Echinococcus granulosus antigen Eg142 and its encoding gene and application

Abstract

An Echinococcus granulosus antigen Eg142, its encoding gene, and its application belong to the field of immunology technology. The present invention utilizes the Eg142 antigen protein, obtained through high-throughput, unbiased screening of a three-day-old Echinococcus granulosus larvae cDNA expression library, to develop a new antigen candidate for a canine Echinococcus granulosus vaccine. This solves the current problems of stagnation in the development of canine Echinococcus granulosus vaccines and the lag in screening for protective antigens. The amino acid sequence of the antigen protein is shown in SEQ ID NO.3, and the nucleotide sequence encoding the Eg142 protein is shown in SEQ ID NO.4. It has been verified that the antigen protein can specifically react with positive serum from dogs infected with Echinococcus granulosus, and has high application value in the development of canine Echinococcus granulosus vaccines.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to an Echinococcus granulosus antigen Eg142 and a coding gene and application thereof. Background Art

[0002] Echinococcosis (also known as hydatid disease) is a zoonotic parasitic disease caused by the larvae (larvae) of the tapeworm Echinococcus. Dogs, as the definitive host, play a central role in the parasite's life cycle. Cystic echinococcosis (CE), caused by infection with the larval stage (hydatid cysts) of the tapeworm Echinococcus granulosus, is a chronic disease in humans. Because many infected individuals lack obvious clinical symptoms, the actual number of infections is often underestimated.

[0003] Cystic echinococcosis is a global disease, posing a serious public health problem and a significant economic burden. Despite the implementation of comprehensive prevention and control measures, including standardized slaughter, these measures have yet to achieve the desired results. The lack of effective control technologies for dogs, the definitive host of the core infection source, has hindered prevention and control efforts. Therefore, vaccination of dogs against Echinococcus has become a critical component of effective disease control.

[0004] The antigenic composition of Echinococcus granulosus is extremely complex, and it cannot be cultured and propagated on a large scale in vitro, which poses a significant challenge to the immunodiagnosis and prevention of echinococcosis. The prevention and control of echinococcosis is a long-term and arduous task, and breakthroughs in control technologies are urgently needed.

[0005] cDNA libraries are essential tools for studying gene expression in specific organs, tissues, or developmental stages. Their construction and screening are core methods for gene cloning and are an effective means of discovering new genes and studying their function. Various methods exist for screening target genes from cDNA expression libraries. Using antisera as probes to screen target clones is a sensitive and reliable technique for obtaining unknown genes. Currently, research has focused on constructing cDNA libraries for various developmental stages of Echinococcus granulosus and cloning and screening antigenic genes for immunodiagnosis and prevention. However, this research has primarily focused on the protoscolex and adult stages. However, the developmental stage from protoscolex eversion to the three-day-old larvae (in vivo infection studies have shown that 86.5% of "sheep" worms begin eversion six hours after infection, and all worms are fully everted by the third day; if the everted protoscolex fail to attach to the small intestine within a short period of time, they are excreted in the feces) is a critical stage in canine Echinococcus granulosus infection.

[0006] Antigen B (AgB) is a heat-resistant, multimeric lipoprotein with a molecular weight of 120–160 kDa, originally discovered and extracted from the cyst fluid of the middle tapeworm stage (echinococcosis) of Echinococcus granulosus. It consists of a minimum monomer of approximately 8 kDa. To date, five AgB subunit genes have been identified: EgB8 / 1, EgB8 / 2, EgB8 / 3, EgB8 / 4, and EgB8 / 5. All have high specificity and sensitivity for the serological diagnosis of echinococcosis and are considered reliable and valuable target antigen candidates. Currently, only AgB1 and AgB2 are known to have been cloned and expressed for the diagnosis of echinococcosis. Further research is needed on AgB3, AgB4, and AgB5 for immunodiagnosis of echinococcosis. Rott et al. evaluated serum samples from a small number of patients with CE, AE, other parasitic diseases, and normal controls using an ELIS assay. They found that rEgAgB8 / 2 was more valuable than rEgAgB8 / 1 for the serological diagnosis of CE patients. AgB1, AgB2, and AgB5 were expressed at low levels in all stages of Echinococcus granulosus. AgB4 was expressed in the cyst wall, in immature and fully developed adult stages, but at low levels. However, AgB3 was expressed at high levels in all stages, especially in the fully developed adult stage, suggesting that EgAgB3 plays an important role in the survival of adult worms in the definitive host. The value of AgB3 as a vaccine for the prevention of cystic echinococcosis has not yet been reported.

[0007] In summary, how to construct a cDNA expression library of 3-day-old larvae of Echinococcus granulosus and screen related antigen genes through it has important scientific significance for the development of genetically engineered diagnostic antigens and vaccines and the early prevention of echinococcosis. Summary of the Invention

[0008] This invention utilizes the Eg142 antigen protein, identified through high-throughput, unbiased screening of a three-day-old Echinococcus granulosus larvae cDNA expression library, to provide a novel antigenic target for the development of candidate antigens for canine Echinococcus granulosus vaccines. This approach addresses the current challenges of stalled vaccine development and the lag in screening for protective antigens in dogs.

[0009] An application of Eg142 protein is to use the Eg142 protein in preparing a vaccine for dogs against Echinococcus granulosus infection. The amino acid sequence of the Eg142 protein is shown in SEQ ID NO.3.

[0010] Furthermore, the screening method of the Eg142 protein is as follows:

[0011] S1: Construction of a cDNA expression library of 3-day-old larvae of Echinococcus granulosus;

[0012] S2: Spread the library onto a plate containing solid culture medium and invert it for incubation. Then remove and invert it for 1-2 hours. Cut a 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 membrane with the colony contact surface facing upwards and place it on a plate containing IPTG-containing solid culture medium. Invert it for incubation. Wrap it with sealing film and store it inverted at 4°C.

[0013] S3: Expose the nitrocellulose membrane to chloroform vapor, then place it on a plate. Add bacterial lysis buffer to immerse the nitrocellulose membrane for lysis, rinse three times with elution buffer, and then block with blocking buffer for 2 hours. Then, incubate with serum obtained from the pseudoscreening library and rinse three times with elution buffer.

[0014] S4: The nitrocellulose membrane obtained above was incubated in an alkaline phosphatase-coupled affinity rabbit anti-dog secondary antibody, and washed three times with elution buffer. After washing, the membrane was developed with 5-bromo-4-chloro-3-indoleyl phosphate / nitro blue tetrazonium chloride substrate. Finally, the reaction was terminated, and a purple color appeared at the positive clone vector antigen-antibody complex.

[0015] S5: Based on the position of the positive ring on the membrane, colonies were picked at the specific locations of the suspected positive clones on the culture plate, inoculated into the culture medium and cultured with shaking; the cultured bacterial liquid was screened for three rounds according to the above method until consistent immune-positive recombinants were obtained, and plasmids were extracted and sequenced for verification. Primers were designed based on the correctly sequenced recombinants for PCR amplification to obtain PCR products; the PCR products were recombinantly expressed to obtain Eg142 protein.

[0016] Furthermore, the exposure time in S3 is 15 min.

[0017] Furthermore, the alkaline phosphatase-coupled affinity rabbit anti-dog secondary antibody described in S4 was diluted at 1:5000.

[0018] The application of the Eg142 protein gene is to use the gene encoding the Eg142 protein to prepare a vaccine for dogs against Echinococcus granulosus infection. The nucleotide sequence of the Eg142 protein gene is shown in SEQ ID NO.4.

[0019] The invention discloses an application of a recombinant expression vector containing an Eg142 protein gene, wherein the application is to use the recombinant expression vector for preparing a vaccine for dogs against Echinococcus granulosus infection.

[0020] An application of a recombinant cell containing the recombinant expression vector is to use the recombinant cell to prepare a vaccine for dogs against Echinococcus granulosus infection.

[0021] A canine anti-Echinococcus granulosus vaccine antigen is prepared from Eg142 protein or Eg142 protein gene.

[0022] Beneficial effects

[0023] The present invention screened the Eg142 protein from a three-day-old larval cDNA expression library against Echinococcus granulosus. Its amino acid sequence is shown in SEQ ID NO. 3, and the nucleotide sequence of the gene encoding Eg142 is shown in SEQ ID NO. 4. This antigenic protein specifically reacts with positive serum from dogs infected with Echinococcus granulosus, and has high application value in the development of canine anti-Echinococcus granulosus vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure is the electrophoresis diagram of total RNA from 3-day-old larvae of Echinococcus granulosus, where M is the relative molecular mass standard of DL2000, and 1 is the total RNA from 3-day-old larvae of Echinococcus granulosus;

[0025] Figure 2 To construct the gel electrophoresis of double-stranded cDNA of 3-day-old larvae of Echinococcus granulosus, where M is the relative molecular mass standard of DL2000, 1 is the first-strand cDNA, and 2 is the double-stranded cDNA;

[0026] Figure 3 Figure 1 is a graph showing the results of monoclonal PCR amplification, where M is the relative molecular mass standard of DL2000, and 1-16 are the PCR results of monoclonal colonies;

[0027] Figure 4 Figure 1 is the PCR amplification result of Eg142 protein and the enzyme digestion identification diagram of pET32a expression vector, where M is the relative molecular mass standard of DL2000, 142 is the PCR amplification product of Eg142, 1 is the target fragment digested by enzyme, and 2 is the pET32a vector after enzyme digestion;

[0028] Figure 5 It is the signal peptide of Eg142 protein;

[0029] Figure 6 This is the secondary structure prediction analysis diagram of Eg142 protein;

[0030] Figure 7 This is the tertiary structure prediction analysis diagram of Eg142 protein;

[0031] Figure 8 The Eg142 protein gene is E. coli BL21 SDS-PAGE results after induced expression and purification in (DE3) cells, where M is the standard molecular weight and 1 is the purification diagram of the induced pET32a-Eg142 protein;

[0032] Figure 9 Figure 3 is the Western blotting result of Eg142 protein, where M is the standard molecular weight, 1 is the His tag antibody, 2 is the result of serum reaction from a dog infected with Echinococcus granulosus, and 3 is the result of serum reaction from a dog not infected with Echinococcus granulosus. DETAILED DESCRIPTION

[0033] Example 1. Construction of a cDNA expression library against 3-day-old larvae of Echinococcus granulosus.

[0034] 1. Materials and methods

[0035] 1.1 Materials.

[0036] 1.1.1 Experimental animals

[0037] Native dogs (aged 6 months to 1.5 years, weighing 10-15 kg) from the low-incidence area of ​​echinococcosis in Xinjiang were placed in iron cages and raised in strict accordance with the standards for the breeding and management of experimental animals.

[0038] 1.1.2 Strains and Reagents

[0039] DNA extraction kits were purchased from Tiangen Biochemical Technology Co., Ltd.; Oligotex mRNA Kits were purchased from Qiagen; E. coli DH10B was purchased from Youningwei Biotechnology; pBluescript sk- vector was purchased from Shanghai Zeye Biotechnology Co., Ltd.; cDNA Library Construction Kit, UltraPure™ Phenol, Chloroform, Isoamyl, and Alcohol were all purchased from Gitech Biotechnology Shanghai Co., Ltd.; and all other chemical reagents were domestically produced analytical grade reagents.

[0040] 1.2 Methods.

[0041] 1.2.1 Isolation of protoscolex.

[0042] Collect diseased organs and protoscolex from designated cattle / sheep slaughterhouses at the county level in areas where echinococcosis is prevalent. Scolex larvae are collected and stained with 0.1% eosin to observe their coloration rate and morphology. They can only be used if their vitality is ≥90%.

[0043] 1.2.2 Infect experimental dogs and collect larvae on day 3.

[0044] A combined deworming regimen of albendazole and praziquantel was used to exclude the possibility of infection with the parasite prior to the experiment. The isolated protoscolex were orally inoculated into experimental dogs, and three-day-old larvae of Echinococcus granulosus were obtained by autopsy according to the time limit.

[0045] 1.2.3 Extraction of total mRNA.

[0046] Total RNA was extracted from three-day-old Echinococcus granulosus larvae using TRIZOL reagent and stored in aliquots at -80°C. mRNA was isolated and purified from the total RNA using Oligotex mRNA Purification Kits according to the kit instructions, and the resulting mRNA was used to construct a cDNA library.

[0047] 1.2.4 Synthesis and purification of double-stranded cDNA.

[0048] Using mRNA as template and oligo(dT)18 as primer, the first-strand cDNA is synthesized under the action of reverse transcriptase;

[0049] join in E. coli .DNA Ligase(10 U / µL) 1 µL, E. coli RNase H (2 U / µL) 1 µL, E. coli DNA Polymerase I (10 U / µL) 4 µL, for second-strand cDNA synthesis;

[0050] Add T4 DNA polymerase to blunt the ends, add a 5' adapter, and ligate the cDNA to a 5' adapter. Pass the double-stranded cDNA through a CHROMA SPIN-400 column for centrifugation to remove adapter fragments and small cDNA fragments. Prepare a 1% low-melting-point agarose gel and run the cDNA product through electrophoresis. Cut the gel to recover fragments larger than approximately 1 kb and dissolve the recovered product in DEPC water.

[0051] 1.2.5 Library construction.

[0052] Using homologous recombination, ligate 7 µL of cDNA obtained in 1.2.4 with 3 µL of pBluescript SK(-) vector. Add 5 µL of all-direct recombinase and 5 µL of deionized water, mix well, and incubate at 25°C for 20 h.

[0053] Add 2 µL of Proteinase K to inactivate the recombinant enzyme, and make up the volume to 100 µL with deionized water. Then add Glycogen (20 µg / µL), 7.5 M NH4OAc, and 100% ethanol, mix well, and incubate at -80°C for at least 1 h. Then, centrifuge at 16,000 rpm for 30 min at 4°C and discard the supernatant.

[0054] Add 150 μL of 70% ethanol (centrifuge at 16,000 r / min for 30 min at 4°C and discard the supernatant). Repeat this step once, remove the supernatant completely to avoid disturbing the cDNA precipitate. Dry the cDNA at room temperature, resuspend the cDNA precipitate in DEPC water, and immediately place it on ice for electroporation into E. coli competent cells ( E. coli DH10B) to obtain the initial cDNA library.

[0055] 1.2.6 Library quality evaluation and amplification preservation.

[0056] Take 10 µL of the converted initial cDNA library and dilute it 100-fold. Then spread 10 µL on an LB plate (containing ampicillin resistance) containing X-gal and ITPG and culture it at 37°C overnight. The next day, count and determine the library capacity. After the initial library capacity is determined, all remaining libraries are plated for amplification. Dilute the initial library appropriately and plate 30,000 colonies per plate on a 25 cm LB agar plate (containing ampicillin resistance) and culture it at 37°C overnight. After the colonies grow to an appropriate size, add about 5 mL of liquid LB medium to each plate for elution. Combine all the eluted culture media and centrifuge (3000 r / min for 10 minutes). Discard the supernatant. The precipitate is the amplified library. Take 10 µL of the amplified library and perform a serial dilution. Add it to 90 µL of LB medium and culture it at 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 The titer of the amplified library was determined by serial dilution and plating, and then stored at -80°C until use.

[0057] 2. Results.

[0058] 2.1 Extraction of total RNA and separation and purification of mRNA.

[0059] The total RNA extracted from 3-day-old larvae of Echinococcus granulosus was detected by 1% agarose gel electrophoresis. Figure 1 Clear 28S and 18S bands were observed, and there may be 5S bands, indicating that the extracted total RNA had good integrity and was not degraded. The OD value of the total RNA was measured by UV spectrophotometer. 260 / OD 280 =2.02. The results showed that the purity met the extraction requirements and satisfied the needs of library construction.

[0060] 2.2 Agarose gel electrophoresis of double-stranded cDNA library.

[0061] The first and second round cDNA products were electrophoresed separately. The electrophoresis results were as follows: Figure 2 As shown, it is diffusely distributed and has a high concentration, which can be used for subsequent experiments.

[0062] 2.3 Library capacity and polymorphism identification.

[0063] Single colonies were counted on plates coated with different dilutions of the library, and the initial library capacity was calculated to be 1.15×10 7 CFU / mL, the library capacity after amplification is 1×10 10 CFU / mL. 16 clones were randomly selected from the plate for colony PCR. Figure 3 As shown, the electrophoresis results showed that there were 16 positive insert fragments in the library, and the positive insertion rate was 100%, indicating that the constructed cDNA library had a wide coverage of mRNA and good diversity.

[0064] Example 2. Screening of Eg142 protein.

[0065] The Echinococcus granulosus Eg142 protein was screened by using a cDNA expression library against 3-day-old larvae of Echinococcus granulosus.

[0066] 1. Experimental materials and methods.

[0067] 1.1 Screening of Eg142 protein from the cDNA expression library of 3-day-old larvae of Echinococcus granulosus.

[0068] Sera from 10 experimentally infected dogs were collected and diluted 1:100 with Trisbuffered saline with Tween-20 (TBST) to screen the cDNA expression library.

[0069] Spread the amplified library evenly on an LB plate (Amp+) and incubate inverted at 37°C overnight. Allow colonies to grow to a diameter of 0.1-0.2 mm. Remove the plate from the incubator and invert at 4°C for 1-2 hours. Cut a 0.45 μm nitrocellulose membrane (Millipore) to the size that matches the culture plate and place it on the surface of the culture medium. Place it in contact with the colonies until it becomes moist. Mark three asymmetric positions with the tip of a syringe. Remove the NC membrane and, with the colony-contact surface facing up, plate it on an LB plate (Amp+) containing IPTG. Incubate inverted at 37°C for 6-8 hours until new colonies form. Wrap the master plate with parafilm and store inverted at 4°C.

[0070] The NC membrane was removed, exposed to chloroform vapor for 15 min, and then placed on a plate. Bacterial lysis buffer was added to immerse the NC membrane. The plate was placed on a shaker at 50 rpm and lysed overnight at room temperature. The elution buffer was changed and allowed to stand at room temperature three times, each time for 30 min. The membrane was blocked with 5% skimmed milk powder in TBST for 2 h, incubated with canine positive serum treated by sham screening for 2 h, and the plate was placed on a shaker and shaken slowly at 50 rpm at room temperature. The NC membrane was placed in an elution buffer containing 1% Triton X, 0.5% sodium deoxycholate, and 0.1% SDS for 1 hour. The NC membrane was washed three times in the elution buffer for 30 minutes each time, with slow shaking at 50 rpm at room temperature. The secondary antibody used was alkaline phosphatase-conjugated rabbit anti-dog IgG (Jackson, America), diluted 1:5000, and incubated on the NC membrane at room temperature for 2 hours. The above washing steps were repeated. After washing, the membrane was developed with 5-bromo-4-chloro-3-indoleyl phosphate / nitro blue tetrazolium chloride substrate. Finally, the reaction was terminated with distilled water. The antigen-antibody complex of the positive clones appeared purple.

[0071] Based on the location of the positive ring on the membrane, pick suspected positive colonies corresponding to the specific location of the clone on the culture plate. Inoculate them into LB medium (Amp+) at 37°C with shaking at 180 rpm for 3 hours. Inoculate these colonies onto LB plates (Amp+) and culture them overnight at 37°C. Repeat the second and third rounds of screening as described above until consistent immunopositive recombinants are obtained.

[0072] 1.2 Amplification and purification of Eg142 protein.

[0073] Amplification primers were designed based on the Eg142 protein sequencing, and BamH I and Xho I restriction sites were introduced at the 5′ ends of the upstream and downstream primers, respectively.

[0074] Upstream primer Eg-142 F: 5′- CGCGGATCCATGCTTCTTTATCATTTTAGTGA -3′;

[0075] Downstream primer Eg-142 R: 5'- CCGCTCGAGCTACTCATCCTCTTTAACCAA-3'.

[0076] The amplification system consisted of 20 μL of 2× Taq PCR Master Mix (10 μL), upstream primer (20 μmol / L) (1 μL), and downstream primer (20 μmol / L) (1 μL), with ddH2O added to make up to 20 μL. PCR amplification was performed with 1 μL of template using a 4-minute initial denaturation at 94°C, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds, followed by an additional 10-minute extension at 72°C. PCR products were identified by electrophoresis on a 1% agarose gel, and target fragments of the correct size were recovered using the Tiangen Gel Fragment Recovery Kit.

[0077] 1.3 Construction and identification of pET32a-Eg142.

[0078] The product and expression vector pET32a were recovered by PCR digestion with BamHI and Xhol, purified separately, and then ligated with T4 DNA ligase overnight at 4°C. The ligated product was transformed into BL21 competent cells and plated on ampicillin-resistant LB plates until colonies appeared. A single colony was then plated onto ampicillin-resistant LB liquid medium and cultured overnight at 37°C on a shaker to obtain a recombinant bacterial suspension. Plasmids were extracted and identified by PCR. Positive plasmids were then sent for sequencing verification.

[0079] 1.4 Purification of recombinant target protein.

[0080] Add the positive bacterial solution to 10 mL of LB liquid culture medium containing ampicillin at a ratio of 1:100, shake the culture at 37°C and 200 rpm, and measure the OD 600 When the pH value reached 0.6-0.8, IPTG was added to a final concentration of 1 mM for induction for 6 hours. The bacterial pellet was collected by centrifugation at 5000 rpm / min for 10 minutes. The pellet was then resuspended in 10 mL of PBS and freeze-thawed three times at -80°C. After ultrasonic disruption, the supernatant and pellet were collected and the target protein was purified using a Sangon protein purification nickel column.

[0081] 1.5 SDS-PAGE and Western Blot Verification.

[0082] The purified Eg142 protein was verified using a His-tagged primary antibody at a dilution of 1:1000 and an AP-conjugated goat anti-mouse secondary antibody at a dilution of 1:5000.

[0083] 2. Results.

[0084] 2.1 PCR amplification of anti-Echinococcus granulosus Eg142 protein.

[0085] The PCR product was detected by 1% agarose gel electrophoresis to obtain a clear specific band of approximately 240 bp in size, which was consistent with the expected fragment size ( Figure 4 ), indicating that the Echinococcus granulosus Eg142 protein was successfully amplified.

[0086] 2.2 Construction of expression vector pET32a-Eg142.

[0087] The recombinant bacterial solution was subjected to bacterial solution PCR to verify the band size, and three bacterial solutions with the correct size were randomly selected and sent for sequencing. The sequence comparison results showed that the sequence was correct.

[0088] The amino acid sequence of the Echinococcus granulosus protein Eg142 was finally screened out as shown in SEQ ID NO.3, and the nucleotide sequence was shown in SEQ ID NO.4. The signal peptide, secondary and tertiary structures of the Eg142 protein were predicted, and the results were as follows: Figure 5-7 .

[0089] SEQ ID NO.3:

[0090] MLLYHFSDDDDDDEVTKTKKGVMKAISEIKHFFQSDPLGKKLVEVMKDVASVCEMVRKKARMALKEYVRKLVKEDE.

[0091] SEQ ID NO.4:

[0092] ATGCTTCTTTATCATTTTAGTGATGATGATGAAGTGACAAAGACGAAGAAGGGTGTGATGAAGGCCATTAGCGAGATTAAGCACTTCTTCCAAAGTGATCCACTGGGTAAAAAGTTGGTTGAAGTAATGAAGGATGTGGCGAGTGTGTGCGAGATGGTGAGGAAGAAGGCACGCATGGCACTGAAGGAGTATGTCAGAAAGTTGGTTAAAGAGGATGAGTAG.

[0093] 2.3 Expression and purification of Eg142 protein.

[0094] The expression and purification results of Eg142 protein are shown in Figure 2. Figure 8 As shown in the figure, a specific protein band with a size of 29.15 KDa appeared after 6 hours of induction at 37°C. The result was consistent with the expectation. The amino acid sequence of the screened Eg142 protein after purification is shown in SEQ ID NO.3.

[0095] 2.4 SDS-PAGE and Western Blot Verification

[0096] The results of SDS-PAGE showed that the recombinant protein was successfully expressed. No obvious target bands appeared in the supernatant after the cells were broken. The recombinant protein was an inclusion body protein. Figure 8 As shown in Figure 2, the purified protein has a single band, the correct size, and good reactivity; Western-blotting was used for detection, as shown in Figure 2. 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.

Claims

1. An application of Eg142 protein, characterized in that: The application is the use of the Eg142 protein in the preparation of a reagent or a kit for diagnosing infection of the terminal host Echinococcus granulosus. The amino acid sequence of the Eg142 protein is shown in SEQ ID NO.

3.

2. Application of Eg142 protein gene, characterized in that: The application is to use the gene encoding the Eg142 protein according to claim 1 in the preparation of a reagent or kit for diagnosing infection of the terminal host Echinococcus granulosus. The nucleotide sequence of the Eg142 protein gene is shown in SEQ ID NO.

4.

3. Use of a recombinant expression vector containing the Eg142 protein gene according to claim 2, characterized in that: The application is the use of the recombinant expression vector in the preparation of a reagent or a kit for diagnosing infection of the terminal host Echinococcus granulosus.

4. Use of a recombinant cell containing the recombinant expression vector according to claim 3, characterized in that: The application is the use of the recombinant cells in preparing a reagent or a kit for diagnosing infection of the terminal host Echinococcus granulosus.

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