A subunit vaccine against Echinococcus suis from Canada, its preparation method and application

Abstract

This invention discloses a subunit vaccine against Echinococcus cantonensis infection and its preparation method; it comprises an immunogen EC95 antigen protein and a pharmaceutically acceptable carrier; wherein the amino acid sequence of the dEC95 protein is as shown in SEQ ID No. 1 or its degenerate sequence. Currently, there is no vaccine against Echinococcus cantonensis infection. This invention uses the dEC95 protein as the antigen, and the prepared subunit vaccine against Echinococcus cantonensis infection has low production cost, simple production process, and many advantages such as safety, high efficiency, and low cost; even when the dEC95 protein antigen content in the anti-Echinococcus cantonensis subunit vaccine is only 40 μg / ml, an antibody titer of over 1:128 can be achieved at week 4 after secondary immunization, and a high antibody titer can be maintained for a long period of time.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and to a subunit vaccine against Echinococcus granulosus infection and its preparation method, particularly to a subunit vaccine against Echinococcus cantonensis infection and its preparation method. Background Technology

[0002] Echinococcosis is a serious zoonotic parasitic disease caused by the hydatid cyst, the larva of the tapeworm genus Echinococcus, which parasitizes the lungs, liver, and other organs of humans and animals. Echinococcosis is widespread and distributed globally. The World Organization for Animal Health (OIE) classifies it as a globally reported infectious disease and belongs to the category of multiple zoonotic diseases. The World Health Organization (WHO) lists it as one of the priority diseases for prediction and emergency response in its global early warning system. Echinococcosis is also one of the five major parasitic diseases planned for prevention and control by the Ministry of Health of China.

[0003] Based on the morphology of the lesions and the differences in infecting pathogens, echinococcosis is mainly divided into two types: cysticercosis (CE) and multilocular echinococcosis. Among them, cysticercosis is the most widespread and affects the largest number of people. The pathogens of CE are currently composed of several Echinococcus tapeworm complexes: Echinococcus granu Losus, Echinococcus canadensis, Echinococcus equi, and Echinococcus ostei. Among them, CE caused by Echinococcus granu Losus G1 type accounts for more than 90%, while CE caused by Echinococcus canadensis G6 type accounts for more than 7%.

[0004] Current research indicates that controlling the prevalence of echinococcosis primarily involves interrupting the developmental stages of Echinococcus granulosus, controlling infection of intermediate hosts such as humans and animals, preventing or treating definitive hosts like dogs, and blocking the widespread dissemination of eggs. Vaccination of intermediate hosts is particularly effective in controlling the prevalence of Echinococcus granulosus. Lightowlers et al. discovered that EG95 is a 24.5 kDa natural oncocytozonal antigen found in Eg, with a full-length 715 bp encoding gene. A 462 bp gene encodes a 16.5 kDa protein containing 153 amino acids, and this protein is the most effective protective antigen among the many proteins screened. A vaccine against sheep Echinococcus granulosus has been successfully developed. However, existing vaccines against sheep Echinococcus granulosus do not offer significant protection against Echinococcus cantonensis G6. Summary of the Invention

[0005] In view of the deficiencies in the existing technology, the purpose of this invention is to provide an anti-Echinococcus canis subunit vaccine and its preparation method, which can prevent infection by Echinococcus canis and has many advantages such as safety, high efficiency, and low cost. The anti-Echinococcus canis subunit vaccine contains the EC95 antigen protein of the immunogen and a pharmaceutically acceptable carrier.

[0006] As one embodiment of the present invention, the amino acid sequence of the dEC95 protein in the anti-Echinococcus cantonensis subunit vaccine of the present invention is shown in SEQ ID No. 1.

[0007] As one embodiment of the present invention, in the anti-Echinococcus cantonensis subunit vaccine of the present invention, the dEC95 protein can be a monomer or a single-chain polymer composed of polypeptides linked together, for example, dimer 2dEC95, trimer 3dEC95, tetramer 4dEC95, and pentamer 5dEC95.

[0008] The anti-Echinococcus canis subunit vaccine of the present invention can achieve an antibody titer of not less than 1:1024 in the fourth week after secondary immunization, and can maintain a high antibody titer for a long period of time.

[0009] As one embodiment of the present invention, the dEC95 antigen protein content in the anti-Echinococcus cantonensis subunit vaccine of the present invention is 20-100 μg / ml.

[0010] In the anti-Echinococcus dacna subunit vaccine, the dEC95 antigen protein content can be selected from 20μg / ml, 30μg / ml, 40μg / ml, 50μg / ml, 60μg / ml, 70μg / ml, 80μg / ml, 90μg / ml, and 100μg / ml.

[0011] Even when the dEC95 protein antigen content in the anti-Echinococcus davidian subunit vaccine is only 40 μg / ml, it can still achieve an antibody titer of no less than 1:128 by the fourth week after secondary immunization, and can maintain a high antibody titer for a long period of time.

[0012] In a preferred embodiment of the present invention, the EC95 protein antigen content in the anti-Echinococcus davidian subunit vaccine of the present invention is 60 μg / ml.

[0013] As one embodiment of the present invention, in the anti-Echinococcus cantonensis subunit vaccine of the present invention, the pharmaceutically acceptable carrier includes an adjuvant, the adjuvant including: (1) white oil, aluminum glue adjuvant, saponin, avrididine, DDA; (2) oil-in-water emulsion, water-in-oil emulsion, water-in-oil-in-water emulsion; or (3) polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives; and one or more of the following: RIBI adjuvant system, Blockco-polymer, SAF-M, monophospholipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-sensitive enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, Montanide ISA 206, Gel adjuvant; preferably, Montanide ISA 50V.

[0014] The adjuvant content is 5%-60% V / V, preferably 20%-60% V / V, and more preferably 50% V / V.

[0015] As one embodiment of the present invention, the pharmaceutically acceptable carrier includes drugs, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives; the immunostimulants include α-interferon, β-interferon, γ-interferon, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin-2 (IL2).

[0016] To prepare such a composition, methods known in the art can be used.

[0017] The present invention also relates to a method for preparing the anti-Echinococcus davidian subunit vaccine, wherein the method comprises: step (1) amplifying and cloning the gene of the 2dEC95 antigen protein of Echinococcus davidianus (as shown in SEQ ID NO.6) into an expression vector to obtain a recombinant expression vector containing the Echinococcus davidianus 2dEC95 antigen protein gene; step (2) transforming or transducing the recombinant expression vector containing the Echinococcus davidianus 2dEC95 antigen protein gene obtained in step (1) into a host to obtain a recombinant containing the recombinant expression vector; step (3) culturing the recombinant obtained in step (2) to express the Echinococcus davidianus 2dEC95 antigen protein; and step (4) purifying the Echinococcus davidianus 2dEC95 antigen protein obtained in step (3), adding an adjuvant, and obtaining the anti-Echinococcus davidianus subunit vaccine.

[0018] In one embodiment of the present invention, the amino acid sequence of the Echinococcus canis dEC95 antigen protein in step (1) is as shown in SEQ ID No. 1 or its degenerate sequence; the host in step (2) is E. coli. The expressed Echinococcus canis 2dEC95 antigen protein in step (3) is an intracellular soluble protein.

[0019] The present invention also relates to the use of the described anti-Echinococcus cantonensis subunit vaccine in the preparation of medicaments for the prevention and / or treatment of Echinococcus cantonensis infection.

[0020] The drugs for the prevention and / or treatment of Echinococcus cantonensis infection as described in this invention are intended for use on sheep, cattle, and camels.

[0021] In this invention, Escherichia coli 24a-2dEC95 was deposited with the China Center for Type Culture Collection on June 23, 2021, at Wuhan University, Wuhan, China, with accession number CCTCC NO:M 2021749.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1) Currently, there is no vaccine against Echinococcus canis infection. This invention uses 2dEC95 protein as antigen to prepare a subunit vaccine against Echinococcus canis infection. The production cost is low, the production process is simple, and it has many advantages such as safety, high efficiency and low cost.

[0024] 2) In this invention, even when the dEC95 protein antigen content in the anti-Echinococcus davidian subunit vaccine is only 40 μg / ml, the antibody titer can reach 1:128 or higher in the 4th week after secondary immunization, and can maintain a high antibody titer for a long period of time. Attached Figure Description

[0025] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0026] Figure 1 : Protein purification results; where lane M: protein standard molecular weight; lane 1: protein sample P-24a-2dEC95; lane 2: protein sample P-24a-2dEG95; lane 3: protein sample P-MAL-2dEC95;

[0027] Figure 2 Competitive ELISA test results. Detailed Implementation

[0028] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0029] Example 1: Construction of Recombinant Vector

[0030] 1.1 Gene Sequence Synthesis. Using *E. coli* as the host bacterium, the codons encoding the recombinant proteins 2dEG95, 2dEC95, and 2aEC95 were optimized in this invention. The optimized sequences were synthesized by Nanjing Genscript Biotech Co., Ltd. Specifically, the amino acids of dEG95 were linked using the "GGGSGGGS" formula to construct the single-stranded homodimer 2dEG95; the amino acids of dEC95 were linked using the "GGGSGGGS" formula to construct the single-stranded homodimer 2dEC95; and the amino acids of aEC95 were linked using the "GGGSGGGS" formula to construct the single-stranded homodimer 2aEC95. The amino acid sequence of 2dEC95 is shown in SEQ ID NO.2, and the amino acid sequence of 2aEC95 is shown in SEQ ID NO.7.

[0031] 1.2 Construction of the recombinant expression vector pET24a-2dEC95.

[0032] (1) Using 2dEC95 as a template, upstream primer Nde IF and downstream primer Xho IR were designed. PCR amplification yielded the gene fragment 2dEC95. The upstream primer had an Nde I restriction endonuclease site and a protective base introduced at its 5' end, with the Nde I site sequence being CATATG. The downstream primer had an Xho I restriction endonuclease site, a stop codon, and a protective base introduced at its 5' end, with the Xho I site sequence being CTCGAG. Primer sequences and PCR reaction procedures are shown in Tables 1 and 2.

[0033] Table 1: PCR primer names and sequences

[0034] Primer name sequence Nde IF 5'-GGTCCATATGCATCACCATCATCACCACCTG-3'SEQ ID NO.4 Xho IR 5'-CCGCTCGAGTTAGACGGTAGATTCTTTTTTTACCAGC-3'SEQ ID NO.5

[0035] Table 2: PCR reaction procedure

[0036]

[0037]

[0038] (2) The amplified gene fragment 2dEC95 was digested with restriction enzymes Nde I and Xho I. The digested gene fragment was recovered and ligated into the pET24a prokaryotic expression vector treated with the same restriction enzymes Nde I and Xho I. The ligation product was transformed into E. coli DH5α competent cells and plated on plates containing 100 μg / ml kanamycin sulfate. The plates were incubated at 37°C. When colonies were clearly visible, single colonies were picked and cultured in 3 ml of liquid medium containing 100 μg / ml kanamycin sulfate at 37°C. The plasmid was then extracted. The recombinant plasmid pET24a-2dEC95 was obtained. The recombinant plasmid was confirmed to be consistent with the target sequence by sequencing.

[0039] 1.3 Recombinant vectors pET24a-2dEG95, pMAL-2dEC95, and pET24a-2aEC95. Referring to the experimental steps in 1.2, (1) the gene sequence 2dEG95 was ligated into the vector pET24a with the restriction enzyme sites Nde I / Xho I to obtain the recombinant vector pET24a-2dEG95. The cloning template used was the 2dEG95 gene. The Nde IF sequence of the primers used is shown in SEQ ID No. 4, and the Xho IR sequence of the primers used is shown in SEQ ID No. 5; (2) the gene sequence 2dEC95 was ligated into the vector pMAL with the restriction enzyme sites Nde I / EcoRI to obtain the recombinant vector pMAL-2dEC95. The cloning template used was the 2dEC95 gene. The Nde IF sequence of the primers used is shown in SEQ ID No. 4, and the EcoRI-R sequence of the primers used is shown in SEQ ID No. 8; (3) the gene sequence 2aEC95 was ligated into the vector pET24a with the restriction enzyme sites Nde I / Xho I. I. The recombinant vector pET24a-2aEC95 was obtained, using the 2aEC95 gene as the cloning template. The Nde IF sequence of the primers used is shown in SEQ ID No. 4, and the aEC95-Xho IR sequence is shown in SEQ ID No. 9. The recombinant plasmid obtained was confirmed to be consistent with the target sequence by sequencing. The amino acid sequence of 2dEG95 is shown in SEQ ID No. 3, and the amino acid sequence of 2aEC95 is shown in SEQ ID No. 7.

[0040] Example 2: Construction of recombinant bacteria

[0041] 2.1 Construction of the original seed. The pET24a-2dEC95, pET24a-2dEG95, pMAL-2dEC95, and pET24a-2aEC95 cultures were transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing the corresponding antibiotics (50 μg / ml ampicillin or 100 μg / ml kanamycin sulfate), and incubated at 37°C. When colonies were clearly visible on the plates, single colonies were picked and placed in 3 ml of liquid medium containing the corresponding antibiotics (50 μg / ml ampicillin or 100 μg / ml kanamycin sulfate), and incubated at 37°C. One ml of the bacterial culture was then added to a final concentration of 8% glycerol and frozen at -80°C to obtain recombinant engineered bacteria 24a-2dEC95, 24a-2dEG95, MAL-2dEC95, and 24a-2aEC95, which served as the original seed bank.

[0042] 2.2 Identification of the original seeds.

[0043] (1) Morphology and biochemical characteristics: The engineered bacteria are Gram-negative short rods. They can ferment and decompose glucose, producing acid and gas; the indole test and methyl red test are both positive; the Voges-Proskauer test (VP test) and citrate test are both negative.

[0044] (2) Culture characteristics: When grown on LB solid medium, it forms smooth colonies that are round, with neat edges, raised, milky white and shiny.

[0045] (3) Purity test: The test was conducted according to the purity test method in the appendix of the Pharmacopoeia of the People's Republic of China, and the result was pure.

[0046] Example 3: Fermentation of recombinant bacteria

[0047] The strain was inoculated into 500 mL of LB medium containing the appropriate antibiotic (50 μg / mL ampicillin or 100 μg / mL kanamycin sulfate) and cultured with shaking at 37°C until OD500. 600 When the OD value is 1.2–1.5, inoculate the seed culture into a 5L fermenter at a 10% inoculation rate for fermentation culture. When the cell OD value reaches 1.2–1.5, the fermentation culture continues. 600 When the pH value reaches 20–25, lower the culture temperature to 28°C and add IPTG to a final concentration of 0.4 mM, inducing for 12–14 hours. Centrifuge to collect approximately 500 g of wet cell weight.

[0048] Resuspend the bacterial cells by adding 10 ml of resuspension buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl) per gram of wet bacterial cells.

[0049] Example 4: Purification of recombinant antigen

[0050] 4.1 The resuspended bacterial solution was broken up using a homogenizer at a pressure of 700 bar, and the process was repeated 4 times.

[0051] 4.2 Centrifuge 28000g of lysis buffer for 40 minutes and collect the supernatant.

[0052] 4.3 Affinity chromatography was used for purification, and the protein chromatography equipment was an AKTA pure 150m protein purifier.

[0053] (1) Regarding recombinant proteins P-24a-2dEC95, P-24a-2dEG95, and P-24a-2aEC95, the chromatography packing material used was Ni Sepharose 6FF, the equilibration buffer was 20 mM Tris-HCl pH 7.5 and 500 mM NaCl, the washing buffer was 30 mM imidazole, and the elution buffer was 500 mM imidazole;

[0054] (2) Regarding the recombinant protein P-MAL-2dEC95, the chromatography packing material used was Dextrin Sepharose HighPerformance, the equilibration buffer was 20mM Tris-HCl, 200mM NaCl, 1mM EDTA, 1mM DTT, pH 7.4, and the elution buffer was 20mM Tris-HCl, 200mM NaCl, 1mM EDTA, 1mM DTT, 10mM maltose, pH 7.4.

[0055] 4.4 Sterile Filtration. In a laminar flow hood, the samples were sterilized by filtering with a 0.22 μm sterile filter and then dispensed into sterile sample vials.

[0056] 4.5 SDS-PAGE electrophoresis analysis of protein purification status, such as... Figure 1 As shown, the results indicate that the target protein was completely bound to the chromatography column and eluted by the elution buffer. The purity of the target protein obtained by one-step purification was greater than 85%.

[0057] Example 5: Physicochemical Analysis of Recombinant Antigen

[0058] 5.1 Sterility test. The sterility test was performed according to the sterility test method in the appendix of the "Veterinary Pharmacopoeia of the People's Republic of China", and the result was sterile.

[0059] 5.2 Antigen protein concentration and purity detection. Gray-scale analysis was used, and samples were subjected to SDS-PAGE electrophoresis. The concentration and purity of each band in the samples were analyzed using a gel imaging system. The results are shown in Table 3.

[0060] Table 3: Protein Sample Purity and Concentration Results

[0061] sample purity(%) Concentration (mg / ml) P-24a-2dEG95 88.50 0.79 P-24a-2dEC95 85.30 0.74 P-MAL-2dEC95 89.60 1.84

[0062] 5.3 The 2017 edition of the "Veterinary Drug Quality Standards" requires an antigen percentage content of ≥15% for the "Echinococcus hydatid disease genetic engineering subunit vaccine". The antigen percentage contents of P-24a-2dEG95 and P-24a-2dEC95 measured by this invention are 5.90 times and 5.69 times the standard, respectively.

[0063] Example 6: Preparation of samples with different adjuvants

[0064] (1) Preparation of water-soluble complex adjuvant GEL vaccine samples.

[0065] The P-24a-2dEC95 antigen was diluted to 100 μg / ml with PBS and mixed with 20% (volume percentage) GEL adjuvant at a volume ratio of 1:1. The mixture was then incubated at 4°C for 12 h to obtain the vaccine sample V-24a-2dEC95-GEL.

[0066] (2) Preparation of water adjuvant QuilA vaccine samples.

[0067] The P-24a-2dEC95 antigen was diluted to 100 μg / ml with PBS and mixed with 1 mg / ml QuilA adjuvant at a volume ratio of 1:1. The mixture was then incubated at 4°C for 12 h to obtain the vaccine sample V-24a-2dEC95-QuilA.

[0068] (3) Preparation of Montanide ISA 50V vaccine samples with oil adjuvant.

[0069] The P-24a-2dEC95 antigen was diluted to 100 μg / ml with PBS and emulsified with Montanide ISA 50V adjuvant at a volume ratio of 1:1. After standing at 4°C for 12 h, the vaccine sample V-24a-2dEC95-50V was obtained.

[0070] Example 7: Immunization Experiment of Lambs with Different Adjuvant Vaccine Samples

[0071] (1) Select 32 negative lambs. The requirements for the lambs are shown in Table 4 below.

[0072] Table 4: Animal Selection Requirements

[0073]

[0074]

[0075] (2) The immunization procedure is shown in Table 5 below.

[0076] Table 5: Immunization Procedures

[0077]

[0078] (3) Antibody level detection. ELISA antibody detection was performed on the collected serum. The results showed that antibodies in all vaccine groups were significantly increased after the second immunization, indicating good immunogenicity. The antibody duration was at least 48 weeks after the second immunization. The oil adjuvant Montanide ISA50V vaccine group was significantly better than the other two vaccine groups, as shown in Tables 6, 7, 8 and 9 below.

[0079] Table 6: Results of V-24a-2dEC95-GEL Antibody Titer Detection

[0080]

[0081] Table 7: Results of V-24a-2dEC95-QuilA Antibody Titer Detection

[0082]

[0083] Table 8: Results of V-24a-2dEC95-50V Antibody Titer Detection

[0084]

[0085] Table 9: Results of PBS Antibody Titer Detection

[0086]

[0087]

[0088] Example 8: Optimization of Emulsification Conditions

[0089] Based on the following optimization experiments, the optimal emulsification conditions were finally selected as a shearing speed of 14,000 rpm, a shearing time of 10 min, and an oil-water ratio of 1:1.

[0090] (1) Optimize the shear force used in the emulsification process. 12000 rpm, 14000 rpm and 16000 rpm were used respectively. According to the uniformity and stability of the emulsified samples, the result was that 14000 rpm was better than 16000 rpm, which was better than 12000 rpm.

[0091] (2) Optimize emulsification time. Under appropriate shearing rates, shearing was performed for 8 min, 10 min, and 12 min respectively. Based on the uniformity and stability of the emulsified samples, the results showed that 10 min was better than 12 min, which was better than 8 min.

[0092] (3) Optimize the oil-water ratio. Oil-water ratios of 0.8:1, 1:1, and 1.2:1 were selected respectively. Based on the uniformity and stability of the emulsified samples, the results showed that 1:1 was superior to 1.2:1, which was superior to 0.8:1. Example 8: Preparation of recombinant vaccines with different antigen contents.

[0093] 8.1 Take an appropriate amount of adjuvant Montanide ISA 50V, autoclave at 121°C for 30 minutes, and set aside.

[0094] 8.2 Take the protein sample P-24a-2dEC95 purified in Example 5 and dilute it with sterile PBS to 80 μg / ml, 120 μg / ml and 160 μg / ml respectively, according to the measured concentration.

[0095] 8.3 Prepare an appropriate amount of adjuvant according to the ratio of oil:water = 1:1 (v:v), place it in a beaker, and immerse the shearing head in the adjuvant.

[0096] 8.4 Pre-emulsification. Begin shearing by slowly adding the aqueous phase to the adjuvant being sheared, ensuring thorough mixing.

[0097] 8.5 Emulsification. Shear at 14000 rpm for 10 minutes, moving the beaker along the stirring head to ensure uniform emulsification.

[0098] 8.6 Analysis. After the emulsion was prepared, it was left at room temperature overnight and then examined. 1 ml of the emulsion was taken and centrifuged at 3000 rpm for 30 min. No aqueous phase precipitated at the bottom, indicating good emulsification.

[0099] 8.7 Vaccine samples V-24a-2dEC95 (40 μg), V-24a-2dEC95 (60 μg), and V-24a-2dEC95 (80 μg) with antigen contents of 40 μg / ml, 60 μg / ml, and 80 μg / ml were prepared.

[0100] Example 9: Stability Analysis of Recombinant Vaccine

[0101] 9.1 The vaccine was placed in glass vials and stored at 4°C, 20°C and 37°C for stability studies.

[0102] 9.2 The criteria for determining the stability of the emulsion are: (1) the height of the precipitated layer of the sample is not greater than 5%; (2) it is kept in a sterile state; (3) after demulsification, the antigen content is not less than 80% compared with the original solution.

[0103] 9.3 The vaccine samples were tested and found to be stable after being stored at 4°C for 18 months, at 20°C for 3 months, and at 37°C for 1 month.

[0104] Example 10: Immunization experiment of lambs with vaccine samples of different antigen contents

[0105] (1) Select 32 negative lambs, and the requirements for the lambs are as shown in Table 10.

[0106] Table 10: Animal Screening Requirements

[0107]

[0108] (2) The immunization procedure is shown in Table 11 below.

[0109] Table 11: Immunization Procedures

[0110]

[0111] (3) Antibody level detection. ELISA antibody detection was performed on the collected serum. The results showed that antibodies in all vaccine groups were significantly increased after the second immunization, indicating good immunogenicity. The antibody duration was at least 48 weeks after the second immunization. The 60μg antigen content vaccine group was significantly better than the other two vaccine groups, as shown in Tables 12, 13, 14 and 15 below.

[0112] Table 12: Results of V-24a-2dEC95 (40μg) antibody titer assay

[0113]

[0114] Table 13: Results of V-24a-2dEC95 (60μg) antibody titer assay

[0115]

[0116] Table 14: Results of V-24a-2dEC95 (80μg) antibody titer assay

[0117]

[0118]

[0119] Table 15: Results of PBS Antibody Titer Detection

[0120]

[0121] Example 11: Preparation of recombinant vaccines with different antigens

[0122] 11.1 Take an appropriate amount of adjuvant Montanide ISA 50V, autoclave at 121 for 30 minutes, and set aside.

[0123] 11.2 Take the protein samples P-24a-2dEC95 and P-24a-2aEC95 purified in Example 4, and dilute them to 120 μg / ml with sterile PBS according to the measured concentration.

[0124] 11.3 Prepare an appropriate amount of adjuvant according to the ratio of oil:water = 1:1 (v:v), place it in a beaker, and immerse the shearing head in the adjuvant.

[0125] 11.4 Pre-emulsification. Begin shearing by slowly adding the aqueous phase to the adjuvant being sheared, ensuring thorough mixing.

[0126] 11.5 Emulsification. Shear at 14000 rpm for 10 minutes, moving the beaker along the stirring head to ensure uniform emulsification.

[0127] 11.6 Analysis. After the emulsion was prepared, it was left at room temperature overnight and then examined. 1 ml of the emulsion was taken and centrifuged at 3000 rpm for 30 min. No aqueous phase precipitated at the bottom, indicating good emulsification.

[0128] The vaccine samples prepared in 11.7 were V-24a-2dEC95 and V-24a-2aEC95, respectively.

[0129] Example 12: Immunization experiment of lambs with different antigen vaccine samples

[0130] (1) Select 24 negative lambs. The requirements for the lambs are shown in Table 16 below.

[0131] Table 16: Animal Screening Requirements

[0132]

[0133] (2) The immunization procedure is shown in Table 17 below.

[0134] Table 17: Immunization Procedures

[0135]

[0136] (3) Antibody level detection. ELISA antibody detection was performed on the collected serum. The results showed that, using the same 60ug antigen, the antibody levels in the vaccine group were significantly increased after the second immunization, indicating good immunogenicity, and the antibody duration was at least 48 weeks after the second immunization; the antibody titers in the V-24a-2dEC95 vaccine group at 48 weeks after the second immunization were all not lower than 1:512, while the antibody titers in the V-24a-2aEC95 vaccine group at 48 weeks after the second immunization were all not higher than 1:128, as shown in Tables 18, 19, and 20 below.

[0137] Table 18: Results of V-24a-2dEC95 Antibody Titer Detection

[0138]

[0139]

[0140] Table 19: Results of V-24a-2aEC95 Antibody Titer Detection

[0141]

[0142] Table 20: Results of PBS Antibody Titer Detection

[0143]

[0144] Example 13: Evaluation of cross-protective effect of antiserum

[0145] It has been reported that there is no cross-protective effect between Echinococcus granulosus G1 and Echinococcus cantonensis G6. This invention designs a competitive ELISA experiment, using antigen P-MAL-2dEC95 as the coating antigen. Anti-V-24a-2dEC95 serum is incubated with the following antigens: P-24a-2dEG95, P-24a-2dEC95, PP-MAL-2dEC95, and PBS. The anti-V-24a-2dEC95 serum is diluted 100 times.

[0146] Competitive ELISA test results, such as Figure 2 As shown in the figure. The results indicate that the EG95 antigen cannot block the binding of anti-EC95 serum to the EC95 antigen, indicating that the vaccine composition composed of Echinococcus granulosus EG95 antigen protein cannot provide complete protection against Echinococcus canis.

[0147] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention. sequence list <110> Shenlian Biopharmaceutical (Shanghai) Co., Ltd. <120> A subunit vaccine against Echinococcus suis from Canada, its preparation method and application <130> DD15189 <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 118 <212> PRT <213> Artificial Sequence <400> 1 Leu Ala Gln Glu Tyr Lys Gly Met Gly Ile Glu Thr Arg Thr Thr Glu 1 5 10 15 Thr Pro Leu Arg Lys His Phe Asn Leu Thr Leu Val Gly Ser Gln Gly 20 25 30 Ile Arg Leu Ser Trp Asp Val Gln His Leu Ser Asp Leu Lys Gly Thr 35 40 45 Asn Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys 50 55 60 Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu 65 70 75 80 Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys 85 90 95 Lys Thr Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro Ala Gly 100 105 110 Lys Lys Glu Ser Thr Val 115 <210> 2 <211> 244 <212> PRT <213> Artificial Sequence <400> 2 Leu Ala Gln Glu Tyr Lys Gly Met Gly Ile Glu Thr Arg Thr Thr Glu 1 5 10 15 Thr Pro Leu Arg Lys His Phe Asn Leu Thr Leu Val Gly Ser Gln Gly 20 25 30 Ile Arg Leu Ser Trp Asp Val Gln His Leu Ser Asp Leu Lys Gly Thr 35 40 45 Asn Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys 50 55 60 Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu 65 70 75 80 Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys 85 90 95 Lys Thr Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro Ala Gly 100 105 110 Lys Lys Glu Ser Thr Val Gly Gly Gly Ser Gly Gly Gly Ser Leu Ala 115 120 125 Gln Glu Tyr Lys Gly Met Gly Ile Glu Thr Arg Thr Thr Glu Thr Pro 130 135 140 Leu Arg Lys His Phe Asn Leu Thr Leu Val Gly Ser Gln Gly Ile Arg 145 150 155 160 Leu Ser Trp Asp Val Gln His Leu Ser Asp Leu Lys Gly Thr Asn Ile 165 170 175 Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys Arg Gln 180 185 190 Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu Lys Pro 195 200 205 Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys Lys Thr 210 215 220 Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro Ala Gly Lys Lys 225 230 235 240 Glu Ser Thr Val <210> 3 <211> 244 <212> PRT <213> Artificial Sequence <400> 3 Leu Ala Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr Thr Glu 1 5 10 15 Thr Pro Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser Gln Gly 20 25 30 Ile Arg Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys Gly Thr 35 40 45 Asp Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys 50 55 60 Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu 65 70 75 80 Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys 85 90 95 Lys Thr Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg Ala Gly 100 105 110 Lys Lys Glu Ser Thr Val Gly Gly Gly Ser Gly Gly Gly Ser Leu Ala 115 120 125 Gln Glu Tyr Lys Gly Met Gly Val Glu Thr Arg Thr Thr Glu Thr Pro 130 135 140 Leu Arg Lys His Phe Asn Leu Thr Pro Val Gly Ser Gln Gly Ile Arg 145 150 155 160 Leu Ser Trp Glu Val Gln His Leu Ser Asp Leu Lys Gly Thr Asp Ile 165 170 175 Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val Tyr Lys Arg Gln 180 185 190 Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly Glu Leu Lys Pro 195 200 205 Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys Ala Lys Lys Thr 210 215 220 Ile Leu Gly Phe Thr Val Asp Ile Glu Thr Pro Arg Ala Gly Lys Lys 225 230 235 240 Glu Ser Thr Val <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <400> 4 ggtccatatg catcaccatc atcaccacct g 31 <210> 5 <211> 36 <212> DNA <213> Artificial Sequence <400> 5 ccgctcgagt tagacggtag attctttttt accagc 36 <210> 6 <211> 756 <212> DNA <213> Artificial Sequence <400> 6 atgcatcacc atcatcacca cctggcacag gaatacaaag gtatgggtat tgaaacccgt 60 accaccgaaa ctccgctgcg taaacacttc aacctgaccc tggtcggcag ccagggtatc 120 cgtctgtctt gggatgttca gcatctgtct gatctgaaag gcaccaacat ctccctgaaa 180 gcagtgaacc cgtctgaccc gctggtctac aaacgtcaaa cggctaaatt ctccgatggt 240 cagctgacca ttggtgaact gaaaccgagc actctgtaca agatgactgt agaagccgtt 300 aaagccaaaa aaactatcct ggaattcact gtcgacattg aaactccgcc ggctggtaaa 360 aaagaatcta ccgtcggcgg cggtagcggc ggcggtagcc tggcacagga atacaaaggt 420 atgggtattg aaacccgtac caccgaaact ccgctgcgta aacacttcaa cctgaccctg 480 gtcggcagcc agggtatccg tctgtcttgg gatgttcagc atctgtctga tctgaaaggc 540 accaacatct ccctgaaagc agtgaacccg tctgacccgc tggtctacaa acgtcaaacg 600 gctaaattct ccgatggtca gctgaccatt ggtgaactga aaccgagcac tctgtacaag 660 atgactgtag aagccgttaa agccaaaaaa actatcctgg aattcactgt cgacattgaa 720 actccgccgg ctggtaaaaa agaatctacc gtctaa 756 <210> 7 <211> 304 <212> PRT <213> Artificial Sequence <400> 7 Leu Phe Ala Thr Ser Val Leu Ala Gln Glu Tyr Lys Gly Met Gly Ile 1 5 10 15 Glu Thr Arg Thr Thr Glu Thr Pro Leu Arg Lys His Phe Asn Leu Thr 20 25 30 Leu Val Gly Ser Gln Gly Ile Arg Leu Ser Trp Asp Val Gln His Leu 35 40 45 Ser Asp Leu Lys Gly Thr Asn Ile Ser Leu Lys Ala Val Asn Pro Ser 50 55 60 Asp Pro Leu Val Tyr Lys Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln 65 70 75 80 Leu Thr Ile Gly Glu Leu Lys Pro Ser Thr Leu Tyr Lys Met Thr Val 85 90 95 Glu Ala Val Lys Ala Lys Lys Thr Ile Leu Glu Phe Thr Val Asp Ile 100 105 110 Glu Thr Pro Pro Ala Gly Lys Lys Glu Ser Thr Val Met Thr Ser Gly 115 120 125 Ser Ala Leu Thr Ser Thr Ile Ala Gly Phe Val Phe Ser Cys Ile Val 130 135 140 Val Val Leu Thr Gly Gly Gly Ser Gly Gly Gly Ser Leu Phe Ala Thr 145 150 155 160 Ser Val Leu Ala Gln Glu Tyr Lys Gly Met Gly Ile Glu Thr Arg Thr 165 170 175 Thr Glu Thr Pro Leu Arg Lys His Phe Asn Leu Thr Leu Val Gly Ser 180 185 190 Gln Gly Ile Arg Leu Ser Trp Asp Val Gln His Leu Ser Asp Leu Lys 195 200 205 Gly Thr Asn Ile Ser Leu Lys Ala Val Asn Pro Ser Asp Pro Leu Val 210 215 220 Tyr Lys Arg Gln Thr Ala Lys Phe Ser Asp Gly Gln Leu Thr Ile Gly 225 230 235 240 Glu Leu Lys Pro Ser Thr Leu Tyr Lys Met Thr Val Glu Ala Val Lys 245 250 255 Ala Lys Lys Thr Ile Leu Glu Phe Thr Val Asp Ile Glu Thr Pro Pro 260 265 270 Ala Gly Lys Lys Glu Ser Thr Val Met Thr Ser Gly Ser Ala Leu Thr 275 280 285 Ser Thr Ile Ala Gly Phe Val Phe Ser Cys Ile Val Val Val Leu Thr 290 295 300 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <400> 8 ccggaattct tagacggtag attctttttt accagc 36 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <400> 9 ccgctcgagt taagtcagaa ctacaacgat gc 32

Claims

1. An anti-Canadian Echinococcus subunit vaccine, characterized in that, The dEC95 antigen protein containing an immunogen and a pharmaceutically acceptable carrier; wherein the dEC95 protein is a single-chain dimer 2dEC95 with an amino acid sequence as shown in SEQ ID NO.

2.

2. The anti-Canadian Echinococcus subunit vaccine according to claim 1, characterized in that, The pharmaceutically acceptable carrier comprises an adjuvant, which comprises: (1) white oil, aluminum adjuvant, saponin, Afuvidine, DDA; or (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion.

3. The anti-Canadian Echinococcus subunit vaccine according to claim 2, characterized in that, The water-in-oil-in-water emulsion comprises Montanide ISA 206; the water-in-oil emulsion comprises Montanide ISA 50V.

4. The anti-Canadian Echinococcus subunit vaccine according to claim 1, characterized in that, The pharmaceutically acceptable carrier comprises an adjuvant, which comprises one or more of polymers of acrylic or methacrylic acid, RIBI adjuvant system, Blockco-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, Gel adjuvant.

5. The anti-Canadian Echinococcus subunit vaccine according to claim 1, characterized in that, The pharmaceutically acceptable carrier comprises an adjuvant, which comprises a copolymer of maleic anhydride and alkenyl derivative.

6. The anti-Canadian Echinococcus subunit vaccine according to claim 2 or 4, characterized in that, The adjuvant content in the anti-Canadian Echinococcus subunit vaccine is 5%-60% V / V.

7. The anti-Canadian Echinococcus subunit vaccine according to claim 1, characterized in that, The pharmaceutically acceptable carrier comprises an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant, and a preservative.

8. A process for the preparation of the anti-Canadian Echinococcus subunit vaccine according to claim 1, characterized by, The method comprises the following steps: S1, the gene of 2dEC95 antigen protein in Canadian Echinococcus is amplified and cloned into an expression vector respectively to obtain a recombinant expression vector containing the gene of 2dEC95 antigen protein in Canadian Echinococcus; S2, the recombinant expression vector containing the gene of 2dEC95 antigen protein in Canadian Echinococcus obtained in step S1 is transformed or transduced into a host to obtain a recombinant containing the recombinant expression vector; S3, the recombinant obtained in step S2 is cultured to express the 2dEC95 antigen protein in Canadian Echinococcus; S4, the 2dEC95 antigen protein in Canadian Echinococcus obtained in step S3 is purified, and a pharmaceutically acceptable carrier is added to obtain the anti-Canadian Echinococcus subunit vaccine.

9. The method of preparing an anti-Canadian Echinococcus subunit vaccine according to claim 8, characterized in that, In step S3, the expressed 2dEC95 antigen protein in Canadian Echinococcus is an intracellular soluble protein.

10. Use of the anti-Canadian Echinococcus subunit vaccine according to claim 1 in the preparation of a medicament for preventing and / or treating Canadian Echinococcus infection.

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