A hypothetical protein gene of Echinococcus granulosus and its encoded protein application

Abstract

A hypothetical protein gene of Echinococcus granulosus and its encoded protein application belong to the field of immunology technology. To address the current stagnation in the development of vaccines for canine anti-Echinococcus granulosus infection and the lagging screening of protective antigens, the present invention first constructs a cDNA expression library of three-day-old Echinococcus granulosus larvae. The library is then immunologically screened using positive serum from dogs infected with Echinococcus granulosus. Through second-generation sequencing and data analysis, an antigenic protein of three-day-old Echinococcus granulosus larvae is screened from the library. The amino acid sequence of the antigenic protein is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1. It has been verified that the antigenic protein can specifically react with positive serum from dogs infected with Echinococcus granulosus, and has high application value in the development of canine anti-Echinococcus granulosus vaccines and diagnostic reagents.

Description

Technical Field

[0001] The invention belongs to the technical field of immunology, and in particular relates to an Echinococcus granulosus hypothetical protein gene and application of the encoded protein thereof. Background Art

[0002] Echinococcosis / hydatid disease is a zoonotic parasitic disease caused by the larvae (intermediate stage) of tapeworms of the genus Echinococcus (Taeniaceae). In my country, the two species that are of public health importance and cause cystic echinococcosis (CE) and alveolar echinococcosis (AE) are Echinococcus granulosus ( Echinococcus granulosus , Eg) and Echinococcus multilocularis ( Echinococcus multilocularis , Em).

[0003] Dogs, the definitive host, are the primary source of echinococcosis in humans and livestock. Vaccinating dogs is the ideal strategy for controlling the prevalence of echinococcosis in my country's pastoral areas. Immunizing dogs would greatly simplify and accelerate echinococcosis control, significantly reducing and controlling the incidence of echinococcosis in both humans and livestock. However, the development of canine vaccines has been stagnant, primarily due to the lack of clear protective immune mechanisms and the lag in identifying protective antigens.

[0004] Initial strategies for vaccination against Echinococcus granulosus have involved extracting crude antigens from various stages of the tapeworm life cycle, including whole adult worms, crude protoscoleces, fertile cysts, and tissue extracts from other stages. These crude antigens can be used to immunize the host to induce immunity, or to extract excretory / secretory products (E / S) that play a key role in parasite metabolism and host immune response. However, these crude antigens are complex, containing multiple antigenic proteins, and exhibit significant batch-to-batch variability, leading to inconsistent clinical outcomes. Furthermore, crude antigens have a high potential for allergic reactions, and limited sample availability leads to high production costs, making them unsuitable for large-scale production and limiting their clinical application. Furthermore, cross-reactivity issues (such as cross-reactivity with sera from dogs infected with other tapeworms and nematodes) and the potential health risks to personnel involved in developing crude antigen vaccines for the terminal host have led to their replacement by other vaccines with the advancement of genetically engineered vaccines.

[0005] Because the number of herding dogs is much smaller than that of livestock, the cost of controlling definitive hosts, dogs, is much lower than that of intermediate hosts. Therefore, we can better control echinococcosis by taking preventive measures to control definitive hosts, which makes the search for candidate antigens for canine vaccines against Echinococcus granulosus infection particularly important. Summary of the Invention

[0006] To address the current issues of stagnant development of vaccines against Echinococcus granulosus infection in dogs and delayed screening of protective antigens, the present invention established a cDNA library of three-day-old larvae of Echinococcus granulosus. Using positive serum from dogs infected with Echinococcus granulosus, and employing multiple rounds of immunological screening technology, single colonies with strong reactogenicity were subjected to second-generation sequencing to identify new antigens, providing new candidate antigens for the development of vaccines against Echinococcus granulosus infection in dogs and specific diagnostic reagents.

[0007] In order to solve the above technical problems and achieve corresponding technical effects, the present invention proposes the following technical solutions:

[0008] The first object of the present invention is to provide an application of a hypothetical protein gene of Echinococcus granulosus, wherein the hypothetical protein gene of Echinococcus granulosus is used to prepare a vaccine for dogs against Echinococcus granulosus infection. The nucleotide sequence of the hypothetical protein gene of Echinococcus granulosus is shown in SEQ ID NO.1.

[0009] A second object of the present invention is to provide an application of the protein encoded by the above-mentioned Echinococcus granulosus hypothetical protein gene, wherein the protein encoded by the Echinococcus granulosus hypothetical protein gene is used to prepare a vaccine for dogs against Echinococcus granulosus infection. The amino acid sequence of the protein encoded by the Echinococcus granulosus hypothetical protein gene is shown in SEQ ID NO.2.

[0010] In one embodiment of the present invention, the method for screening the protein encoded by the above-mentioned Echinococcus granulosus hypothetical protein gene comprises the following steps:

[0011] 1) Construction of a cDNA expression library of 3-day-old larvae of Echinococcus granulosus canis;

[0012] 2) Spread the library evenly on LB solid medium plates and incubate inverted at 37°C overnight to obtain colonies;

[0013] 3) Invert the colony at 4°C for 1-2 hours. Cut a 0.45 μm nitrocellulose membrane to the size that matches the culture plate and place it on the surface of the culture medium. Mark three asymmetric positions with a toothpick. Remove the nitrocellulose membrane, leaving the colony contact surface facing up, and place it on an LB plate containing IPTG and Amp resistance. Incubate inverted at 37°C for 6-8 hours. After incubation, wrap the culture plate with parafilm and store inverted at 4°C.

[0014] 4) Remove the nitrocellulose membrane from the clean bench and expose it to chloroform vapor for 15 minutes. Place it on a plate and immerse the membrane in bacterial lysis buffer. Lyse the membrane overnight at room temperature. Change the elution buffer three times for 30 minutes each time and then block with blocking buffer at room temperature for 2 hours.

[0015] 5) Incubate the blocked nitrocellulose membrane with canine positive serum at room temperature for 2 h. Place the nitrocellulose membrane in elution buffer containing 1% Triton-X, 0.5% sodium deoxycholate, and 0.1% SDS for 1 h. Wash the nitrocellulose membrane three times in elution buffer for 30 min each.

[0016] 6) Place the nitrocellulose membrane obtained in step 5) in a 1:5000 dilution of alkaline phosphatase AP-labeled affinity-purified rabbit anti-dog IgG secondary antibody and incubate at room temperature for 2 h. After washing the nitrocellulose membrane with the elution buffer described in step 5), develop the color using 5-bromo-4-chloro-3-indoleyl phosphate / nitro blue tetrazolyl ammonium chloride as a substrate to determine the location of the positive color signal on the nitrocellulose membrane. Finally, terminate the reaction with distilled water. A purple color will appear at the site of the positive clone vector antigen-antibody complex.

[0017] 7) Based on the location of the positive ring on the nitrocellulose membrane, select a colony corresponding to the specific location of the suspected positive clone on the culture plate. Inoculate the colony into LB medium containing Amp at 37°C and shake at 180 rpm for 3 hours. Inoculate this bacterial suspension onto an LB plate containing Amp and culture overnight at 37°C. Perform a second and third round of screening until consistent immunopositive recombinants are obtained. Extract the plasmid and verify it by sequencing. Design primers based on the sequenced recombinants for PCR amplification to obtain the PCR product.

[0018] 8) Recombinantly expressing the PCR product to obtain a highly purified Echinococcus granulosus hypothetical protein. A third object of the present invention is to provide a use of a recombinant expression vector containing the Echinococcus granulosus hypothetical protein gene, wherein the recombinant expression vector is used to prepare a candidate gene for a vaccine against Echinococcus granulosus, the definitive host of dogs.

[0019] The fourth object of the present invention is to provide a use of a recombinant expression vector containing the above-mentioned Echinococcus granulosus hypothetical protein gene, wherein the recombinant expression vector is used to prepare a vaccine for dogs against Echinococcus granulosus infection.

[0020] A fifth object of the present invention is to provide a use of a recombinant cell containing the above-mentioned Echinococcus granulosus hypothetical protein gene or the above-mentioned recombinant expression vector, wherein the recombinant cell is used to prepare a vaccine for dogs against Echinococcus granulosus infection.

[0021] The sixth object of the present invention is to provide a use of a fusion protein containing the protein encoded by the above-mentioned Echinococcus granulosus hypothetical protein gene, wherein the fusion protein is used to prepare a vaccine for dogs against Echinococcus granulosus infection.

[0022] The seventh object of the present invention is to provide a use of a fusion protein containing the protein encoded by the above-mentioned Echinococcus granulosus hypothetical protein gene, wherein the fusion protein is used to prepare a reagent or kit for diagnosing infection of the terminal host Echinococcus granulosus.

[0023] Beneficial effects of the present invention:

[0024] The present invention discloses an antigenic protein of three-day-old Echinococcus granulosus larvae, obtained through high-throughput, unbiased screening using a three-day-old Echinococcus granulosus larvae cDNA expression library. The amino acid sequence of the protein is shown in SEQ ID NO. 2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 1. The present invention first constructs a three-day-old Echinococcus granulosus larvae cDNA expression library. The library is then immunologically screened using positive serum from dogs infected with Echinococcus granulosus. Through next-generation sequencing and data analysis, the antigenic protein of three-day-old Echinococcus granulosus larvae is screened from the library. Verification shows that the antigenic protein specifically reacts with positive serum from dogs infected with Echinococcus granulosus, demonstrating its high application value in the development of canine anti-Echinococcus granulosus vaccines and diagnostic reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the map of the modified pBlueScript II SK(-) vector;

[0026] Figure 2 This is a diagram showing the result of BLAST alignment of a hypothetical protein gene of Echinococcus granulosus screened in Example 2 on NCBI;

[0027] Figure 3 This is a secondary structure analysis result of a protein encoded by a hypothetical protein gene of Echinococcus granulosus screened in Example 2; wherein, Figure 3 The h in the figure represents an α-helix, the e represents an extended chain, and the c represents a random coil;

[0028] Figure 4 This is a structural domain analysis diagram of a protein encoded by a hypothetical protein gene of Echinococcus granulosus screened in Example 2;

[0029] Figure 5 The figure shows the PCR identification results of the full-length nucleotide sequence of the hypothetical protein gene amplified from the protoscolecus of Echinococcus granulosus; Figure 5 M in the table represents a molecular weight standard, and 1 represents a hypothetical protein gene;

[0030] Figure 6 is an agarose gel electrophoresis diagram of the single enzyme digestion product of the pET28a expression vector; wherein, Figure 6 M in the figure is a molecular weight standard, 1 is a pET28a undigested vector control, and 2 is a pET28a vector digested with BamHI.

[0031] Figure 7 A hypothetical protein gene of Echinococcus granulosus screened in Example 2 is E. coli The results of 10% SDS-PAGE after induced expression in BL21 (DE3) cells; Figure 7 M in the table is the standard molecular weight, 1 is the pET28a empty vector control induced by IPTG, 2 is the pET28a-Eg174 expression precipitate induced by IPTG, and 3 is the uninduced pET28a-Eg174 precipitate control;

[0032] Figure 8 A hypothetical protein gene of Echinococcus granulosus screened in Example 2 is E. coli The results of 10% SDS-PAGE after induction expression and purification in BL21 (DE3) cells; Figure 8 M in the table is the standard molecular weight, and 1 is the purified PET28a-Eg174 protein;

[0033] Figure 9 The figure is a Western blotting diagram of the reaction between the purified Echinococcus granulosus hypothetical protein and anti-His tag antibody; Figure 9 Where M is the standard molecular weight and 1 is the sample to be tested;

[0034] Figure 10 The Western blotting diagram of the purified hypothetical protein of Echinococcus granulosus reacting with the positive serum of dog against Echinococcus granulosus adult worms; Figure 10 Where M is the standard molecular weight and 2 is the sample to be tested;

[0035] Figure 11 The figure is a Western blotting diagram of the reaction between the purified Echinococcus granulosus hypothetical protein and negative serum; Figure 11 Where M is the standard molecular weight and 3 is the sample to be tested. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the spirit of the contents disclosed in the present invention will be described in detail below in conjunction with specific embodiments and the accompanying drawings. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify the contents of the present invention based on the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0037] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.

[0038] The experimental methods involved in the following examples are conventional methods unless otherwise specified, and the materials, reagents, enzymes, etc. used are all available from commercial channels unless otherwise specified.

[0039] The present invention uses E. coli BL21 (DE3) can be purchased from Sangon Biotech (Shanghai) Co., Ltd. through commercial channels.

[0040] Example 1: Construction of a cDNA library of 3-day-old larvae of Echinococcus granulosus

[0041] The Echinococcus granulosus protoscoleces used in the present invention were collected from the livers of sheep infected with Echinococcus granulosus, slaughtered at county-level designated cattle and sheep slaughterhouses in areas endemic for echinococcosis. Construction of a cDNA library from three-day-old larvae of Echinococcus granulosus included the following steps: first, isolating the protoscoleces of Echinococcus granulosus, artificially orally infecting experimental dogs, and then killing the three-day-old larvae at designated time limits to obtain total RNA. mRNA was then isolated and purified, followed by first-strand cDNA synthesis; second-strand cDNA synthesis was then performed; the double-stranded cDNA was ligated into a pBlueScript II SK(-) vector, and then electroporated into competent Escherichia coli cells.

[0042] 1. Total RNA extraction and mRNA purification

[0043] Three-day-old larvae of Echinococcus granulosus stored in liquid nitrogen were placed in a 1.5 mL centrifuge tube. Total RNA was extracted from the three-day-old larvae of Echinococcus granulosus using TRIZOL reagent, and mRNA was isolated and purified from the total RNA using Oligotex mRNA Kits.

[0044] 2. cDNA synthesis and purification

[0045] (1) Synthesis of the first strand of cDNA

[0046] In a 0.2 mL RNase-free centrifuge tube, add the following reaction system: E. coli .DNA Ligase(10 U / µL) 1 µL, E. coli RNase H (2 U / µL) 1 µL, E. coliAdd 4 µL of DNA Polymerase I (10 U / µL), 22.5 µL of mRNA sample (4.5 µg), and 2 µL of 3' RT Primer (1.5 μg / µL). Place the reaction tube in a PCR instrument and incubate at 70°C for 7 minutes. Immediately place on ice. Prepare the first-strand reaction in a new 0.2 mL RNase-free tube: 10 µL of 5× RT Buffer, 5 µL of water, 2.5 µL of 10 mM dNTPs, and 5 µL of RT enzyme. After the primer reaction in the first tube cools to 45°C, incubate at 45°C for 2 minutes. Add the reaction mixture from the second tube and mix thoroughly, avoiding bubbles. Transfer the reaction mixture to a new 1.5 mL RNase-free tube and add the following reagents, mix thoroughly, and incubate at -80°C for at least 1 hour: 1 µL of 20 μg / µL glycogen, 25 µL of 7.5 M NH₄OAc, and 187 µL of anhydrous ethanol. Centrifuge the cryoprecipitated first-strand product at 16,000 × g for 30 minutes at 4°C, discard the supernatant, add 150 µL of RNase-free 70% ethanol, centrifuge at 16,000 × g for 3 minutes at 4°C, and discard the supernatant. Repeat this process once. Dry the cDNA at room temperature, dissolve the precipitate in 20 µL of DEPC-free water, and place on ice until needed.

[0047] (2) Synthesis of the second strand of cDNA

[0048] The following reagents were added to the above reaction solution: 91 µL of DEPC-treated water, 30 µL of 5× Second Strand Buffer, 3 µL of 10 mM (each) dNTPs, and 6 µL of Second Strand Enzyme Mix, for a total volume of 130 µL. The reaction mixture was incubated at 16°C for 2 hours. 2 µL of T4 DNA Polymerase was added, and the reaction mixture was incubated at 16°C for 5 minutes. 10 µL of 0.5 M EDTA (pH 8.0) was added, and 160 µL of a mixture of phenol, chloroform, and isoamyl alcohol (volume ratio of phenol, chloroform, and isoamyl alcohol: 25:24:1) was added. The reaction mixture was mixed thoroughly for 30 seconds. The reaction mixture was centrifuged at 14,000 rpm at room temperature for 5 minutes. The supernatant was carefully transferred to a new centrifuge tube, precipitated with ethanol, and dissolved in 40 µL of DEPC water.

[0049] Add 5' adapters (three reading frames, one copy for each reading frame, for a total of three copies) using a system consisting of 34 µL cDNA, 5 µL 10× T4 ligase Buffer, 10 µL 5' adapter (1 µg / µL), and 1 µL T4 DNA Ligase (40 U / µL, NEB) in a total volume of 50 µL. Mix well and incubate at 16°C for 20 hours. Add 2 µL 10 mM dNTPs and 2 µL T4 DNA polymerase, and incubate at 16°C for 20 min to blunt the ends.

[0050] (3) Recovering cDNA fragments of target length

[0051] The cDNA product was electrophoresed on a 1% low-melting-point agarose gel and the approximately 1 kbp fragment was recovered. The gel was incubated at 70°C for 10 min, then transferred to 45°C. 10× buffer and 5 µL of lysozyme were added and incubated at 45°C for 3 h. The product was then centrifuged at 12,000 g for 15 min at 4°C. The supernatant was aspirated, ethanol precipitated, and the product was recovered by resolving in 14 µL of DEPC-containing water.

[0052] (4) Ligation of cDNA and vector

[0053] Using homologous recombination, 7 µL of cDNA from the previous step was combined with 3 µL of the modified pBlueScript II SK(-) vector (a 1884 bp fragment (SEQ ID NO. 5) was inserted into the original vector XhoI: CTCGAG-HindIII: AAGCTT restriction enzyme cutting site, so that the size of the modified vector was 4818 bp, as shown in Figure 5). Figure 1 ) and add 5 µL of all-direct recombinase and 5 µL of water, mix thoroughly, and incubate at 25°C for 20 h. To this reaction mixture, add 2 µL of Proteinase K to inactivate the recombinase and 78 µL of sterile water to bring the total volume to 100 µL. Then, add 1 µL of 20 µg / µL of glycogen, 50 µL of 7.5 M NH₄OAc, and 375 µL of anhydrous ethanol, mix thoroughly, and incubate at -80°C for at least 1 hour. Centrifuge at 16,000 rpm at 4°C for 30 min, carefully remove the supernatant, add 150 µL of 70% ethanol, and centrifuge at 16,000 rpm at 4°C for 3 min. Repeat this step once, removing all the supernatant, avoiding disturbing the cDNA pellet. Air-dry the cDNA at room temperature for 10 min. Resuspend the cDNA pellet in 10 µL of DEPC-treated water and pipette up and down 30-40 times. The cDNA was collected by instant centrifugation for 2 s and immediately placed on ice.

[0054] SEQ ID NO.5:

[0055]

[0056] (5) Electrotransformation of E. coli competent cells

[0057] Precool a 1 mm electroporation cuvette at -80°C for 30 min. On ice, add 2.5 µL of the recombinant product and 50 µL of competent E. coli cells to the cuvette. Place on ice for 45 min and electroporate on an electroporator (voltage: 2.9 kV, resistance: 200 Ω, power: 25 μF). After electroporation, quickly add 1 mL of LB medium to the cuvette, then transfer the tube to a new 15 mL centrifuge tube, make up the volume to 5 mL, and incubate at 37°C, 250 rpm for at least 1 h.

[0058] After the culture is completed, the culture was diluted 10, 100, 1000, and 10,000 times, and 10 μL of the dilution was taken to plate. The remaining culture can be stored at 4°C overnight, or glycerol is added to the final concentration of 20% and stored at -80°C.

[0059] (6) Library quality assessment

[0060] The quality of the library was determined by the following formula: CFU / mL = number of clones on the plate / 10 µL × 100 × 1 × 10 3 μL; total CFU in the library = CFU / mL × total volume of the library solution (mL).

[0061] After identification, the constructed library had a capacity of 1×10 10 CFU / mL.

[0062] (7) Insert size identification

[0063] Single clones on the plate were picked, PCR amplified, and the size of the PCR product was detected by electrophoresis. The PCR primers were universal primers of the vector, M13F: 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO. 3); M13R: 5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO. 4).

[0064] Example 2: Immunoscreening of cDNA libraries

[0065] The immunological screening of the cDNA library provided by the present invention first uses a pseudo-screening method to adsorb and purify a probe from positive serum from dogs infected with Echinococcus granulosus to remove non-specific cross-reactive antibody components, primarily the relatively low-abundance anti-Escherichia coli antibody component, ultimately producing an ideal antibody probe for specific immunological screening. Specific immunological screening is then performed, specifically using a pooled sample of positive serum from 10 dogs infected with Echinococcus granulosus to immunologically screen a cDNA library of three-day-old Echinococcus granulosus larvae. Positive clones containing the Echinococcus granulosus gene with high sensitivity and strong reactivity are selected. The recombinant vector is then sent to a biotechnology company for sequencing to obtain the target gene sequence. Finally, the positive clones containing the cDNA insertion are amplified and sequenced, and the sequences are searched for homology in the NCBI database. The sequenced amino acid sequences of the target genes are then subjected to bioinformatics analysis and immunogenicity analysis.

[0066] 1. Processing of canine positive serum for library screening

[0067] Pick E. coli BL21 (DE3) monoclonal cells were cultured in 100 mL of LB medium at 37°C overnight. The culture was centrifuged at 5000 × g for 10 min at 4°C and the medium was removed. The cells were resuspended in 3 mL of Tris-HCl (50 mM, pH 8.0) and EDTA (10 mM, pH 8.0), frozen and thawed three times, and ultrasonically disrupted until the cell suspension became clear. The cells were centrifuged at 5000 × g for 15 min at 4°C and the supernatant was collected. E. coli BL21 (DE3) lysis buffer. Take 100 μL of Echinococcus granulosus dog positive mixed serum and add 1 mL E. coli BL21 (DE3) lysate was adsorbed overnight at room temperature and centrifuged at 5000 × g for 10 min. The precipitate was removed and the supernatant was diluted with antibody diluent to a final serum concentration of 1:100. Sodium azide was added to a final concentration of 0.02% and stored at 4°C.

[0068] 2. Immunoscreening of cDNA libraries

[0069] 2.1 Initial screening

[0070] 2.1.1 Bacterial culture

[0071] The amplified library was evenly spread on an LB plate (Amp+), inverted and cultured at 37°C overnight. When the colonies grew to a diameter of 0.1-0.2 mm, the plate was removed from the incubator and inverted at 4°C for 1-2 h.

[0072] 2.1.2 Film application

[0073] A nitrocellulose membrane (NC membrane, Millipore, 0.45 μm) was cut into a size that matched the culture plate and spread on the surface of the culture medium. It was kept in contact with the colonies until it became wet, and marked at three asymmetric positions with the tip of a syringe.

[0074] 2.1.3 Inducible expression

[0075] Remove the NC membrane and, with the colony-contacting surface facing up, plate onto an LB plate (Amp+) containing IPTG. Incubate the plate upside down at 37°C for 6-8 hours. Incubate the plate at 37°C for approximately 6 hours until new colonies appear. Wrap the master plate with parafilm and store upside down at 4°C.

[0076] 2.1.4 Fixation

[0077] The NC membrane was taken out from the clean bench, exposed to chloroform vapor for 15 min, and then placed on a petri dish.

[0078] 2.1.5 Lysis and elution

[0079] Add bacterial lysis buffer to immerse the NC membrane, place the plate on a shaker at 50 rpm and lyse overnight at room temperature, change the elution buffer and let it stand at room temperature three times, each time for 30 minutes.

[0080] 2.1.6 Closure

[0081] Block the membrane with 5% skim milk powder in TBST at room temperature for 2 h.

[0082] 2.1.7 Detection of positive clones expressing target fusion protein

[0083] The membrane was incubated with sham-screened canine positive serum for 2 hours, with the plate shaken gently at 50 rpm at room temperature. The NC membrane was placed in elution buffer containing 1% Triton-X, 0.5% sodium deoxycholate, and 0.1% SDS for 1 hour. The membrane was then washed three times in elution buffer, 30 minutes each time, with gentle shaking at 50 rpm at room temperature. The secondary antibody, alkaline phosphatase AP-conjugated affinity-purified rabbit anti-dog IgG (Jackson, USA), was diluted 1:5000 and incubated on the NC membrane for 2 hours at room temperature. The membrane washing steps were repeated. After washing, the membrane was developed using BCIP (5-bromo-4-chloro-3-indoleyl phosphate) / NBT (nitro blue tetrazolium chloride) as a substrate. The reaction was terminated with distilled water. Positive clones exhibited a purple-purple coloration in the presence of antigen-antibody complexes.

[0084] 2.1.8 Positive clone positioning

[0085] According to the position of the positive ring on the membrane, a single colony was picked at the specific position of the suspected positive clone on the culture plate, inoculated into LB medium (Amp+), and shaken at 180 rpm at 37°C for 3 h.

[0086] 3. Rescreening

[0087] The above colonies were inoculated onto LB plates (Amp+) and cultured overnight at 37°C. A second round of screening was performed as described above to obtain a single positive clone.

[0088] 4. Three sieves

[0089] Repeat the rescreening steps for the third round of screening until consistent immune-positive recombinants are obtained and 100% positive clones appear on the plate.

[0090] Using the above method, the present invention screened for a hypothetical protein gene from Echinococcus granulosus (designated Eg174 in the present invention), the nucleotide sequence of which is shown in SEQ ID NO. 1. The target gene obtained in the present invention is 468 bp in length and encodes 155 amino acids. The amino acid sequence is shown in SEQ ID NO. 2. In the nucleotide sequence, ATG at positions 1-3 corresponds to the mRNA start codon, and TAA at positions 466-468 corresponds to the mRNA stop codon. The molecular weight of the encoded Echinococcus granulosus hypothetical protein is approximately 17.43 kilodaltons. BLAST comparison analysis of the target gene sequence in NCBI revealed that the encoded protein is a hypothetical Echinococcus granulosus protein.

[0091] Example 3: Extraction, detection and analysis of PBluescript recombinant plasmid

[0092] Pick a single colony of positive clones and place it in LB (containing 100 μg / mL Amp) liquid medium, shake and culture at 37°C, 200 rpm overnight. The next day, use Tiangen's plasmid extraction kit to extract plasmid DNA, and elute the plasmid DNA with 30-50 μL elution buffer. Send the extracted plasmid to Shenggong Biotechnology Co., Ltd. for sequence determination, using M13F and M13R as universal primers. Delete the upstream and downstream restriction sites and perform BLAST comparison and homology analysis on the sequence in NCBI. HTTP: / / www.ncbi.nlm.nih.gov / orffinder / ORF analysis was performed on the website to determine the largest open reading frame; https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_sopma.html Site secondary structure analysis (e.g. Figure 2 、 Figure 3 and Figure 4 ). Figure 2 It can be seen that the base sequence of the plasmid gene of the positive clone single colony was subjected to BLAST analysis in NCBI, and the sequence of the protein encoded by it was identical to the amino acid sequence 1-155 of SEQ ID NO.2. Figure 3As shown in the figure, secondary structure analysis of the protein encoded by the gene contained in the positive clones obtained by screening showed that α-helices accounted for 58.71%, random coils accounted for 36.13%, and extended chains accounted for 5.16%. Flexible structures such as protein turns and random coils are relatively loose and easily twisted, coiled, and appear on the surface of the protein, so they are more likely to become surface antigens. Nearly half of the secondary structure of this sequence is random coils, indicating that this sequence has the structural basis for containing B cell epitopes. Figure 4 As shown, the domain prediction analysis of the gene encoding protein screened by the present invention (SMART: Main page (embl.de)) shows that the sequence contains two transmembrane regions, which are 76-93AA and 113-135AA respectively.

[0093] SEQ ID NO.1:

[0094] atggcatcccgcgagagaaggcatagtagccccctcgcaactgattggtcggctgtagaccaatcacgcagcgaagccgaccaatggaatcgttgcaaaaccctgcctgtccgtcgc acgcttaaggctcatctactagcgcctcgtgtagctacgttcgcgcactatctctctctgcctcgctttactctctctccgtgttcgtgcacaaaccgaaccatgtcagttctttgt cttcttctcattgccctcgcgcatttcgcctcagcagtcacactgaaagagggcttggcggacaaggagaagtacatattctatgatacctcacccttcaatgtcggtttgcatgcc ggtttggcactgctaattacctatggtgttttgggaacgttcaccccctccgttatgattattacaaaggcgttggagaaaaggaggaaacgacgctcccgcaccatgtcacactaa

[0095] SEQ ID NO.2:

[0096] MASRERRRHSSPLATDWSAVDQSRSEADQWNRCKTLPVRRTLKAHLLAPRVATFAHYLSLPRFTLSPCSCTNRTMSVLCLLLIALAHFASAVTLKEGLADKEKYIFYDTSPFNVGLHAGLALLITYGVLGTFTPSVMIITKALEKRRKRRSRTMSH

[0097] Example 4: Cloning and transformation of target genes

[0098] RNA was extracted from Echinococcus granulosus protoscolecus and reverse transcribed into cDNA. The reverse transcription system was as follows: 8 × g DNAEraser Premix 2 µL, RNA sample 1 µL, RNase-free H2O 13 µL, 5 × RT Premix 4 µL, total volume 20 µL; the reverse transcription reaction conditions were as follows: 42°C for 2 min, 37°C for 10 min, and 85°C for 5 s.

[0099] Based on the sequence of the target gene, seamless cloning primers were designed and amplified by PCR. The PCR system was as follows: 25 µL of PrimeSTAR Max Premix (2×), 5 µL of cDNA, 1 µL of the upstream primer (nucleotide sequence shown in SEQ ID NO. 6), 1 µL of the downstream primer (nucleotide sequence shown in SEQ ID NO. 7), and 18 µL of sterile deionized water, for a total volume of 50 µL. The PCR program was as follows: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 s, annealing at 55°C for 10 s, and extension at 72°C for 10 s, for a total of 35 cycles; and a final extension at 72°C for 5 min. The PCR amplification products were subjected to agarose gel electrophoresis. The results are shown in Figure 2. Figure 5 As shown, the full-length nucleotide sequence of the amplified hypothetical protein gene was consistent with the expected size. After electrophoresis, the target fragment was cut out from the gel and purified using the Tiangen Agarose Gel DNA Recovery Kit.

[0100] SEQ ID NO.6:

[0101] 5'-GGACAGCAAATGGGTCGCGGATCCATGGCATCCCGCGAGAGAA-3';

[0102] SEQ ID NO.7:

[0103] 5'-GTCGACGGAGCTCGAATTCGGATCCGTGTGACATGGTGCGGGAG-3'.

[0104] The expression vector pET28a was digested with the nuclease BamHI. The digestion system was as follows: 10× Buffer 5 µL, BamHI 1 µL, pET-28a vector 4 µL, sterile deionized water 40 µL, total volume 50 µL; digestion reaction conditions: 37°C for 2 h. The digestion products were subjected to 1.0% agarose gel electrophoresis. The results were as follows: Figure 6 As shown, it proves that the vector has been completely cut, and the remaining enzyme digestion products are purified using Tiangen's common DNA product purification kit.

[0105] The purified target gene and expression vector were ligated using a seamless cloning reagent. The ligation system was as follows: 5 µL of 2×MultiFSeamless Assembly Mix, 1 µL of pET28a, 1 µL of target gene, 3 µL of sterile deionized water, and a total volume of 10 µL. The ligation reaction conditions were 50°C for 30 min.

[0106] Take 5 µL of the ligation product and transform E. coli DH5a cells, the specific method is: 5 μL of ligation product and 100 μL E. coli Mix the DH5a competent cells, place them on ice for 30 min, then heat shock them in a 42°C water bath for 45 s, place them on ice for 2-3 min, then add 900 µL of LB medium without antibiotics, culture them at 37°C with shaking at 200 rpm for 1 h, centrifuge them at 4000 rpm for 3 min, discard 800 µL, resuspend them in the remaining 200 µL liquid, take 50 µL of the bacterial solution and spread it on an LB (Kan+) plate, and culture it upside down at 37°C incubator overnight.

[0107] Pick 5-10 single colonies from the plate and place them in 1 mL LB (Kan+) liquid medium. Incubate at 37°C, 200 rpm, and shake overnight. Send to Shenggong Biotechnology Co., Ltd. for sequencing to check whether the connection is successful. Extract the plasmid from the successfully connected recombinant vector using the plasmid extraction kit from Tiangen Company. Take 5 µL of the plasmid and transform it according to the above method. E. coli BL21(DE3) cells.

[0108] Example 5: Expression and purification of recombinant protein

[0109] 1. Small-scale expression of recombinant proteins

[0110] Take the transformed recombinant vector and the one containing the pET28a empty vector respectively E. coliA single BL21 (DE3) colony was cultured in 3 mL LB medium (50 µg / mL Kan) at 37°C and 200 rpm overnight, and then 500 µL of the bacterial solution was added to 50 mL fresh LB medium (50 µg / mL Kan) and cultured at 200 rpm for 2 h. IPTG was then added to a final concentration of 1 mM and induced at 37°C and 200 rpm for 4 h. The cells were collected by centrifugation and washed twice with PBS. E. coli For the uninduced control, a single BL21 (DE3) colony was collected by centrifugation, washed twice with PBS solution, and repeatedly frozen and thawed three times in liquid nitrogen and a 37°C water bath. 1 mL of high-pressure PBS solution was added and sonicated for 10 min, 3 s each time, with an interval of 5 s. The bacterial suspension after ultrasonic disruption was centrifuged again and the precipitate was collected. The three samples were subjected to 10% SDS-PAGE electrophoresis to observe whether the recombinant protein was expressed.

[0111] Depend on Figure 7 It can be seen that the precipitate obtained after induction of Escherichia coli containing the recombinant vector pET28a-Eg174 has an obvious induced expression band at 15-25 kDa, and the size is consistent with the predicted molecular weight of the recombinant protein, proving that the recombinant protein is successfully expressed.

[0112] 2. Large-scale expression of recombinant proteins

[0113] Take the expression E. coli A single BL21 (DE3) colony was cultured in 100 mL of LB (50 μg / mL Kan) medium at 37°C, shaking at 200 rpm overnight. Then, 100 mL of the culture was added to 1000 mL of fresh LB (50 μg / mL Kan) medium and cultured at 37°C, shaking at 200 rpm for 2 h. IPTG was then added to a final concentration of 1 mM and expression was induced at 37°C, 180 rpm for 6 h. The cells were harvested by centrifugation, washed twice with PBS, and then freeze-thawed three times in liquid nitrogen and a 37°C water bath. 10 mL of high-pressure PBS was added and sonicated three times for 3 s each, with 5 s intervals between cycles, for a total of 30 min. The sonicated cells were then centrifuged again, and the precipitate was collected and subjected to 10% SDS-PAGE electrophoresis to verify protein expression.

[0114] 3. Purification of recombinant protein

[0115] Since the protein exists in inclusion bodies, the precipitate was resuspended in an appropriate amount of 8 M urea and sonicated 3 times, 3 s each time, with an interval of 5 s, for a total of 30 min. The sonicated solution was placed at 4°C, 6000 rpm, and centrifuged for 20 min. The supernatant was collected and subjected to 10% SDS-PAGE electrophoresis. The gel after electrophoresis was soaked in pre-cooled KCL (0.25 M) for 8 min, the target band was cut out, and it was soaked in pure water for 8 min until it was colorless and transparent. The gel strip was taken out of the pure water, crushed with sterilized tweezers and placed in a centrifuge tube. An appropriate amount of PBS was added, and it was placed on a rotary mixer at 4°C overnight. The supernatant was centrifuged the next day. The purity of the centrifuged protein was analyzed by SDS-PAGE, as shown in the following figure. Figure 8 As shown, the purified protein had a single band and high purity. The purified protein concentration was determined using the BCA Protein Concentration Assay Kit (Enhanced) from Beyotime, and the resulting recombinant protein was used as an antigen.

[0116] Example 6: Immunogenicity Analysis of Recombinant Protein

[0117] Immunological analysis of the antigen was performed by Western blotting. The specific steps were as follows: 20 μg of antigen was loaded per lane, and canine positive serum was diluted 1:1000 as the primary antibody. Horseradish peroxidase-conjugated rabbit anti-dog IgG (Promega, China) was diluted 1:5000 in TBST (TBS Tween-20) as the secondary antibody. An ultra-high sensitivity ECL kit (Biosharp) was used as the chemiluminescent substrate.

[0118] like Figure 9 As shown, the purified recombinant protein reacted with anti-His tag antibody and specifically bound to canine positive serum against adult Echinococcus granulosus, while no reactive band was found at the target protein with negative serum, indicating that the recombinant protein had good antigenicity.

[0119] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An application of a hypothetical protein gene of Echinococcus granulosus, characterized in that: The application is to use the Echinococcus granulosus hypothetical protein gene to prepare a vaccine for dogs against Echinococcus granulosus infection. The nucleotide sequence of the Echinococcus granulosus hypothetical protein gene is shown in SEQ ID NO.

1.

2. Use of the protein encoded by the Echinococcus granulosus hypothetical protein gene according to claim 1, characterized in that: The application is to use the protein encoded by the Echinococcus granulosus hypothetical protein gene to prepare a vaccine for dogs against Echinococcus granulosus infection. The amino acid sequence of the protein encoded by the Echinococcus granulosus hypothetical protein gene is shown in SEQ ID NO.

2.

3. Use of a recombinant expression vector containing the Echinococcus granulosus hypothetical protein gene according to claim 1, characterized in that: The application is to use the recombinant expression vector to prepare a vaccine for dogs against Echinococcus granulosus infection.

4. Use of a recombinant cell containing the Echinococcus granulosus hypothetical protein gene according to claim 1, characterized in that: The application is to use the recombinant cells to prepare a vaccine for dogs against Echinococcus granulosus infection.

5. Use of a recombinant cell containing the recombinant expression vector according to claim 3, characterized in that: The application is to use the recombinant cells to prepare a vaccine for dogs against Echinococcus granulosus infection.

6. The use of the protein encoded by the Echinococcus granulosus hypothetical protein gene according to claim 2, characterized in that: The application is to use the protein to prepare a kit for diagnosing infection of the terminal host Echinococcus granulosus.

Images