An antigen p34 of cysticercus of bovine and its use

Abstract

The application discloses an antigen p34 of Taenia saginata and application thereof, and belongs to the technical field of biochemistry. The application aims to provide a detection method and a treatment and prevention method of Taenia saginata disease. The application provides an antigen of Taenia saginata, and the amino acid sequence of the antigen of Taenia saginata is shown as SEQ ID NO. 4. The antigen provides a new candidate antigen for new vaccine research and development and development of specific diagnostic reagents.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to an antigen p34 of bovine cysticercosis 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] The present invention provides an antigen of bovine cysticercosis, Alpha- and gamma-adaptin-binding protein p34, the amino acid sequence of which is shown in SEQ ID NO.4.

[0008] The present invention provides a gene encoding the aforementioned antigen Alpha- and gamma-adaptin-binding protein p34.

[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 antigen Alpha-and gamma-adaptin-binding protein p34.

[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% fluorescent microsphere-labeled goat anti-rabbit IgG antibody, and 0.4% fluorescent microsphere-labeled bovine cysticercosis antigen; the amino acid sequence of the bovine cysticercosis antigen is shown in SEQ ID NO.4. Further specifying, the coupling ratio of fluorescent microspheres to goat anti-rabbit IgG antibody is 1:2, and the coupling ratio of fluorescent microspheres to bovine cysticercosis antigen is 1:20; the detection line coated on the nitrocellulose membrane is mouse anti-bovine IgG antibody, and the control line is coated with rabbit anti-goat IgG antibody; the coating concentration is 5 μL / mm.

[0013] This invention provides the application of the above-mentioned antigen Alpha- and gamma-adaptin-binding protein p34, the above-mentioned encoding 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.

[0014] This invention provides the application of Alpha- and gamma-adaptin-binding protein p34 in the preparation of a vaccine against bovine cysticercosis, wherein the amino acid sequence of Alpha- and gamma-adaptin-binding protein p34 is shown in SEQ ID NO.4.

[0015] This invention provides the use of a recombinant vector or recombinant host cell containing a gene encoding Alpha- and gamma-adaptin-binding protein p34 in the preparation of a vaccine against bovine cysticercosis, wherein the gene encoding Alpha- and gamma-adaptin-binding protein p34 is shown in SEQ ID NO.3.

[0016] 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

[0017] Figure 1 Positive clones formed from cDNA libraries on LB solid medium.

[0018] Figure 2 The image shows the BLAST alignment of the p34 protein gene (Alpha- and gamma-adaptin-binding protein) of the bovine cysticercosis larvae according to the present invention with the amino acid sequence that has the highest homology, performed on NCBI.

[0019] Figure 3 This is a secondary structure diagram of the protein encoded by the p34 protein gene of the bovine cysticercosis Alpha- and gamma-adaptin-binding protein, according to the present invention.

[0020] Figure 4 This is a prediction analysis diagram of the antigenic epitope of the bovine cysticercosis Alpha- and gamma-adaptin-binding protein p34.

[0021] Figure 5 This is a diagram showing the predicted signal peptide of the protein encoded by the p34 protein gene of the bovine cysticercosis Alpha- and gamma-adaptin-binding protein, according to the present invention.

[0022] Figure 6This is a diagram showing the predicted transmembrane region of the protein encoded by the p34 protein gene of the bovine cysticercosis Alpha- and gamma-adaptin-binding protein according to the present invention.

[0023] Figure 7 The results of PCR amplification of the p34 protein gene, which is an Alpha- and gamma-adaptin-binding protein, are shown. M represents the molecular weight standard, and 1 and 2 represent the PCR amplification products.

[0024] Figure 8 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).

[0025] Figure 9 This image shows a 10% SDS-PAGE image of the *Alpha- and gamma-adaptin-binding protein p34* gene of *Taenia solium* BL21(DE3) cells after induction of expression. M: standard molecular weight; 1: uninduced whole cells of *PET28a*-Alpha- and gamma-adaptin-binding protein p34; 2: IPTG-induced supernatant of *pET28a*-Alpha- and gamma-adaptin-binding protein p34 expression; 3: IPTG-induced *PET28a*-Alpha- and gamma-adaptin-binding protein p34 expression precipitate; 4: uninduced empty *PET28a* vector; 5: induced *PET28a* empty vector supernatant; 6: induced *pET28a* empty vector precipitate. Figure 10 This is a 10% SDS-PAGE image of the *Taenia solium* Alpha- and gamma-adaptin-binding protein p34 gene after induction and purification in *E. coli* BL21(DE3) cells, where M: standard molecular weight; 1: IPTG-induced pET28a-Alpha- and gamma-adaptin-binding protein p34 expression precipitate; 2-5: purified pET28a-Alpha- and gamma-adaptin-binding protein p34 protein.

[0026] Figure 11 This is a Western blotting diagram of the p34 protein gene of the bovine cysticercosis Alpha- and gamma-adaptin-binding protein, as described in this invention. M: standard molecular weight; A: His-tagged antibody; B: positive serum from bovines infected with bovine cysticercosis; : negative serum from bovines not infected with bovine cysticercosis. Detailed Implementation

[0027] Example 1: Construction of a cDNA library of Beef Tapeworm Cysts 1. Total RNA extraction and mRNA purification A sample of beef tapeworm cysticerci, 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 hexacanth cysts using TRIZOL reagent, and mRNA was isolated and purified from the total RNA using the Oligotex mRNA Kits.

[0028] 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.

[0029] 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.

[0030] (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.

[0031] Add a 5' connector (3 barcode reader frames, one barcode reader frame connected to each frame, for a total of 3 barcode reader frames). 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.

[0032] (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.

[0033] (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.

[0034] Add 2 µL Proteinase K to inactivate 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.

[0035] (5) Electroporation of competent Escherichia coli cells Pre-cool the 1 mm electric rotary cup at -80℃ for 30 min. On ice, add 2.5 µL of recombinant product and 50 µL of competent cells to an electroporation cuvette and incubate on ice for 45 min. Electroporation was performed on the electroporator (Voltage 2.9 kV, Resistance 200 Ω, Capacity 25 μF). Immediately after electroporation, 1 mL of LB medium was added to the electroporator cuvette, and then transferred to a new 15 mL centrifuge tube. The volume was brought up to 5 mL, and the tube was incubated at 37°C and 225-250 rpm for at least 1 h.

[0036] 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.

[0037] (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 the 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).

[0038] 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℃.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 2.1.6 Closed 5% skim milk powder was sealed in TBST at room temperature for 2 hours.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] Repeat the above film washing steps.

[0049] 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.

[0050] The reaction was terminated with distilled water.

[0051] Positive clones showed a purplish-red color at the antigen-antibody complex site.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] The extracted plasmid was sent to Sangon Biotech Co., Ltd. for sequencing. Using universal primers M13F and M13R, upstream and downstream restriction enzyme sites were deleted. The sequence was then subjected to BLAST alignment and homology analysis on NCBI, and ORF analysis was performed on the website HTTP: / / www.ncbi.nlm.nih.gov / orffinder / to determine the largest open reading frame (ORF). Figure 1 Secondary structure analysis was performed on the NetSurfP 3.0 - DTU Health Tech - Bioinformatic Services website. Figure 3 ).Depend on Figure 3 It is evident that BLAST analysis of the gene sequence of this invention in NCBI shows that the sequence encoding the protein is identical to amino acid sequence 1-198 of SEQ ID NO: 3. For example... Figure 3As shown, secondary structure analysis was performed on the protein encoded by the gene of this invention. Flexible structures such as protein turns and random coils are relatively loose, easily twisting and coiling, and readily appearing on the protein surface, thus having a high probability of becoming surface antigens. Nearly half of the secondary structure of this sequence is random coils, indicating that this sequence has a structural basis containing B-cell epitopes. Figure 4 As shown, B-cell antigen 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 (…). Figure 5 ) and transmembrane region Figure 6 .

[0057] Bovine cysticercus Alpha- and gamma-adaptin-binding protein p34 gene: (SEQ ID NO.3) ; Bovine cysticercus Alpha- and gamma-adaptin-binding protein p34: (SEQ ID NO.4) MDTKDAEAIIICFDTNGNSESWSVACDWLKLGEEEDIPVQLLVCDSLSNESLRAEVFKEATKNHFEVVQLSPHSDEIEEDEEYGVARITAALVAHQWPNLALKNTKTAISESAPTNRSQQKSNVVNPMEKKANQKNNSDEDDEDSDGEVFNELFPKLMEMRSKGASMDLEGRRKMAEKMTVRFWRALKLDEEEIRGLSDDGED.

[0058] Implementation Case 4: Cloning and Transformation of the Target Gene 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 7 and Figure 8 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.

[0059] 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. coliMix 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.

[0060] 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.

[0061] 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.

[0062] 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 9 As shown.

[0063] 3. Purification of recombinant proteins Since the protein is present in inclusion bodies, the precipitate was resuspended in an appropriate amount of inclusion body lysis buffer: >20 mL / g bacteria (wet weight), and stirred overnight at 4°C with a magnetic stirrer.

[0064] The lysed solution was centrifuged at 4°C, 6000 rpm for 20 min, and the supernatant was collected. The 6×histidine fusion protein was expressed in *E. coli* BL21 cells, purified by affinity of the 6×His tag using NI-NTA resin, and eluted under denaturing conditions (urea) according to the supplier's (Qiagen, GmbH, Hilden, Germany) instructions. Figure 10 ).

[0065] 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.

[0066] 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.

[0067] 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 9 As shown, since the pET28a vector contains a histidine tag, the target protein size is approximately 27.91 kDa, which is consistent with the theoretical value.

[0068] Recombinant proteins were purified and collected using Ni column affinity chromatography, such as... Figure 10 As shown, there is a single protein band at a molecular weight of 27.91 kDa.

[0069] 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 11 ).

[0070] Example 7. 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-rabbit 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 coupling ratio of fluorescent microspheres to goat anti-rabbit IgG antibody is 1:2, and the coupling ratio of fluorescent microspheres to bovine cysticercosis antigen is 1:20. The detection line coated on the nitrocellulose membrane is coated with mouse anti-bovine IgG antibody, and the control line is coated with rabbit anti-goat IgG antibody. The coating concentration is 5 μL / mm. Positive sera for bovine cysticercosis showed a positive reaction, while negative sera showed a negative reaction.

Claims

1. An antigenic protein p34 of bovine cysticercosis, characterized in that, The amino acid sequence of the antigen Alpha- and gamma-adaptin-binding protein p34 of the bovine cysticercosis is shown in SEQ ID NO.

4.

2. A gene encoding the antigen Alpha- and gamma-adaptin-binding protein p34 as described in claim 1.

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 antigen Alpha- and gamma-adaptin-binding protein p34 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-rabbit 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.

7. The reagent kit according to claim 6, characterized in that, The ratio of fluorescent microspheres to goat anti-rabbit 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-bovine IgG antibody, and the control line was coated with rabbit anti-goat IgG antibody. The coating concentration was 5 μL / mm.

8. The use of the antigen Alpha- and gamma-adaptin-binding protein p34 of claim 1, the encoding gene of claim 2, the recombinant vector of claim 3, or the recombinant host cell of claim 4 in the preparation of a kit for diagnosing or detecting bovine cysticercosis or anti-bovine cysticercosis antibodies.

9. The application of an Alpha- and gamma-adaptin-binding protein p34 in the preparation of a vaccine against bovine cysticercosis, characterized in that, The amino acid sequence of the Alpha- and gamma-adaptin-binding protein p34 is shown in SEQ ID NO.

4.

10. The use of a recombinant vector or recombinant host cell containing a gene encoding the alpha- and gamma-adaptin-binding protein p34 in the preparation of a vaccine against bovine cysticercosis, characterized in that, The gene encoding the Alpha-and gamma-adaptin-binding protein p34 is shown in SEQ ID NO.3.

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