CN114196691B - Gene, protein, vaccine and application for preparing multi-epitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep - Google Patents
Gene, protein, vaccine and application for preparing multi-epitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
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- C07K14/4355—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from cestodes
- C07K14/43554—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from cestodes from Taenia
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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- Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry
Abstract
The invention provides a gene, protein, vaccine and application for preparing multi-epitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep, belonging to the technical field of animal vaccine preparation, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 1. The multi-epitope recombinant vaccine prepared by the gene provided by the invention has good immunogenicity and safety, high target protein expression, high antigen purity, simple preparation process, no use of urea, safety and environmental protection, and improved production efficiency, and has important industrial and research values.
Description
Technical Field
The invention belongs to the technical field of animal vaccine preparation, and particularly relates to a gene, a protein, a vaccine and application for preparing a multi-epitope recombinant vaccine for preventing and treating bovine and ovine echinococcosis.
Background
Echinococcosis commonly known as echinococcosis (Hydatidosis) is a parasitic disease caused by the fact that echinococcosis granulosa (Echinococcus granulosus, EG) larvae of echinococcum are parasitic disease of serious human and livestock in various animals such as human beings, cattle, sheep and the like. The disease is popular worldwide, and has a heavy popularity in pasture areas and agriculture and pasture areas in the western part of China. The focus of echinococcosis is mainly concentrated on the liver and the lung, causes great damage to human health, not only causes incapacitation of patients, but also brings great economic burden to families and society of the patients. The epidemic of the echinococcosis is more serious in epidemic areas, the number of domestic animals suffering from the echinococcosis is more than 5000 tens of thousands per year, the direct economic loss caused by death of the domestic animals and viscera waste (mainly cattle and sheep) exceeds 30 hundred million yuan, serious economic loss is caused for animal husbandry, and the immunity of the echinococcosis is brought into forced immunity in China.
The control of echinococcosis mainly takes immune pre-prevention, only commercial echinococcosis vaccine for sheep at present takes EG95 protein as immune antigen for producing vaccine, the vaccine is expressed in insoluble inclusion bodies in thalli such as escherichia coli, yeast and the like, the vaccine can be prepared through a plurality of steps such as denaturation and renaturation, the production process is very complex, the antigen loss in the middle process is serious, and urea denaturation generates a large amount of ammonia nitrogen wastewater, so that the environmental protection treatment difficulty is increased.
Disclosure of Invention
Therefore, the invention aims to provide a gene, protein, vaccine and application for preparing multi-epitope recombinant vaccine for preventing and treating bovine and sheep echinococcosis, the protein content obtained by adopting the gene provided by the invention is high, and urea is not used for preparing the vaccine.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a gene for preparing a multi-epitope recombinant vaccine for preventing and treating bovine and sheep echinococcosis, and the nucleotide sequence of the gene is shown as SEQ ID No. 1.
The invention also provides the protein encoded by the gene according to the technical scheme, and the amino acid sequence of the protein is shown as SEQ ID No. 2.
The invention also provides a recombinant plasmid, which is obtained by connecting the genes in the technical scheme into a vector.
Preferably, the gene contains EcoR I and Sac I cleavage sites at both ends, and is ligated to a vector.
Preferably, the carrier comprises a PET-32a carrier.
The invention also provides a preparation method of the protein in the technical scheme, which comprises the following steps:
1) Transforming the recombinant plasmid into an expression strain, and culturing to obtain a fermentation strain;
2) Fermenting the fermentation strain obtained in the step 1), and obtaining the OD (OD) of a fermentation broth 450 When the value is 0.4, adding IPTG to induce expression, and collecting fermentation thalli;
3) Crushing and centrifuging the fermentation thalli obtained in the step 2) to obtain a supernatant, and subjecting the supernatant to Ni 2+ Purifying by using an NTA affinity chromatography system to obtain the protein.
Preferably, the final concentration of IPTG in the fermentation broth of step 2) is 0.5mM;
the conditions for inducing expression include: the speed of oscillation was 150rpm, the temperature was 25℃and the time was 10 hours.
The invention also provides a multi-epitope recombinant vaccine for preventing and treating bovine and sheep echinococcosis, and the components of the vaccine comprise the protein, the immune adjuvant and the freeze-drying protective agent in the technical scheme.
Preferably, the volume ratio of the mass of the protein, the mass of the immunoadjuvant and the lyoprotectant is 2500 ug/25 mg/0.517 ml.
The invention also provides application of the vaccine, protein or recombinant plasmid in preparing the multi-epitope recombinant vaccine for preventing and treating bovine and sheep echinococcosis.
The invention has the beneficial effects that:
the multi-epitope recombinant vaccine prepared by the gene provided by the invention has good immunogenicity and safety, high target protein expression, high antigen purity, simple preparation process, no use of urea, safety and environmental protection, and improved production efficiency, and has important industrial and research values.
The protein induced by the commercialized vaccine is an inclusion body, and urea denaturation treatment is needed to be purified; the protein induced by the invention is soluble protein, has good immunogenicity, and can be directly prepared into vaccine after purification.
Drawings
FIG. 1 shows the gray scale analysis of the purified target protein, wherein M: a Marker; 1.2, 3: purifying the protein; 4: gray level analysis of target protein content;
FIG. 2 is a 2 week secondary lamb antibody test;
FIG. 3 shows the detection of the calf antibody in the second-day 2 week period.
Detailed Description
The invention provides a gene for preparing a multi-epitope recombinant vaccine for preventing and treating bovine and sheep echinococcosis, wherein the nucleotide sequence of the gene is shown as SEQ ID No.1, and the gene is specifically as follows:
SEQ ID No.1：
the invention screens the full-length gene (Genebank: AY 421719.1) of the echinococcosis EG95 and the B cell epitope of the AgB8/1 full-length gene (Genebank: AF 143813.1) by bioinformatics software, and utilizes flexible linker to connect the screened 4 segments with stronger antigenicity (the thickened part is the 4 segments with stronger antigenicity obtained by screening) in series to obtain the gene for preparing the multi-epitope recombinant vaccine for preventing and treating bovine and sheep echinococcosis.
The invention also provides the protein coded by the gene in the technical scheme, the amino acid sequence of the protein is shown as SEQ ID No.2, and the protein is specifically as follows:
SEQ ID No.2：
KGMGIETRTTETPLRKHFNLTPVGSGGGGSGGGGSGGGGSVNPSDPLVYKRQTAKFSDEAAAKEAAAKEAAAKIETPRAGKKESTVMTSGSAGGGGSGGGGSGGGGSDLLKELEEVFQLLRKKLRMA。
the invention also provides a recombinant plasmid, which is obtained by connecting the genes in the technical scheme into a vector. In the present invention, the gene preferably contains EcoRI and SacI cleavage sites, ligated into a vector. In the present invention, the carrier preferably comprises a PET-32a carrier, and the source of the PET-32a carrier is not particularly limited, and the carrier can be prepared by a conventional preparation method or a commercially available product. The conditions for the cleavage and the method for the ligation are not particularly limited, and those skilled in the art can use conventional procedures.
The invention also provides a preparation method of the protein in the technical scheme, which comprises the following steps:
1) Transforming the recombinant plasmid into an expression strain, and culturing to obtain a fermentation strain;
2) Fermenting the fermentation strain obtained in the step 1), and obtaining the OD (OD) of a fermentation broth 450 When the value is 0.4, adding IPTG to induce expression, and collecting fermentation thalli;
3) Crushing and centrifuging the fermentation thalli obtained in the step 2) to obtain a supernatant, and subjecting the supernatant to Ni 2+ Purifying by using an NTA affinity chromatography system to obtain the protein.
The recombinant plasmid is transformed into an expression strain and then cultured to obtain a fermentation strain. In the invention, the expression strain is preferably a Rossrta (DE 3) expression strain, has chemically transformed competent cells prepared on the basis of a common escherichia coli BL21 strain, supplements tRNA corresponding to 6 rare codons (AUA, AGG, AGA, CUA, CCC, GGA) lacking in escherichia coli, and provides the expression level of exogenous genes in a prokaryotic system. In the present invention, the medium used for the culture preferably includes LB liquid medium. In the present invention, the conditions of the culture preferably include: the temperature of the culture was 37℃and the time of the culture was 16 hours. The method of transforming the recombinant plasmid into the expression strain is not particularly limited, and may be performed by those skilled in the art according to conventional procedures.
The invention ferments the obtained fermentation strain, and when the OD of the fermentation liquor is equal to the OD 450 When the value is 0.4, IPTG is added to induce expression, and zymophyte is collected. In the present invention, the inoculum size of the fermentation strain is preferably 1%. In the present invention, the conditions of the fermentation preferably include: the speed of oscillation was 150rpm and the temperature was 37 ℃. In the present invention, the final concentration of IPTG in the fermentation broth is preferably 0.5mM; the conditions for inducing expression preferably include: the speed of oscillation was 150rpm, the temperature was 25℃and the time was 10 hours.
The invention breaks and centrifugates the obtained zymophyte to obtain supernatant, and the supernatant is processed by Ni 2+ Purifying by using an NTA affinity chromatography system to obtain the protein. In the invention, the fermentation thalli is preferably washed by Buffer A, resuspended by Buffer A according to the mass-volume ratio of 1g to 10ml and then ultrasonically crushed. In the present invention, the conditions of the ultrasonic disruption preferably include: the power is 300W, the work is 3s, the interval is 3s, and the ultrasonic treatment is 120min. In the present invention, the conditions of the centrifugation preferably include: the centrifugation temperature was 4℃and the centrifugation force was 12000g, and the centrifugation time was 20min.
In the present invention, the Ni 2+ The conditions for purification of the NTA affinity chromatography system preferably comprise: after balancing the nickel column by using a balancing Buffer solution Buffer A with the volume of 5 times of the nickel column, circularly loading the supernatant to ensure that the total loading volume is 3 times of the volume of the protein sample; and eluting the mixed protein by using 10 times of nickel column volume Buffer B after the sample loading is finished, and finally eluting the target protein by using eluent Buffer C. In the invention, the formula of Buffer A is as follows: taking 29.22g of NaCl and 20ml of 1M Tris-HCL with pH of 8.0, adding water to fix the volume to 1000 ml. The formula of Buffer B is: taking 29.22g of NaCl, 20ml of 1M Tris-HCL with pH of 8.0 and 1.7g of imidazole, and adding water to fix the volume to 1000 ml. The formula of Buffer C is: taking 29.22g of NaCl, 20ml of 1M Tris-HCL with pH of 8.0, 17g of imidazole, and adding water to fix the volume to 1000 ml.
The invention also provides a multi-epitope recombinant vaccine for preventing and treating bovine and sheep echinococcosis, and the components of the vaccine comprise the protein, the immune adjuvant and the freeze-drying protective agent in the technical scheme.
In the present invention, the mass of the protein, the mass of the immunoadjuvant to the volume ratio of the lyoprotectant is preferably 2500 ug/25 mg/0.517 ml. In the present invention, the immunoadjuvant preferably includes the immunoadjuvant Quil a (saponin Sigma aldrich-S7900). In the present invention, the lyoprotectant preferably comprises a sucrose solution and a mannitol solution, wherein the mass percentage of the sucrose solution is preferably 15%, the mass percentage of the mannitol solution is preferably 6.25%, and the volume ratio of the sucrose solution to the mannitol solution is 0.417:0.1. The preparation method of the vaccine is not particularly limited, the components are mixed and frozen for 24 hours at the temperature of minus 80 ℃, and the vaccine is obtained by freeze-drying by a freeze dryer.
In the present invention, the method of using the vaccine preferably comprises: when the animal is a bovine, the dosage is 500 ug/head; when the animal is a sheep, a dose of 100 ug/head is used.
The invention also provides application of the gene, the protein or the recombinant plasmid in preparing the multi-epitope recombinant vaccine for preventing and treating bovine and sheep echinococcosis. The kind and preparation method of the vaccine are not particularly limited, and may be selected by those skilled in the art according to the conventional vaccine.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Construction of multi-epitope recombinant plasmid, protein purification and vaccine preparation
1. Construction of recombinant plasmid with multiple epitopes
Screening the B cell epitope of the echinococcosis EG95 full-length gene (Genebank: AY 421719.1) and AgB1 full-length gene (Genebank: AF 143813.1) by bioinformatics software, connecting the screened 4 segments of fragments with stronger antigenicity in series by using flexible linker, wherein the enzyme cutting sites at two ends are EcoRI and SacI, the connecting carrier is PET-32a, and sending the recombinant plasmid with multiple epitopes artificially synthesized by biological company (the DNA sequence and the amino acid sequence after the flexible linker is respectively shown as SEQ ID No.1 and SEQ ID No. 2).
2. Recombinant protein purification
Transforming the recombinant plasmid into Rosseta (DE 3) expression strain, culturing for 16h at 37 ℃ in a incubator, picking up single bacterial colony to 10ml LB liquid medium containing Amp, and culturing under shaking at 37 ℃ at 150r/min to obtain fermentation strain; inoculating 1% fermentation strain into 1L LB liquid culture medium, and shake culturing at 150 r/min; when the bacterial liquid OD 450 When the value reaches 0.4, IPTG with the final concentration of 0.5mM is added to induce expression, 150r/min shaking culture is performed, the induction temperature is 25 ℃, and the induction is performed for 10 hours. Washing the fermentation thalli once by using Buffer A, adding 10 times of Buffer A according to the weight-volume ratio into the sediment for resuspension, and then carrying out ultrasonic crushing; ultrasonic power 300W, working for 3s, intermittent for 3s,120min. Centrifuging 12000g at 4deg.C for 20min to obtain supernatant, and treating with Ni 2+ -NTA affinity chromatography system purification. After balancing the nickel column by using a balancing Buffer solution Buffer A with the volume of 5 times of the nickel column, circularly loading the supernatant to ensure that the total loading volume is 3 times of the volume of the protein sample; and eluting the mixed protein by using 10 times of nickel column volume Buffer B after the sample loading is finished, and finally eluting the target protein by using eluent Buffer C. (the formula of Buffer A is that 29.22g of NaCl, 20ml of 1M Tris-HCL with pH8.0 and water are taken and added to a volume of 1000ml, the formula of Buffer B is that 29.22g of NaCl, 20ml of 1M Tris-HCL with pH8.0 and 1.7g of imidazole are taken and added to a volume of 1000ml, the formula of Buffer C is that 29.22g of NaCl, 20ml of 1M Tris-HCL with pH8.0 and 17g of imidazole are taken and added to a volume of 1000 ml.
The purified recombinant protein was assayed for total protein content by Bradford method. According to the measured total protein content, each sample was diluted to 1mg/mL, mixed with 5×loading Buffer at a ratio of 4:1, and boiled for 5 minutes. 10. Mu.L of the prepared samples were subjected to SDS-PAGE gel electrophoresis, 3 replicates were set for each sample, and the results were subjected to gray scale analysis by Bandscan software and the target protein content was determined. Results: the concentration of the purified recombinant protein is 2.1mg/ml, and the content of the target protein is 80.09% (shown in figure 1).
3. Vaccine preparation
The purified recombinant protein, immune adjuvant Quil A and freeze-drying protective agent (15% sucrose and 6.25% mannitol) are taken to prepare the vaccine according to the component requirements of the table 1 (the immunization dose of the cattle is 5 times of the immunization dose of the sheep, so that the vaccine is 100 mug/head and 25 head/bottle for immunization of the sheep, 500 mug/head and 5 head/bottle for immunization of the cattle), each bottle is divided into 2.5mL, frozen for 24 hours at the temperature of minus 80 ℃ and then freeze-dried by a freeze dryer to prepare the freeze-dried vaccine.
Table 1 vaccine formulation
| Purified protein | Quil A | 15% sucrose | 6.25% mannitol | |
| Vaccine | 2500μg | 25mg | 0.417mL | 0.1mL |
The results of the example 1 show that the multi-epitope recombinant plasmid is successfully constructed, the expression quantity of the induced protein is good, the purity of the purified protein is high, and the purified protein is used for preparing the freeze-dried vaccine.
Example 2
Immunogenicity test of multi-epitope recombinant vaccine for echinococcosis (artemia) of cattle and sheep
1. Immunogenicity test of multi-epitope recombinant vaccine (sheep)
Labeling ear marks of 10 healthy lambs with 2-4 months of age, which are negative in detection result of echinococcosis serum antibody, randomly dividing the healthy lambs into 2 groups, subcutaneously injecting 1 head (1 ml) of prepared vaccine (prepared in example 1) into the neck of each sheep of an immune group, and injecting the same volume of diluent into a control group; 28 days after the first immunization, the immunization was boosted once in the same manner at the same dose; serum was collected for 14 days from the second immunization and antibody detection was performed.
Results: the results of the detection are shown in Table 2, and the results of the detection of the sheep serum of the control group are all negative (the OD average value of the antibody is 0.197). The immune group sheep all showed antibody reaction, the serum detection results were positive (antibody OD >0.3 was positive), the average antibody OD of the immune group was 1.694, and there was a significant difference compared to the control group, as shown in fig. 2.
TABLE 2 antibody detection results (lamb)
2. Immunogenicity test of multiple epitope recombinant vaccine (cattle)
Labeling the healthy calves of 3-6 months of age with negative detection results of the echinococcosis serum antibodies, randomly dividing the healthy calves into 2 groups, subcutaneously injecting 1 head (2 ml) of prepared vaccine (prepared in example 1) into the neck of each sheep of the immune group, and injecting the diluent of the same volume into the control group; 28 days after the first immunization, the immunization was boosted once in the same manner at the same dose; serum was collected for 14 days from the second immunization and antibody detection was performed.
Results: the test results are shown in Table 3, and the test results of the control group bovine serum are negative (antibody OD average value 0.188). The immune group cattle all showed antibody reaction, the serum detection results were positive (the OD value of the antibody was >0.3 positive), the average OD value of the antibody in the immune group was 1.245, and the difference was significant compared with the control group, as shown in fig. 3.
TABLE 3 antibody detection results (Calf)
The results of example 2 show that the prepared freeze-dried vaccine has good immunogenicity, and the cattle and sheep generate higher antibody level after immunization, so that good protection effect against echinococcosis can be provided.
Example 3
Safety test of bovine and sheep echinococcosis multi-epitope recombinant vaccine (prepared in example 1)
1. Safety inspection of multiple epitope recombinant vaccine (sheep)
Screening 5 healthy lambs and healthy primary pregnant females of 2-4 months old with a echinococcosis serum antibody detection result as a negative, subcutaneously injecting 2 parts (2 ml) of prepared vaccine into the neck of each sheep, and setting the same type of healthy lambs of 2-4 months old without vaccine as a control group. 28 days after the first immunization, the immunization was boosted once in the same manner at the same dose.
The results of the experimental sheep observations, including local and systemic reactions such as body temperature, spirit, appetite, etc., were shown in Table 4, as observed 7d after inoculation. As can be seen from table 4: therefore, the spirit, the body temperature, the appetite and the like of the experimental sheep are not abnormal. Vaccinated lambs did not see any abnormal response caused by vaccination; the pregnant sow is vaccinated for normal pregnancy without abortion. Proved that the multi-epitope recombinant vaccine is safe to the immunization of sheep.
Table 4 sheep vaccine safety test results
2. Safety inspection of multiple epitope recombinant vaccine (cattle)
Screening 5 healthy calves and healthy primary pregnant cows of 3-6 months of age with negative detection results of echinococcosis serum antibodies, subcutaneously injecting 2 parts (4 ml) of prepared vaccine into the neck of each cow, and setting the same type of healthy calves and healthy primary pregnant cows of 3-6 months of age without vaccine as a control group. 28 days after the first immunization, the immunization was boosted once in the same manner at the same dose.
The results of the experimental sheep observations, including local and systemic reactions such as body temperature, spirit, appetite, etc., were shown in Table 5, as observed 7d after inoculation. As can be seen from table 5: therefore, the spirit, the body temperature, the appetite and the like of the tested cattle are not abnormal. The vaccinated calf does not see abnormal reaction caused by vaccination; the vaccinated pregnant cows were pregnant normally with no abortion. Proved that the multi-epitope recombinant vaccine is safe to the immunization of cattle.
TABLE 5 safety test results for bovine vaccine
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Chongqing Australian biological products Co., ltd
<120> Gene, protein, vaccine and use for preparing multi-epitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep
<141> 2021-12-28
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Claims (10)
1. A gene for preparing multi-epitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep is characterized in that the nucleotide sequence of the gene is shown as SEQ ID No. 1.
2. The protein encoded by the gene of claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID No. 2.
3. A recombinant plasmid obtained by ligating the gene of claim 1 into a vector.
4. The recombinant plasmid according to claim 3, wherein the gene comprises EcoR I and Sac I cleavage sites at both ends, and is ligated to a vector.
5. The recombinant plasmid according to claim 3 or 4, wherein the vector comprises a PET-32a vector.
6. A method for producing the protein according to claim 2, comprising the steps of:
1) Transforming the recombinant plasmid according to any one of claims 3 to 5 into an expression strain, and culturing to obtain a fermentation strain;
2) Fermenting the fermentation strain obtained in the step 1), and obtaining the OD (OD) of a fermentation broth 450 When the value is 0.4, adding IPTG to induce expression, and collecting fermentation thalli;
3) Crushing and centrifuging the fermentation thalli obtained in the step 2) to obtain a supernatant, and subjecting the supernatant to Ni 2+ Purifying by using an NTA affinity chromatography system to obtain the protein.
7. The method of claim 6, wherein the final concentration of IPTG in the fermentation broth in step 2) is 0.5mM;
the conditions for inducing expression include: the speed of oscillation was 150rpm, the temperature was 25℃and the time was 10 hours.
8. A multi-epitope recombinant vaccine for preventing and treating bovine and sheep echinococcosis, which is characterized in that the components of the vaccine comprise the protein, the immunoadjuvant and the freeze-drying protective agent as claimed in claim 2.
9. The vaccine of claim 8, wherein the ratio of the mass of the protein, the mass of the immunoadjuvant to the volume of the lyoprotectant is 2500 ug/25 mg/0.517 ml.
10. Use of the gene of claim 1, the protein of claim 2 or the recombinant plasmid of claim 3 for preparing multi-epitope recombinant vaccine for preventing and treating bovine and ovine echinococcosis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111681190.4A CN114196691B (en) | 2021-12-28 | 2021-12-28 | Gene, protein, vaccine and application for preparing multi-epitope recombinant vaccine for preventing and treating echinococcosis of cattle and sheep |
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