Echinococcus granulosus antigen Eg73 as well as coding gene and application thereof

Abstract

The invention discloses an echinococcus granulosus antigen Eg73 as well as a coding gene and application thereof, and belongs to the technical field of immunology. According to the present invention, the Eg73 antigen protein is screened by using the cDNA expression library of the three-day larva of the echinococcus granulosus with high throughput and no prejudice, the preparation method and the application of the antigen protein are further provided, and the purpose of the present invention is to provide the new antigen target for the development of the canine anti-echinococcus granulosus vaccine candidate antigen. The problems that at present, development of vaccines for resisting canine echinococcus granulosus infection lags before, and screening of protective antigens lags are solved, the amino acid sequence of the antigen protein is as shown in SEQ ID NO.1, and the nucleotide sequence for coding the antigen protein is as shown in SEQ ID NO.2. Through verification, the antigen protein can specifically react with positive serum of dogs infected with echinococcus granulosus, and has very high application value in research and development of canine anti-echinococcus granulosus vaccines and diagnostic reagents.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunology, and particularly relates to an Echinococcus granulosus antigen Eg73, its encoding gene and application. Background Art

[0002] Echinococcosis is one of the seriously harmful zoonotic parasitic diseases. Echinococcus granulosus is the main pathogen of cystic echinococcosis. Immunoprevention and control is an effective means to control the prevalence of this disease. Due to limited antigen sources from the worm body and lagging protective antigen screening technologies, etc., the widespread application of vaccines for the definitive host dogs is restricted. The 3-day-old larva of Echinococcus granulosus is a key link in parasitism in the definitive host dog. Obtaining immune protective antigen genes from the 3-day-old larval development stage to develop molecular vaccines is of great significance for the early prevention and control of echinococcosis.

[0003] Multiple methods have been used for the development of candidate vaccines against Echinococcus granulosus in dogs, including obtaining crude antigens or excretory / secretory (E / S) antigens by in vitro culture, radiation irradiation, DNA vaccines, recombinant protein vaccines, and live attenuated Salmonella vaccines. Although considerable efforts have been made in the results of recombinant vaccines recently for the development of effective vaccines against Echinococcus granulosus in dogs, so far, no consensus has been reached on a single and suitable vaccine for the definitive host. Therefore, it is very necessary to continue to search for new Echinococcus granulosus antigens. Summary of the Invention

[0004] The present invention uses high-throughput and unbiased screening of a cDNA expression library of 3-day-old larvae of Echinococcus granulosus to obtain the Eg73 antigen protein, and further provides a preparation method and application of this antigen protein, aiming to provide a new antigen target for the development of candidate antigens for vaccines against Echinococcus granulosus in dogs. It solves the problem that the research and development of vaccines against Echinococcus granulosus infection in dogs has stagnated and the screening of protective antigens has lagged behind.

[0005] An application of an Eg73 protein, wherein the application is to use the Eg73 protein to prepare a vaccine against Echinococcus granulosus infection in dogs, and the amino acid sequence of the Eg73 protein is as shown in SEQ ID NO.1.

[0006] Furthermore, the screening method of the Eg73 protein is as follows: S1: Construct a cDNA expression library of 3-day-old larvae of Echinococcus granulosus; S2: Use the false screening method to process the library to screen serum probes; S3: Coat the library on the plate of solid medium, take it out after inverted culture for 1 h, cut the nitrocellulose membrane to the specification matching the culture plate and lay it on the surface of the medium, make marks at 3 asymmetric positions with toothpicks, take off the membrane with the colony contact surface facing up, lay it on the plate of solid medium containing IPTG, perform inverted culture, wrap the culture plate with sealing film after incubation and store it upside down at 4°C; S4: Expose the nitrocellulose membrane to chloroform vapor, then place it in a petri dish, add bacterial lysis buffer to submerge the nitrocellulose membrane and lyse it at room temperature, wash it 3 times with elution buffer, then block it with blocking solution for 2 h, incubate it with the serum probe described in S2 together, and wash it 3 times with elution buffer; S5: Incubate the nitrocellulose membrane obtained above in alkaline phosphatase-conjugated affinity rabbit anti-dog secondary antibody, wash it 3 times with elution buffer, use 5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium chloride as the substrate for color development after membrane washing, and finally terminate the reaction. A purple color appears at the antigen-antibody complex of the positive clone vector. S6: According to the position of the positive ring on the membrane, pick the colonies at the specific positions corresponding to the suspected positive clones on the culture plate and inoculate them into the medium for shaking culture; perform three rounds of screening on the cultured bacterial liquid according to the aforementioned method until consistent immunopositive recombinants are obtained, extract the plasmid for sequencing verification, and design primers for PCR amplification according to the correctly sequenced recombinants to obtain PCR products; perform recombinant expression on the said PCR products to obtain Eg73 protein.

[0007] Further, the exposure time in S4 is 15 min.

[0008] Further, the termination reaction in S5 is to terminate the reaction with deionized water.

[0009] Application of Eg73 protein gene, the said application is to use the gene encoding Eg73 protein for preparing a vaccine against Echinococcus granulosus infection in dogs, and the nucleotide sequence of the Eg73 protein gene is as shown in SEQ ID NO.2.

[0010] Application of a recombinant expression vector containing Eg73 protein gene, the said application is to use the recombinant expression vector for preparing a vaccine against Echinococcus granulosus infection in dogs.

[0011] Application of a recombinant cell containing the above recombinant expression vector, the said application is to use the recombinant cell for preparing a vaccine against Echinococcus granulosus infection in dogs.

[0012] A vaccine antigen against Echinococcus granulosus in dogs, the said antigen is prepared from Eg73 protein or Eg73 protein gene.

[0013] Beneficial effects The present invention screens for novel antigens recognized by the immune system by establishing a recombinant cDNA expression library. This method can identify previously unknown specific antigens. Compared with the prior art that requires prior knowledge of the target antigen, this method can identify antigens that trigger protective immune responses.

[0014] The present invention screens for the Eg73 protein through the above screening. The amino acid sequence of the Eg73 protein is shown in SEQ ID NO.1, and the nucleotide sequence encoding the Eg73 protein is shown in SEQ ID NO.2. This antigen protein can specifically react with the positive serum of dogs infected with Echinococcus granulosus, and has high application value in the research and development of canine anti-Echinococcus granulosus vaccines and diagnostic reagents. Brief Description of the Drawings

[0015] Figure 1 It is a diagram for signal peptide prediction and analysis.

[0016] Figure 2 It is a diagram for secondary structure prediction and analysis.

[0017] Figure 3 It is a diagram for tertiary structure prediction and analysis.

[0018] Figure 4 It is for PCR amplification of the gene fragment of Eg73.

[0019] Figure 5 It is a diagram for the construction of the pET28a expression vector, where M: standard molecular weight; 1: target fragment cut by enzymes; 2: pET28a vector control after enzyme digestion.

[0020] Figure 6 It is for the Eg73 protein in E.coli BL21 It is a diagram of the 10% SDS-PAGE detection results after the induced expression of the Eg73 protein in (DE3) cells. Where M: standard molecular weight; 1: uninduced pET28a empty vector; 2: pET28a empty vector induced by IPTG; 3: supernatant of the pET28a empty vector induced by IPTG; 4: precipitate of the pET28a empty vector induced by IPTG; 5: whole cell protein of the Eg73 protein without induction; 6: whole cell protein of the Eg73 protein induced by IPTG; 7: supernatant of the Eg73 protein induced by IPTG; 8: precipitate of the Eg73 protein induced by IPTG.

[0021] Figure 7 It is for the Eg73 protein in E.coli BL21 It is a diagram of the 10% SDS-PAGE detection results after the induced expression and purification of the Eg73 protein in (DE3) cells. Where M: standard molecular weight; 1: purification diagram of the induced pET28a-Eg73 protein.

[0022] Figure 8Western blotting image of Eg73 protein, where M: standard molecular weight; 1: His-tag antibody; 2: reaction with positive serum of dogs infected with Echinococcus granulosus; 3: reaction with negative serum of dogs not infected with Echinococcus granulosus. Detailed implementation methods

[0023] The present invention will be further described in detail below in conjunction with embodiments. The following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0024] Example 1. Construction of a cDNA expression library of 3-day-old larvae of Echinococcus granulosus.

[0025] 1. Extract RNA using the CTAB method.

[0026] (1) Add 1 mL of 2% CTAB extraction buffer to a 1.5 mL centrifuge tube (RNase-Free) in advance, and then add 20 μL of 2% β-mercaptoethanol. After gently mixing, incubate in a water bath at 65 °C for 5 min to obtain a homogenization buffer.

[0027] (2) Take the 3-day-old larvae of Echinococcus granulosus pre-preserved in liquid nitrogen and place them in a 1.5 mL centrifuge tube. Centrifuge at 12000 rpm for 5 min at room temperature, aspirate the supernatant, and collect the worm bodies.

[0028] (3) Add the homogenization buffer to the collected worm bodies (0.05 g - 0.1 g), and use an electric grinder to lyse the worm bodies (if the worm bodies are frozen, the buffer can be directly added to the frozen worm bodies and homogenization can be started immediately. At the beginning of homogenization, the homogenizer should be adjusted to a lower speed and the plug should be inserted into the frozen worm bodies to break them and suspend them from the bottom of the test tube. After the large frozen pieces are dispersed, the homogenizer should be immediately adjusted to the maximum speed for homogenization). Aliquot the homogenate into pre-warmed centrifuge tubes containing homogenization buffer, invert or vortex gently to mix, and then incubate in a water bath at 65 °C for 10 - 15 min. During the water bath, gently invert and mix several times.

[0029] (4) After the water bath is completed, let it stand. After the centrifuge tube cools to room temperature, add an equal volume of phenol / chloroform / isoamyl alcohol mixture (mixed volume ratio is 25:24:1), gently invert up and down for 3 - 5 min until the liquid is fully mixed, centrifuge at 4 °C, 10000 rpm for 10 - 15 min. Then aspirate the supernatant into a new RNase-Free centrifuge tube, add an equal volume of chloroform / isoamyl alcohol mixture (mixed volume ratio is 24:1) and extract 2 - 3 times until there is no precipitate at the interface. Aspirate the supernatant into a new centrifuge tube, repeat the above extraction steps to precipitate the protein, and retain the supernatant.

[0030] (5) Transfer the supernatant to a new RNase-Free centrifuge tube, add 0.6 volume of 8 M LiCI or 1 / 2 volume of 6 M LiCI (both need to be pre-cooled at 4 °C in advance), mix well, and precipitate at -20 °C for 4 h or sediment at 4 °C overnight to denature the RNA.

[0031] (6) Centrifuge at 4 °C and 12,000 rpm for 20 min to precipitate the RNA, discard the supernatant to remove DNA, collect the precipitate, then add 200 μL - 500 μL of 75% ethanol, and repeatedly rinse the precipitate to remove impurities. Centrifuge at 4 °C and 12,000 rpm for 10 min, slowly discard the ethanol supernatant, dry the residual liquid in the centrifuge tube, and dry the precipitate on the ultra-clean bench to obtain total RNA. Appropriately adjust the drying time to ensure that the ethanol is completely volatilized. The drying time should not be too long, otherwise it is not conducive to RNA dissolution. Then add 20 μL of DEPC water to dissolve at room temperature, take part for agarose gel electrophoresis and ultraviolet spectrophotometer to detect the quality of total RNA, and store the rest at -80 °C for later use.

[0032] 2. mRNA isolation and purification.

[0033] Use the Oligotex mRNA Kits (Qiagen) isolation and purification kit to isolate and purify mRNA from total RNA. The specific method is operated according to the kit instructions.

[0034] 3. First-strand cDNA synthesis.

[0035] (1) Add 4.5 μg of the above-mentioned isolated mRNA to a 0.2 mL RNase free centrifuge tube, then add DEPC water to a total volume of 25.5 μL, and then add 2 μL of 3’RT enzyme (1.5 μg / μL).

[0036] (2) Place the centrifuge tube on a PCR instrument, treat at 70 °C for 7 min, then add 1 μL of biotin-attB2-Oligo(dT) Primer (5'-Biotin–GGCGGCCGCACAACTTTGTACAAGAAAGTTGGGT(T)19-3', 30 pmol), mix well (react at 65 °C for 5 min), then cool down to 45 °C and incubate for 2 min, and immediately place on ice to obtain mixture 1.

[0037] (3) During the previous step, prepare the first-strand reaction system in a new 0.2 mL RNase-free centrifuge tube. The reaction system consists of 10 μL of 5X RT Buffer, 5 μL of DEPC water, 2.5 μL of 10 mM dNTPs, and 5 μL of RT enzyme (invitrogen), and place it on ice for later use to obtain Mixture 2.

[0038] (4) Mix Mixture 1 and Mixture 2 thoroughly, avoiding the generation of bubbles. Then add 1 μL of Glycogen (20 μg / μL), 25 μL of 7.5 M NH4OAc, and 187 μL of 100% ethanol to synthesize the first-strand cDNA.

[0039] 4. Second-strand cDNA synthesis.

[0040] Add 91 μL of DEPC water, 30 μL of 5X Second Strand Buffer, 3 μL of 10 mM (each) dNTPs, and 6 μL of Second Strand Enzyme Mix to the first-strand cDNA reaction solution, react at 16 °C for 2 h, then add 2 μL of T4DNA Polymerase, react at 16 °C for 5 min, then add 10 μL of 0.5 M EDTA (pH 8.0), add 160 μL of phenol / chloroform / isoamyl alcohol mixture (mixed volume ratio is 25:24:1), mix well for 30 s; centrifuge at 14000 rpm at room temperature for 5 min, transfer the supernatant to a new centrifuge tube, precipitate with ethanol, and dissolve the precipitate in 40 μL of DEPC water for storage and later use to obtain double-stranded cDNA.

[0041] 5. Add 5' adapter.

[0042] Set up the reaction system according to Table 1, mix well, then react at 16 °C for 16 - 24 h, then add 2 μL of 10 mM dNTP and 2 μL of T4DNA polymerase, place at 16 °C for 20 min to fill in the ends and obtain the cDNA with the adapter.

[0043] Table 1 Reaction system for adding 5' adapter

[0044] 6. Ligation of cDNA and vector.

[0045] Using homologous recombination, mix the ligated cDNA (7 μL) with the pBluescript SK(-) vector (3 μL), add 5 μL of all-direct recombinase and 5 μL of water, mix well, and incubate at 25 °C for 20 h. Add 2 μL of Proteinase K to inactivate the recombinase, add 60 μL of sterile water to the reaction system to make the total volume 100 μL. Then, add 1 μL of Glycogen (20 μg / μL), 50 μL of 7.5 M NH4OAc, and 375 μL of 100% ethanol in sequence, mix well and place at -80 °C for at least 1 h. Centrifuge at 4 °C, 16000 rpm for 30 min, carefully remove the supernatant, add 150 μL of 70% ethanol, centrifuge at 4 °C, 16000 rpm for 3 min; repeat the steps of adding ethanol and centrifugation once, remove all the supernatant, avoid touching the cDNA precipitate, air-dry the cDNA at room temperature for 5 - 10 min, resuspend the cDNA precipitate with 10 µL of DEPC water, and pipette 30 - 40 times. Centrifuge instantaneously for 2 s to collect the recombinant product and immediately place it on ice.

[0046] 7. Electrotransform Escherichia coli competent cells.

[0047] Place a 1 mm electroporation cuvette (Bio-Rad) at -80 °C for pre-cooling for 30 min; on ice, add 2.5 μL of the recombinant product and 50 μL of competent cells to the electroporation cuvette, place on ice for 45 min, and perform electroporation under the conditions of Voltage 2.9 kV, Resistance 200 Ω, Capacity 25 μF. Immediately after electroporation, add 1 mL of LB medium to the electroporation cuvette, then transfer it to a new 15 mL centrifuge tube, make up the volume to 5 mL, incubate at 37 °C, 225 - 250 rpm for at least 1 h. After incubation, store the culture at 4 °C overnight, or add glycerol with a final concentration of 20% and store at -80 °C for later use.

[0048] 8. Library quality identification.

[0049] Take 10 μL of the transformed bacterial stock solution, dilute it 100 times, then take 10 μL from it and spread it on an LB plate (containing ampicillin resistance). Count the colonies the next day. A total of about 230 colonies grew, so the library capacity is 230 / 10 * 100 * 1000 = 2.3 * 10 6 CFU / mL. For the 5 mL of the original transformed bacterial solution, its total library capacity is: 2.3 * 5 * 10 6 CFU = 1.15 * 10 7 CFU. Pick single colonies on the plate, use PCR amplification, and detect the size of the PCR inserted fragment by electrophoresis. The PCR primer sequences are as follows: M13F: TGTAAAACGACGGCCAGT; M13R: CAGGAAACAGCTATGACC.

[0050] The average length of the inserted fragments is about 1 - 1.2 Kbp, and the positive rate of the library is 100%. Example 2: Application of the 3-day larval expression library of Echinococcus granulosus.

[0051] 1. The library screening serum probe was processed by the false screening method.

[0052] Before immunoscreening in this application, the library screening serum probe was processed by the false screening method. By adsorbing and purifying the serum of dogs infected with Echinococcus granulosus, the non-specific cross-reactive antibody components (mainly removing the relatively low-titer anti-E. coli antibodies) were removed, and finally an ideal antibody probe that could be used for immunoscreening the constructed library was obtained.

[0053] 2. The expression library was screened using the serum of dogs infected with Echinococcus granulosus.

[0054] (1) The library prepared in Example 1 was evenly spread on the plate of LB solid medium (containing ampicillin resistance), incubated overnight at 37 °C in an inverted position, then the plate was taken out of the incubator and incubated at 4 °C in an inverted position for 1 h.

[0055] (2) The nitrocellulose membrane (NC membrane) was cut to a size matching the culture plate and laid on the surface of the medium. It was allowed to come into contact with the colonies until it became wet, and marks were made at 3 asymmetric positions with the tip of a 20 mL syringe.

[0056] (3) The NC membrane was gently lifted off with the colony contact surface facing up and laid on the plate of LB solid medium containing IPTG. It was incubated at 37 °C in an inverted position for 6 - 8 h until new colonies grew on the LB solid culture plate. The plate was wrapped with sealing film and stored in an inverted position at 4 °C.

[0057] (4) The NC membrane was taken out, exposed to chloroform vapor for 15 min and then placed in a petri dish. Bacterial lysis buffer was added to submerge the NC membrane, and the petri dish was placed on a shaker and lysed slowly at room temperature. The membrane was washed 3 times with elution buffer, and then blocked with TBST containing 5% non-fat milk powder for 2 h.

[0058] (5) After the incubation was completed, it was incubated with the serum probe screened by the false screening method for 2 h. The NC membrane was washed 3 times with elution buffer, and then the alkaline phosphatase-conjugated affinity rabbit anti-dog secondary antibody was diluted (diluted at 1:5000), added to the NC membrane and incubated at room temperature, and then the NC membrane was washed 3 times with elution buffer; After washing the membrane, 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium chloride were used as substrates for color development, and deionized water was used to terminate the color development. A purple color appeared at the antigen-antibody complex of the positive clone vector. According to the position of the positive clone on the membrane, the suspected positive colonies were corresponding to the specific positions of the clones on the culture plate and picked, inoculated into LB medium, shaken at 37 °C and 180 rpm for 3 h, and the above steps were repeated for screening three times until 100% positive clones appeared on the plate. The positive clone single colonies were picked and sent to Sangon Biotech Co., Ltd. for sequence determination. The sequences were aligned in NCBI, and then signal peptide, secondary structure and tertiary structure analyses were carried out. The results are as Figures 1-3 , and it was named Eg73 protein.

[0059] Signal peptide analysis website: http: / / www.cbs.dtu.dk / services / SignalP-3.0 / ; Secondary structure and tertiary structure analysis websites: https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_sopma.html; https: / / swissmodel.expasy.org / interactive.

[0060] Example 3: Expression, purification and application of the protein.

[0061] 1. Construction of the protein expression plasmid.

[0062] (1)PCR amplification of the target gene.

[0063] Through screening the library and sequencing analysis, an Eg73 protein of Echinococcus granulosus was screened, and different fragment sizes of this protein were screened. The full-length sequence had a low expression level, so a partial truncated sequence of this protein was expressed. The amino acid sequence of this protein is as SEQ ID NO.1, and the nucleotide sequence encoding this protein is as SEQ ID NO.2. Primers for amplifying the Eg73 gene were designed, and the primers contained nucleotide sequences binding to the template, restriction enzyme sites and protective bases. Using the genome of 3-day-old larvae of Echinococcus granulosus as the template, the Eg73 gene fragment was amplified by PCR. The amplification results are as Figure 4As shown, the target band is clear and distinct, and the band size meets expectations. After gene amplification by agarose gel electrophoresis, the DNA of this fragment was recovered using the Tiangen Gel Extraction Kit and the DNA concentration was measured. The DNA of the gene fragment was double digested with BamHⅠ / XhoⅠ (Takara), and the DNA was recovered by cutting the gel through agarose gel electrophoresis. After measuring the concentration with a spectrophotometer, it was stored at -20°C for later use.

[0064] PCR amplification system for Eg73 gene fragment: Template (genome of Echinococcus granulosus three-day larvae) 1 μL, Eg73 primer F 1 μL, Eg73 primer R 1 μL, 2X PCR Master Mix 12.5 μL, and ddH2O 4.5 μL. The primer sequences are as follows (the underlined parts are restriction enzyme sites): Eg73 primer F: 5’-CGC GGATCC ATGAAAGTGGAAAGCTTTCCGGTG-3’ Eg73 primer R: 5’-CCG CTCGAG TTACGGCAGCGCGTTCATCT-3’ PCR amplification conditions for Eg73 gene fragment: 95°C for 5 min, 95°C for 30 s, 59°C for 30 s, 72°C for 1 min, 35 cycles, and extension at 72°C for 10 min.

[0065] SEQ ID NO.1: HMKVESFPVEWPITPIPCVEALATVVMASSDEVGKPAAVEEARLTQEEVKLGTQPSLQDHPKLDPASQYPLSICNYWAVRQQELMACQHDLKSRLHSQEAEARLETCISQMLAAGEVREQQVRQYFHENEHLLKVDLGTSGRLQMLNPLTSPTPNEGENDDGDDEDDSEMVATLLQLMRDNERLEALNASHIENIVSERDSCAHLKVQLRLNSYVAPPPLPSLQMNALP.

[0066] SEQ ID NO.2: atgaaagtgg aaagctttcc ggtggaatgg ccgattaccc cgattccgtgcgtggaagcg ctggcgaccg tggtgatggc gagcagcgat gaagtgggca aaccggcggc ggtggaagaagcgcgcatta cccaggaaga agtgaaactg ggcacccagc cgagcctgca ggatcatccg aaactggatccggcgagcca gtatccgctg agcatttgca actattgggc ggtgcgccag caggaactga tggcgtgccagcatgatctg aaaagccgca ttcatagcca ggaagcggaa attgcgcgca ttgaaacctg cattagccagatgattgcgg cgggcgaagt gcgcgaacag caggtgcgcc agtattttca tgaaaacgaa catctgctgaaagtggatct gggcaccagc ggccgcctgc agatgctgaa cccgctgacc agcccgaccc cgaacgaaggcgaaaacgaa gataacgatg gcgatgatga agatgatgat gatgatgaaa gcgaaatggt ggcgaccctgctgcagctga tgcgcgataa cgaacgcctg gaagcgctga acgcgagcca tattgaaaac attgtgagcgaacgcgatag ctgcgcgcat ctgaaagtgc agctgcgcct gaacagctat gtggcgccgc cgccgctgccgagcctgcag atgaacgcgc tgccgtaa。

[0067] (2) Construct the plasmid of Eg73 gene.

[0068] After digesting the pET28a plasmid vector, ligate it with the DNA of the target fragment ( Figure 5 ), and the ligation system is 3 μL of pET28a vector, 1 μL of DNA, 1 μL of T4 ligase, 4 μL of 10× buffer and 11 μL of ddH2O. Incubate the ligation system in a metal bath instrument at 16 °C overnight to obtain the ligation product. Add the ligation product to BL21In (DH5α) competent cells, place them on ice for 30 min of ice bath, heat shock at 42 °C for 45 - 60 s. After heat shock, place the competent cells on ice for 2 min. Add 700 μL of LB antibiotic-free liquid medium to the competent cells, put them in a shaker at 37 °C and incubate at 180 rpm for 1 h. After incubation, centrifuge at 3500 rpm for 3 min, discard 600 μL of the supernatant, resuspend the pellet with the remaining 100 μL of supernatant. After resuspension, spread the bacterial solution onto a plate of LB solid medium containing 50 μg / mL kanamycin, invert the plate and culture it overnight in an incubator at 37 °C. The next day, pick a single colony into 1 mL of LB liquid medium containing 30 μg / mL kanamycin, culture for 12 h and then transfer it to 10 mL of LB liquid medium containing 30 μg / mL kanamycin for overnight expansion culture. Extract the plasmid and perform PCR identification using Eg73 primer F and Eg73 primer R. If there is a target band amplified by PCR and its size meets the expectation, it proves that the expression plasmid is successfully ligated, and sequence the verified correct plasmid.

[0069] 2. Expression and purification of the protein.

[0070] Add 10 μL of the plasmid with correct sequencing to the expression competent BL21 (DE3), place it on ice for 30 min of ice bath, heat shock at 42 °C for 45 s. After heat shock, immediately place the competent cells on ice for 2 min. Add 700 μL of LB liquid medium to the competent cells, put them in a shaker at 37 °C and incubate at 180 rpm for 60 min. After incubation, centrifuge at 5000 rpm for 3 min, discard 600 μL of the supernatant, resuspend the pellet with the remaining 100 μL of supernatant, then spread the bacterial solution onto a plate of LB solid medium containing 30 μg / mL kanamycin, culture in an incubator at 37 °C for 12 h, pick a single colony into a bacterial flask containing 10 mL of medium, add kanamycin to make its final concentration 30 μg / mL, shake culture for about 8 hours, transfer the bacterial solution at a ratio of 1:100 (volume ratio) to 1 L of LB liquid medium, and put it in a shaker at 37 °C and shake the bacteria until OD 600The value is approximately 0.6, and IPTG is added to make its final concentration 1 mmol / L, and induced expression is carried out for 6 - 8 h. Pour the cultured bacteria into a centrifuge bottle, centrifuge at 4000 rpm for 30 min, discard the supernatant and collect the precipitate, and wash the cell precipitate twice with 1×PBS (PH = 7.4). Resuspend the bacterial cell precipitate with binding buffer (8M Urea, 50mM Tris, 300mM Nacl, 0.1% Triton X - 100, PH = 8.0), use an ultrasonic cell disruptor to break the protein at low temperature until the liquid becomes clear, then collect the broken protein, centrifuge at 10000 rpm for 10 min to collect the protein supernatant, and filter the supernatant with a 0.22μm filter. Take Ni - NTA packing material to load the column, and purify the protein by passing the filtered protein supernatant through a His - tagged protein purification pre - column. After the protein solution has drained, replace it with washing buffer (8 M Urea, 50 mM Tris, 300 mM Nacl, 50 mM Imidazole, adjusted to pH 8.0 with concentrated hydrochloric acid) to wash the miscellaneous proteins. After about 10 minutes of washing, change the buffer to elution buffer (8 M Urea, 50 mM Tris, 300 mM Nacl, 500 mM Imidazole, adjusted to pH 8.0 with concentrated hydrochloric acid) to elute the target protein bound to the His column, collect the effluent, dialyze the purified fraction into protein storage buffer (50 mM Tris, 300mM NaCl, 10% Glycerol, 2 mM DTT, pH 8.0), concentrate, filter and sterilize, aliquot at 1 mL / tube, and store at - 80°C to obtain the purified protein.

[0071] 3. Verify the purified protein by SDS - PAGE and Western blot.

[0072] Add 20 μL of 5× protein loading buffer to the purified protein, then mix well by vortex oscillation and heat at 100 °C in a metal bath for 10 min. Take out two prepared 12% separating gels and 5% stacking gels. One is used for SDS - PAGE to verify the purified protein, and the other is used for Western blot verification. First, run the stacking gel at 80 V and then switch the voltage to 120V to run the separating gel. After the protein gel is run, one is directly placed in Coomassie Brilliant Blue staining solution for 2 h, and then transferred to the decolorizing solution for decolorization ( Figure 6 ). And the other protein gel is used for Western blot verification.

[0073] Cut the SDS-PAGE protein gel block, and transfer the protein to the NC membrane by wet transfer at a current of 300 mA for 70 min. Wash the membrane 3 times with PBST, with an interval of 5 min each time. Block the membrane with 5% skim milk powder at room temperature for 2 h. Wash the membrane 3 times with PBST, with an interval of 5 min each time. Use the positive serum of dogs infected with Echinococcus granulosus and His-tag antibody as the primary antibody, and incubate overnight at 4 °C (the serum is diluted 1:1000 with PBST). Wash the membrane 5 times with PBST, with an interval of 5 min each time. Use the enzyme-labeled rabbit anti-dog IgG and rabbit anti-mouse as the secondary antibody and incubate at room temperature for 2 h (diluted 1:5000 with PBST). Wash the membrane 5 times with TBST, with an interval of 5 min each time (negative serum is used as a control). Mix equal volumes of ECL chemiluminescent developing solution A and B and use a chemiluminescence instrument to develop the color in the dark. The results of the purified protein are as Figure 7 shown. It was found by SDS-PAGE that the purified Eg73 protein was approximately 26.33 kDa, which was consistent with the predicted result, and was verified by Western blot ( Figure 8 ). There was no reactive band with the negative serum at the target protein, and the purified protein had good immunoreactivity with the positive serum of dogs infected with Echinococcus granulosus.

Claims

1. Use of an Eg73 protein, characterized in that, The application is to use the Eg73 protein for preparing a vaccine against Echinococcus granulosus infection in dogs, and the amino acid sequence of the Eg73 protein is as shown in SEQ ID NO.

1.

2. The application according to claim 1, wherein The screening method of the Eg73 protein is as follows: S1: Construct a cDNA expression library of Echinococcus granulosus larvae at 3 days old; S2: Use the false screening method to process the library to screen serum probes; S3: Coat the library on a plate of solid medium, take it out after inverted culture for 1 h, cut the nitrocellulose membrane to a size matching the culture plate and lay it on the surface of the medium, make marks at 3 asymmetric positions with a toothpick, remove the membrane and place the colony contact surface upwards, lay it on a plate of solid medium containing IPTG, perform inverted culture, wrap the culture plate with sealing film after incubation and store it inverted at 4°C; S4: Expose the nitrocellulose membrane to chloroform vapor, then place it in a petri dish, add bacterial lysis buffer to submerge the nitrocellulose membrane and lyse it at room temperature, rinse it 3 times with elution buffer, then block it with blocking solution for 2 h, incubate it together with the serum probe described in S2, and rinse it 3 times with elution buffer; S5: Incubate the obtained nitrocellulose membrane in alkaline phosphatase-conjugated affinity rabbit anti-dog secondary antibody, rinse it 3 times with elution buffer, use 5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium chloride as a substrate for color development after membrane washing, and finally terminate the reaction. A purple color appears at the antigen-antibody complex of the positive clone vector; S6: According to the position of the positive ring on the membrane, pick the colonies at the specific positions corresponding to the suspected positive clones on the culture plate and inoculate them into the medium for shaking culture; perform three rounds of screening on the cultured bacterial liquid according to the aforementioned method until a consistent immunopositive recombinant is obtained, extract the plasmid for sequencing verification, and design primers according to the correctly sequenced recombinant for PCR amplification to obtain a PCR product; Recombine and express the said PCR product to obtain the Eg73 protein.

3. The application according to claim 2, wherein The exposure time described in S4 is 15 min.

4. The application according to claim 2, wherein The termination of the reaction described in S5 is to terminate the reaction with deionized water.

5. Use of the Eg73 protein gene, characterized in that, The application is to use the gene encoding the Eg73 protein as claimed in claim 1 for preparing a vaccine against Echinococcus granulosus infection in dogs, and the nucleotide sequence of the Eg73 protein gene is as shown in SEQ ID NO.

2.

6. Use of a recombinant expression vector containing the gene according to claim 5, characterized in that, The application is to use the recombinant expression vector for preparing a vaccine against Echinococcus granulosus infection in dogs.

7. Use of a recombinant cell containing the recombinant expression vector according to claim 6, characterized in that, The application is to use the recombinant cell for preparing a vaccine against Echinococcus granulosus infection in dogs.

8. A canine anti-Echinococcus granulosus vaccine antigen, characterized in that, The antigen is prepared from the Eg73 protein as claimed in claim 1 or the Eg73 protein gene as claimed in claim 5.

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