KR100628023B1 - Recombinant Surface Antigen Derived from Sporozoites of Pathogens Responsible for Coccidiosis and Vaccines Comprising the Same - Google Patents

Abstract

The present invention relates to a technique for expressing spores surface antigenic protein of Eimeria spp. That causes avian coccidiosis in plant cells, and more particularly, novel polynucleotides encoding spores surface antigens. The present invention relates to a vector comprising the plant, the plant into which the vector is introduced, and a method for producing a surface antigen protein using the plant, and to a vaccine for avian coccidium comprising the surface antigen protein of the recombinant coccidi protozoan spore obtained from the plant.

Chickens, Coccidiosis, Aimeric Protozoa, Spore Body, Surface Antigen, Plant, Transient Transfection, Transient Expression, Stable Expression, Transformation

Description

Recombinant Surface Antigen Derived from Sporozoites of Pathogens Responsible for Coccidiosis and Vaccines Comprising the Same}             

1 is a genetic map of a vector comprising the novel polynucleotide of the present invention encoding the spores surface antigen protein of coccidium protozoa.

Figure 2 is a photograph of the results of electrophoresis of coccidium protozoan surface antigen protein expressed in transgenic plants, ie transient transfected plants, on SDS-polyacrylamide gels. M, molecular weight marker; Lane W, protein of wild tobacco; Lane T1, coccidium protozoan surface antigen G1 expressed in the transgenic plant; And lane T2, coccidium protozoan surface antigen G2 expressed in the transgenic plant.

Figure 3 is a photograph showing the results of Western blot analysis of coccidium protozoan surface antigen expressed in the transgenic plant. M, molecular weight marker; Lane 1, protein of wild tobacco; Lane 2, coccidial protozoan surface antigen G1 expressed in transgenic tobacco; And lane 3, coccidium protozoan surface antigen G2 expressed in the transgenic plant.

4 is a graph showing the antigenicity of recombinant coccidial protozoan surface antigen expressed in transgenic plants. Absorbance value at 405 nm on vertical axis; Horizontal axis, dilution factor of monoclonal antibody against coccidial protozoan surface antigen; 4, coccidium protozoan surface antigen protein G1 isolated and purified from transgenic tobacco; Midline, recombinant coccidial protozoan surface antigen protein as a positive control; Below, the protein of wild type tobacco as a negative control.

5 is an ELISA plate photograph showing the antigenicity of recombinant coccidial protozoan surface antigen expressed in transgenic plants. Landscape: (+), positive control expressed in E. coli ; (-), Wild type tobacco; 1-10, Coccidium protozoan surface antigen G1 expressed in transgenic tobacco. Length: 1-7, antibody dilution factor; (-) PBS buffer.

The present invention relates to a technique for expressing spores surface antigenic protein of Eimeria spp. That causes avian coccidiosis in plant cells, and more particularly, novel polynucleotides encoding spores surface antigens. The present invention relates to a vector comprising the plant, the plant into which the vector is introduced, and a method for producing the surface antigen protein using the plant, and a chicken coccidial vaccine comprising the surface antigen protein of the recombinant coccidi protozoan spore obtained from the plant.

Plant genetic engineering, despite its short history, has made remarkable progress, enabling the development of transgenic plants with new traits by introducing useful genes from plants and microorganisms into plants. The transformed plant thus developed can be used as a production system for the production of high value-added useful recombinant proteins using various functions of plants and the advantages of higher organisms.

Plants are of particular interest for mass production systems of medical and industrial proteins. This is because it can be easily harvested and processed by using the existing agricultural foundation established, and thus a large amount of desired biomass can be secured. In addition, unlike bacteria, which were mainly used as protein expression systems, plants have eukaryotic protein synthesis pathways where post-detox modification processes are essential for the activity of mammalian proteins (Cabanes-Macheteau et al ., Glycobiolgy 9: 365-372). (1999)), proteins expressed in plants are almost identical to proteins expressed in mammals, unlike proteins expressed in bacteria.

Meanwhile, coccidiosis is a disease that occurs in poultry including livestock such as cattle and pigs, and chickens. In particular, algae coccidiosis is a parasitic disease caused by protozoa of Eimeria, which belongs to spores. Is a disease that causes economic losses estimated at more than $ 500 million annually worldwide.

There are 9 species of coccidial parasites in chickens, 5 of which are Eimeria acervulina, Eimeria brunett, Eimeria maxima, Eimeria necatrix, and Eimeria tenella .

Eimeria necatrix and Eimeria tenella belong to the pathogenic species, which are parasitic in the middle part of the caecum and small intestine of chickens. However, Eimeria species, which are relatively weak in pathogenicity, usually develop into a chronic form with soft stools. However, acute coccidiosis is suppressed by the anticoccidial agent for feed addition, but the type causing chronic coccidiosis is resistant to drugs, resulting in lower feed efficiency, poor weight gain, reduced immunity, and intestinal infection caused by bacteria. It is more important because it gives.

Currently, various anticoccidial agents have been developed and show good effects, but there are often cases in which farms are not as effective as expected. Although numerous drugs have been developed and used alone or in combination to prevent or treat coccidiosis, coccidiosis is still prevalent in the poultry industry. In Korea, no exception, poultry farms that are raised on a large scale, despite the addition of prophylactic agents to the feed, coccidiosis develops and are being treated again. In particular, broiler chickens are fed a feed containing anticoccidial until shipment, which can lead to serious residual problems. Therefore, long-term prevention is required using vaccines, not drug treatment with temporary anticoccidial preparations.

The administration of various anticoccidial agents for the occurrence and prevention of the disease is effective in preventing the occurrence of the disease, but as mentioned above, the use of drugs due to the emergence of resistant protozoa and the safety of livestock products is increasing. In order to solve the problems of these existing agents, studies have been conducted to use the pathogenic protozoan or attenuated attenuated liquor for the disease in the United States, Europe and Korea. Attenuated live protozoa vaccines have been developed and are commercially available to prevent infection by the two most damaging protozoa in Korea.

 However, the vaccines using these protozoan directly have difficulty in precisely adjusting their dosage, and in the case of attenuated live protozoa vaccines, they may be idled for several years until it is a good condition to form spores in the presence of inadvertent eggs. Shortened life cycles of spores forming within 12 to 48 hours under moderate conditions (humidity and temperature) can lead to massive reproduction of coccidial protozoa, causing millions of eggs to be produced by the infected family, causing further problems. have. In order to solve these problems, it is essential to develop a subunit vaccine that uses a highly conserved epitope site of antigenic proteins present on the surface of the protozoa rather than directly using the protozoan.

The present inventors have made diligent research efforts to solve the problems of the prior art described above. As a result, the recombinant coccidium protozoa obtained from plants by expressing the spore body surface antigen protein of Eimeria spp. That causes avian coccidiosis in plant cells The present invention was completed by discovering that the surface antigen protein of induces an excellent immunization response.

It is therefore an object of the present invention to provide a novel polynucleotide encoding the spore body surface antigen of the coccidium protozoa.

Another object of the present invention is to provide a plant expression vector comprising the spore body surface antigen of the coccidium protozoa.

It is still another object of the present invention to provide a method for producing a transient transfected plant which temporarily expresses the spores surface antigen of the coccidium protozoa.

Another object of the present invention is to provide a transient transfected plant which expresses the spore body surface antigen of the coccidium protozoa temporarily.

It is another object of the present invention to provide a method for preparing spore body surface antigen of coccidium protozoa.

Another object of the present invention is to provide a method for producing a transgenic plant stably expressing the spores surface antigen of the coccidium protozoa.

Another object of the present invention is to provide a vaccine against avian coccidiosis.

Another object of the present invention is to provide a method for inducing immunization against coccidiosis.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides a polynucleotide encoding a spherosomal surface antigen of a coccidial protozoan comprising the nucleotide sequence of SEQ ID NO: 51-700 in SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 38-430 in SEQ ID NO: 3 To provide.

The novel polynucleotides of the present invention encode spore body surface antigens of the coccidium protozoa, which have modified nucleotide sequences to optimize expression in plants without amino acid substitutions.

The design of the polynucleotides of the invention is carried out by the following criteria: (i) to make the GC content at least 50%, (ii) to have the plant's preferred codons, and (i) to remove intron-like sequences. will be. Such plant-compatible sequences can increase the translation rate of genes (Kusnadi et al ., Biotechnol. Prog. 14: 149-155 (1998)). In addition, the intron-like sequence in the foreign gene to be introduced is removed in order to rule out the possibility that the arrangement of the transgene is recognized as an intron in the host plant, resulting in cleavage in the plant nucleus and a failure to produce the desired protein.

The novel polynucleotides of the present invention comprise not only the nucleotide sequence of SEQ ID NO: 51-700 in SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 38-430 in SEQ ID NO: 3, but also a nucleotide sequence showing substantial identity to the nucleotide sequence described above. It is interpreted as. The above substantial identity is at least 90% of the phases when the nucleotide sequence of the present invention and any other sequence are arranged to correspond as closely as possible and the sequence is analyzed using algorithms commonly used in the art. By nucleotide sequence, showing homology, more preferably 95% homology, most preferably 98% homology.

According to another aspect of the present invention, the present invention provides an antibody comprising (i) the nucleotide sequence of SEQ ID NO: 51-700 in SEQ ID NO: 1 or SEQ ID NO: 38-430 in SEQ ID NO: 3; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3'-non-detoxification site that acts on plant cells to cause 3'-end polyadenylation of the RNA molecule, thereby providing a plant expression vector of a spore body surface antigen of a recombinant coccidial protozoan. do.

According to a preferred embodiment of the present invention, a promoter suitable for the present invention may be any one commonly used in the art for gene introduction of a plant, for example, the cauliflower mosaic virus (CaMV) 35S promoter , Nophalin synthase (nos) promoter, pigwarm mosaic virus 35S promoter, sugacran basilisform virus promoter, commelina yellow mottle virus promoter, ribulose-1,5-bis-phosphate carboxylase small subunit ( photoinducible promoter of ssRUBISCO), rice cytosol triosphosphate isomerase (TPI) promoter, adenine phosphoribosyltransferase (APRT) promoter of arabidopsis and octopine synthase promoter.

According to a preferred embodiment of the present invention, the 3'-non-detoxification site causing the polyadenylation suitable for the present invention is derived from the nopalin synthase gene of Agrobacterium turmeration (nos 3 'end) ( Bevan et al ., Nucleic Acids Research , 11 (2): 369-385 (1983)), derived from the Octopine Scenetase Gene of Agrobacterium Tumation, and of the Protease Inhibitor I or II Gene of Tomato or Potatoes 3 'terminal portion and CaMV 35S terminator.

Optionally, the vector additionally carries a gene encoding a reporter molecule (eg, luciferase and β-glucuronidase). In addition, the vector of the present invention may be selected as an indicator for antibiotics (e.g. neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, and the like) and resistance genes (e.g. neomycin phosphotransferase ( nptII ), hygro Mycin phosphotransferase ( hpt ), and the like).

According to the invention, the production of plants incorporating genes encoding the spores surface antigens of the coccidium protozoa is largely achieved in two ways: transient transfected plants and transgenic plants.

As used herein, the term "transient transfected plant" means that the introduced gene is not transferred to the next generation as the plant into which the foreign gene has been introduced. In transiently transfected plants, foreign genes are generally present separately from the chromosome of the host cell. In contrast, the term “transgenic plant” refers to a plant having a foreign gene that is transmitted to the next generation. In transgenic plants, foreign genes are generally integrated into the chromosome of the host cell, becoming the genetic repertoire of the host cell, and reliably delivered to the next generation.

Therefore, according to another aspect of the present invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention to plant cells of the plant; And (b) provides a method for producing a transient transfected plant to temporarily express the spores surface antigen of the recombinant coccidium protozoa comprising the step of checking whether the gene has been introduced into the plant cell.

According to another aspect of the present invention, the present invention provides a transient transfected plant which temporarily expresses the spore body surface antigen of the recombinant coccidium protozoa produced by the above method.

According to another aspect of the present invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention into plant cells; (b) confirming whether the gene has been introduced into the plant cell; And (c) obtaining the spores surface antigen of the recombinant coccidial protozoa from a plant comprising the plant cell into which the gene has been introduced.

In the methods of the invention, transient transfection of plant cells can be carried out according to conventional methods known in the art (Rainer Fisher et al ., Biotechnol. Appl. Biochem. , 30: 113-116 (1999) ). Transient gene expression in plants is faster than in transformants with stable expression, and the result of protein expression can be confirmed as soon as possible. Therefore, whether the target protein obtained in plants prior to large-scale production using stable transgenic plants is functioning normally. Suitable for checking

Therefore, the present inventors expressed, isolated and purified the spores surface antigens of the coccidium protozoa using a transient gene expression method for efficient mass production of the spores surface antigens of the coccidium protozoa having biological activity.

Transient gene expression methods are suitable approaches to test the functionality and stable expression of target genes before manufacturing plant transformants for mass production, which has the advantage of saving time and money. In particular, plant transformants produced by stable transformation show a chromosomal positional effect that is affected by the position at which the foreign gene is inserted. Transient transfection can rule out this effect. It is well suited for the efficient expression and isolation of foreign genes.

In transient transfection, there are three types of methods for transferring foreign genes to plant cells: particle bombardment method for coating and introducing naked DNA onto particles (Christou, Trends Plant Sci. 1: 423-431 (1996)), agroinfiltration to introduce Agrobacterium containing expression vectors into plant tissues by vacuum infiltration (Kapila et al ., Plant Sci. , 122) . : 101-108 (1996)), and viral vectors using modified plant viral vectors (Scholthof et al ., Annu. Rev. Phytopathol. 34: 299-323 (1996)).

The transformation efficiencies of these three methods vary, so that in the case of DNA introduction by particle guns, genes are generally introduced into only a small number of cells and function only when DNA reaches the cell nucleus for transcription. This method can be used to test the stability of recombinant proteins prior to stable transformation, but is not suitable for mass expression of recombinant proteins.

The agroinfiltration method allows genes to be introduced into more cells than the particle gun. T-DNAs containing the target genes are actively introduced into the nucleus with the help of several bacterial proteins. In this method, the introduced T-DNA is not integrated into the host cell's chromosome and continuously expressed, but is present in the nucleus independently and expresses the transient expression of the gene of interest. It is suitable for obtaining protein quickly.

In addition, the method using a viral vector introduces a foreign gene into the genome of a viral plant pathogene to be controlled by a strong promoter, and when the recombinant viral vector including the foreign gene is infected with a plant, the foreign gene introduced is During plant virus replication, they are amplified with high efficiency and then expressed to produce foreign proteins. However, when using a viral vector, the size of the protein to be produced is limited to less than 30 kDa, so the size of the gene to be introduced is difficult to use.

Therefore, in the present invention, the agroin fill method is most preferable. The agroin filtration method is generally carried out with leaves.

When the present invention is practiced by the agroinfiltration method, more preferably, an Agrobacterium tumefaciens -binary vector system is used.

In a method using the Agrobacterium system, one specific embodiment includes the following steps: (a ') Agrobacterium tumefaciens containing a vector having a sequence of Infiltrating tissue of: SEQ ID NO: 51-700 in SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 38-430 in SEQ ID NO: 3; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3′-non-detoxification site that acts on plant cells to cause 3′-end polyadenylation of the RNA molecule; (b ') confirming the expression of the protein in the infiltrated leaves.

In one specific embodiment described above, suitable leaves include any tissue derived from the germinated seed, preferably the leaves produced before the peduncle rises, most preferably the leaves are too young or not mature. The younger the leaves, the higher the expression level, but the tissues are more susceptible to yellowing or dying. Seed germination is carried out under suitable dark / light conditions using a suitable medium.

Introduction of the gene into plant cells is carried out with Agrobacterium trimers, including Ti plasmids (Depicker et al ., In Genetic Engineering of Plants, Plenum Press, New York (1983)). More preferably, binary vector systems such as pBin19, pRD400, pRD320 and pHS737 are used (An et al ., "Binary vectors" In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York (1986); Jain et al) , Biochem. Soc. Trans. 28: 958-961 (2000).

Gene introduction into the plant cells in the leaves by Agrobacterium tufasions includes methods known in the art. Most preferably, the gene introduction process includes infiltration of a culture of Agrobacterium tumeration into the tissues of the leaves.

The infiltration process may be performed by a vacuum method or a syringe method. One specific embodiment when using a vacuum is as follows. The plant leaves are placed in a culture of Agrobacterium trimmers and vacuum is applied for a short time. Quickly release the vacuum, disinfect the leaves with sterile water, and place them on a damp piece of paper with the top of the leaves facing down. The leaves are then stored at about 22 ° C. under light conditions of about 16 hours. Then, after about 2-3 hours, the target protein expressed is identified.

On the other hand, a specific embodiment when using a syringe is as follows. The back of the plant leaves is infused with a syringe of Agrobacterium turmeration using a syringe, the plant is incubated for about 3-5 days, and then the expressed target protein is identified.

Agrobacterium tuberation cultures preferably include acetosyringone, to aid in the penetration of Agrobacterium into plants.

Plants transgenic according to the present invention are confirmed the expression of the protein of interest by methods known in the art. For example, PCR can be performed using DNA samples obtained from tissues of transgenic plants to identify foreign genes present in plant cells. Alternatively, Northern or Southern blotting can be performed to confirm gene introduction (Maniatis et al ., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). On the other hand, if the vector used to introduce the gene has a β-glucuronidase gene, a part of the leaf tissue into which the gene is introduced is selected from X-gluc (5-Bromo-4-Chloro-3-Indole-β-D- By immersing in a substrate solution such as Glucuronic Acid), the color development can be easily observed by visual observation (Jefferson, Plant Mol. Biol. Rep. , 5: 387 (1987)).

The method of the invention is applied to a variety of plants, but preferably to tobacco, melons, cucumbers, watermelons and rapeseeds.

The spores surface antigen of the coccidium protozoa can be obtained from the tissue of the transgenic plant of the present invention, ie, the transiently transfected plant (eg, leaves), and the extract obtained from the tissue can be obtained through a conventional purification process. In the present invention, the purification step may be carried out by employing a purification method commonly used in the art. For example, solubility fractionation using ammonium sulfate or PEG, ultrafiltration separation according to molecular weight, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. Various methods may be used and are usually purified using a combination of the above methods.

In a specific embodiment of the present invention, when 6 x His is present at the N-terminus of the spore body surface antigen protein of the recombinant coccididium protozoa of the present invention, a Ni-NTA His-binding resin column is used for Spore body surface antigen proteins can be isolated and purified quickly and easily.

According to the method of the present invention, by using a transient transfected plant, not only can the spore body surface antigen protein of the recombinant coccidial protozoan be produced at a low cost in a short time, but also the spore body surface antigen of the produced coccidial protozoa is natural-generated. ) Exhibits the same biological properties and functions as the spore body surface antigen of the coccidium protozoa.

Another approach for preparing plants incorporating genes encoding spores surface antigens of coccidium protozoa is to use transgenic plants that stably express the spores surface antigens of coccidium protozoa.

Therefore, according to another aspect of the present invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention to plant cells; (b) selecting the transformed plant cells; And (c) regenerating the plant from the transformed plant cells to obtain a transformed plant, thereby providing a method for producing a transformed plant stably expressing the spore body surface antigen of the recombinant coccidial protozoan.

According to another aspect of the present invention, the present invention provides a transgenic plant stably expressing the spore body surface antigen of the recombinant coccidium protozoa produced by the method of the present invention described above.

According to another aspect of the invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention to plant cells; (b) selecting the transformed plant cells; (c) regenerating the plant from the transformed plant cells to obtain a transformed plant; And (d) obtaining the spores surface antigen of the recombinant coccidial protozoa from the transformed plant.

According to another aspect of the present invention, the present invention provides spore body surface antigen of recombinant coccidial protozoa prepared by the above method.

In the method of the present invention, the transformation of plant cells can be carried out according to conventional methods known in the art, which are performed by electroporation (Neumann et al ., EMBO J. , 1: 841 (1982) and Saul et. al ., Plant Molecular Biology Manual , Vol. A1, Kluwer Academic Publishers, Dordrecht (1988), particle gun method (Yang et al ., Proc. Natl. Acad. Sci. , 87: 9568-9572 (1990) and Christou , Trends Plant Sci. 1: 423-431 (1996)) and Agrobacterium-mediated transformation (US Pat. Nos. 5,004,863, 5,349,124 and 5,416,011). Of these, Agrobacterium-mediated transformation is most preferred. Agrobacterium-mediated transformation is generally carried out with leaf discs and other tissues (cotyledon and hypocotyls). Agrobacterium-mediated transformation is most efficient in dicotyledonous plants.

Selection of transformed plant cells can be carried out by exposing the transformed culture to a selection agent (eg metabolic inhibitors, antibiotics and herbicides). Plant cells that are transformed and stably contain marker genes that confer selectivity resistance are grown and divided in the cultures described above. Exemplary labels include, but are not limited to, glycophosphate resistance genes and neomycin phosphotransferase (nptII) systems.

Methods of developing or regenerating plants from plant protoplasts or various implants are well known in the art (Bhojwani et al ., Plant Tissue Culture: Theory and Practice , Elsevier Science, New York, (1983); and Lindsey, Ed. , Plant Tissue Culture Manual , Kluwer Academic Publishers, Dordrecht, The Netherlands, (1991)). The transformed root shoots formed are imbedded in a suitable plant growth medium. Development or regeneration of plants comprising foreign genes introduced by Agrobacterium can be accomplished according to methods known in the art (US Pat. Nos. 5,004,863, 5,349,124 and 5,416,011).

On the other hand, the present inventors have made extensive research efforts to develop novel transgenic plants (eg, tobacco, melons, cucumbers, watermelons and rapeseeds), and as a result, have established the most efficient method for the transformation of specific plants. Such a method is disclosed in PCT / KR02 / 01461, PCT / KR02 / 01462 and PCT / KR02 / 01463, which patent documents are incorporated herein by reference.

The method of the invention is applied to a variety of plants, but preferably to tobacco, melons, cucumbers, watermelons and rapeseeds.

In the present invention, the preferred transformation method is carried out using the Agrobacterium system, more preferably using the Agrobacterium tumefaciens -binary vector system.

In a method using the Agrobacterium system, one specific embodiment includes the following steps: (a ') an Agrobacterium tumer embedded in a genome DNA of a plant cell and having a vector having the sequence Infecting the plant plant with Agrobacterium tumefaciens : (i) the nucleotide sequence of SEQ ID NO: 51-700 in SEQ ID NO: 1 or SEQ ID NO: 38-430 in SEQ ID NO: 3; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on plant cells to form RNA molecules; (Iii) a 3'-non-detoxification site that acts on plant cells to cause 3'-end polyadenylation of the RNA molecule; (b ') re-differentiating the infected implant in a regeneration medium to obtain a redifferentiated shoot; And (c ') culturing the re-differentiated shoots in a rooting medium to obtain a transformed plant.

In one specific embodiment described above, suitable implants for transformation include any tissue derived from the germinated seed, preferably cotyledon and hypocotyl, most preferably cotyledon. Seed germination is carried out under suitable dark / light conditions using a suitable medium. Transformation of plant cells is carried out with Agrobacterium trimers containing Ti plasmids (Depicker et al ., Plant cell transformation by Agrobacterium plasmids.In Genetic Engineering of Plants, Plenum Press, New York (1983)).

More preferably, binary vector systems such as pBin19, pRD400 and pRD320 are used (An et al ., Binary vectors "In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York (1986)). Binary suitable for the present invention The vector comprises (i) a promoter acting on a plant, (ii) a structural gene operably linked to said promoter, and (iii) a polyadenylation signal sequence Optionally, said vector comprises a reporter molecule (e.g., lucifer Additionally carries genes encoding laase and glucuronidase) Examples of promoters used in binary vectors include, but are not limited to, the CaMV 35S promoter, 1 promoter, 2 promoter and the nopaline synthase (nos) promoter. It is not limited.

Infection of implants with Agrobacterium trimmers includes methods known in the art. Most preferably, the infection process includes co-culturing the cotyledons by impregnating the cotyledon in a culture of Agrobacterium tumerification. As a result, Agrobacterium trimmers are infected into plants.

Explants transformed with Agrobacterium trimerization are re-differentiated in regeneration medium, which successfully causes regeneration of shoots. Finally, transgenic plants are produced by the rooting of shoots that have been regenerated in the rooting medium.

Plants transformed according to the present invention is confirmed whether or not transformed by methods known in the art. For example, PCR can be performed using DNA samples obtained from tissues of transformed plants to identify foreign genes inserted into the genomes of the transformed plants. Alternatively, Northern or Southern blotting can be performed to confirm transformation (Maniatis et al ., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)).

The spore body surface antigen of the coccidial protozoan expressed in the transformant can be obtained from tissues derived from various organs (e.g., stems, leaves, roots, fruits, seeds, etc.) of the transformed plant. It can be obtained through the purification process. In the present invention, the purification step may be carried out by employing a purification method commonly used in the art. For example, solubility fractionation using ammonium sulfate or PEG, ultrafiltration separation according to molecular weight, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. Various methods may be used and are usually purified using a combination of the above methods.

According to the method of the present invention, not only the spores surface antigens of the coccidium protozoa can be produced in large quantities at low cost, but the produced spores surface antigens of the coccidium protozoa are the same as the spores surface antigens of the natural (occurring) coccidium protozoa. Biological properties and function. Therefore, the spore body surface antigen of the coccidium protozoa of the present invention can be usefully used for the development of a vaccine for preventing chicken coccidiosis.

According to another aspect of the present invention, the present invention provides a spore body surface antigen of recombinant coccidial protozoa prepared using the above-described transgenic plant.

According to another aspect of the present invention, the present invention provides a composition comprising (a) the spore body surface antigen of the recombinant coccidium protozoa described above; And (b) provides a vaccine against avian coccidiosis, comprising a pharmaceutically acceptable carrier.

According to another aspect of the present invention, the present invention provides a method of inoculating an alga with the vaccine to induce immunization against coccidiosis.

According to a preferred embodiment of the present invention, the alga to which the vaccine composition of the present invention is applied includes, but is not limited to, algae on which coccidiosis develops, such as chicken, duck, turkey, quail, pheasant, ostrich, goose, and the like. Most preferably chicken.

In a preferred embodiment of the present invention, Eimeria spp. To which the vaccine composition of the present invention acts is Eimeria acervulina ( Eimeria acervulina ), Eimeria tenella ( Eimeria tenella ), Eimeria maxima , Eimeria coccidia , Eimeria mitis , Eimeria praecox , Eimeria brunetti , Emieria necattrix ( Eimeria necatrix a), this Almeria US Bhatia (Eimeria mivati) and this in Almeria and going (Eimeria hagani), and most preferably this Almeria ah Sergio burina (Eimeria acervulina) comprising a.

The vaccine composition of the present invention includes any carrier or diluent commonly used in vaccine preparation. Pharmaceutically acceptable carriers included in the vaccine composition of the present invention are conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, and the like. It is not. In addition to the above components, the vaccine composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

According to a preferred embodiment of the present invention, the vaccine composition of the present invention further comprises Complete Freund's Adjuvant (CFA) or Incomplete Freund's Adjuvant (IFA).

The vaccine composition of the present invention may be administered orally or parenterally, most preferably parenterally. Preferred parenteral administration methods are intramuscular (in particular thoracic muscle) injection and intravenous injection.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and reaction sensitivity of the chicken, Usually a skilled veterinarian can easily determine and prescribe a dosage effective for the desired immunity. According to a preferred embodiment of the present invention, when parenteral administration of the vaccine composition of the present invention, a suitable dosage is 5-50 μg, preferably 25-50 μg of surface antigen per kg bird.

The vaccine composition of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those skilled in the art. Or by incorporating into a multi-dose container. The formulations here may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of extracts, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

In the immunization method of the present invention, the vaccination preferably includes one or more booster vaccination procedures.

The spore body surface antigens of the coccidium protozoa prepared according to the present invention are similar to the natural surface antigens formed in algae by coccidium protozoa, and have excellent immunity to the coccidial protozoa.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, and the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example 1 Synthesis of Coccidial Protozoan Surface Antigen

The same nucleotide sequence encoding 220 (surface antigen G1) or 130 (surface antigen G2) amino acids in the same manner as the spores surface antigen gene of the native coccidium protozoan, but encodes the spores surface antigen of the existing coccidium protozoan Alternatively, the optimal nucleotide sequence designed by the plant was designed. The design criteria for the new gene is first, to make the GC content above 50%, to have the plant's preferred codons, and to remove intron-like sequences. The nucleotide sequences thus designed were chemically synthesized at the Plant Biotechnology Institute (hereinafter referred to as "PBI") and the National Research Center (SK, Canada). DNAs encoding the spherosomal surface antigen proteins G1 and G2 of synthesized coccidium protozoa are described in SEQ ID NO: 1 and 3 sequences.

Example 2: Preparation of vector for plant expression of coccidium protozoan surface antigen gene

The synthetic genes encoding the spores surface antigens G1 and G2 of the recombinant coccidium protozoa, Eimeria acervulina , consisted of 716 bp and 446 bp, respectively, and the Xba I and 3'-ends at the 5'-end. It has been synthesized to have a recognition site of Bam HI.

PHS737 (PBI, National Research Center, Canada), a plant expression binary vector, was digested with restriction enzymes Xba I and Bam HI enzymes, digested, and isolated and purified. The synthetic genes of the 716 bp and the 446 bp and the cleaved pHS737 vector were mixed, and the reaction was performed at 16 ° C. for at least 1 hour after adding ligation buffer (KOSCO, Korea) and T4 ligase (KOSCO, Korea). I was. The reaction was then transformed into competent cells treated with CaCl ₂ ( E. coli strain DH5α) (Promega, USA) and antibiotic resistant strains were selected in LB medium containing the antibiotic kanamycin (100 mg / ml). . In the gene map of Fig. 1, G1 and G2 are the nucleotide sequences described in SEQ ID NO: 1 and 3, respectively, 35S is the Cauliflower Mosaic Virus 35S promoter, Tnos and Pnos are the termination sequence and promoter sequence of the nos gene, LB and RB are the left and right borders of T-DNA, MCS is the multiple cloning position, nptII is the neomycin for spottransferase II sequence, and KM is the kanamycin resistance gene.

Plasmid DNA was isolated from the two selected clones using an alkaline method, and the forward primer 5'-AAT CTA GAA ATG GCT ACT AT-3 'and the reverse primer 5'-AAG GAT CCC CGA GCT TGA GA-3' Polymerase chain reaction (PCR). The enzyme used in the PCR amplification was Taq polymerase (Solgent, Korea), and the temperature conditions were set as follows: predenature for 2 minutes at 96 ° C, denature for 1 minute at 94 ° C, and connect for 1 minute at 55 ° C. The reaction was repeated 35 times at 72 ° C. for 2 minutes, and further extended at 72 ° C. for 10 minutes.

The amplification product was then analyzed on a 1.0% agarose gel to confirm that the desired insert sequences G1 and G2 were in each plasmid.

Example 3 Introduction of Coccidium Protozoan Surface Antigen Gene into Plant

Example 3-1 Preparation of Agrobacterium tumefaciens GV3101 Culture

Agrobacterium tuberization GV3101 (Mp90) (Plan -cell-rep. , 15 (11): 799-803 (1996) by conjugation of the recombinant pHS737 / G1 vector and pHS737 / G2 vector prepared in Example 2 Respectively). E. coli S17-1 and Agrobacterium tuber GV3101 (Mp90) with recombinant pHS737 / G1 vector or pHS737 / G2 vector were cultured in liquid LB medium to logarithmic phase, respectively. Each of these cells were mixed together 1: 1 in an Eppendorf tube and then centrifuged for 30 seconds to concentrate the cells, followed by stationary culture at 1-2C for 1-2 days. The Eppendorf tube containing the cells over time was gently shaken to float the concentrated cells, and then plated in LB solid medium to which 50 mg / L kanamycin and 30 mg / L gentamicin were added. Colonies were selected after incubation for one day (Alt-Morbe et al ., J. Bacteriol. 178: 4248-4257 (1996)). The strains into which the vectors were introduced were named Agrobacterium turmerics GV3101-pHS737 / G1 and GV3101-pHS737 / G2.

Agrobacterium tumeration strains having a recombinant pHS737 / G1 vector or pHS737 / G2 vector were inoculated in liquid LB medium and incubated at 28 ° C. for 2 days to be used as seed culture. The fully cultured spawn was added to 1/50 of the final culture volume and antibiotics (50 mg / L Kanamycin) in liquid LB medium and incubated for about 18 hours until the absorbance reached 1.5 at 600 nm. The culture solution was centrifuged at 3500 rpm for 15 minutes at room temperature to remove the supernatant, and then the cells were suspended in a 10 mM MgCl ₂ solution (containing 0.2 ppm of BA, containing 150 μM of acetosyringone) and allowed to acclimate at room temperature for at least 3 hours. It was used for infection of plants.

Example 3-2 Preparation of Plant Cells and Agroinfiltration

Seeds of the first sterilized tobacco ( Nicotina bentamiana ) were sown and incubated for at least 3 weeks and then purified in a greenhouse. Agrobacterium tamperate GV3101 (Mp90) containing a recombinant pHS737 / G1 vector or pHS737 / G2 vector having a spheroid surface antigen gene of coccidium protozoa on the leaves of the purified plant was cultured for infection by the method shown in Example 3-1. After preparing the solution, the needle was inserted into a 1 ml syringe (Note Co., Ltd.) with the needle removed, and then injected until it spreads evenly on the back of the leaf. If the fungus is injected into the whole leaf, the leaf is heavy and the plant itself may fall down or the leaf disease may break. Plants infused with Agrobacterium return to their original condition after 10 minutes to 1 hour. Plants injected with Agrobacterium were harvested within 5 days after 3 days of culture and analyzed using the method of Example 4.

Example 4 Label Factor Analysis for Confirming Transgene Expression in Transgenic Plants

Confirmation of gene expression of the plant secured in Example 3 was carried out as follows: beta-glucuronidase (β-glucuronidase, GUS) After extracting the leaves to confirm the expression of the reporter gene, GUS assay solution 200 μl of (X-gluc (5-Bromo-4-Chloro-3 3-Indole-β-D-Glucuronic Acid), 1 mg / ml) is placed in a container with leaves and thoroughly vacuumed using a vacuum. The vacuum was maintained until it was. After that, the appearance of color was visually observed to confirm the introduction and expression of the gene.

When the gene of the leaf to which the gene is introduced is expressed, the beta-glucuronidase, a reporter gene, is produced, and the enzyme beta-glucuronidase is produced. When the X-gluc solution is added, the beta-glucuronidase is added. As the X-gluc is broken down, pigments are produced, causing the cells to appear blue. In the present invention, the leaf into which the gene was introduced was expressed as a reporter gene, beta-glucuronidase, which was expressed in blue, indicating that the gene was introduced and expressed.

Example 5 Isolation and Purification of Recombinant Coccidium Protozoan Surface Antigen Protein from Transgenic Plants

In a mortar containing an appropriate amount of liquid nitrogen, 1 g of tobacco leaves with the genes introduced were cut and crushed. Finely ground was added 2 ml of protease inhibitor-containing extract buffer (250 mM sucrose, 1 M Hepes, 1 mM MgCl ₂ and 1 mM DTT) per 0.5 g of plant weight. The extract was centrifuged at 12,000 rpm and 4 ° C. for at least 30 minutes, then the supernatant was removed and transferred to a new tube. The lysed cell solution was added to a Probond column (Quiagen GmbH, Germany) containing nickel resin and gently shaken for 10 minutes at room temperature, while the spore body surface antigen protein of the recombinant coccidial protozoa in the cell solution was well adhered to the nickel resin. It was allowed to adsorb.

The resin to which the spores surface antigen protein of the recombinant coccidium protozoa was adsorbed was washed twice with the same amount of pH 7.8 adsorption buffer solution (20 mM sodium phosphate and 500 mM sodium chloride) to remove the plant cell-derived protein adsorbed on the resin. An equal amount of pH 6.0 wash buffer solution (20 mM sodium phosphate, 500 mM sodium chloride) was added to the washed resin, and the mixture was gently shaken at room temperature to wash the resin again, and the same amount of pH 4.0 elution buffer solution (20 mM sodium) was added. Phosphate, 500 mM sodium chloride) was added to elute the spores surface antigen G1 or G2 protein of the recombinant coccidium protozoa.

Recombinant coccidial protozoan surface antigen proteins expressed in transgenic plants have six histidine residues at the amino terminus and are easily isolated by binding to a cationically nickel resin. Through this process, only the recombinant coccidial protozoan surface antigen protein from which all plant cell proteins were removed was purified. The eluted protein was concentrated using Centricon (Amicon Co., U.S.A.).

As shown in FIG. 2, the recombinant coccidium protozoan surface antigen protein was electrophoresed on an SDS-PAGE gel and stained with Coomassie blue, resulting in 24.2 kD and 14.3 kD recombinant coccidial protoplast surface antigens G1 and G2 not detected in wild-type tobacco. The protein was detected in the cell eluate, and especially when eluted with a nickel resin column, it was confirmed that the pure recombinant coccidial protozoan surface antigen protein from which all plant cell proteins were removed.

A recombinant assay kit (Bio-Rad) was added to the recombinant coccidium protozoan surface antigen protein purified by the above method, and the absorbance was measured at 595 nm using a UV-spectrophotometer, followed by BSA (Bovine Serum Albumin). ) Protein quantitation was performed as compared to the standard. The extracted protein was adjusted to the same amount, and each sample was subjected to 8% polyacrylamide gel electrophoresis, and Western blot analysis was performed (see Kaufma et al ., Molecular and Cellular Methods in Biology and Medicine , 108). -121, CRC press). Western blot analysis was performed as follows. The protein in the SDS-PAGE gel was transferred to PVDF membrane using Semi-Dry Transfer Units (Hoefer, USA). After confirming that all proteins in the gel had migrated to the PVDF membrane, the PVDF membrane was dried. This PVDF membrane was reacted with 0.5% BSA / TBS (Tris-Buffered Saline) solution containing monoclonal antibody (Biosource Inc. Canada) that specifically reacts with His-tag for 1 hour, followed by 5 minutes of PVDF membrane with TBS solution. Wash three times. Then, after reacting with the peroxidase labeled secondary antibody (peroxidase labelled-anti mouse IgG, KPL, USA) for 1 hour, the PVDF membrane was washed three times for 10 minutes with TBS solution. Finally, the PVDF membrane was added to the left buffer solution to which the ABTS coloring substrate solution (KPL, USA) was added, and the resultant was reacted for 30 minutes at room temperature and the result was read. As shown in FIG. 3, Western blot analysis showed that the recombinant coccidium protozoan surface antigens G1 and G2 proteins of 24.2 kD and 14.3 kD were identified in the same manner as the above result.

Example 6: Determination of expression of recombinant coccidium protozoan surface antigen protein

In order to confirm the function of the recombinant coccidi protozoan surface antigen protein, G1, isolated and purified from the leaves of tobacco expressing the gene introduced by agroinfiltase, the antigen-antibody response to the recombinant coccidial protozoan surface antigen protein was tested. Measurements were made using well microtiter plates.

Spore body surface antigen protein G1 of recombinant Coccidium protozoan purified on 96-well microtiter plate was serially diluted in phosphate buffer solution, and 100 μl per well was dispensed, and the antigen was allowed to stand at 4 ° C. for 8 hours or more. Fixed. The plate containing the protein antigen adsorbed was washed three times with washing buffer (0.05% Tween 20, 0.01 M Phosphate, pH 7.6) to remove the antigen that was not adsorbed on the plate. The polyclonal antibody (self-produced) was diluted in a dilution solution (0.1% BSA, 0.05% Tween 20, 20 mM Trisma base, 150 mM NaCl) in a predetermined dilution multiple, divided into 100 μl per well and reacted at room temperature for at least 1 hour. -Induced antibody response. Then, each well was washed three times with washing buffer, and then the blocking buffer was dispensed into each well, and reacted at room temperature for 1 hour, followed by washing in the same manner as above. Peroxidase labeled secondary antibody (peroxidase labelled-anti rabbit IgG, KPL, U.S.A.) was aliquoted and reacted and washed in the same manner as above. Finally, each well was reacted for 30 minutes at room temperature by adding ABTS color substrate (KPL, USA), followed by reading at 405 nm using an ELISA reader (ELISA Reader, Titertek, USA). 4 is shown. The vertical axis shows absorbance values at 405 nm and the horizontal axis shows the dilution factor of the purified recombinant coccidium protozoan surface antigen. This is compared with the protein isolated from wild-type plants. As shown in FIG. 4, the antigenicity of the recombinant coccidial protozoan surface antigen protein purified from the transgenic plant was excellent, and the protein isolated from the wild-type plant was not shown at all.

In addition, as can be seen in Figure 5 showing the ELISA results, the recombinant coccidi protozoan surface antigen purified from the transformed tobacco by the present invention shows that it is antigenic to the antibody against the spores surface antigen of the natural coccidium protozoa It can be seen that it can exhibit a physiological function similar to that of spores on the surface antigens of natural coccidium protozoa.

Example 7: Immunogenic Activity of Recombinant Coccidium Protozoan Surface Antigen Protein

Example 7-1 Antibody Induction of Coccidium Protozoan Surface Antigens

As in Example 5, the immunogenic ability of the coccidium protozoan surface antigen protein isolated and purified from plant cells was tested using chicken.

As experimental animals, 10 chickens of 4-week-old broiler chickens were used for each test group, and the experimental group contained the complete Freund's Adjuvant as a supplement for 10 μg / kg body weight of coccidial protozoan surface antigen protein purified from plants per animal. Shaking 50 ml of CFA, Sigma, USA) or Incomplete Freund's Adjuvant (IFA, Sigma, USA) with saline, and the control group with 50 µl of CFA or IFA mixed with saline. Then, the syringe was administered to the vein of the chicken 5 times a week for 5 weeks. One week after the last inoculation, all the chickens were taken from the immune serum, allowed to stand at room temperature for 1 hour to coagulate, and centrifuged at 25 ° C. and 8,000 rpm for 10 minutes to obtain only plasma components. Anticoccidial surface antigen G1 or G2 antibodies contained in this plasma were searched by radioimmunoassay (Ausab Kit; Abbott, USA) as follows.

The composition of the solution (total 1 ml) administered at each time period is as follows.

(1) 1, 2, 3 and 4 doses

             Experimental group: Control group:

Surface antigen 10-100 ㎍ / ㎏ weight-

  50 μl CFA 50 μl

 Proper amount of saline

(2) At the fifth dose, the adjuvant was changed from CFA to IFA.

Example 7-2 Radioimmunoassay

Beads bearing surface antigen G1 were placed in the wells, and the plasma obtained from 10 experimental groups and 10 control groups was diluted 100-fold and added to submerse beads, thereby inducing an 18-hour antigen-antibody reaction at room temperature. Samples in the wells were removed using an inhaler and the beads were washed four times with deionized water. 200 씩 of the reaction solution containing the surface antigen G1 protein labeled with I ¹²⁵ was added to the washed beads and allowed to react at room temperature for 4 hours. After the reaction, the beads were washed four times with deionized water, placed in a test tube, and measured by a gamma counter (Wallac, USA). The results are shown in Table 1 below.

As shown in Table 1, all of the plasma obtained from the 10 chickens of the experimental group showed a positive reaction, even if there is an individual difference, it can be seen that all showed an immune response.

division Anti-G1 antibody Anti-G2 antibody   Experimental group P1 4321.3 3983.1 P2 3592.4 3688.6 P3 6920.1 5832.8 P4 5421.5 4144.2 P5 4823.6 4853.1 P6 4867.2 3891.4 P7 3987.5 3906.8 P8 6103.2 4122.1 P9 5815.3 4782.1 P10 5991.4 3664.7   Control C1 100.3 101.1 C2 81.2 90.4 C3 124.5 106.8 C4 150.6 115.2 C5 194.5 132.1 C6 98.4 128.1 C7 113.4 98.5 C8 107.2 109.2 C9 98.5 111.9 C10 132.3 102.3

As described in detail above, the present invention provides a novel polynucleotide encoding the spores surface antigen of the coccidium protozoa and a plant expression vector of the spores surface antigen of the recombinant coccidium protozoa comprising the same. The present invention also provides a transient transfected plant that temporarily expresses the spores surface antigen of the recombinant coccidium protozoa and a transformed plant that stably expresses the spores surface antigen of the recombinant coccidial protozoa. On the other hand, the present invention provides a method for producing a spore body surface antigen of recombinant coccidium protozoa. According to the present invention, not only the spores surface antigens of the coccidium protozoa can be produced in large quantities at low cost, and the produced spores surface antigens of the coccidium protozoans exhibit the same biological properties and functions as those of the native coccidium protozoa Induces an excellent immunization response to coccidium protozoa.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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Claims (11)

A polynucleotide encoding the spherosomal surface antigen of a coccidial protozoa represented by SEQ ID NO: 51-700 in SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NOs: 38-430 in SEQ ID NO: 3. (Iii) the polynucleotide of claim 1; (Ii) a promoter operably linked to the polynucleotide of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3'-non-detoxification site that acts on plant cells to cause 3'-terminal polyadenylation of the RNA molecule, and the plant expression vector of the spores surface antigen of the recombinant coccidial protozoan. A method for preparing a transient transfected plant that temporarily expresses a spore body surface antigen of a recombinant coccidium protozoa comprising the following steps: (a) introducing the plant expression vector of claim 2 into plant cells of a mature plant leaf; And (b) confirming whether the gene has been introduced into the plant cell. A transient transfected plant prepared by the method of claim 3, wherein said transgenic plant transiently expresses the spore body surface antigen of recombinant coccidium protozoa, selected from the group consisting of tobacco, melon, cucumber, watermelon and rapeseed. A method for preparing spore body surface antigen of recombinant coccidial protozoa comprising the following steps: (a) introducing the plant expression vector of claim 2 into plant cells of a mature plant leaf; (b) confirming whether the gene has been introduced into the plant cell; And (c) obtaining the spore body surface antigen of the recombinant coccidium protozoan from the plant including the plant cell into which the gene is introduced. A method for producing a transformed plant stably expressing the surface antigen of the spores of recombinant coccidium protozoa comprising the following steps: (a) introducing the plant expression vector of claim 2 into explant plant cells of a leaf disk, cotyledon or hypocotyl; (b) selecting the transformed plant cells; And (c) regenerating the plant from the transformed plant cells to obtain a transformed plant. A transgenic plant prepared by the method of claim 6, which stably expresses the spore body surface antigen of the recombinant coccidi protozoan selected from the group consisting of tobacco, melon, cucumber, watermelon and rapeseed. A method for preparing spore body surface antigen of recombinant coccidial protozoa comprising the following steps: (a) introducing the plant expression vector of claim 2 into explant plant cells of a leaf disk, cotyledon or hypocotyl; (b) selecting the transformed plant cells; (c) regenerating the plant from the transformed plant cells to obtain a transformed plant; And (d) obtaining the spore body surface antigen of recombinant coccidial protozoa from the transgenic plant. Spore body surface antigen of recombinant coccidial protozoa prepared by the method of claim 5 or 8. (a) spore body surface antigen of recombinant coccidial protozoa prepared by the method of claim 5 or 8; And (b) a pharmaceutically acceptable carrier. A method of inducing immunization against coccidiosis by inoculating a bird of the vaccine of claim 10.

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