CN106075416A - The design of a kind of novel Echinococcus moltilocularis subunit vaccine, preparation method and application - Google Patents

Abstract

The present invention provides a kind of novel Echinococcus moltilocularis subunit vaccine, and its active component is a polypeptide, and mainly surface antigen Emy162 and Mucosal Adjuvants b subunit of cholera toxin (CTB) by Echinococcus moltilocularis are constituted.The present invention mainly synthesizes Echinococcus moltilocularis surface antigen Emy162 gene order by gene synthesis technology, then by the gene order phase coupling of this gene with b subunit of cholera toxin, forms a fusion gene.Escherichia coli prokaryotic expression system is utilized to express this fusion gene, after protein purification, it is thus achieved that Echinococcus moltilocularis subunit vaccine.This Echinococcus moltilocularis subunit vaccine can induce body and produce for Echinococcus moltilocularis T cell and B cell immunne response and high titre specific antibody humoral immunoresponse(HI), can be used for preventing and to treat Echinococcus multilocularis diseases related.

Description

Design, preparation method and application of a novel multilocular echinococcosis subunit vaccine

technical field

The invention belongs to the field of biomedicine, and in particular relates to a design and preparation method and application of a novel multilocular hydatid subunit vaccine.

Background technique

Alveolar Echinococcosis leads to alveolar echinococcosis. Alveolar Echinococcus grows in the intermediate host liver by budding or infiltrating proliferation, produces new vesicles, grows into the liver tissue, and the outer corner of the cyst wall The cortex is very thin and often incomplete, and there is no obvious boundary between the cyst and the surrounding tissue. Continuous leakage of the cyst fluid can contact the liver tissue, causing local liver tissue lesions, hyperplasia, liver fibrosis, atrophy, degeneration, and necrosis. Liver cancer-like metastasis can be transferred to the lung, brain, breast and other organs. It is known as "worm cancer" and has a very poor prognosis. Alveolar echinococcosis occurs in high-altitude areas of the northern hemisphere worldwide. The Sanjiangyuan area of my country is a rare and high-incidence area of multilocular echinococcosis in the world. Multilocular echinococcosis has the characteristics of wide incidence range, high degree of malignancy, difficult early diagnosis, poor prognosis, and easy recurrence after operation. There are currently no satisfactory therapeutic strategies for multilocular echinococcosis. Most patients in pastoral areas seek medical treatment when symptoms appear, but the disease is often at an advanced stage, and the opportunity for surgery is lost. Even if partial or half liver resection is performed, the postoperative recurrence rate is also high. Antibiotic therapy such as mebendazole and albendazole has a certain therapeutic effect, but due to the need for long-term continuous treatment, the emergence of drug resistance and the high recurrence rate after treatment have limitations.

Surface antigens are the basic functional units that stimulate the body to generate an immune response, and many surface antigens can be used in the development of vaccines. Subunit vaccines are prepared based on surface antigens and have a unique vaccine design idea, which is a new direction for the development of vaccines for the prevention and treatment of infectious diseases, malignant tumors, and autoimmune diseases. Subunit vaccines have the following advantages: (1) Subunit vaccines reduce or eliminate pyrogens, allergens and other harmful reactants that are difficult to remove from conventional live vaccines or inactivated vaccines; and because subunit vaccines cannot replicate in vivo, It has no risk of causing disease to the host and is the safest and most stable genetic engineering vaccine. (2) Subunit vaccines contain only one or several types of pathogens that are necessary for the production of protective immune responses, but do not contain other genetic information of pathogens, so they do not contain infectious components, so they do not need to be inactivated, nor can they be inactivated. Non-pathogenic, with good stability. (3) Subunit vaccines can be expressed using prokaryotic expression systems such as Escherichia coli and yeast. Therefore, the subunit vaccine has a high yield, is easy to purify, and is convenient for industrial production. In the field of Echinococcus multilocularis vaccine research, subunit vaccines also have great advantages compared with other vaccines. For immune diseases, it has higher immune specificity and safety.

The search for Echinococcus multilocularis antigens has always been one of the current research hotspots. At present, the research on echinococcosis mainly focuses on the diagnostic antigens of Echinococcosis multilocularis, and there are few researches on the antigens for preventing and inhibiting Echinococcus multilocularis infection; In addition, the immune escape mechanism of the host infected with Echinococcus multilocularis is complicated, and there is no immunotherapy method for Echinococcus multilocularis. The study found that Emy162 has a strong immune effect, and is stably expressed in all stages of the life cycle of Echinococcus (protoscole, cultured Echinococcus, immature adults and mature adults), and is involved in the adhesion and movement of Echinococcus. important role in physiology.

In this project, the surface antigen Emy162 of Echinococcus multilocularis is genetically fused with the mucosal immune adjuvant cholera toxin B subunit, expressed and purified in a prokaryotic system to obtain the subunit vaccine CTB-Emy162, which is used to immunize BALB/C mice. Cell proliferation, specific ELISA and other methods were used to confirm the immunogenicity and immune specificity of the subunit vaccine.

The idea of the present invention is as follows: according to the immune protection mechanism of the body against Echinococcus multilocularis, the surface antigen Emy-162 of Echinococcus multilocularis with good immunogenicity is screened out, and molecular biology technology and mucosal immune adjuvant cholera Toxin B subunit (CTB) was coupled to design a scientifically rational and structurally novel multilocular Echinococcus subunit vaccine CTB-Emy162. The immunogenicity and immunospecificity of multilocular Echinococcus subunit vaccine CTB-Emy162 were detected by mouse splenic lymphocyte proliferation, ELISA and other immunological methods. Studies have shown that Echinococcus multilocularis subunit vaccine CTB-Emy162 can stimulate the T cell and B cell immune responses produced by BALB/c mice and effectively inhibit the high-titer specific antibodies of Echinococcus multilocularis activity, which has a good effect. Immunogenicity and immunospecificity.

Contents of the invention

The first aspect of the present invention is to provide a novel subunit vaccine of Echinococcus multilocularis.

The second aspect of the present invention provides a preparation method of the subunit vaccine of Echinococcus multilocularis.

The third aspect of the present invention discloses the use of the subunit vaccine of Echinococcus multilocularis.

The first aspect of the present invention is to provide a subunit vaccine against Echinococcus multilocularis CTB-Emy162. The Echinococcus multilocularis subunit vaccine is mainly composed of Emy162 and cholera toxin B subunit. The overall amino acid sequence of the Echinococcus multilocularis subunit vaccine CTB-Emy162 is shown in (Sequence 1), and the amino acid sequence of the surface antigen Emy162 is shown in (Sequence 3) shown.

The novel Echinococcus multilocularis subunit vaccine CTB-Emy162 of the present invention has the following advantages: (1) Emy162 is the surface antigen of Echinococcus multilocularis, is recognized as an ideal candidate antigen for Echinococcus multilocularis vaccine and can obtain good results Protective effects. Echinococcus multilocularis subunit vaccine CTB-Emy162 can induce T cell and B cell immune response and specific antibody humoral immune response. (3) The molecular weight of the Emy162 epitope peptide is very small, and the immunogenicity is very low. The Echinococcus multilocularis subunit vaccine CTB-Emy162 of the present invention adopts the design idea of "coupling intramolecular immune adjuvant" to enhance the surface antigen Immunogenicity of Emy162, thereby inducing high-titer specific antibody production.

The second aspect of the present invention is to provide the preparation method of multilocular hydatid subunit vaccine, and its technical route is described in detail as follows:

(1) Structural design of the novel multilocular Echinococcus subunit vaccine antigen molecule

According to the immune protection mechanism of the body against Echinococcus multilocularis, the surface antigen Emy-162 of Echinococcus multilocularis with good immunogenicity was reasonably selected and coupled with the mucosal immune adjuvant cholera toxin B subunit (CTB) , designed a scientifically rational and novel structure of multilocular echinococcosis subunit vaccine CTB-Emy162.

(2) Construction of recombinant expression plasmid pET28a-CTB-Emy162 (containing fusion gene CTB-Emy162)

Firstly, a recombinant expression vector pET-CTB containing the cholera toxin B subunit (CTB) gene was constructed; then a nucleotide sequence of the surface antigen Emy162 of Echinococcus multilocularis was synthesized by gene synthesis technology, and inserted into the recombinant expression vector pET - In CTB, construct the recombinant expression vector pET28a-CTB-Emy162.

(3) Prokaryotic expression and purification of fusion protein CTB-Emy162

The recombinant expression vector pET28a-CTB-Emy162 was transformed into Escherichia coli BL21(DE3), and the recombinant genetic engineering strain BL21(DE3)/pET28a-CTB-Emy162 was constructed. The expression was induced by IPTG and high-purity fusion protein CTB-Emy162 was obtained by Ni-NTA nickel ion affinity chromatography, which is the subunit vaccine of Echinococcus multilocularis.

The third aspect of the present invention provides the application of the subunit vaccine of Echinococcus multilocularis. Echinococcus multilocularis subunit vaccine CTB-Emy162 can be used to prevent and treat diseases related to Echinococcus multilocularis infection.

Description of drawings

Figure 1: Molecular structural design features of the novel multilocular echinococcosis subunit vaccine.

Figure 2: The PCR result of Emy162 gene.

Lane 1: DNA Marker; Lane 2 is the result of Emy162 DNA fragment after PCR

Figure 3: Double enzyme digestion identification of the recombinant expression vector pET28a-CTB-Emy162.

Lane 1: DNA Marker; Lane 2: pET28a-CTB-Emy162 plasmid digested with Nco I and Xho I

Figure 4: Plasmid map of the recombinant expression vector pET28a-CTB-Emy162.

The fusion gene CTB-Emy162 inserted between Nco I and Xho I

Figure 5: Prokaryotic expression of Echinococcus multilocularis fusion protein CTB-Emy162.

Lane 1: BL21(DE3)/ pET-28a whole protein; Lane 2: Protein Marker; Lane 3: BL21(DE3)/pET-28a-CTB-Emy162 whole protein; Lane 4: BL21(DE3)/ pET- 28a-CTB-Emy162 soluble protein; Lane 5: BL21(DE3)/CTB-Emy162 inclusion body protein.

Figure 6: Ni-NTA affinity chromatography purification of Echinococcus multilocularis fusion protein CTB-Emy162.

Lane 1: CTB-Emy162 protein sample before purification; Lane 2: CTB-Emy162 protein sample after Ni-NTA purification.

Figure 7: Detection of anti-CTB-Emy162 and E _7-13 , E _129-129 antibodies induced by multilocular Echinococcus subunit vaccine CTB-Emy162.

Echinococcus multilocularis subunit vaccine CTB-Emy162 can induce higher titer antibodies against CTB-Emy162 and E _7-13 , E _{129-129 epitopes} of Emy162.

Figure 8: Antibody titer detection of CTB-Emy162 subunit vaccine against Echinococcus multilocularis.

Figure 9: The proliferative response of spleen lymphocytes of mice sensitized with multilocular Echinococcus subunit vaccine CTB-Emy162 to antigen stimulation.

Figure 10: Detection of anti-Echinococcus multilocularis antibody types induced by subunit vaccine CTB-Emy162 of Echinococcus multilocularis.

detailed description

Material

1. IPTG solution: Weigh 1.2g of IPTG into a 50ml centrifuge tube, add 40ml of sterile water, mix well and dissolve, then dilute to 50ml. Sterilize by filtration with a 0.22 μm filter, aliquot in small portions, and store at -20°C.

2. Kana penicillin (Kana) stock solution (1g/mL): Weigh 1g of Kana penicillin (Kana) and dissolve it in 1mL sterile water to prepare a stock solution with a concentration of 1g/mL, and filter it through a 0.22µm bacterial filter Bacteria, the solution was stored in a -20°C refrigerator.

3. Medium: (1) LB liquid medium: Weigh 10g of tryptone, 5g of yeast extract and 5g of NaCl, add distilled water to 1000ml, adjust the pH to 7.4, and sterilize by autoclaving. (2) LB solid medium: 1.5g agar powder/100ml LB culture medium, after autoclaving, pour the plate.

4. DNA electrophoresis buffer (50 x TAE): Weigh 242g Tris, 37.2g Na ₂ EDTA·2H ₂ O and 57.1ml glacial acetic acid, add water to 1000ml, and dilute 50 times before use.

5. SDS-PAGE electrophoresis buffer (5X): Weigh 15.1g of Tris powder, 94g of glycine, and 5.0g of SDS; add about 800ml of deionized water, stir to dissolve; add deionized water to make up to 1L, and store at room temperature; note : When adding water, let the water flow down the wall slowly to avoid a lot of foam due to SDS.

6. Coomassie Brilliant Blue R-250 dye solution: 0.25g Coomassie Brilliant Blue R-250 was dissolved in 100ml decolorization solution. (3) Fixative: Dilute 500ml of ethanol and 100ml of glacial acetic acid to 1000ml with distilled water. (4) Decolorization solution: Dilute 250ml of ethanol and 80ml of glacial acetic acid to 1000ml with distilled water. (5) Preservation solution: 25ml 87% glycerin dissolved in 225ml decolorization solution.

9. Experimental animals: (2) BALB/c mice: SPF grade, male, 6-7 weeks old, purchased from Beijing, license number: SCXK (Beijing) 2012-0001.

12. Main reagents for lymphocyte proliferation experiment (1) Mouse spleen lymphocyte separation medium (Tianjin Haoyang Biotechnology Co., Ltd.) (2) RPMI-1640 complete culture medium: add 10% fetal bovine serum to RPMI-1640 basic culture medium , 100U/ml penicillin, 100μg/ml streptomycin.

Example 1: Molecular structure design of multilocular Echinococcus subunit vaccine CTB-Emy162

On the basis of obtaining the amino acid sequence of Emy162, the intramolecular mucosal immune adjuvant CTB was added on this basis, and a fusion protein of Echinococcus multilocularis with a scientific and reasonable structure was designed to effectively improve the mucosal immune response in vivo.

Results: The molecular structure design features and ideas of the multilocular hydatid subunit vaccine CTB-Emy162 are shown in (Figure 1).

Example 2: Construction of recombinant expression vector (containing fusion gene CTB-Emy162)

(1) Gene synthesis of the nucleotide sequence of the surface antigen Emy162 of Echinococcus multilocularis

The amino acid sequence of Emy162 obtained earlier was transformed into the corresponding nucleotide sequence according to the codon preference principle of Escherichia coli, and Nanjing GenScript Biotechnology Co., Ltd. was entrusted to carry out the gene synthesis. The synthesized Emy162 was subjected to PCR, and the reaction system was as follows:

Emy162 (0.5mg/mL) 1 µL Emy162 primer 1 µL Mix (0.1mg/mL) 10µL ddH ₂ O (RNase-free) 8 µL Total 20 µL

The PCR reaction conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 sec, annealing at 72°C for 30 sec, extension at 72°C for 40 sec, a total of 30 cycles, and a final extension at 72°C for 10 min. Then use 1% agarose gel electrophoresis to observe the electrophoresis results (Figure 2). The PCR-amplified Emy162 gene was recovered and purified using the DNA Fragment Gel Recovery and Purification Kit (Beijing Tiangen Biotechnology Co., Ltd.) for use in subsequent experiments.

(2) Construction of the recombinant expression vector pET28a-CTB (containing the choleraxin B subunit gene)

The recombinant plasmid pET-CTB-UE (gifted by Professor Guo Le) was extracted by the plasmid mini-extraction kit (Tiangen), and double-digested with Kpn I/ Xho I respectively. The double-digestion reaction system is as follows:

pET-CTB-UE 24 µL 0.1% BSA 4 µL Kpn I (15U/µL) 2µL Xho I (15U/µL) 2µL K Buffer 4µL L Buffer 4µL Total 40 µL

Reaction for 2 h at 37°C, followed by electrophoresis on a 1% agarose gel. The multi-epitope peptide UE gene and pET28a-CTB expression vector double digestion products were recovered by using the agarose gel DNA recovery and purification kit respectively, and pET28a-CTB was used for subsequent experiments.

(3) Ligation of the surface antigen Emy162 gene of Echinococcus multilocularis and pET28a-CTB expression vector

The recovered pET28a-CTB linearized plasmid vector and the Emy162 gene recovered after PCR were ligated into a closed circular DNA molecule through complementary cohesive ends under the action of T4 DNA ligase (overnight at 4°C) to form a recombinant expression plasmid pET28a-CTB-Emy162. The T4 DNA ligase ligation system is as follows:

pET28a-CTB linearized vector 2 µL Emy162 gene 14 µL 10×T4 DNA Ligase Buffer 2 µL T4-DNA Ligase (3U/µL) 2 µL Total 20 µL

Results: The recombinant plasmid pET28a-CTB-Emy162 to be detected was digested with Nco I/ Xho I, reacted at 37°C for 2 hours, and detected by 1% agarose gel electrophoresis. The theoretical size of CTB-Emy162 is consistent (shown in Figure 3).

Send pET28a-CTB-Emy162 to Nanjing GenScript Biotechnology Co., Ltd., and use T7 promoter and terminator universal primers for gene sequencing, proving that the nucleotide sequence of the fusion gene CTB-Emy162 in pET28a-CTB-Emy162 is completely correct, and No frameshift mutations. The plasmid map of the recombinant expression vector pET28a-CTB-Emy162 is shown in (Figure 4).

Example 3 Prokaryotic expression of fusion protein CTB-Emy162 of Echinococcus multilocularis

The 100% successful recombinant expression plasmid pET28a-CTB-Emy162 was transformed into E.coli BL21(DE3) strain. On the pre-prepared LB plate containing 50ug/mL Kana, inoculate the loop-streaked genetically engineered strain pET28a-CTB-Emy162/BL21(DE3), place it upside down in a 37°C incubator, and culture it overnight for 12-16 hours, then pick a single Colonies were inoculated in LB liquid medium containing 50µg/mL Kana, cultured at 37°C, 190rpm, for 6-8h. Inoculate recombinant bacteria with 1% inoculum in LB liquid medium containing 50 µg/mL Kana, shake overnight at 37°C and 180 rpm, and inoculate the bacterial solution at 1% inoculum in LB containing 50 µg/mL Kana the next morning In liquid medium, after shaking the flask at 37°C and 190rpm for 9h, add IPTG to make the final concentration reach 1mmol/L, induce expression at 28°C and 190rpm, and use the empty vector strain pET28a/BL21(DE3) as a negative control.

Results: The genetically engineered recombinant strain pET28a-CTB-Emy162/BL21(DE3) was induced and expressed at pH 7, IPTG concentration 1 mmol/L, induction temperature 28°C, and induction time 9 hours. Compared with the control strain, the genetically engineered recombinant strain pET28a-CTB-Emy162/BL21(DE3) had the target protein band at about 33KD, which was consistent with the theoretical size of the recombinant fusion protein CTB-Emy162 of Echinococcus multilocularis (Figure 5) . The recombinant fusion protein CTB-Emy162 of Echinococcus multilocularis exists in the inclusion body protein.

Example 4: Purification of multi-epitope peptide fusion protein CTB-EMY162 from Echinococcus multilocularis

(1) Purification of Ni-NTA nickel ion affinity chromatography column

Mix the Ni-NTA filler thoroughly and add it to the chromatography column. The solution can flow out slowly by gravity. After it is completely added, put the upper sieve plate into the column; add an appropriate amount of deionized water to the packed column After washing the ethanol, add 8 times the column volume of Binding buffer to balance. After the balance is completed, the sample can be loaded; after collecting the bacteria, add 1-5ml Binding Buffer per 100mg of bacteria, and ultrasonically lyse the bacteria in an ice bath; centrifuge at 12000rpm, Discard the supernatant, resuspend the pellet in BindingBuffer, and perform ultrasonic treatment until the inclusion bodies are cleaned (in a relatively clean milky white shape); resuspend the pellet in Binding Buffer containing 8M urea, and bathe on ice for 1 hour to make the inclusion bodies Dissolve; load the cell lysate onto the column at a flow rate of 10 times the column volume/hour; wash the column with 15 times the column volume of Wash buffer and collect the flow-through peak; use 5 times the column volume of Elution Buffer to elute and collect the elution Peak; wash with 3 column volumes of Binding Buffer and 5 column volumes of deionized water in sequence, equilibrate with 3 column volumes of 20% ethanol, and store at 4°C.

Results: After purification by Ni-NTA nickel ion affinity chromatography column, the collected protein peaks were analyzed by SDS-PAGE. It can be found that the target protein is concentrated in the protein peaks eluted by 50mM imidazole and 250mM imidazole. The purity of the target protein eluted at 250 mM imidazole by gel imaging detector was above 85% (Figure 6).

Example 5: Study on immunogenicity and immune specificity of multi-epitope vaccine CTB-EMY162 of Echinococcus multilocularis

(1) Immunization of BALB/c mice

Experimental grouping: SPF grade BALB/c mice were randomly divided into two groups, namely the CTB-Emy162 immunization group and the PBS immunization group. There are 12 BALB/c mice in each group, 24 in total, and the detailed grouping is shown in the figure below:

Table 1: Grouping and immunization scheme of SPF BALB/c mice

test group number way of immunization Number of immunizations immune dose immune adjuvant CTB-Emy162 12 intraperitoneal injection 4 times 100μg/piece Freund's adjuvant PBS 12 intraperitoneal injection 4 times 100μg/piece Freund's adjuvant

Immunization method: Disinfect the abdomen of mice with 75% alcohol, and then intraperitoneally inject the mixed emulsifier of recombinant protein and Freund's complete adjuvant; boost immunization once every other week, add Freund's incomplete adjuvant in the 2nd and 3rd weeks, and add Freund's incomplete adjuvant in the second week Immunization was boosted by direct injection of recombinant protein solution at 4 weeks.

Collection of antiserum: On the fifth day after the last immunization, the mice’s eyeballs were picked for blood collection, and the blood was collected until the serum was completely separated, then centrifuged at 3000rpm for 5 minutes to pack the serum, and stored at -80°C for later use.

(2) ELISA detection of specific antibodies in antiserum

Antigen Emy162, Emy162-specific epitope small peptides (E _7-13 , E _129-139 ), and PBS were diluted to 5 μg/ml with coating solution, 100 μl/well was coated on an ELISA plate, and left overnight at 4°C. After washing 4 times with washing solution, add 300 μl of blocking solution to each well, and block at 37°C for 2 hours. After washing 4 times with washing solution, the antiserum (mouse anti-CTB-Emy162 antiserum, and mouse anti-CTB antiserum) and mouse negative serum were diluted and added to the ELISA plate, 100μl/well, incubated at 37°C for 60min . After washing 4 times with washing solution, add HRP-labeled goat anti-mouse IgG (1:2000), 100 μl/well, and incubate at 37°C for 1 hour. After washing 4 times with washing solution, add TMB substrate chromogenic solution, react at room temperature in the dark for 15 minutes, and add 50 μl stop solution to terminate the reaction. The OD ₄₅₀ value of each well was measured with a microplate reader. The titer of the sample is defined as the dilution corresponding to the well whose OD value is greater than 2.1 times of the set negative control OD value.

Results: The coating concentration of Emy162 was 5 μg/ml. Through ELISA detection, CTB-EMY162 subunit vaccine could produce IgG antibody against Emy162 antigen, while PBS immunization group could not produce IgG antibody against Emy162 antigen; E _7-13 and E The coating concentration of _129-139 was 5 μg/ml. As detected by ELISA, the CTB-EMY162 subunit vaccine could produce IgG antibodies against Emy162-specific epitope peptides E _7-13 and E _129-139 , while PBS could not produce IgG antibodies against Emy162 IgG antibodies to specific epitope peptides E _7-13 and E _129-139 (Figure 7, 8). The above results indicate that the subunit vaccine CTB-Emy162 can stimulate the body to produce high-titer specific antibodies against Emy162, and has good immunogenicity.

(3) Mouse spleen lymphocyte proliferation experiment

The mice immunized with the antigen were killed by dislocation, soaked in 75% alcohol for 5 minutes, and then moved into the ultra-clean workbench. Carefully cut the abdominal skin of the mouse with scissors, then cut the abdominal cavity of the mouse, and remove the mouse spleen (dark red) with forceps. Put 4~5 ml of EZ-Sep™ Mouse 1X Lymphocyte Separation Solution in a 20 ml sterile beaker; fix the nylon mesh with tweezers, then gently grind the mouse spleen with a syringe plunger to allow the dispersed single cells to permeate Nylon net into the lymphocyte separation medium; immediately transfer the separation medium with spleen cells suspended to a centrifuge tube, and cover with about 200 μl of 1640 medium before centrifugation; centrifuge at 800 g for 30 min. After centrifugation, lymphocytes will float up and collect under the 1640 overlay. Prepare a certain concentration of cell suspension (5 x 10 ⁴ /ml) with RPMI-1640 medium containing 10% calf serum. In a 96-well flat-bottom culture plate, add 100 μl cells to each well, and add CTB-Emy162, E _7-13 , E _129-139 , PBS (20 μg/ml) at the same time, the final volume is 200 μl/well, and do 3 parallels for each group hole. Place the added 96-well flat-bottomed culture plate in a 37°C 5% CO ₂ incubator for 48 hours, then add 30 μl of MTT solution (5 mg/ml) to each well, and continue to cultivate for 4 hours, then gently suck out the supernatant, each well Add 100 μl of DMSO, shake and mix well, and read the OD ₅₇₀ value with a microplate reader.

Results: Spleen lymphocytes of mice sensitized by CTB-Emy162, stimulated by CTB-Emy162, E _7-13 , and E _129-139 , could all undergo obvious lymphocyte proliferation, while The spleen lymphocytes of mice in the PBS immunized group did not undergo lymphocyte proliferation when stimulated by the above antigens, indicating that the Echinococcus multilocularis subunit vaccine CTB-Emy162 maintains its immunological properties and can stimulate the body to produce specific cellular immune responses (Figure 9).

(4) ELISA detection of specific antibody typing in antiserum

Antigen Emy162 and PBS were diluted to 5 μg/ml with coating solution, and 100 μl/well was coated on an ELISA plate, overnight at 4°C. After washing 4 times with washing solution, add 300 μl of blocking solution to each well, and block at 37°C for 2 hours. After washing 4 times with washing solution, the antiserum and mouse negative serum were serially diluted and added to the ELISA plate, 100 μl/well, and incubated at 37°C for 60min. After washing 4 times with washing solution, add HRP-labeled goat anti-mouse IgA, IgG1, IgG2a, dilute with antibody diluent, dilution ratio (1:2000), 100 μl/well, incubate at 37°C for 1h. After washing 4 times with washing solution, add TMB substrate chromogenic solution, react at room temperature in the dark for 15 minutes, and add 50 μl stop solution to terminate the reaction. The OD ₄₅₀ value of each well was measured with a microplate reader.

Results: As shown in (Figure 10), compared with the control group immunized with PBS, Echinococcus multilocularis subunit vaccine CTB-EMY162 could produce IgG1, IgGA and IgG2a antibodies against Emy162.

Claims (9)

1. A multilocular hydatid subunit vaccine, the active ingredient of which is a polypeptide, mainly composed of cholera toxin B subunit (CTB) and hydatid surface antigen Emy162, and its amino acid sequence is shown in sequence 1. 2. As claimed in claim 1, a subunit vaccine of Echinococcus multilocularis CTB-Emy162, the nucleotide sequence of which is shown in Sequence 2. 3. The amino acid sequence of Emy162, a surface antigen of Echinococcus multilocularis, is shown in Sequence 3. 4. As claimed in claim 3, the nucleotide sequence of the surface antigen Emy162 of Echinococcus multilocularis is shown in Sequence 4. 5. An expression vector comprising the nucleotide sequence described in claims 2 and 4, a transgenic cell line and a host bacterium. 6. As claimed in claim 1, the spacer sequence between cholera toxin B subunit (CTB) and Echinococcus multilocularis surface antigen Emy162 is DPRVPSS. 7. A method for preparing a subunit vaccine of Echinococcus multilocularis, by synthesizing the nucleotide sequence of the surface antigen Emy162 of Echinococcus multilocularis by gene synthesis technology, and fusing it downstream of the cholera toxin B subunit (CTB) gene , construct the recombinant expression vector pET28a-CTB-Emy162 containing the fusion gene CTB-Emy162 and its recombinant genetically engineered bacterium BL-21 (DE3), After the recombinant gene engineering strain is fermented, it is purified by Ni-NTA nickel ion exchange chromatography to obtain the fusion protein CTB-Emy162 of the vaccine. 8. The subunit vaccine of Echinococcus multilocularis according to claims 1 and 6, characterized in that it can stimulate the body to produce an immune response against Echinococcus multilocularis T cells and effectively inhibit the high-titer Echinococcus multilocularis subunit vaccine. Sexual antibodies. 9. The subunit vaccine of Echinococcus multilocularis according to claims 1 and 7, characterized in that it can be used as a drug combination for prevention and treatment of Echinococcus multilocularis infection.