CN115737792A - Echinococcus canadensis subunit vaccine and preparation method and application thereof - Google Patents

Abstract

The invention discloses a subunit vaccine for resisting echinococcus canadensis infection and a preparation method thereof; an EC95 antigenic protein comprising an immunogen and a pharmaceutically acceptable carrier; wherein, the amino acid sequence of the dEC protein is shown as SEQ ID No.1 or the degenerate sequence thereof. At present, no vaccine for resisting echinococcus canadensis infection exists, the 2dEC protein is adopted as the antigen, and the prepared subunit vaccine for resisting echinococcus canadensis infection is low in production cost, simple in production process, safe, efficient, low in cost and the like; even when the antigen content of the dEC protein in the anti-echinococcus canadensis subunit vaccine is only 40 mu g/ml, the 4 th week after the secondary immunization can achieve the antibody titer of more than 1.

Description

Echinococcus canadensis subunit vaccine and preparation method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, relates to an echinococcus infection-resistant subunit vaccine and a preparation method thereof, and particularly relates to an echinococcus canadensis infection-resistant subunit vaccine and a preparation method thereof.

Background

Echinococcosis (Echinococcosis) is a serious zoonosis caused by the parasitism of Echinococcus tapeworm larvae, namely echinococcus (hydatid cysts), in human and animal tissues and organs such as lung and liver. Echinococcosis is popular and is distributed worldwide, and the World Animal Health Organization (World Organization for Animal Health; french: office international de zootics, OIE) classifies echinococcosis as a globally informed infectious disease and a common disease for various animals; the World Health Organization (WHO) ranks echinococcosis as one of the diseases that are preferentially predicted and urgently treated by the global early warning system. Echinococcosis is also one of the five parasitic diseases planned by the ministry of health of china.

According to the difference of the form of the focus and the pathogen of infection, echinococcosis is mainly divided into echinococcosis granulosa (CE) and echinococcosis multocida, wherein the echinococcosis granulosa is distributed most widely and the number of patients is the most. The causative agent of CE is currently composed of several echinococcus complex species: echinococcus granuLosus (Echinococcus granuLosus), echinococcus canadensis (Echinococcus canadensis), echinococcus equina, echinococcus austenitalis, wherein the CE caused by Echinococcus granuLosus type G1 exceeds 90%, and the CE caused by Echinococcus canadensis type G6 exceeds 7%.

The current research shows that the current echinococcosis disease epidemic is mainly controlled by cutting off the echinococcus development link, controlling the infection of echinococcosis by human, livestock and other intermediate hosts, preventing or expelling parasites to treat canine and other final hosts, and blocking the large-scale spreading of the eggs. Among them, epidemic Lightowers, etc. which can effectively control echinococcosis granulosa for intermediate host vaccination found that EG95 is a natural oncosphere antigen with molecular weight of 24.5kDa in Eg, and its coding gene has a total length of 715bp, wherein 462bp gene can code 153 amino acid proteins with molecular weight of 16.5kDa, and is the most effective protective antigen among numerous proteins screened, and successfully developed vaccines against echinococcosis granulosa in sheep. However, the existing vaccine for resisting echinococcosis granulosus ovis has no obvious protection on echinococcosis canadensis type G6.

Disclosure of Invention

In view of the defects in the prior art, the invention aims to provide an anti-echinococcus canadensis subunit vaccine and a preparation method thereof, which can prevent the infection of echinococcus canadensis and have the advantages of safety, high efficiency, low cost and the like. Wherein the anti-echinococcus canadensis subunit vaccine comprises an immunogenic EC95 antigen protein and a pharmaceutically acceptable carrier.

As an embodiment of the invention, in the echinococcus canadensis-resistant subunit vaccine, the amino acid sequence of the dEC protein is shown in SEQ ID No. 1.

In one embodiment of the invention, in the anti-echinococcus canadensis subunit vaccine of the invention, the dEC protein can be a monomer, or a single-chain polymer formed by connecting polypeptides in series, such as dimer 2dEC, trimer 3dEC, tetramer 4dEC, and pentamer 5 dEC.

The anti-echinococcus canadensis subunit vaccine can reach an antibody titer of not less than 1 to 1024 at 4 weeks after secondary immunization, and can maintain a high titer antibody titer for a long period of time.

As an embodiment of the invention, the anti-echinococcus canadensis subunit vaccine of the invention has the dEC antigen protein content of 20-100 mu g/ml.

In the anti-echinococcus canadensis subunit vaccine, the dEC95 antigen protein content can be selected from 20 μ g/ml, 30 μ g/ml, 40 μ g/ml, 50 μ g/ml, 60 μ g/ml, 70 μ g/ml, 80 μ g/ml, 90 μ g/ml and 100 μ g/ml.

Even when the antigen content of the dEC protein in the anti-echinococcus canadensis subunit vaccine is only 40 mu g/ml, the antibody titer of not less than 1.

As a preferred embodiment of the invention, in the echinococcus canadensis subunit vaccine, the EC95 protein antigen content in the echinococcus canadensis subunit vaccine is 60 μ g/ml.

As an embodiment of the present invention, in the echinococcus canadensis subunit vaccine of the present invention, the pharmaceutically acceptable carrier comprises an adjuvant, and the adjuvant comprises: (1) White oil, alumina gel adjuvant, saponin, alfvudine, DDA; (2) Water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, blockco-polymer, SAF-M, monophosphoryl lipid A, avridine lipid-amine adjuvant, escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, montanide ISA 206, and Gel adjuvant; preferably, montanide ISA 50V.

The adjuvant content is 5% -60% V/V, preferably from 20% -60% V/V, more preferably 50% V/V.

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 immunostimulant includes alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 2 (IL 2).

To prepare such compositions, methods well known in the art may be used.

The invention also relates to a method for preparing the anti-echinococcus canadensis subunit vaccine, wherein the method comprises the following steps: respectively amplifying and cloning genes (shown as SEQ ID NO. 6) of 2dEC antigen protein in echinococcus canadensis to an expression vector to obtain a recombinant expression vector containing 2dEC antigen protein gene of the echinococcus canadensis; step (2) transforming or transducing the recombinant expression vector containing the echinococcus canadensis 2dEC antigen protein gene obtained in the step (1) into a host to obtain a recombinant containing the recombinant expression vector; culturing the recombinant obtained in the step (2) in the step (3) to express echinococcus canadensis 2dEC antigen protein; and (4) purifying the echinococcus canadensis 2dEC antigen protein obtained in the step (3), and adding an adjuvant to obtain the echinococcus canadensis subunit vaccine.

As an embodiment of the invention, in the method of the invention, the Echinococcus canadensis dEC antigenic protein amino acid sequence in step (1) is shown in SEQ ID No.1 or its degenerate sequence; coli in the step (2). The echinococcus canadensis 2dEC antigen protein expressed in the step (3) is an intracellular soluble protein.

The invention also relates to application of the echinococcus canadensis resistant subunit vaccine in preparation of a medicine for preventing and/or treating echinococcus canadensis infection.

The administration objects of the medicine for preventing and/or treating the echinococcus canadensis infection comprise sheep, cattle and camels.

In the invention, escherichia coli 24a-2dEC (Escherichia coli 24a-2 dEC) has been submitted to China center for type culture Collection at 23.6.2021, with the collection address of Wuhan university in Wuhan, china and the collection number of CCTCC NO: M2021749.

Compared with the prior art, the invention has the following beneficial effects:

1) At present, no vaccine for resisting echinococcus canadensis infection exists, the 2dEC protein is adopted as the antigen, and the prepared subunit vaccine for resisting echinococcus canadensis infection is low in production cost, simple in production process, safe, efficient, low in cost and the like;

2) In the invention, even when the antigen content of the dEC protein in the anti-echinococcus canadensis subunit vaccine is only 40 mug/ml, the antibody titer of more than 1.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1: protein purification results; wherein, lane M: protein standard molecular weight; lane 1: protein sample P-24a-2 dEC; lane 2: protein sample P-24a-2dEG95; lane 3: protein sample P-MAL-2 dEC;

FIG. 2 is a schematic diagram: competition ELISA test results.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will aid those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any manner. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.

Example 1: construction of recombinant vectors

1.1 Synthesis of the Gene sequence. Coli is used as a host bacterium, the invention optimizes the codons of the base sequences of the coding recombinant proteins 2dEG95, 2dEC and 2aEC, and the optimized base sequences are synthesized by Nanjing Kingsler Biotech limited. Wherein dEG95 amino acids are connected through GGGSGGGS to construct a single-chain homodimer 2dEG95; dEC95 amino acids are connected through 'GGGSGGGS' to construct single-chain homodimer 2dEC, aEC amino acids are connected through 'GGGSGGGS' to construct single-chain homodimer 2 aEC. The amino acid sequence of 2dEC is shown in SEQ ID NO.2, and the amino acid sequence of 2aEC is shown in SEQ ID NO. 7.

1.2 construction of recombinant expression vector pET24a-2 dEC.

(1) 2dEC is used as a template, an upstream primer Nde I-F and a downstream primer Xho I-R are designed, a gene fragment 2dEC is obtained by PCR amplification, a restriction enzyme Nde I site and a protective base are introduced into the 5' end of the upstream primer, wherein the sequence of the Nde I site is CATATG; the 5' end of the downstream primer is introduced with a restriction endonuclease Xho I site, a stop codon and a protective base, wherein the sequence of the Xho I site is CTCGAG. The primer sequences and the PCR reaction procedure are shown in tables 1 and 2.

Table 1: PCR primer name and sequence

Primer name	Sequence of
		Nde I-F	5’-GGTCCATATGCATCACCATCATCACCACCTG-3’SEQ ID NO.4
Xho I-R	5’-CCGCTCGAGTTA GACGGTAGATTCTTTTTTACCAGC-3’SEQ ID NO.5

Table 2: PCR reaction procedure

(2) The amplified gene fragment 2dEC was digested with endonuclease Nde I and endonuclease Xho I, the digested gene fragment was recovered, ligated to pET24a prokaryotic expression vector treated with the same endonuclease Nde I and endonuclease Xho I, and the ligation product was transformed into e.coli DH5 α competent cells, spread on a plate containing 100 μ g/ml kanamycin sulfate, cultured at 37 ℃, and when colonies on the plate were clearly visible, single colonies were picked up in 3ml liquid medium containing 100 μ g/ml kanamycin sulfate, cultured at 37 ℃, and then plasmids were extracted. The recombinant plasmid pET24a-2dEC is obtained, and the recombinant plasmid is confirmed to be consistent with a target sequence through sequencing verification.

1.3 recombinant vectors pET24a-2dEG95, pMAL-2dEC, pET24a-2aEC95. Referring to the experimental procedure of 1.2, (1) the gene sequence 2dEG95 was ligated to the vector pET24a with Nde I/Xho I as the cleavage site to obtain a recombinant vector pET24a-2dEG95, using the cloning template 2dEG95 gene, the primers Nde I-F shown in SEQ ID No.4 and Xho I-R shown in SEQ ID No.5, respectively; (2) Connecting a gene sequence 2dEC to a vector pMAL, wherein the restriction enzyme site is NdeI/EcoRI, obtaining a recombinant vector pMAL-2dEC, the used cloning template is a 2dEC gene, the sequence of a used primer Nde I-F is shown as SEQ ID No.4, and the sequence of a primer EcoRI-R is shown as SEQ ID No. 8; (3) The gene sequence 2aEC was ligated to the vector pET24a with Nde I/Xho I as cleavage site to obtain recombinant vector pET24a-2aEC, using the cloning template 2aEC gene, nde I-F as primer shown in SEQ ID No.4 and aEC-95 Xho I-R as primer shown in SEQ ID No. 9. The recombinant plasmid obtained above was confirmed to be identical to the target sequence by sequencing verification. Wherein, the amino acid sequence of 2dEG95 is shown in SEQ ID NO.3, and the amino acid sequence of 2aEC is shown in SEQ ID NO. 7.

Example 2: construction of recombinant bacterium

2.1 construction of Primary seeds. pET24a-2dEC, pET24a-2dEG95, pMAL-2dEC and pET24a-2aEC were prepared as described above. E.coli BL21 (DE 3) competent cells were transformed, plated on LB medium plates containing the corresponding antibiotics (50. Mu.g/ml ampicillin or 100. Mu.g/ml kanamycin sulfate), cultured at 37 ℃ and, when colonies on the plates were clearly visible, single colonies were picked up on 3ml liquid medium containing the corresponding antibiotics (50. Mu.g/ml ampicillin or 100. Mu.g/ml kanamycin sulfate), cultured at 37 ℃, 1ml of the resulting bacterial solution was taken out therefrom, glycerol was added to the final concentration of 8%, and frozen storage was carried out at-80 ℃ to obtain recombinant engineered bacteria 24a-2dEC, 24a-2dEG95, MAL-2dEC, 24a-2aEC, respectively, as original stocks.

2.2 identification of primordial seeds.

(1) Morphological and biochemical characteristics: the engineering bacteria are gram-negative brevibacterium. Can ferment and decompose glucose, and produce acid and gas; the indole test and the methyl red test are both positive; the Voges-Proskauer two's test (V.P test), citrate test were negative.

(2) Culture characteristics: grow on LB solid culture medium to form round, neat, raised, milky and glossy smooth colony.

(3) Pure inspection: the test was carried out according to the pure test method in appendix of the pharmacopoeia of the people's republic of China, and the result is pure.

Example 3: fermentation of recombinant bacteria

The strain was inoculated into 500mL of LB medium containing the corresponding antibiotic (50. Mu.g/mL ampicillin or 100. Mu.g/mL kanamycin sulfate) and shake-cultured at 37 ℃ to OD ₆₀₀ When the value is 1.2-1.5, inoculating the seed solution into a 5L fermentation tank according to the inoculation amount of 10%, and performing fermentation culture when the thallus OD is ₆₀₀ When the value reaches 20-25 ℃, the culture temperature is reduced to 28 ℃, and IPTG is added to the final concentration of 0.4mM at the same time, and the induction is carried out for 12-14 h. The wet weight of the cells was collected by centrifugation to give about 500g.

The cells were resuspended in 10 ml of resuspension buffer (20 mM Tris-HCl pH 7.5, 500mM NaCl) per gram of wet cells.

Example 4: purification of recombinant antigens

4.1 crushing the resuspended liquid by a homogenizer at a pressure of 700bar for 4 times.

4.2 lysate 28000g was centrifuged for 40 minutes and the supernatant was collected.

4.3 adopting an affinity chromatography mode for purification, wherein the protein chromatography equipment is an AKTA pure 150m protein purification instrument.

(1) For the recombinant proteins P-24a-2dEC, P-24a-2dEG95 and P-24a-2aEC, ni Sepharose 6FF is adopted as a chromatographic packing, 20mM Tris-HCl pH 7.5 is adopted as an equilibrium solution, 500mM NaCl is adopted as a washing solution, 30mM imidazole is adopted as a washing solution, and 500mM imidazole is adopted as an eluent;

(2) As for the recombinant protein P-MAL-2dEC, dextran Sepharose High Performance, 20mM Tris-HCl,200mM NaCl,1mM EDTA,1mM DTT, pH 7.4 as an equilibrium solution, 20mM Tris-HCl,200mM NaCl,1mM EDTA,1mM DTT,10mM maltose, pH 7.4 as an eluent were used.

4.4 sterile filtration. In the clean bench, the samples were sterile filtered with a 0.22 μm sterile filter and dispensed into sterile sample vials.

4.5SDS-PAGE analyzing the protein purification, as shown in FIG. 1, the results showed that the target protein was completely bound to the column and eluted by the eluent. The purity of the target protein obtained by one-step purification is more than 85%.

Example 5: physicochemical analysis of recombinant antigens

5.1 sterility test. The test was carried out according to the sterility test method in appendix of the pharmacopoeia of the people's republic of China.

5.2 detecting the concentration and purity of the antigen protein. Samples were run by grey scale analysis for SDS-PAGE and analyzed by gel imager for concentration and purity of each band. The results are shown in Table 3.

Table 3: protein sample purity and concentration results

Sample (I)	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

5.3 The quality standard of Echinococcus ovis (hydatid) disease genetic engineering subunit vaccine in 'veterinary drug quality standard' of 2017 edition requires that the antigen percentage content is more than or equal to 15%, and the antigen percentage contents of P-24a-2dEG95 and P-24a-2dEC measured by the invention are respectively 5.90 times and 5.69 times of the standard.

Example 6: preparation of samples of different adjuvants

(1) And preparing a water-soluble compound adjuvant GEL vaccine sample.

P-24a-2dEC antigen is diluted to 100 mu g/ml by PBS, mixed with 20% (volume percentage) GEL adjuvant according to the volume ratio of 1:1, and kept stand for 12h at 4 ℃ to obtain a vaccine sample V-24a-2 dEC-GEL.

(2) Water adjuvant QuilA vaccine sample preparation.

P-24a-2dEC antigen is diluted to 100 mu g/ml by PBS, mixed with 1mg/ml QuilA adjuvant according to the volume ratio of 1:1, and kept stand for 12h at 4 ℃ to obtain a vaccine sample V-24a-2 dEC-QuilA.

(3) Oil adjuvant Montanide ISA 50V vaccine sample preparation.

P-24a-2dEC antigen is diluted to 100 mu g/ml by PBS, emulsified with Montanide ISA 50V adjuvant according to the volume ratio of 1:1, and kept stand for 12h at 4 ℃ to obtain a vaccine sample V-24a-2 dEC-50V.

Example 7: lamb immunity experiment of vaccine samples with different adjuvants

(1) 32 negative lambs were screened and the sheep were asked as in Table 4 below.

Table 4: requirement for animal screening

(2) The immunization protocol is shown in Table 5 below.

Table 5: immune operation procedure

(3) And (5) detecting the antibody level. ELISA antibody detection was performed on the collected sera. The results show that the antibodies of the vaccine groups after the second immunization are both remarkably improved, the immunogenicity is good, the antibody duration is at least 48 weeks after the second immunization, and the oil adjuvant Montanide ISA 50V vaccine group is obviously superior to the other two vaccine groups, and the specific results are shown in the following tables 6, 7, 8 and 9.

Table 6: v-24a-2 dEC-GEL antibody titer detection result

Table 7: v-24a-2 dEC-QuilA antibody titer detection result

Table 8: v-24a-2 dEC-50V antibody titer detection result

Table 9: PBS antibody titer detection result

Example 8: optimization of emulsification conditions

According to the following optimization experiment, the shear rotation speed of 14000rpm, the shear time of 10min and the oil-water ratio of 1:1 are finally selected as the optimal emulsification conditions.

(1) The shear force used in the emulsification process is optimized. 12000rpm, 14000rpm and 16000rpm were used respectively, and as a result, 14000rpm was superior to 16000rpm and 12000rpm were judged from the uniformity and stability of the emulsified sample.

(2) The emulsification time is optimized. And respectively shearing for 8min, 10min and 12min at a proper shearing speed, and judging according to the uniformity and stability of the emulsified sample, wherein the result that 10min is better than 12min and is better than 8min.

(3) The proportion of oil and water is optimized. The oil-water ratio is respectively selected from 0.8, 1:1 and 1.2, and the result is that 1:1 is superior to 1.2. Example 8: preparing recombinant vaccines with different antigen contents.

8.1 taking a proper amount of adjuvant Montanide ISA 50V, and autoclaving for 30min at 121 deg.C for use.

8.2 the protein sample P-24a-2dEC purified in example 5 was taken and diluted to 80. Mu.g/ml, 120. Mu.g/ml, 160. Mu.g/ml with sterile PBS, respectively, depending on the measured concentration.

8.3 prepare the appropriate amount of adjuvant in the ratio of oil to water =1:1 (v: v), put in a beaker, and dip the shearing head into the adjuvant.

8.4 Pre-emulsification. Shear is initiated and the water phase is slowly added to the shearing adjuvant to ensure uniform mixing.

8.5 emulsifying. Shear at 14000rpm for 10 minutes and move the beaker along the stirring head to ensure uniform emulsification.

8.6 analysis. After the emulsion was prepared, it was left at room temperature overnight and then examined. 1ml of the emulsion was centrifuged at 3000rpm for 30min, and as a result, no water phase was precipitated at the bottom, indicating good emulsification.

8.7 vaccine samples V-24a-2dEC (40. Mu.g), V-24a-2dEC (60. Mu.g), V-24a-2dEC (80. Mu.g) with antigen contents of 40. Mu.g/ml, 60. Mu.g/ml, and 80. Mu.g/ml were prepared.

Example 9: recombinant vaccine stability analysis

9.1 the vaccines were placed in glass vials and stored at 4 deg.C, 20 deg.C and 37 deg.C, respectively, for stability studies.

9.2 the judgment criteria of the emulsion stability are: (1) the height of a sample precipitation layer is not more than 5%; (2) maintaining sterility; (3) After demulsification, compared with the stock solution, the antigen content is not lower than 80%.

9.3 the tested vaccine samples are stored for 18 months at 4 ℃,3 months at 20 ℃ and 1 month at 37 ℃, and the vaccine samples are stable.

Example 10: lamb immunity experiment of vaccine samples with different antigen contents

(1) 32 negative lambs were screened and the sheep were as shown in Table 10.

Table 10: requirement for animal screening

(2) The immunization protocol is as follows in Table 11.

Table 11: immune operation procedure

(3) And (5) detecting the antibody level. ELISA antibody detection was performed on the collected sera. The results show that the antibodies of the vaccine groups are remarkably improved after the second immunization, the immunogenicity is good, the antibody duration is at least 48 weeks after the second immunization, and the vaccine groups with the antigen content of 60 mu g are obviously superior to the other two vaccine groups, and the specific results are shown in the following tables 12, 13, 14 and 15.

Table 12: v-24a-2dEC (40 mug) antibody titer detection result

Table 13: v-24a-2dEC (60 mug) antibody titer detection result

Table 14: v-24a-2dEC (80 mug) antibody titer detection result

Table 15: PBS antibody titer detection result

Example 11: preparation of recombinant vaccines with different antigens

11.1 taking a proper amount of adjuvant Montanide ISA 50V, and autoclaving for 30min at 121 deg.C for use.

11.2 protein samples P-24a-2dEC and P-24a-2aEC purified in example 4 were taken and diluted to 120. Mu.g/ml with sterile PBS, respectively, depending on the concentrations determined.

11.3 appropriate amount of adjuvant was prepared in the ratio of oil to water =1:1 (v: v), put in a beaker, and dip the cutting head into the adjuvant.

11.4 Pre-emulsification. Shear is initiated and the water phase is slowly added to the shearing adjuvant to ensure uniform mixing.

11.5 emulsifying. Shear at 14000rpm for 10 minutes and move the beaker along the stirring head to ensure uniform emulsification.

11.6 analysis. After the emulsion was prepared, it was left at room temperature overnight and then examined. 1ml of the emulsion was centrifuged at 3000rpm for 30min, resulting in no aqueous phase separating out at the bottom, indicating good emulsification.

11.7 vaccine samples were prepared as V-24a-2dEC and V-24a-2aEC, respectively.

Example 12: lamb immunity experiment with different antigen vaccine samples

(1) 24 negative lambs were screened and the sheep were asked as in Table 16 below.

Table 16: requirement for animal screening

(2) The immunization protocol is shown in Table 17 below.

Table 17: immune operation procedure

(3) And (5) detecting the antibody level. ELISA antibody detection was performed on the collected sera. The results show that: the 60ug of antigen is also used, the antibody of the vaccine group after the second immunization is obviously improved, the immunogenicity is good, and the duration of the antibody is at least 48 weeks after the second immunization; the antibody titers 48 weeks after the secondary immunization of the V-24a-2dEC vaccine group were all no less than 1:512, whereas the V-24a-2aEC vaccine group was totally no higher than 1:128, as shown in table 18, table 19 and table 20 below.

Table 18: v-24a-2dEC antibody titer detection result

Table 19: v-24a-2aEC antibody titer detection result

Table 20: PBS antibody titer detection result

Example 13: evaluation of Cross-protective Effect of antiserum

The invention discloses a competitive ELISA experiment, wherein an antigen P-MAL-2dEC is used as a coating antigen, and an anti-V-24 a-2dEC serum is used as a dilution multiple of P-24a-2dEG95, P-24a-2dEC, P-P-MAL-2dEC, PBS and an anti-V-24 a-2dEC serum which are respectively incubated with the following antigens by 100 times.

The results of the competition ELISA assay are shown in FIG. 2. The results show that the EG95 antigen can not block the combination of anti-EC 95 serum and EC95 antigen, and indicate that the vaccine composition consisting of the echinococcus granulosus EG95 antigen protein in the narrow sense can not generate complete protective effect on the echinococcus canadensis.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Sequence listing

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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 (Artificial Sequence)

<400> 4

ggtccatatg catcaccatc atcaccacct g 31

<210> 5

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ccgctcgagt tagacggtag attctttttt accagc 36

<210> 6

<211> 756

<212> DNA

<213> Artificial Sequence (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 (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 (Artificial Sequence)

<400> 8

ccggaattct tagacggtag attctttttt accagc 36

<210> 9

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ccgctcgagt taagtcagaa ctacaacgat gc 32

Claims (10)

1. An anti-echinococcus canadensis subunit vaccine, which is characterized by comprising an immunogenic EC95 antigenic protein and a pharmaceutically acceptable carrier; wherein, the amino acid sequence of the dEC protein is shown as SEQ ID No.1 or the degenerate sequence thereof.

2. The anti-echinococcus canadensis subunit vaccine of claim 1, wherein the dEC protein is a monomer or a single-chain multimer linked in tandem with a polypeptide.

3. The anti-echinococcus canadensis subunit vaccine of claim 2, wherein the single-chain multimer is dimer 2dEC95, trimer 3dEC, tetramer 4dEC, or pentamer 5dEC95.

4. The echinococcus canadensis-resistant subunit vaccine of claim 1, wherein the echinococcus canadensis-resistant subunit vaccine has a dEC95 antigenic protein content of 20-100 μ g/ml.

5. The echinococcus canadensis subunit vaccine of claim 1, wherein the pharmaceutically acceptable carrier comprises an adjuvant comprising: (1) White oil, alumina gel adjuvant, saponin, alfvudine, DDA; (2) Water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, blockco-polymer, SAF-M, monophosphoryl lipid A, avridine lipid-amine adjuvant, escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, montanide ISA 206, and Gel adjuvant; preferably, montanide ISA 50V.

6. The echinococcus canadensis-resistant subunit vaccine of claim 5, wherein the echinococcus canadensis-resistant subunit vaccine has an adjuvant content of 5% -60% V/V.

7. The echinococcus canadensis subunit vaccine of claim 1, wherein the pharmaceutically acceptable carrier comprises a drug, an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant, and a preservative; the immunostimulant comprises one or more of alpha-interferon, beta-interferon, gamma-interferon, granulocyte-macrophage colony stimulating factor GM-CSF, macrophage colony stimulating factor M-CSF and interleukin 2IL 2.

8. A method for preparing the echinococcus canadensis subunit vaccine according to claim 1, wherein the method comprises the following steps:

s1, respectively amplifying and cloning genes of 2dEC antigen protein in echinococcus canadensis to an expression vector to obtain a recombinant expression vector containing 2dEC antigen protein gene of the echinococcus canadensis;

s2, transforming or transducing the recombinant expression vector containing the echinococcus canadensis 2dEC antigen protein gene obtained in the step S1 into a host to obtain a recombinant containing the recombinant expression vector;

s3, culturing the recombinants obtained in the step S2 to express echinococcus canadensis 2dEC antigen protein;

s4, purifying the echinococcus canadensis 2dEC antigen protein obtained in the step S3, and adding a pharmaceutically acceptable carrier to obtain the echinococcus canadensis subunit vaccine.

9. The method for preparing the echinococcus canadensis subunit vaccine according to claim 8, wherein in step S3, the expressed echinococcus canadensis 2dEC antigenic protein is an intracellular soluble protein.

10. Use of the echinococcus canadensis subunit vaccine according to claim 1 in the preparation of a medicament for the prevention and/or treatment of echinococcus canadensis infection.

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