Cpges protein based on immunological screening of cDNA prokaryotic expression library of cattle cysticercus and application thereof

Abstract

This invention discloses a cPGES protein based on immunological screening of a bovine cysticercosis cDNA prokaryotic expression library and its applications, belonging to the field of biochemistry. The purpose of this invention is to provide a method for the detection, treatment, and prevention of bovine cysticercosis. This invention provides a bovine cysticercosis antigen, the amino acid sequence of which is shown in SEQ ID NO.4. This provides a new candidate antigen for the development of novel vaccines and specific diagnostic reagents.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a cPGES protein based on immunological screening of a bovine cysticercosis cDNA prokaryotic expression library and its application. Background Technology

[0002] Bovine cysticercus (Cysicercus bovis) is a foodborne zoonotic parasite belonging to the family Taeniaceae. The larvae primarily parasitize bovine animals (such as cattle, buffalo, yaks, and zebu), causing cysticercosis (commonly known as bovine cysticercosis). When people consume raw or undercooked beef or beef products contaminated with bovine cysticercus, the cysticercus develops in the small intestine, leading to bovine tapeworm infection.

[0003] Cattle, as intermediate hosts, are the main source of human infection with bovine tapeworm. Vaccination of cattle is the most ideal measure to control the prevalence of bovine cysticercosis in my country. Immunization of cattle would greatly simplify control methods, accelerate the control process, and significantly reduce and control the transmission of bovine tapeworm between humans and animals. However, the development of bovine vaccines is currently stalled, primarily due to the unclear protective immune mechanisms and the lag in the screening of protective antigens.

[0004] Currently, there is a lack of effective immunological diagnostic and testing techniques and effective preventive vaccines for bovine cysticercosis, resulting in a continued upward trend in the global prevalence of the disease. In my country, research on the epidemiology, surveillance, diagnosis, testing, and vaccines related to bovine cysticercosis is severely lacking, or even nonexistent.

[0005] Bovine cysticercosis poses a significant threat to beef cattle farming and beef food safety. Currently, prevention and control primarily rely on a comprehensive approach, including drug deworming (administered praziquantel powder or tablets), health education (popularizing dietary habits in endemic areas, advocating for safe beef cooked at high temperatures, and advising against raw beef consumption), and sanitation improvement (harmless treatment of human excrement). With the development of molecular biology techniques, research in parasitic immunology has made continuous progress. Breakthroughs in antigen isolation and molecular cloning have enabled pre- and post-mortem immunological diagnosis and testing, providing ample technical support for effective vaccine development. Based on this, the applicant has established a cDNA library of bovine cysticercosis larvae and oncocytozoa. Using bovine serum infected with bovine cysticercosis, and employing multi-round immunological screening techniques, highly reactive single colonies were identified through next-generation sequencing to identify novel antigens, providing new candidate antigens for novel vaccine development and the development of specific diagnostic reagents. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting, treating, and preventing bovine cysticercosis.

[0007] This invention provides a cPGES protein from bovine cysticercosis, the amino acid sequence of which is shown in SEQ ID NO.4.

[0008] The present invention provides a gene encoding the above-mentioned cPGES protein, characterized in that the gene is as shown in SEQ ID NO.3.

[0009] The present invention provides a recombinant vector containing the above-mentioned genes.

[0010] The present invention provides a recombinant host cell containing the above-mentioned genes.

[0011] This invention provides a kit for detecting bovine cysticercosis, the kit containing the aforementioned cPGES protein.

[0012] Further specifying, the kit includes a sample buffer, a PVC base plate, a sample pad, a nitrocellulose membrane, and an absorbent pad; the sample buffer consists of 0.01M PBS, 10% sucrose, 1% BSA, 1.4% Tween-20, 0.2% fluorescently labeled goat anti-bovine IgG antibody, and 0.4% fluorescently labeled bovine cysticercosis cPGES protein; the amino acid sequence of the bovine cysticercosis cPGES protein is shown in SEQ ID NO.4.

[0013] Further specified, the coupling ratio of fluorescent microspheres to goat anti-bovine IgG antibody was 1:2, and the coupling ratio of fluorescent microspheres to bovine cysticercosis antigen was 1:20; the detection line coated on the nitrocellulose membrane was mouse anti-human IgG antibody, and the control line coated on the nitrocellulose membrane was goat anti-bovine IgG antibody; the coating concentration was 5 μL / mm.

[0014] This invention provides the application of the above-mentioned cPGES protein of bovine cysticercosis, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant host cell in the preparation of a kit for diagnosing or detecting bovine cysticercosis or anti-bovine cysticercosis antibodies.

[0015] This invention provides the application of cPGES protein from bovine cysticercosis in the preparation of a vaccine against bovine cysticercosis, wherein the amino acid sequence of the cPGES protein is shown in SEQ ID NO.4.

[0016] This invention provides the application of a recombinant vector or recombinant host cell containing a gene encoding the cPGES protein of bovine cysticercosis in the preparation of a vaccine against bovine cysticercosis, wherein the gene encoding the cPGES protein is shown in SEQ ID NO.3.

[0017] Beneficial effects: The purified protein was verified by Western blotting. Bovine cysticercosis negative and positive sera were diluted 1:1000, incubated overnight at 4°C, washed three times with TBST, and then incubated for 1 hour at room temperature with 1:10000 diluted HRP-goat anti-bovine IgG. After washing, the protein was developed. Bovine cysticercosis positive serum showed bands of consistent size, while negative serum showed no bands. The protein specifically bound to bovine cysticercosis-resistant positive serum, but showed no reaction with negative serum at the target protein site, indicating that the recombinant protein has good antigenicity. Attached Figure Description

[0018] Figure 1 The results of PCR amplification of the cytosolic prostaglandin E synthase protein gene are shown, where M: molecular weight standard; 1 and 2: PCR amplification products.

[0019] Figure 2 The diagram shows the construction of the pET28a expression vector, where M: molecular weight standard; 1: undigested plasmid control; 2, 3, 4, 5: digested pET28a plasmid; 6: blank control (no plasmid added to the system).

[0020] Figure 3 This is a 10% SDS-PAGE image of the bovine cysticercosis cytosolic prostaglandin E synthase (cPGES) protein gene induced in E. coli BL21(DE3) cells according to the present invention. M: standard molecular weight; 1: uninduced PET28a empty vector; 2: induced PET28a empty vector supernatant; 3: induced pET28a empty vector precipitate; 4: uninduced PET28a-cPGES whole cell; 5: IPTG-induced pET28a-cPGES expression supernatant; 6: IPTG-induced PET28a-cPGES expression precipitate.

[0021] Figure 4 This is a 10% SDS-PAGE image of a bovine cysticercosis cytosolic prostaglandin E synthase protein gene after induction and purification in E. coli BL21(DE3) cells. M: standard molecular weight; 1: IPTG-induced pET28a-cPGES expression supernatant; 2: transfer buffer; 3: first elution buffer; 4: last elution buffer; 5-8: PET28a-cPGES protein purified by the first to fourth elutions.

[0022] Figure 5This is a Western blotting diagram of the gene of the bovine cysticercosis cytosolic prostaglandin E synthase protein according to the present invention, where M: standard molecular weight; A: His-tagged antibody; B: positive serum from bovines infected with bovine cysticercosis; and C: negative serum from bovines not infected with bovine cysticercosis. Detailed Implementation

[0023] Example 1. Construction of a cDNA library of *Taenia solium* hexacyteres. 1. Total RNA extraction and mRNA purification A sample of beef tapeworm hexacytes, which had been pre-preserved in liquid nitrogen, was placed in a 1.5 mL centrifuge tube. Total RNA was extracted from the beef tapeworm hexacytes using TRIZOL reagent, and mRNA was isolated and purified from the total RNA using the Oligotex mRNA Kits.

[0024] 2. cDNA Synthesis and Purification (1) Synthesis of the first strand of cDNA The following reaction mixture was added to a 0.2 mL RNase-free centrifuge tube: E. coli DNA Ligase (10 U / µL) 1 µL E. coli RNase H (2 U / µL) 1 µL.

[0025] E. coli .DNA Polymerase I(10 U / µL)4 µL mRNA sample (4.5 µg) 22.5 µL 3' RT Primer (1.5 μg / μL)2 μL Place the reaction tube on the PCR instrument and incubate at 70°C for 7 min, then immediately place it on ice. Prepare the first chain reaction system in a new 0.2 mL RNase-free tube: 5×RT Buffer 10 µL 5 µL of water 10 mM dNTPs 2.5 µL RT enzyme 5 µL After the primer reaction tube in the first tube cools to 45°C, maintain incubation at 45°C for 2 min. Add the reaction mixture from the second tube and mix well, avoiding the formation of bubbles. Transfer the reaction product to a new 1.5 mL RNase-free tube, add the following reagents, mix well, and incubate at -80°C for at least 1 h: Glycogen (20 μg / μL) 1 µL 7.5 M NH4OAc25 µL 100% ethanol 187 µL Centrifuge the first-stranded product precipitated at low temperature at 16,000 × g at 4 °C for 30 min, discard the supernatant, add 150 μL of RNase-free 70% ethanol, centrifuge at 16,000 × g at 4 °C for 3 min, discard the supernatant, repeat once, dry the cDNA at room temperature, dissolve the precipitate in 20 μL LEPC water, and place on ice for later use.

[0026] (2) Synthesis of the second strand of cDNA Add the following reagents to the above reaction solution: DEPC-treated water 91 µL 5×Second Strand Buffer 30 µL 10 mM (each) dNTPs 3 µL Second Strand Enzyme Mix 6 µL Total Volume: 130 µL At 16°C for 2 hours, add 2 µL of T4 DNA Polymerase, at 16°C for 5 min, add 10 µL of 0.5 M EDTA (pH 8.0), add 160 µL of phenol:chloroform:isoamyl alcohol (25:24:1), mix thoroughly for 30 s; centrifuge at 14,000 rpm at room temperature for 5 min, carefully transfer the supernatant to a new centrifuge tube, precipitate with ethanol, and dissolve in 40 µL of DEPC water.

[0027] Add a 5' connector (3 barcode reader frames, each frame connected to one copy, for a total of 3 copies): 34 µL of cDNA 10×T4 ligase buffer 5 µL 5' Adapter (1µg / µL) 10 µL T4 DNA Ligase(40 U / µL, NEB)1 µL Total Volume 50 µL After mixing, incubate at 16°C for 16-24 hours, then add 2 µL of 10 mM dNTP and 2 µL of T4 DNA polymerase, and incubate at 16°C for 20 minutes to fill in the ends.

[0028] (3) Recover cDNA fragments of the target length cDNA products were electrophoresed using a 1% low-melting-point agarose gel, and fragments larger than approximately 1 kbp were recovered by gel excision. The gel was incubated at 70°C for 10 min, then transferred to 45°C, with the addition of 10× buffer and 5 µL of lysozyme, and incubated at 45°C for 3–4 h. The gel was then centrifuged at 12000 g for 15 min at 4°C. The supernatant was collected, ethanol was used to precipitate the product, and it was dissolved and recovered using 14 µL of DEPC-treated water.

[0029] (4) Ligation of cDNA to vector Using homologous recombination, 7 µL of cDNA from the previous step was mixed with 3 µL of the modified pDEST17 vector, followed by 5 µL of all-direct recombinase and 5 µL of water. The mixture was then incubated at 25°C for 20 h.

[0030] Add 2 µL of Proteinase K to inactivate the recombinase; Add 78 µL of sterile water to the reaction system to make the total volume 100 µL; Glycogen (20 μg / μl) 1 µL 7.5 M NH4OAc 50 µL 100% ethanol 375 µL Mix thoroughly and incubate at -80°C for at least 1 hour. Centrifuge at 16,000 rpm for 30 min at 4°C. Carefully remove the supernatant. Add 150 µL of 70% ethanol and centrifuge at 16,000 rpm for 3 min at 4°C. Repeat this step once, removing all supernatant while avoiding disturbing the cDNA precipitate. Allow the cDNA to air dry at room temperature for 5-10 min. Resuspend the cDNA precipitate in 10 µL of DEPC water by pipetting 30-40 times. Collect the cDNA by brief centrifugation for 2 s and immediately place on ice.

[0031] (5) Electroporation of competent Escherichia coli cells Pre-cool the 1 mm electroporation cuvette at -80℃ for 30 min. On ice, add 2.5 µL of recombinant product and 50 µL of competent cells to the cuvette and place it on ice for 45 min. Electroporate the cuvette (Voltage 2.9 kV, Resistance 200 Ω, Capacity 25 μF). Immediately after electroporation, add 1 mL of LB medium to the cuvette and transfer it to a new 15 mL centrifuge tube. Make up the volume to 5 mL and incubate at 37℃ and 225-250 rpm for at least 1 h.

[0032] After the culture is completed, dilute the culture by 10, 100, 1000, and 10000 times, and take 10 µL of each dilution to plate. The remaining culture can be stored at 4°C overnight, or add glycerol to a final concentration of 20% and store at -80°C.

[0033] (6) Document quality assessment CFU / mL = (Number of clones on plate / 10 µL) × 100 × 1 × 10 3 µL; Total CFU of the library = CFU / mL × Total volume of bacterial culture in the library (mL); 7) Insertion fragment size determination Single clones were picked from the plate, amplified by PCR, and the size of the PCR product was detected by electrophoresis. The PCR primers were universal primers for the vector: M13F (TGTAAAACGACGGCCAGT, SEQ ID NO.1) and M13R (CAGGAAACA GCTATGACC, SEQ ID NO.2).

[0034] Implementation Case 2. Immune Screening of cDNA Libraries 1. Processing of bovine positive serum for library screening Pick E. coli (BL21 DE3) single clones were cultured overnight at 37°C in 100 mL LB medium. The culture was centrifuged at 4°C, 5000×g for 10 min to remove the medium. The cells were resuspended in 3 mL Tris-HCl (50 mM, pH 8.0) and EDTA (10 mM, pH 8.0), and the mixture was subjected to three freeze-thaw cycles. The cells were then sonicated until the culture became clear. The culture was centrifuged at 4°C, 5000×g for 15 min, and the supernatant was collected. E. coli (BL21 DE3) lysis buffer. Take 100 µL of bovine cysticercosis-positive mixed serum and add 1 mL of the buffer. E. coli (BL21 DE3) lysis buffer was adsorbed overnight at room temperature, centrifuged at 5000×g for 10 min to remove the precipitate, and the supernatant was diluted with antibody diluent to make the final serum concentration 1:100. Sodium azide was added to a final concentration of 0.02%, and the solution was stored at 4℃.

[0035] 2. Immune screening of cDNA libraries 2.1 Initial screening 2.1.1 Bacterial Culture Spread the amplified library evenly on LB plates (Amp+), incubate upside down at 37°C overnight until the colonies grow to a diameter of 0.1-0.2 mm, then remove the plates from the incubator and incubate upside down at 4°C for 1-2 hours.

[0036] 2.1.2 Applying the film Nitrocellulose membrane, or NC membrane (Millipore, 0.45μm), is cut to the size that matches the culture plate and laid on the surface of the culture medium. It is brought into contact with the colonies until it becomes wet, and then marked at three asymmetrical positions with the tip of a syringe needle.

[0037] 2.1.3 Induced Expression Remove the NC membrane, with the colony-to-cell contact side facing up, and plate it onto an LB plate (Amp+) containing IPTG. Incubate upside down at 37°C for 6-8 hours. Continue incubating the plate at 37°C for approximately 6 hours until new colonies appear. Seal the plate with sealing film and store it upside down at 4°C.

[0038] 2.1.4 Fixed Remove the NC membrane from the clean bench, expose it in chloroform vapor for 15 min, and then place it in a petri dish.

[0039] 2.1.5 Pyrolysis Elution Immerse the NC membrane in bacterial lysis buffer, place the plate on a shaker at 50 rpm overnight at room temperature for lysis, change the elution buffer and let it stand at room temperature 3 times, 30 min each time.

[0040] 2.1.6 Closed 5% skim milk powder was sealed in TBST at room temperature for 2 hours.

[0041] 2.1.7 Detection of positive clones expressing the target fusion protein Incubate with bovine positive serum treated by the sham screening method for 2 h, then place the petri dish on a shaker and shake slowly at room temperature at 50 rpm.

[0042] The NC membrane was placed in an elution buffer containing 1% Triton-X, 0.5% sodium deoxycholate and 0.1% SDS for 1 h. The NC membrane was washed 3 times in the elution buffer for 30 min each time, and then slowly shaken at 50 rpm on a shaker at room temperature.

[0043] The secondary antibody was alkaline phosphatase AP-labeled affinity-purified goat anti-bovine IgG (Jackson, USA), diluted 1:5000, and incubated in an NC membrane at room temperature for 2 h.

[0044] Repeat the above film washing steps.

[0045] After washing the membrane, BCIP (5-bromo-4-chloro-3-indoleyl phosphate) / NBT (nitro blue tetranitrile ammonium chloride) was used as the substrate for color development.

[0046] The reaction was terminated with distilled water.

[0047] Positive clones showed a purplish-red color at the antigen-antibody complex site.

[0048] 2.1.8 Localization of positive clones Based on the location of the positive ring on the membrane, a single colony was picked from the specific location of the suspected positive clone on the culture plate and inoculated into LB medium (Amp+) and shaken at 37°C and 180 rpm for 3 h.

[0049] 3. Secondary screening The colonies were inoculated onto LB plates (Amp+) and incubated overnight at 37°C. A second round of screening was performed as described above to obtain single positive clones.

[0050] 4. Three-screen The screening process was repeated for a third round until consistent immunopositive recombinants were obtained, with 100% positive clones appearing on the plate.

[0051] Implementation Case 3: Extraction, Detection, and Analysis of pDEST17 Recombinant Plasmid Positive clones were picked and cultured in LB broth (containing 100 μg / mL Amp) overnight at 37°C with shaking at 200 rpm. The next day, plasmid DNA was extracted using a plasmid extraction kit from Tiangen Biotech, and the plasmid DNA was eluted with 30-50 μL of elution buffer.

[0052] The extracted plasmid was sent to Sangon Biotech Co., Ltd. for sequencing. Using universal primers M13F and M13R, upstream and downstream restriction sites were deleted. The sequence was then subjected to BLAST alignment and homology analysis in NCBI. ORF analysis was performed on the website HTTP: / / www.ncbi.nlm.nih.gov / orffinder / to determine the largest open reading frame. Secondary structure analysis was performed on the NetSurfP 3.0 - DTU Health Tech - Bioinformatic Services website. The base sequence of the gene of this invention was BLAST analyzed in NCBI, and the sequence of the encoded protein was identical to the amino acid sequence 1-198 of SEQ ID NO: 3.

[0053] Secondary structure analysis of the protein encoded by the gene of this invention revealed that the protein's flexible structures, such as turns and random coils, are relatively loose, easily twisted and coiled, and tend to appear on the protein surface, thus making it more likely to become a surface antigen. Furthermore, nearly half of the secondary structures of this sequence are random coils, indicating that this sequence has a structural basis containing B-cell epitopes.

[0054] B-cell antigenic epitopes were predicted for the gene-encoded protein of this invention, and multiple epitopes 0-26 and 129-196 were found in the sequence. BLAST analysis of the gene sequence of this invention using NCBI did not reveal any highly homologous proteins. The predicted target protein lacked a signal peptide and transmembrane region.

[0055] Cytosolic prostaglandin E synthase gene of Taenia saginata cysticercus: SEQ ID NO.3 ATGAGCCTTCTAGAATCGACCTATCGCAGTTATTCGTCGAGCCTCGCAACCATGTCCGGTGATGCTGTTTGTGCTGCGCGTCATCCCGAAATACTTTGGGCACAGCGGCCTAATTGCGTCTACTTAACAGTTGCTCTTTCTGACGTTAAGGATGAGGCGATTGATATTAAACCCGAGTCTTTTCATTTCAAAGGAAGAGTAGACACTCCGACACCAACAAATTACGAACTAACGTTTGATTTTTATGCTGAAGTCGACCCCAATAAGGCGGTCAGGCAGATTGGTGATCGGGTAATAACCTATTGCGTTGAGAAGAAGGAATCTGGTGTTTGGCCTCGGCTTCTCAAGGATGCATCAAAGCAACCTTGGTTAAAGACAGATTTTAGCAGGTGGAAGGATTTGGATGATTCTGATTCGGAGGGCGAGGGTGGCCTCGGTGGTTTCGGTGGATCTGGCGGGAACTTTCAGGATATGCTCTCAATGATGGGCAACAAGGATTTGGGGGAGGAGGATGATGAGGAAGACTCGGATGATGATGAACTCCCGGACCTCGAAGATCCTGCGAAGCCCAAGGCGGAGGAGGTGAATTCGTAA; Cytosolic prostaglandin E synthase protein of Taenia saginata cysticercus: SEQ ID NO.4 MSLLESTYRSYSSSLATMSGDAVCAARHPEILWAQRPNCVYLTVALSDVKDEAIDIKPES FHFKGRVDTPTPTNYELTFDFYAEVDPNKAVRQIGDRVITYCVEKKESGVWPRLLKDASK QPWLKTDFSRWKDLDDSDSEGEGGLGGFGGSGGNFQDMLSMMGNKDLGEEDDEEDSDDDELPDLEDPAKPKAEEVNS。

[0056] Implementation Case 4. Cloning and Transformation of Target Genes Based on the sequence of the target gene, primers for the homologous recombination vector were designed and constructed using biosynthetic methods. Endonucleases (BamHI and XhoI) were selected to linearize the pET28a-vector. The target gene was then ligated into pET28a using homologous recombination technology to construct the prokaryotic expression vector pET28a-H2. The enzyme digestion system is as follows: Buffer 1 µL BamHI1 µL XhoI1 µL PET-28a empty carrier 1 µg Sterilized deionized water to 10 µL Total volume 10 µL Enzyme digestion reaction conditions: 37℃ for 2 hours like Figure 1 and Figure 2 As shown, the enzyme digestion products were subjected to 1% agarose gel electrophoresis. The target fragment and expression vector were excised from the gel under UV light and purified using the agarose gel DNA recovery kit from Tiangen Biotech. The target gene and expression vector were then ligated using homologous recombinase. The ligation system is as follows: 2×mix5 µL PET28a2 µL Target gene 3 µL Total volume 10 µL Connection reaction conditions: 50℃, 45 min.

[0057] Take 10 µL of the ligation product for conversion E. coli DH5a cells, specifically, were prepared by mixing 10 µL of the ligation product with 100 µL of... E. coli Mix the DH5a competent cells, incubate on ice for 30 min, then heat shock in a 42℃ water bath for 90 s, then place on ice for 2 min, then add 900 µL of preheated antibiotic-free LB medium, and incubate at 37℃ with shaking at 180 rpm for 1 h. Take 100 µL of the bacterial culture, spread it on an LB (Kan+) plate, and incubate it upside down at 37℃ overnight.

[0058] Pick 5-10 single colonies from the plate and place them in 1 mL LB (Kan+) liquid medium. Incubate overnight at 37°C with shaking at 200 rpm. Send the culture to Sangon Biotech Ltd. for sequencing to check if the ligation was successful. Extract the plasmid from the successfully ligated recombinant vector using the plasmid extraction kit from Tiangen Biotech Ltd. Take 10 µL of plasmid and transform it according to the above method. E. coli BL21 (DE3) cells.

[0059] Implementation Case 5. Expression and Purification of Recombinant Proteins 1. Low-level expression of recombinant proteins Take the transformed recombinant vector and the empty vector containing pET28a respectively. E. coli A single colony of BL21(DE3) was cultured overnight at 37°C with shaking at 200 rpm in 3 mL LB medium (100 µg / mL Kan). Then, 500 µL of the bacterial culture was added to 50 mL of fresh LB medium (100 µg / mL Kan) and cultured for 2 h with shaking at 200 rpm. IPTG was then added to a final concentration of 1 mM, and expression was induced at 37°C with shaking at 200 rpm for 8 h. The bacterial cells were collected by centrifugation, washed twice with PBS, and both were simultaneously subjected to 10% SDS-PAGE electrophoresis to observe whether the recombinant protein was expressed.

[0060] 2. High-level expression of recombinant proteins Take the definite expression E. coli Single colonies of BL21(DE3) were cultured overnight at 37°C with shaking at 200 rpm in 100 mL LB (100 μg / mL Kan) medium. Then, 100 mL of the bacterial culture was added to 1000 mL of fresh LB (100 µg / mL Kan) medium and cultured for 2 h with shaking at 37°C and 200 rpm. IPTG was then added to a final concentration of 1 mM, and expression was induced at 37°C and 180 rpm for 8 h. The cells were collected by centrifugation, washed twice with PBS, and then resuspended in 10 mL of lysis buffer. The cells were sonicated three times for 3 seconds each time, with 5-second intervals, for a total of 30 min. A portion of the whole colony was sonicated again, centrifuged once more, and the supernatant and precipitate were collected for 10% SDS-PAGE electrophoresis to examine protein expression. Figure 3 As shown.

[0061] 3. Purification of recombinant proteins Since the protein was present in the supernatant, the lysed bacterial culture was centrifuged at 4°C, 12000 rpm for 10 min, and the supernatant was collected. The 6×histidine fusion protein was expressed in *E. coli* BL21 cells and purified by NI-NTA resin based on affinity for the 6×His tag, followed by elution. Figure 4 ).

[0062] The protein concentration of the sample was measured using the BCA protein concentration assay kit (enhanced version) from Beyotime Corporation, and the obtained recombinant protein was used as an antigen.

[0063] Implementation Case 6. Immunogenicity Analysis of Recombinant Proteins The antigen was identified using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Bovine positive serum was diluted 1:1000 with the antigen at a concentration of 20 μg / lane for detection. Goat anti-bovine IgG horseradish peroxidase (Bioss, China) was diluted 1:5000 in TBST containing 0.05% Tween-20 (TBS Tween-20) as a secondary antibody. A high-sensitivity ECL kit (Biosharpl) was used as the chemiluminescent substrate.

[0064] result: SDS-PAGE results showed that the recombinant protein was successfully expressed, and a clear target band appeared in the supernatant after bacterial cell lysis. Figure 4 As shown, due to the histidine tag in the pET28a vector, the target protein size is approximately 26.54 kDa, consistent with the theoretical value. The recombinant protein was purified and collected using Ni column affinity chromatography, revealing a single protein band at a molecular weight of 26.54 kDa.

[0065] Western blotting analysis of the purified recombinant protein showed that it reacted with the anti-His tag antibody and specifically bound to bovine positive serum containing *Taenia solium*. No band was observed at the target protein in the negative serum, indicating that the recombinant protein possesses good antigenicity. In summary, bovine cysticercosis negative and positive sera were diluted 1:1000, incubated overnight at 4°C, washed three times with TBST, and then incubated for 1 hour at room temperature with 1:10000 diluted HRP-goat anti-bovine IgG. After washing, development was performed. The bovine cysticercosis positive serum showed bands of consistent size, while the negative serum showed no bands. Figure 5 This indicates that the recombinant protein has good antigenicity.

[0066] Example 7. Reagent Kit The kit includes sample buffer, a PVC base plate, a sample pad, a nitrocellulose membrane, and an absorbent pad. The sample buffer consists of 0.01M PBS, 10% sucrose, 1% BSA, 1.4% Tween-20, 0.2% fluorescently labeled goat anti-bovine IgG antibody, and 0.4% fluorescently labeled bovine cysticercosis antigen. The amino acid sequence of the bovine cysticercosis antigen is shown in SEQ ID NO. 4. The performance is consistent with that in Example 6.

Claims

1. A cPGES protein from bovine cysticercosis, characterized in that, The amino acid sequence of the cPGES protein of the bovine cysticercosis is shown in SEQ ID NO.

4.

2. A gene encoding the cPGES protein of claim 1, characterized in that, The gene is shown in SEQ ID NO.

3.

3. A recombinant vector containing the gene described in claim 2.

4. A recombinant host cell containing the gene of claim 2.

5. A reagent kit for detecting bovine cysticercosis, characterized in that, The kit contains the cPGES protein as described in claim 1.

6. The reagent kit according to claim 5, characterized in that, The kit includes a sample buffer, a PVC base plate, a sample pad, a nitrocellulose membrane, and an absorbent pad. The sample buffer consists of 0.01M PBS, 10% sucrose, 1% BSA, 1.4% Tween-20, 0.2% fluorescently labeled goat anti-bovine IgG antibody, and 0.4% fluorescently labeled bovine cysticercosis cPGES protein. The amino acid sequence of the bovine cysticercosis cPGES protein is shown in SEQ ID NO.

4.

7. The reagent kit according to claim 6, characterized in that, The ratio of fluorescent microspheres to goat anti-bovine IgG antibody was 1:2, and the ratio of fluorescent microspheres to bovine cysticercosis antigen was 1:

20. The detection line coated on the nitrocellulose membrane was mouse anti-human IgG antibody, and the control line coated on the nitrocellulose membrane was goat anti-bovine IgG antibody. The coating concentration for both was 5 μL / mm.

8. The use of the cPGES protein of bovine cysticercosis according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3, or the recombinant host cell according to claim 4 in the preparation of a kit for diagnosing or detecting bovine cysticercosis or anti-bovine cysticercosis antibodies.

9. The use of the cPGES protein of bovine cysticercosis as described in claim 1 in the preparation of a vaccine against bovine cysticercosis.

10. The use of a recombinant vector or recombinant host cell containing the gene of claim 2 in the preparation of a vaccine against bovine cysticercosis.

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