, orange scatter plots represent known positive sera, box plots represent the distribution of OD450 values, and horizontal dashed lines represent the optimal judgment values ​​determined by ROC curve analysis.

[0038] Figure 16 The receiver operating characteristic curve for iELISA of recombinant PGK1 protein is shown. Detailed Implementation

[0039] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0040] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0041] Unless otherwise specified, all instruments, reagents and consumables used in this invention are purchased from the market.

[0042] The parasite samples and serum used in this invention: Second-stage and third-stage larvae of *Gnaphalium affineum* were collected from a slaughterhouse in Baoding City, Hebei Province. Positive horse serum samples from horses infected with *Gnaphalium affineum* were collected from: 70 positive horse serum samples from family ranches in the Evenk Autonomous Banner of Hulunbuir City, Inner Mongolia, from horses confirmed to be infected with *Gnaphalium affineum* by rectal examination; and 50 samples from a slaughterhouse in Baoding City, Hebei Province, from which numerous *Gnaphalium affineum* larvae were found parasitized upon necropsy. A portion of the confirmed positive serum samples were mixed in equal volumes to form the standard positive serum.

[0043] Negative horse serum samples from horses not infected with equine gastric fly myiasis: Six negative horse serum samples were collected from one-month-old foals at the Ejin Horo Banner Horse Breeding Farm in Ordos City, Inner Mongolia; 24 samples were collected from a slaughterhouse in Baoding City, Hebei Province. No *Megalobrachial meiasis* larvae were found after necropsy of these horses. These horses had been dewormed five months prior to slaughter and had not received any further treatment. Positive horse serum samples from horses infected with *Strongyloides equine*, *Parascaris equine*, and *Taenia equinea*, and two sheep serum samples infected with other parasites were collected from the Veterinary College of Inner Mongolia Agricultural University and a slaughterhouse in Baoding City, Hebei Province. 220 horse serum samples awaiting field testing were collected from family ranches in Ewenki Autonomous Banner of Hulunbuir City, Siziwang Banner of Ulanqab City, East Ujimqin Banner of Xilingol League, and Keshiketeng Banner of Chifeng City, Inner Mongolia.

[0044] All experimental procedures and animal experimental protocols conducted in this study were approved by the Laboratory Animal Welfare and Ethics Committee of Inner Mongolia Agricultural University (NND2023067), and the protocols complied with the approved guidelines.

[0045] Example 1

[0046] *Gnaphalium brevicornum* is a species of horse stomach fly. In this embodiment, third-stage larvae of *Gnaphalium brevicornum* were used as experimental material, cultured into adult flies in the laboratory, and sequenced. Third-stage larvae must be used when constructing the genome because second-stage larvae cannot complete the third-stage development through laboratory culture. Third-stage larvae emerge as adult flies for sequencing of the adult genome (however, second-stage larvae are used in recombinant protein preparation, as this is the most active period of growth and development in *Gnaphalium brevicornum*).

[0047] Using adult fly genomic DNA as material, second-generation short-read genome survey sequencing, PacBioHiFi high-accuracy long-read sequencing, Oxford Nanopore Technologies (ONT) ultra-long-read sequencing, and Hi-C chromatin conformation capture sequencing data were obtained. Survey data was used to estimate genome size, heterozygosity, and the proportion of repetitive sequences; PacBio HiFi data was used to construct a high-accuracy initial assembly; ONT ultra-long reads were used to traverse long repetitive regions and improve assembly continuity; and Hi-C data was used to locate, sequence, and orient the assembled sequences to chromosomes. PacBio Kinnex Iso-Seq full-length transcriptome sequencing data were also obtained to assist in gene structure annotation. The complementary data were used to construct a chromosome-level reference genome for *Gastropoda melanogaster*. Subsequently, RNA sequencing was performed at four developmental stages: egg, larva, pupa, and adult. Gene expression quantification and functional annotation were performed using the self-constructed reference genome as a comparison framework, obtaining a reference transcriptome expression profile that allows for cross-stage comparisons, providing a basis for confirming the gene structure of candidate antigens, comparing stage expression, and subsequent screening.

[0048] When screening candidate antigens, genes that were expressed in all four developmental stages and had a basis for expression in the parasitic larval stage were first retained to reduce the risk of limited sample applicability due to transient expression in only a single stage. Then, a comprehensive evaluation was conducted considering factors such as protein molecular weight, isoelectric point, instability index, transmembrane domain, signal peptide, hydrophobicity, surface accessibility, potential antigenic epitopes, feasibility of prokaryotic soluble expression, homology, and potential cross-reactivity risk. The PGK1 candidate coding sequence was first screened from whole-genome and four-stage transcriptome data of *Gastropoda melanogaster* and ORF prediction. Then, PCR amplification, cloning, and sequencing were performed using cDNA from second-stage *Gastropoda melanogaster* larvae as a template, ultimately confirming the PGK1 gene coding sequence as SEQ ID NO. 3. SEQ ID NO. 4 is the amino acid sequence obtained by translating SEQ ID NO. 3 according to its open reading frame.

[0049] After multidimensional screening, PGK1 was selected as a candidate antigen, and its immunoreactivity and detection performance were further verified by recombinant expression, Western blot, and equine serum iELISA.

[0050] Example 2

[0051] This embodiment provides a recombinant protein of phosphoglycerate kinase PGK1 from the black-bellied gastric fly. The preparation of this recombinant PGK1 protein includes the following steps: S1: Total RNA Extraction and First-Strand cDNA Synthesis: Total RNA was extracted from second-stage larvae of *Gnaphalium affine* using the TaKaRa RNAiso Plus RNA extraction reagent according to the manufacturer's instructions. The integrity and concentration of the extracted total RNA were assessed using agarose gel electrophoresis and a micro spectrophotometer. The qualified total RNA was then used to synthesize the first strand of cDNA using a reverse transcription kit.

[0052] Gene coding region amplification results: PCR amplification of the PGK1 gene was performed using cDNA from second-stage larvae of the black-bellied gastropod botfly as a template (three parallel samples were set up for the PGK1 amplification product). The results are shown below. Figure 1 Left image. Agarose gel electrophoresis shows a single primary target band of approximately 1248 bp, consistent with the expected size; sequencing results of the PCR product are consistent with the coding sequence shown in SEQ ID NO.3. This result demonstrates that the designed primers can obtain the target coding sequence from the cDNA of second-stage larvae of *Gnaphalium affine*, but does not, based on this, classify the highly conserved PGK1 gene itself as a species-specific gene.

[0053] S2: Primers were designed using Oligo 7 software based on the PGK1 coding sequence of *Gnaphalium affine* obtained through screening. EcoRI and XhoI restriction sites were added to the primers according to the multiple cloning site and fusion tag reading frame of the pET-30a(+) expression vector, and primer sequences were synthesized. Primer sequences are shown in Table 1.

[0054] Table 1 Primer sequences of candidate genes

[0055] S3: PCR amplification was performed using cDNA from *Gnaphalium affine*, *Gnaphalium affine*, *Botrytis cinerea*, *Parascaris equine*, and *Strongyloides equine* as templates, respectively, using PGK1 primers. The total reaction volume was 50 μL; the amplification program was 94℃ pre-denaturation for 5 min, followed by 35 cycles of 94℃ for 30 s, 68℃ for 30 s, and 72℃ for 30 s, with a final extension at 72℃ for 7 min. PCR products were analyzed by 2% agarose gel electrophoresis. The reaction system is shown in Table 2.

[0056] Table 2 PCR amplification reaction system

[0057] After amplification, the PCR products were subjected to 2% agarose gel electrophoresis. The target band was excised, purified, and recovered. The purified product was sent to BGI Genomics (Beijing) Co., Ltd. for sequencing. The sequencing results were compared with the target gene reference sequence. After confirming the correctness of the amplified fragment sequence, it was used for the subsequent construction of cloning and expression vectors. See Figure 1 Right figure: Under the primers and amplification conditions used in this embodiment, only the cDNA of *Gastropoda melanogaster* showed a 1248 bp target band, while the cDNAs of *Gastropoda nigra*, *Gastropoda spp.*, *Gastropoda spp.*, *Gastropoda spp.*, and *Gastropoda spp.* did not show corresponding bands.

[0058] S4: Ligate the purified PCR product with the pMD19-T cloning vector. The ligation reaction consisted of: 1 μL pMD19-T, 2 μL gel-recovered target gene product, 2 μL sterile ddH2O, and 5 μL Solution I. After mixing, ligation was carried out at 16°C for 16 h. The ligation product was then transformed into TOP10 competent cells: 10 μL of the ligation product was mixed with 100 μL of competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 45 s, and then placed on ice for 2 min. 700 μL of antibiotic-free LB liquid medium was added to a centrifuge tube, and the cells were incubated at 37°C with shaking at 220 r / min for 1.5 h. The bacterial culture was then plated on LB agar plates containing Amp and incubated at 37°C for 16 h. Single colonies were picked and inoculated into 50 mL of LB liquid medium containing Amp resistance, and the culture was shaken at 200 r / min for 12–16 h.

[0059] PCR amplification was performed using the cultured bacterial solution as a template. The amplification procedure was the same as step S2, and the reaction system was the same as in Table 2. After amplification, the results were detected by 2% agarose gel electrophoresis. Figure 2 (Left figure). Plasmids extracted from PCR-positive bacteria were subjected to double enzyme digestion; the reaction system is shown in Table 3. After digestion, the digestion products were detected by 1% agarose gel electrophoresis. Figure 2 (Right figure). The recombinant clones that were initially identified as positive by PCR and double enzyme digestion were sent to BGI Genomics (Beijing) Co., Ltd. for sequencing, and the results matched the PGK1 sequence shown in SEQ ID NO.3.

[0060] Table 3. Double enzyme digestion protocol and reaction system for the target gene

[0061] Note: The amount of each restriction endonuclease added in each double digestion reaction system is 2 μL.

[0062] S5: The pMD19-PGK1 recombinant clone strain, whose sequencing verification was correct, was inoculated into LB medium containing Amp and cultured at 37°C for 16 h. The pMD19-PGK1 recombinant clone plasmid was then extracted and processed... EcoR I and Xho The PGK1 target fragment was recovered after double digestion with enzyme I; the strain containing the pET-30a(+) vector was inoculated into LB medium containing Kan and cultured at 37℃ for 16 h. The pET-30a(+) expression vector plasmid was extracted and processed... EcoR I and Xho The linearized pET-30a(+) vector fragment was recovered after double digestion with enzyme I. The recovered PGK1 target fragment was ligated into the linearized pET-30a(+) expression vector. The ligation reaction system consisted of 6 μL of the target fragment, 1 μL of T4 DNA ligase, 1 μL of 10×T4 DNA Buffer, and 2 μL of pET-30a(+) expression vector, for a total volume of 10 μL. Ligation was carried out at 16℃ for 16 h.

[0063] The ligation product was transformed into BL21(DE3) competent cells, which were then revived and plated on LB agar plates containing Kans. PCR identification was performed using the cultured bacterial culture as a template, and plasmids were extracted for further analysis. Eco RI and Xho I. Double enzyme digestion identification. Figure 3 Electrophoresis results showed that the PGK1 gene amplified a target fragment band of approximately 1248 bp, consistent with the expected size. Figure 4 The 1.5% agarose gel electrophoresis results showed that a specific band of the same size as the target gene could be excised from the plasmid. Simultaneously, a band of the same size as the pET-30a(+) vector backbone was also observed, indicating successful insertion of the target gene into the pET-30a(+) vector, resulting in a recombinant expression bacterium (denoted as BL21(pET-PGK1) recombinant expression bacterium). Recombinant expression bacteria with positive results in both identifications were selected for sequencing confirmation.

[0064] S6: Inoculate the BL21(pET-PGK1) recombinant expression bacteria into LB medium containing Kan, culture until OD600 is 0.4-0.6, and then add IPTG to induce expression.

[0065] After induction, the bacteria were collected by centrifugation, resuspended in TBS buffer, and lysozyme solution was added at 1 / 10 of the bacterial resuspension volume. The mixture was sonicated for 15 min and centrifuged at 12000×g for 30 min at 4℃. The soluble supernatant containing the PGK1 recombinant protein was collected, filtered through a 0.45 μm pore size filter, and then purified using Ni... 2+ Affinity chromatography column purification.

[0066] Example 3

[0067] Based on the operation in Example 2, this example optimizes the specific parameters in the preparation of recombinant PGK1 protein.

[0068] (1) Optimization of induction temperature: The recombinant expression bacteria were inoculated into 100 mL of LB medium containing Kan at an inoculation ratio of 1:100 and cultured at 37℃ and 220 r / min for 3 h. When the OD600 value reached 0.4 to 0.6, IPTG was added at 16℃, 25℃ and 37℃ to a final concentration of 1 mmol / L (the concentration in the recombinant expression bacteria culture system). The culture was then cultured at 180 r / min for 6 h. After induction, the bacteria were centrifuged at 12000×g for 10 min to collect the bacteria. Protein loading buffer was added and the bacteria were placed in a metal bath at 100℃ for 10 min to allow the protein to denature fully. The expression of the recombinant protein was detected by 12% SDS-PAGE electrophoresis.

[0069] The results showed that, compared with BL21(DE3) empty bacteria, BL21(pET-30a(+)) empty vector bacteria before induction, BL21(pET-30a(+)) empty vector bacteria after induction, and BL21(pET-PGK1) before induction, BL21(pET-PGK1) after induction showed a distinct band at approximately 52 kDa, which was consistent with the expected molecular weight. Figure 5 The results showed that the target protein band intensity was higher at 16℃ than at 25℃ and 37℃, therefore 16℃ was determined as the induction temperature for subsequent expression.

[0070] (2) Optimization of induction time: When the recombinant expression bacteria are cultured in the same way as in (1) until the OD600 value reaches 0.4-0.6, 5 mL of bacterial solution is taken as the control bacterial solution before induction (0 h), and IPTG is added to the final concentration of 1 mmol / L. The bacteria are continuously induced at 37℃ and 180 r / min. 2 mL of bacterial solution is taken at each time point after induction: 2 h, 4 h, 6 h, 8 h and 12 h. Protein loading buffer is added and the bacteria are placed at 100℃ for 10 min to fully denature. The expression of recombinant protein is detected by 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel).

[0071] The results showed that the BL21(pET-PGK1) recombinant expression bacteria successfully expressed a specific protein band at the expected size and location after induction. The induced protein expression level increased sequentially with increasing induction time. Figure 6 As shown, the expression level of BL21(pET-PGK1) recombinant expression bacteria reached its peak after 12 h of induction, and the optimal induction time for recombinant expression bacteria was 12 h.

[0072] (3) IPTG concentration optimization: When the recombinant expression bacteria were cultured in the same way as in (1) until the OD600 value reached 0.4-0.6, 5 mL was taken as the control bacterial solution before induction (0 h). The bacterial solution was aliquoted into 12 mL shaking tubes, with 7 mL in each tube. Then, IPTG inducer was added to each tube of bacterial solution to the final concentrations of 0.05, 0.1, 0.2, 0.6, 1.0, and 2.0 mmol / L, respectively. After induction culture at 37℃ and 180 r / min for 6 h, protein loading buffer was added and the mixture was placed at 100℃ for 10 min for complete denaturation. The expression of recombinant protein was detected by electrophoresis.

[0073] according to Figure 7 As shown, the BL21(pET-PGK1) recombinant expression strain can fully activate the transcriptional activity of the T7 expression system and achieve the maximum induction efficiency when the IPTG concentration is 0.1 mmol / L. Therefore, the optimal IPTG induction concentration for the BL21(pET-PGK1) recombinant expression strain is 0.1 mmol / L.

[0074] Comparative Example Not all genes screened according to the method in Example 1 were successfully expressed. For example, among the candidate antigens screened was the IDGF6 candidate antigen gene from *Gnaphalium affine*, whose coding sequence is shown in SEQ ID NO. 5 and the corresponding amino acid sequence is shown in SEQ ID NO. 6.

[0075] Following the preparation method in Example 2 and the optimized induction conditions in Example 3, IPTG was added. After induction, the bacterial cells were collected by centrifugation, resuspended, and then lysozyme was added followed by sonication. After centrifugation, the supernatant and precipitate of the bacterial lysis were collected separately, and the expression of the IDGF6 recombinant protein was detected by SDS-PAGE. The SDS-PAGE results showed that only very weak bands were observed in the supernatant and precipitate after induction, which was insufficient to obtain an effective expression level for subsequent purification and iELISA detection. Therefore, this study did not select the IDGF6 recombinant protein for further antigen purification and the establishment of serological detection methods.

[0076] Example 4

[0077] This embodiment describes the extraction and purification of soluble PGK1 recombinant protein, following the same basic steps as in Example 2, except that step S6 uses the optimized induction conditions from Example 3: The BL21(pET-PGK1) recombinant expression bacteria were inoculated into culture medium and cultured. When the OD600 reached 0.4–0.6, IPTG was added at 16℃ to a final concentration of 0.1 mmol / L, and the induction time was 12 h. After induction, the bacteria were collected by centrifugation. The precipitate was resuspended in TBS solution, and lysozyme solution was added at a ratio of 1 / 10 of the bacterial resuspension volume. The mixture was ultrasonically disrupted for 15 min, centrifuged at 12000×g at 4℃ for 30 min, and the soluble protein supernatant was collected. After filtration, the supernatant was purified using Nitrogen hydrochloride (Nitrogen hydrochloride). 2+ Affinity chromatography column purification.

[0078] Identification of recombinant protein expression: BL21(pET-PGK1) recombinant expression bacteria were induced to express the protein in large quantities. After lysozyme treatment, the bacterial cells were lysed, and the supernatant and precipitate were separated. The expression form of the recombinant protein was detected by SDS-PAGE gel electrophoresis. The results showed that the recombinant protein was expressed in both the supernatant and inclusion bodies. The expression level of recombinant PGK1 in the supernatant was significantly higher than that in the inclusion bodies, thus proving that PGK1 was expressed primarily in the supernatant. Figure 8 ).

[0079] Recombinant protein purification: Based on the expression characteristics of soluble proteins, the supernatant after centrifugation of the BL21(pET-PGK1) recombinant expression bacterial culture was filtered through a 0.45 μm pore size filter and directly added to His GraviTrap™ Affinity Columns with Ni 2+ The packing material was fully incorporated, and protein purification was performed according to the manufacturer's instructions. SDS-PAGE results showed that the purified recombinant protein exhibited a clear target band, and contaminating proteins were significantly reduced, indicating good purification results. Figure 9 ).

[0080] Recombinant protein Western blot identification results: His tag identification was performed using Mouse Anti-6×His Tag IgG as the primary antibody and HRP-Goat Anti-Mouse IgG as the secondary antibody. A specific band appeared at approximately 52 kDa, indicating that the His-tagged PGK1 recombinant protein was successfully expressed. Figure 10 Immunoreactivity was verified using serum from infected and uninfected horses (both infected with *Gnaphalium affine*) as the primary antibody and HRP-labeled goat anti-horse IgG as the secondary antibody. A specific band appeared at approximately 52 kDa in the positive horse serum group, while no corresponding band was observed in the uninfected horse serum group. Figure 11 ). Figure 11 Lanes 5 and 6 were for observation of sheep serum infected with other parasites; no bands were observed, further confirming the specificity of this method for infected horse serum. Cross-reactivity was evaluated using the iELISA results of 43 positive horse serum samples with other parasites from Example 6.

[0081] Example 5

[0082] This embodiment provides a kit containing an ELISA plate coated with recombinant protein of PGK1 phosphoglycerate kinase from the black-bellied gastric fly, as well as blocking solution and enzyme-labeled secondary antibody; The basic operating procedure for the iELISA kit using recombinant PGK1 protein as the coating antigen is as follows, for the detection of in vitro serum samples: 1) Dilute the purified PGK1 recombinant protein with antigen coating buffer, add 100 μL / well to a 96-well microplate, and coat overnight at 4°C; discard the liquid in the plate the next day, wash 3 times with PBST and pat dry.

[0083] 2) Add blocking buffer at 100 μL / well and incubate overnight at 4°C; discard the liquid in the plate, wash 3 times with PBST and pat dry.

[0084] 3) Dilute the isolated horse serum and add 100 μL / well to the microplate, incubate at 37°C for 2 h; discard the liquid in the plate, wash 3 times with PBST and pat dry.

[0085] 4) Add HRP-labeled goat anti-horse IgG at 100 μL / well and incubate at 37°C for 1 h; discard the liquid in the plate, wash 3 times with PBST and pat dry.

[0086] 5) Add 100 μL of TMB colorimetric solution to each well and develop color for 15 min in the dark; add 100 μL of stop solution to each well and measure the OD450 value.

[0087] Based on the basic operating procedures of iELISA, this embodiment optimizes the reagent kit parameters.

[0088] Determination of optimal antigen coating concentration and serum dilution: The recombinant protein was quantitatively diluted with antigen coating buffer using a checkerboard titration method. The initial concentration of PGK1 protein was 5.08 μg / μL. Dilutes were made to 0.375, 0.75, 1.5, 3.0, 6.0, and 12.0 μg / mL at a loading volume of 100 μL per well, and added to 96-well ELISA plates. Coating was performed overnight at 4°C. The next day, standard positive and negative sera were serially diluted at 1:25, 1:50, 1:100, 1:200, and 1:400, respectively. HRP-labeled goat anti-horse IgG was diluted 1:10000. OD450 values ​​were measured according to the iELISA procedure. The optimal antigen coating concentration and serum dilution for iELISA were determined by calculating the P / N ratio of standard positive serum (P) to standard negative serum (N), and by combining the P / N ratio, positive OD450 value, and negative background.

[0089] The results showed that the OD450 value of positive serum generally increased with increasing antigen coating concentration. When the antigen concentration reached a certain threshold, the OD450 value of negative serum also increased, leading to a decrease in the P / N ratio. Significant differences in OD450 and P / N values ​​were observed under different antigen coating concentrations and serum dilutions, as detailed in Table 4. Based on the iELISA results, the optimal coating combination for PGK1 recombinant protein was determined using OD450 and P / N values ​​under different conditions. The optimal antigen coating concentration and serum dilution were 1.5 μg / mL and 1:50, respectively. Although the P / N value was high at an antigen coating concentration of 3 μg / mL and a serum dilution of 1:200, the OD450 value of positive serum was only 0.360, indicating a weak reaction signal. Considering both reaction intensity and negative background, 1.5 μg / mL and 1:50 were determined to be the optimal combination.

[0090] Table 4 Optimization of iELISA antigen coating concentration and serum dilution based on PGK1 recombinant antigen

[0091] Note: P represents the OD450 value of standard positive serum, N represents the OD450 value of standard negative serum, and P / N represents the ratio of the OD450 value of standard positive serum to the OD450 value of standard negative serum.

[0092] Determination of the optimal working concentration of the secondary antibody: Based on the determined optimal antigen coating concentration and serum dilution, HRP-labeled goat anti-horse IgG was used as the secondary antibody and serially diluted at 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, and 1:20000. The OD450 value was measured by iELISA, and the P / N value of each dilution was calculated. The optimal working concentration of the secondary antibody was determined by combining the P / N value, positive OD450 value, and negative background. The results are shown in Table 5. The optimal working concentration of the secondary antibody for PGK1 recombinant protein iELISA was determined to be 1:2000.

[0093] Table 5. Determination of secondary antibody dilution for recombinant antigen PGK1 iELISA

[0094] Determination of optimal antigen coating conditions: Five groups of ELISA plates were coated based on the determined optimal antigen coating concentration and serum dilution. The first group was coated overnight at 4°C, the second to fourth groups were coated at 37°C for 1 h, 2 h, and 3 h respectively, and the fifth group was coated overnight at 27°C. After coating, iELISA assays were performed to measure the results, and the P / N values ​​at different coating temperatures were calculated. The optimal coating conditions were determined by combining the P / N values, positive OD450 values, and negative background.

[0095] The results showed that different coating temperatures and times significantly affected the detection efficiency of recombinant protein iELISA. Under coating conditions of 27℃, the OD450 value of PGK1 positive serum was at a high level, but the background value of negative serum was also significantly increased, while the P / N value decreased (Table 6). Comparing the OD450 and P / N values ​​under different coating conditions, the recombinant antigen performed best under overnight coating conditions at 4℃. Figure 12 Under these conditions, the positive serum reaction is stronger, the negative background is lower, and the P / N value is higher than that under other coating conditions.

[0096] Table 6 Results of determination of optimal antigen coating conditions

[0097] Note: P represents the OD450 value of standard positive serum, N represents the OD450 value of standard negative serum, and P / N represents the ratio of the OD450 value of standard positive serum to the OD450 value of standard negative serum.

[0098] Determination of optimal blocking solution and blocking time: Based on the determined optimal antigen coating concentration and serum dilution, the ELISA plate was coated with blocking solutions of 1% skim milk powder, 3% skim milk powder, 5% skim milk powder, 1% BSA, 3% BSA, 5% BSA, 10% fetal bovine serum, and commercial rapid blocking solution (all blocking solutions were diluted with TBST buffer). The OD450 value was measured by iELISA test and the P / N value was calculated. The optimal blocking solution was determined by combining the P / N value, positive OD450 value, and negative background.

[0099] Based on the optimal antigen coating concentration, serum dilution, and blocking buffer, the coated ELISA plates were blocked at 37°C for 1 h, 2 h, 4 h, and overnight at 4°C. After blocking, iELISA was performed to measure the OD450 value and calculate the P / N value. The optimal blocking time was determined by combining the P / N value, positive OD450 value, and negative background.

[0100] The results showed that, under the blocking condition of 5% BSA, the average OD450 value of positive serum for PGK1-iELISA was 1.132, the average OD450 value of negative serum was 0.249, and the maximum P / N value was 4.56. The P / N value of the 5% skim milk powder group was 3.65, the P / N value of the 3% BSA group was 3.50, and the P / N values ​​of the other blocking solution groups were all low. Therefore, 5% BSA was determined to be the optimal blocking solution for PGK1-iELISA (Table 7). Figure 13 (Left image).

[0101] After determining the optimal blocking solution, the effects of different blocking times (1 h, 2 h, 4 h, and overnight at 4°C) on the iELISA detection results were further compared. The results showed that the average P / N values ​​of PGK1-iELISA under the four blocking conditions were 3.51, 3.81, 5.10, and 5.19, respectively, with the highest P / N value observed at 4°C overnight (Table 8). Considering the positive reaction intensity, negative background value, and P / N value, the optimal blocking time for PGK1-iELISA was overnight at 4°C. Figure 13 (Right image).

[0102] Table 7 Optimization results of different blocking solutions for PGK1 recombinant antigen iELISA

[0103] Table 8 Results of Optimized Blocking Time for PGK1 Recombinant Antigen iELISA

[0104] The optimal reaction time of the primary antibody and the optimal color development time of the TMB substrate were determined as follows: Recombinant antigen PGK1 was coated onto the ELISA plate at the optimal coating concentration. Positive and negative sera were added to the ELISA plate at the optimal primary antibody (serum) dilution concentration. The plates were incubated at 37°C for 30 min, 1 h, 2 h, and 4 h before iELISA assays. Results showed that the P / N value of PGK1 reached its maximum (5.58–6.23) at 2 h incubation, significantly higher than the 30 min and 1 h groups (4.43–4.87). The P / N value decreased slightly at 4 h incubation (4.09–4.35). The recombinant antigen was then subjected to iELISA assays under all the optimized conditions described above. OD450 values ​​were measured at 5 min, 10 min, 15 min, and 20 min after adding the TMB chromogenic solution to screen for the optimal color development time. The results showed that the P / N value of PGK1 reached its highest level (5.74–6.13) at 15 min of color development, significantly higher than that of the 10 min group (4.59–4.88) and the 20 min group (5.27–5.62). In summary, the optimal incubation time with primary antibody for recombinant antigen PGK1 was 2 h, and the optimal TMB color development time was 15 min. Figure 14 ).

[0105] ROC curve analysis and determination of cut-off value: 120 positive serum samples from horses infected with *Gnaphalium affine* and 30 negative serum samples from horses not infected with *Gnaphalium affine* were selected and tested under the optimized iELISA conditions described above. The OD450 value was measured for ROC curve analysis and determination of the cut-off value. The results are shown in Table 9.

[0106] Traditional statistical methods were used to calculate the mean and standard deviation (SD) of OD450 values ​​from 30 negative serum samples, with mean+2SD and mean+3SD serving as reference cutoff values. Receiver operating characteristic (ROC) curve analysis was performed using SPSS 26.0 software, and the area under the curve (AUC) was calculated to evaluate the iELISA method's ability to distinguish between positive and negative sera. The Youden index was calculated based on the sensitivity and specificity corresponding to different OD450 thresholds, using the formula: Youden index = sensitivity + specificity - 1. The OD450 value corresponding to the highest Youden index corresponds to the ROC recommended cutoff value. The cutoff value for the PGK1 recombinant antigen was determined by combining mean+2SD, mean+3SD, ROC cutoff value, and the distribution of OD450 values ​​in positive and negative sera. A sample was considered negative if its OD450 value was less than the cutoff value, and positive if its OD450 value was greater than or equal to the cutoff value.

[0107] Table 9. Raw data of serum sample OD450 values ​​used to determine the PGK1-iELISA checkpoint.

[0108] The results are shown in Table 10. There was a significant difference in the OD450 values ​​of positive and negative sera detected by iELISA for PGK1 recombinant protein. The OD450 values ​​of positive serum were generally higher than those of negative serum, and a relatively clear distribution boundary was formed on both sides of the cutoff value. Figure 15 The area under the ROC curve is 0.999, and the 95% confidence interval is 0.996–1.000. Figure 16 The recommended ROC threshold is 0.492, corresponding to a Youden index of 0.975. Combining the results of negative serum mean plus two standard deviations, mean plus three standard deviations, and ROC analysis, the final threshold is determined to be 0.492. This threshold is only applicable to the in vitro detection performance evaluation of isolated horse serum samples.

[0109] Table 10 Determination of iELISA values ​​for recombinant PGK1 protein

[0110] Example 6 This embodiment provides a kit comprising an ELISA plate coated with recombinant protein PGK1 (phosphoglycerate kinase) from *Gnaphalium affine*, a blocking buffer, and an ELISA-labeled secondary antibody. The coating concentration of the recombinant protein is 1.5 μg / mL, the blocking buffer is 5% BSA, and the ELISA-labeled secondary antibody is HRP-labeled goat anti-horse IgG at a working dilution of 1:2000. TMB chromogenic reagent is used, and color development is performed for 15 min in the dark. The cutoff value is 0.492; a sample is considered negative if its OD450 value is less than the cutoff value, and positive if its OD450 value is greater than or equal to the cutoff value. Forty-three serum samples positive for other parasitic infections were tested using iELISA to evaluate cross-reactivity.

[0111] Based on the final judgment value determined by the ROC curve, the PGK1 recombinant protein iELISA detected 117 positive samples out of 120 known positive serum samples of *Gnaphalium affine* infection, with a positive concordance rate of 97.50%; all 30 known negative serum samples were negative, with a negative concordance rate of 100.00%.

[0112] The cross-reactivity results are shown in Table 11. No positive reactions were observed in any of the 43 positive horse serum samples for *Strongyloides equine*, *Parascaris equine*, and *Taenia equinea*. The cross-reactivity rate of the tested samples was 0% (0 / 43), with a specificity of 100%. This result indicates that the PGK1 recombinant protein iELISA has low cross-reactivity within the range of positive sera for common equine parasites tested in this example.

[0113] Table 11 Results of iELISA Cross-Reactivity Evaluation of PGK1 Recombinant Protein

[0114] Repeatability results of iELISA assay: Strongly positive, moderately positive, and weakly positive sera from known *Gnaphalium affine* infections, along with three negative sera, were selected for intra-assay and inter-assay repeatability tests. Each serum sample was tested in triplicate. (See Table 12). In the three assays, the intra-assay CV ranged from 0.13% to 8.64%, with an average intra-assay CV of 1.07% to 5.19%, and the inter-assay CV ranged from 2.47% to 9.52%. All CVs were below 10%, indicating that PGK1-iELISA has good repeatability.

[0115] Table 12 Results of PGK1-iELISA repeatability test

[0116] Example 7: Field Sample Detection Results The kit from Example 6 was used to perform in vitro iELISA detection on 220 field horse serum samples. The results are shown in Table 13. 198 samples had values ​​above the cutoff, and 22 samples had values ​​below the cutoff, resulting in a positive detection rate of 90.00% for infection-related antibodies. This result indicates that the recombinant protein and detection system described herein can be used for the detection of *Gastropoda melanogaster* infection-related antibodies in isolated field horse serum samples.

[0117] Table 13 Results of PGK1 recombinant antigen detection in field serum samples

[0118] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Any improvements and modifications made by those skilled in the art without departing from the spirit and essence of the invention should fall within the protection scope of the invention.

Claims

1. A *Gnaphalium affine* phosphoglycerate kinase PGK1 gene, characterized in that, The gene sequence of the melanogaster phosphoglycerate kinase PGK1 is shown in SEQ ID NO.

3.

2. A recombinant protein of *Gnaphalium gravidarum* phosphoglycerate kinase PGK1, characterized in that, The amino acid sequence of the recombinant protein PGK1 phosphoglycerate kinase from the black-bellied gastric fly is shown in SEQ ID NO.

4.

3. The recombinant protein PGK1 phosphoglycerate kinase from *Gnaphalium gravidarum* according to claim 2, characterized in that, It is prepared by the following steps: S1: Total RNA was extracted from second-stage larvae of the black-bellied gastric fly and the first strand of cDNA was synthesized using a reverse transcription kit. S2: Primers PGK1 F ​​and PGK1 R were designed targeting the PGK1 gene, with sequences SEQ ID NO. 1 and SEQ ID NO. 2, respectively. The primer sequences were then modified by adding... Eco RI and Xho I restriction site, synthesize PGK1 primer; S3: Using cDNA from *Gnaphalium affine*, *Gnaphalium affine*, *Gnaphalium affine*, *Parascaridium equine*, and *Strongyloides equine* as templates, PCR amplification and specificity verification were performed using the PGK1 primers, confirming that the primers could only amplify the target fragment from *Gnaphalium affine* cDNA. S4: The PCR product of the cDNA of the black-bellied gastric fly was ligated with the cloning vector pMD19-T. The ligation product was transformed into TOP10 competent cells and cultured. The cultured bacteria were used for PCR identification. Plasmids were extracted from the PCR-positive bacteria and double enzyme digestion was performed for identification. The recombinant clones that were initially identified as positive by PCR and double enzyme digestion products were sequenced. S5: The recombinant cloning bacterial strain and the pET-30a(+) expression vector strain were cultured separately, and the recombinant cloning plasmid and pET-30a(+) expression plasmid were extracted separately. The target gene fragment and the linearized pET-30a(+) vector fragment were recovered by double enzyme digestion. The target gene fragment and the linearized pET-30a(+) vector fragment were ligated. The ligation product was transformed into competent cells, cultured, and screened to identify the PGK1 recombinant expression strain. S6: When the PGK1 recombinant expression bacteria reach an OD600 value of 0.4-0.6, IPTG is added for induction. The induced bacterial cells are collected, resuspended in buffer, treated with lysozyme, sonicated, and centrifuged. The soluble supernatant containing the recombinant protein is collected and processed using Ni... 2+ Affinity chromatography column purification.

4. The recombinant protein PGK1 phosphoglycerate kinase from *Gnaphalium gravidarum* according to claim 3, characterized in that, In step S6, the final concentration of IPTG added to the system for culturing the PGK1 recombinant expression bacteria is 0.1 mmol / L.

5. The recombinant protein PGK1 phosphoglycerate kinase from *Gnaphalium gravidarum* according to claim 3, characterized in that, In step S6, the induction temperature is 16℃ and the induction time is 12 h.

6. The recombinant protein PGK1 phosphoglycerate kinase from *Gnaphalium gravidarum* according to claim 3, characterized in that, In step S6, the bacterial cells were resuspended in TBS buffer, and lysozyme solution was added at 1 / 10 of the bacterial resuspension volume. The mixture was then sonicated and filtered through a 0.45 μm pore size filter to obtain the soluble supernatant, which was then subjected to Ni... 2+ Affinity column chromatography.

7. A kit for detecting antibodies against *Gnaphalium affine* infection in isolated horse serum, characterized in that, An enzyme-labeled plate containing the recombinant protein of the melanogaster phosphoglycerate kinase PGK1 as described in any one of claims 2 to 6.

8. The reagent kit according to claim 7, characterized in that, The coating concentration of the recombinant protein PGK1 phosphoglycerate kinase from the black-bellied gastric fly was 1.5 μg / mL.

9. The reagent kit according to claim 7, characterized in that, The kit also includes a blocking solution and an enzyme-labeled secondary antibody; the blocking solution is 5% BSA, and the enzyme-labeled secondary antibody is HRP-labeled goat anti-horse IgG diluted at 1:2000; the kit has a cutoff value of 0.492.

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