# Patent

## Title
DNA encoding an antigenic protein of Eimeria apical membrane antigen 1 and use thereof

## Abstract

This invention provides a novel DNA for preventing avian coccidiosis and use thereof. The novel DNA encodes an antigenic protein of Eimeria apical membrane antigen 1, and is used for DNA vaccine and/or for vaccine comprising the recombinant protein deduced from the said DNA sequence as a main ingredient. The chickens immunized with vaccines described herein can be induced immunity against coccidiosis.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is in the field of avian coccidiosis and is related to a DNA molecule encoding an antigenic protein of Eimeria apical membrane antigen 1 and use thereof.
Coccidiosis is an intestinal disorder of poultry and causes an assortment of problems in the infected host. These problems range from poor feed conversion ratios in light infections to acute death in heavier infections.
Coccidiosis is caused by protozoans belonging to the genus Eimeria. The members of this genus in poultry are E. acervulina, E. tenella, E. maxima, E. necatrix, E. brunetti, E. mitis and E. praecox. Some investigators include E. mivati and E. hagani into the member. All of these species have similar life cycles but display different tissue specificity and pathogenicity. A broiler chicken will be subjected to a great deal of damage by E. acervulina or E. maxima because they parasitize large portions of the small intestine, where food digestion plays a major role.
Coccidiosis can be controlled by the administration of anti-coccidial agents. However, drug resistant strains arise at a frequent rate and the cost of development of new drugs is quite high. In addition, a number of these agents leave residues in the meat, which might give problems on consumer.
Attempts have been made to prevent the disease by vaccinating chickens with live attenuated strains of Eimeria or inactivated parasites. These live attenuated strains such as precocious lines are obtained by inoculating chickens with oocysts of a wild Eimeria species and collecting the very first parasite that are excreted as a result of infection (J Parasitol. 1975, 61: 1083-1090). However, such attenuated live vaccines produce fewer parasites and give an appreciable disease effect to vaccinated chickens. On the other hand, a protection level using the latter (inactivated vaccine) is far from complete. Furthermore, the disadvantage of these vaccines is expensive to be produced because a large-scale production of these vaccines needs a lot of live chickens.
An alternative solution would be to produce, by genetic engineering, the protective antigens of Eimeria parasites. Once developed, these immunogens could be produced cheaply in a prokaryotic or eukaryotic culture system in an unlimited supply and used to vaccinate chickens against coccidiosis.
2. Related Art
Several protective antigen genes of Eimeria have been reported. For examples, Jenkins et al. reported screening using a rabbit serum against the membrane fraction of E. acervulina, and a part of the cDNA encoding a 250-kDa protein in parasite surface (Exp. Parasitol. 1988; 66: 96-107, U.S. Pat. No. 5,122,471). Some Eimeria antigen genes were screened using monoclonal antibodies to Eimeria parasites instead of antisera (U.S. Pat. Nos. 5,028,694, 5,279,960, 5,814,320, 5,449,768). However, these antigens could elicit only partial protection against Eimeria infection to chickens immunized with a recombinant protein or recombinant virus expressing the antigen (U.S. Pat. Nos. 5,387,414, 5,403,581, 5,602,033, 6,001,363).
Plasmodium that causes human malaria is closely related to Eimeria, and both parasites belong to the phylum of Apicomplexa. Malaria is one of three major infection diseases of human, and there are an estimated 500 million infected persons, with 1-2 million dying annually. Therefore, many research teams focus on developing malaria vaccine to control infection. Plasmodium apical membrane antigen 1 is currently, one of the most promising antigens for Malaria vaccine (Molecular Microbiology (2004) 52, 159-168, The Journal of Immunology 172 (2004) 6167-6174, and Infect. Immun. 72 (2004) 4464-4470). The Eimeria apical membrane antigen 1 (AMA-1) gene is expressed only small amount in the specific organ of Eimeria. Therefore the purification of the antigen was difficult, and full amino acid sequence was unknown. Because there was no purified AMA-1, it was difficult to get the AMA-1 gene by the general screening method that is to obtain an antibody against the antigen and to screen a cDNA library with the antibody.
As described above, there is no satisfied vaccine against Avian Coccidiosis. In addition that, a DNA molecule encoding Eimeria AMA-1 protein has not been cloned and known in terms of usefulness for vaccine antigen.
SUMMARY OF THE INVENTION
Accordingly, an object of the present invention is to provide a means for identifying and cloning of cDNA encoding Eimeria AMA-1. A further object of the present invention is to provide a DNA molecule encoding Eimeria AMA-1 and use thereof. Still further objects of the present invention are to provide methods and vaccines useful for protecting chickens against avian coccidiosis.
The present invention provides a DNA molecule encoding an antigenic protein of Eimeria AMA-1, specifically Eimeria tenella AMA-1. More especially, the DNA sequence encodes the Eimeria tenella AMA-1 protein comprising of the amino acid sequence shown in SEQ. ID. No. 2.
Additionally, the present invention provides a DNA molecule encoding an antigenic protein of Eimeria AMA-1, wherein the antigenic protein comprises of an amino acid sequence, which is modified by insertion, deletion, and/or substitution of one or more amino acids in the sequence shown in SEQ ID No. 2, or wherein the antigenic protein with more than 90% identity to the amino acid sequence shown in SEQ ID No. 2.
Moreover, the present invention provides a DNA molecule encoding an antigenic protein of Eimeria AMA-1, wherein said the DNA molecule is hybridized to the region of open reading frame in the DNA shown in SEQ ID No.1 under high stringent condition.
A DNA vector containing the DNA molecule described above is also disclosed herein, and can be used as DNA vaccine for avian coccidiosis.
The present invention provides an antigenic protein of Eimeria AMA-1 and a method of producing it.
The protein is also useful for protecting chickens against Avian coccidiosis.
DETAILED DESCRIPTION OF THE INVENTION
Antigenic Protein of Eimeria Apical Membrane Antigen 1
Eimeria apical membrane antigen 1 (AMA-1) of the present invention is derived from Eimeria, avian parasite, and does, more specifically, comprise the amino acid sequence of SEQ ID No.2.
However, the antigenic protein of Eimeria AMA-1 is not limited only to AMA-1, but includes the protein that comprises the amino acid sequence modified by substitution, deletion, addition and/or insertion of one or more amino acids in SEQ ID No.2, so long as it could induce the similar antibodies into chicken as those induced into chicken immunized with Eimeria AMA-1. The sample of the said antigenic protein is one having the same epitope as Eimeria tenella AMA-1 comprising the amino acid sequence shown in SEQ ID No. 2, and one example is the recombinant protein described in Example 3, which is containing from the 57th to 276th amino acid of SEQ ID. No. 2.
The numbers of the above amino acid modification are preferable, less than 10%, more preferably less than 5% to the whole of SEQ ID No.2, and are most preferably less than 10 amino acids.
The antigenic protein of Eimeria AMA-1 of the present invention is also encoded to the DNA molecule that is hybridized to the DNA molecule of the open reading frame in SEQ ID No.1 under high stringent condition. In this case, the high stringent condition is equivalent to “0.2×SSC and 68° C.”
Moreover, antigenic proteins of Eimeria AMA-1 of the present invention include the protein comprising amino acid sequence that is more than 90% identity to the amino acid sequence shown in SEQ ID No. 2, and being antigenic protein of Eimeria tenella AMA-1. Preferably, the identity is more than 95% at least.
DNA Molecule Encoding an Antigenic Protein of Eimeria AMA-1
The DNA molecule of the present invention encodes an antigenic protein of the aforesaid AMA-1 of Avian parasite Eimeria. A specific example of a DNA of the prevent invention is the one encoding the Eimeria AMA-1 comprising amino acid sequence shown in SEQ ID No. 2. More specifically, a DNA molecule of the present invention is that having a nucleotide sequence shown in SEQ ID No. 1.
However, the DNA of the present invention is not limited only to that having the sequence of SEQ ID No. 1, but includes a DNA encoding a protein that comprises the amino acid sequence modified by substitution, deletion, addition and/or insertion of one or more amino acids in SEQ ID No. 2, so long as the protein could induce the similar antibodies into chicken as those induced into chicken immunized with Eimeria AMA-1.
The numbers of the above amino acid modification are preferably, less than 10%, more preferably less than 5% to the whole of SEQ ID No. 2, and are most preferably less than 10 amino acids.
The DNA molecules encoding an antigenic protein of Eimeria AMA-1 of the present invention also include a DNA molecule that is hybridized to the DNA molecule of the open reading frame in SEQ ID No. 1 under high stringent condition. In this case, the high stringent condition is equivalent to “0.2×SSC and 68° C.”
Moreover, the DNA molecules encoding an antigenic protein of Eimeria AMA-1 of the present invention include a DNA molecule encoding the protein having the amino acid sequence that is more than 90% (more preferably more than 95%) identity to the amino acid sequence shown in SEQ ID No. 2, and being antigenic protein of Eimeria tenella AMA-1. In this case, identity is calculated using Basic Local Alignment Search Tool (BLAST).
One of the methods to get the above DNA molecule that is hybridized to the DNA molecule of the open reading frame in SEQ ID No.1 under high stringent condition, or that is encoding the protein of amino acid sequence that is more than 90% identity to the amino acid sequence shown in SEQ ID No. 2 is modification to the DNA molecule of SEQ ID No. 1. To modify the DNA molecule, site-specific mutation using primer and/or random mutations are known in the art: see e.g. Sambrook et al Chapter 13 of Molecular Cloning: A Laboratory Manual. 3rd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 2001.
Vector and Construction of it
A DNA vector of the present invention is the vector containing the DNA molecule as described above. To get a DNA vector of the present invention, DNA having an artificial nucleotide sequence can be connected to the DNA molecule of the prevent invention. The sample of the DNA for connection is a linker used popularly in genetic engineering. The linker is at least one or two more nucleotide(s) that is/are not naturally connected to the DNA molecule of the present invention, and be appropriately designed depending on the site of the vector to be inserted.
The DNA vector in which the DNA molecule of the present invention is integrated can be chosen from a plasmid such as pBR322, pBR325, pUC7, pUC8, pUC18, pUC19, pBluescript or pGEM, a cosmid such as pHC79, or a phage such as λ or M13 phage. The vector is digested with appropriate restriction enzyme(s) and the DNA molecule of the present invention or other necessary DNA such as linker is inserted therein by the standard procedure.
A DNA vector to construct the expression vector for an antigenic protein of Eimeria AMA-1 is not limited, and may be chosen from the aforesaid vectors. A DNA vector to express the objective protein as a fusion protein with a Tag, by which the objective protein could be affinity-purified, is more suitable. For examples of such a vector, pGEX vector (AMERSHAM BIOSCIENCES Corp.) or pQE vector (QIAGEN Inc.) is commercially available. One example is pGEX-6p-3 described in EXAMPLE 3.
The transcriptional regulation region like a promoter or terminator is necessary to be included in the expression vector. The suitable promoter differs in host-by-host cell. For example, lac, tac or T5 promoter is used as promoter in the case of E. coli as host cell. OAX1 or GAPDH promoter in the case of Yeast, polyhedrin promoter in the case of insect cell, or cmv or β-actin promoter in the mammalian cells are suitable.
Host Cell
Using the resulting expression vector, a variety of host cells can be appropriately transformed to obtain a microorganism or cells capable of producing an antigenic protein of Eimeria AMA-1 or a recombinant protein comprising a part of SEQ ID No.2 and a Tag sequence.
Host cells used in herein can be chosen in terms of compatibility of the expression vector, suitability of the products, etc. and may be either prokaryotic or eukaryotic cells. Specific examples of the host cells include bacteria such as the genus Escherichia (e.g., E. coli) or the genus Salmonella (e.g., Salmonella typhimurium), and lower eukaryotic cells such as yeast (e.g., Saccharomyces cerevisiae) or fungi (e.g., Penicillium).
And examples of the host cells in higher eukaryotic cells are insect cells, Chinese hamster ovary (CHO) cells, CEF cells, or human cell lines (e.g., HeLa).
To transduce an expression vector said above into some host cells, appropriate methods well known to one skilled in the art according the host cells can be used.
Expression of an Antigenic Protein of Eimeria AMA-1
The host cells transformed with an appropriate expression vector can be cultured and proliferated under incubation conditions well known to one skilled in the art.
For example, the transformed E. coli can be well grown in LB medium at 37° C. under aerobic conditions. In producing an antigenic protein of Eimeria AMA-1, the condition for the induction of the protein can be chosen according to the used promoter. In the case of E. coli lactose promoter and operator system, as a specific example, it is achieved by adding an appropriate amount of isopropyl-1-thio-β-D-galactopyranoside (IPTG) to a culture medium.
A method to purify the antigenic protein of Eimeria AMA-1 is not particularly limited, but any known method is applicable to the purification in combination with techniques well known in this field. When the antigenic protein of Eimeria AMA-1 is expressed as a fusion protein containing some Tag that can be used in purification through an affinity column, the affinity column is a very convenient tool. For example, the antigenic protein of Eimeria AMA-1 expressed as fusion with glutathione S-transferase (GST) using pGEX vector could be purified easily through Glutathione Sepharose 4B column (AMERSHAM BIOSCIENCES Corp).
Vaccines Against Coccidiosis
Vaccines against coccidiosis of the present invention include DNA vaccine and protein vaccine. The DNA vaccine of the present invention can induce the immunity into chickens which are immunized with it. On the contrary, protein vaccine contains the antigenic protein of Eimeria AMA-1 of the present invention, which can induce the immunity into chickens.
The DNA vaccine of the present invention contains the DNA molecule of the present invention, and specifically can be as a main ingredient the recombinant DNA vector such as plasmid into which the DNA molecule of the present invention is inserted. The said recombinant DNA vector can be the expression vector for an antigenic protein of Eimeria AMA-1 said above, and is not limited only to it as far as the DNA vaccine can express the antigenic protein gene of Eimeria AMA-1 in the immunized chicken.
The DNA vaccine can contain any other ingredient besides the DNA molecule encoding the antigenic protein of Eimeria AMA-1. As the said other ingredient, CpG oligonucleotide, which is recognized by toll-like receptor on the cell surface and can activate cell-mediated immunity, or stabilizer additive can be used after being solubilized in the phosphate buffer saline (PBS).
The administration method of the DNA vaccine of the present invention is not particularly limited, and a general method of intramuscular, intravenous or subcutaneous injection is listed. The DNA vaccine is administered with 50 μl of 0.1 to 10 mg/ml, more preferably of 0.5 to 5 mg/ml per a bird.
The protein vaccine of the present invention consists chiefly of the antigenic protein of Eimeria AMA-1, and can contain any ingredients such as saline, adjuvant, and/or preservatives as long as the DNA vaccine can be safely immunized to chickens.
The administration method of the protein vaccine of the present invention is not particularly limited as far as it can induce immune responses to the administered chicken. It is preferable that the protein vaccine is injected with adjuvant, because it can induce strong immune responses to the injected chicken. Adjuvant can be selected among numerous ones well known in this art. For example, oil adjuvant or aluminum hydroxide. The amount of the injecting protein vaccine is not also limited. For example, protein suspended with PBS of more than 0.02 ml of more than 0.1 mg/ml can be injected, and in this case, the booster immunization is preferable.



BRIEF DESCRIPTION OF THE DRAWING FIGURES
FIG. 1 Results of tBLASTn search of Eimeria EST database using N-terminal amino acid sequence of PfAMA-1, in which BG725003 corresponds to SEQ ID NO: 16 and BG724708 corresponds to SEQ ID NO: 17
FIG. 2 Results of tBLASTn search of Eimeria tenella genome database using amino acid sequence of TgAMA-1. In FIGS. 2A-C, TgAMA1 corresponds to SEQ ID NO: 18 and c001002545 Contig 1 corresponds to SEQ ID NO: 19. In FIG. 2D, c008400755.Contig1, a genome sequence of Eimeria tenella, corresponds to SEQ ID NO: 20.
FIG. 3A illustrates portions of c001002545.Contig1, a sequence of the Eimeria tenella genome, and corresponds to nucleotides 1 to 1,196 of SEQ ID NO: 19.
FIG. 3B illustrates c0008400755.Contig1, a genome sequence of Eimeria tenella, and corresponds to SEQ ID NO: 20.
FIG. 4 Results of homology search using amino acid sequence of Eimeria tenella AMA-1, in which EtAMA1 corresponds to SEO ID NO: 2 and TgAMA1, the amino acid sequence of Toxoplasma gondii AMA-1 protein, corresponds to SEQ ID NO: 18.
FIG. 5 Construction of the plasmid pGEX-EtAMA



LIST OF THE SEQ ID SEQUENCES
SEQ ID No. 1: cDNA Sequence of Eimeria tenella AMA-1gene
SEQ ID No. 2: Amino acid sequence of Eimeria tenella AMA-1 protein
SEQ ID No. 3: PCR primer BG708-F
SEQ ID No. 4: PCR primer BG003-R
SEQ ID No. 5: PCR primer BG003-F
SEQ ID No. 6: PCR primer c545-1F
SEQ ID No. 7: PCR primer c545-2R
SEQ ID No. 8: PCR primer c755-4F
SEQ ID No. 9: PCR primer c755-4R
SEQ ID No. 10: PCR Primer for site-specific mutagenesis EcoAMA1
SEQ ID No. 11: PCR Primer for site-specific mutagenesis AMA1Sal
SEQ ID No. 12: PCR Primer for site-specific mutagenesis XbaAMA1
SEQ ID No. 13: PCR Primer for site-specific mutagenesis AMA1endSal
SEQ ID No. 14: Oligonucleotide for adjuvant CpG
SEQ ID No. 15: Oligonucleotide for adjuvant CpG-30
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The plasmid construction was essentially performed by the standard molecular biology techniques (Molecular Cloning: A Laboratory Manual. 3rd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 2001). DNA restriction fragments were electrophoresed on agarose gels and purified with QIAquick Gel Extraction Kit (QIAGEN, Cat # 28704).
Polymerase chain reaction (PCR) was conducted, unless there is particular mention in the statement, using ExTaq polymerase (Product #: RR001A, TaKaRa Bio, Shiga, Japan) under the condition of 30 temperature cycles consisting of denaturation at 94° C. for 1 minute, annealing at 55° C. for 2 minute, and extention at 72° C. for 3 minute.
EXAMPLE 1
Cloning of cDNA Encoding Eimeria AMA-1
1.1 Preparation of Eimeria Tenella cDNA Library
Sporozoites were prepared from 1×108 oocysts of Japanese field isolated strain of Eimeria tenella (sharing from Research Institute for Animal Science, Kanagawa, Japan) by the common procedure of this field, washed with PBS, and lysed with 0.5 ml of lysis buffer (4M guanidine thiocyanate, 25 mM sodium citrate, 0.5% sodium lauryl sarcosinate, and 0.1 M β-mercaptoethanol). Messenger RNA was purified on an oligo(dT) column (FastTrack 2.0 mRNA Isolation Kit, Invitrogen Carlsbad, Calif.) and used as a template for cDNA synthesis (cDNA Synthesis Kit, Product #: 6120, Takara Bio, Shiga, Japan). After double stranded cDNA synthesis, cDNA library was prepared using cDNA library Kit (Product #: 6119, TaKara Bio, Shiga, Japan) and cassette adapter in the kit.
1.2 Searching of Eimeria EST Database and Cloning 5′ End of Eimeria AMA-1 Gene
Malaria parasite, Plasmodium falciparum AMA-1 (PfAMA-1) gene and protein sequences had been published yet. Eimeria EST database was searched with translating BLAST (tBLASTn) tool (E. tenella OmniBlast of the Sanger Institute) using N-terminal 392 amino acid sequence of PfAMA-1 (Protein database Accession #: AAA29475), and resulted findings shown in FIG. 1.
High score EST sequences shown in FIG. 1A were aligned mutually. The results indicated that a part (394 bp) of BG725003 was identical to that of BG724708 as shown in FIG. 1B. Two PCR primers (BG708-F and BG003-R) were designed from the part before and after the identical region (underlined sequence in FIG. 1B). Sequences of these PCR primers were shown in SEQ ID No. 3 and 4, respectively. PCR was conducted using these PCR primers (BG708-F and BG003-R) and cDNA library described above as a template.
The PCR amplified a DNA fragment of about 0.6 kbp, which was ligated into the plasmid vector of PCR Script Amp cloning Kit (STRATAGENE, Product #: 211188). The resultant transformants were cultured and each plasmid was purified. The insert of each plasmid was sequenced, and a plasmid having the DNA fragment which encoded 5′ non-translational region of 181 bp and N-terminal region (151 amino acids) of open reading frame (ORF) homologous to PfAMA-1 was found. The plasmid was named as pPCR-EtAMA1-N.
1.3 Cloning of Middle and 3′-End Regions of Sequence-Unknown Eimeria AMA-1
3′ rapid amplification of cDNA end (3′ RACE) was conducted using a new primer (BG003-F; SEQ ID No. 5) which existed in 3′-end of the insert of pPCR-EtAMA1-N, and the oligo (dT) primer being present in the cDNA Synthesis Kit described above (Takara Bio, Product #: 6120), but unfortunately no DNA was amplified. Therefore, as the next trial, Eimeria EST database was searched with translating BLAST (tBLASTn) tool (E. tenella OmniBlast of the Sanger Institute) using the amino acid sequence of 150th to 240th of Toxoplasma gondii AMA-1 (TgAMA-1) (Protein database Accession #: AAB65410), and consequently it was found that the amino acid sequence deduced from 753rd to 989th nucleotide sequence of c001002545. Contig1 was homologous to that of TgAMA-1 as shown in FIG. 2A.
Furthermore Eimeria EST database was also searched with translating BLAST (tBLASTn) tool (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/e—tenella/omni) using the amino acid sequence of 240th to 420th of TgAMA-1, and consequently it was found that the amino acid sequence deduced from 990th to 1046th nucleotide sequence of c001002545.Contig1 was homologous to that from 260th to 276th of TgAMA-1 as shown in FIG. 2B. The amino acid sequence from 260th to 276th of TgAMA-1 was found to be homologous to that deduced from 1255th to 1305th of c001002545.Contig1 as shown in FIG. 2C. Moreover, the amino acid sequence from 336th to 395th of TgAMA-1 was homologous to that deduced from 711st to 878th nucleotide sequence of c008400755.Contig1 as shown in FIG. 2D.
Considering existence of intron from the above results and the rule of splicing signal, an exon-intron structure of Eimeria AMA-1 gene was presumed as shown in FIG. 3. From this presumption, four primers (c545-1F, c545-2R, c755-4F, c755-4R: Each sequence is SEQ ID No. 6 to 9, respectively) were designed.
PCR was conducted using cDNA library described in EXAMPLE 1.1 as a template, and PCR primer set of c545-1F and c545-2R. The DNA fragment of about 450 bp was amplified. In the case of PCR primer set of c545-1F and c755-4R, the DNA fragment of about 850 bp was amplified, inserted into pBluescript SK(+) plasmid vector (STRATAGENE, product #: 212205), and sequenced. As the inserted DNA seemed to encode a central region of Eimeria AMA-1, the plasmid was named as pBS-EtAMA1-M.
Next, 3′ RACE was conducted using cDNA library described in EXAMPLE 1.1 as a template, a primer set of c755-4F and the oligodT primer in the cDNA Synthesis Kit described above (Takara Bio, Product #: 6120), and consequently a DNA fragment of about 630 bp was amplified. The DNA fragment was also inserted into pBluescript SK(+), and sequenced. The sequence data indicated that the insert was the DNA containing C-terminus region of Eimeria AMA-1 and 3′ non-translational region. The inserted plasmid was named as pBS-EtAMA1-C.
The inserts of three plasmids, pPCR-EtAMA1-N, pBS-EtAMA1-M, and pBS-EtAMA1-C were 5′-terminus, center, and 3′-terminus of cDNA encoding Eimeria tenella AMA-1, respectively, from their sequence data. As three inserts had overlapping ends, where there were restriction enzyme sites, they were connected to one continuous cDNA (sequence of which is shown in SEQ ID No.1) using these sites. The plasmid containing the continuous cDNA was named as pBS-EtAMA1.
EXAMPLE 2
Nucleotide/Amino Acid Sequence Analysis of Eimeria AMA-1
The DNA molecule of SEQ ID No. 1 has total 2,002 nucleotides, which contain 5′ non-translational region of 126 bp, one open reading frame of 536 amino acids, and 3′ non-translational region including polyA signal. The amino acid sequence is shown in SEQ ID No. 2.
Protein-protein BLAST (BLASTp) search (E. tenella OmniBlast of the Sanger Institute) was conducted using the amino acid sequence shown in SEQ ID No. 2. No identical protein was found in Protein Data Bank, but the most similar protein was TgAMA-1 (E value of 9e-92 and 52% homology). The BLASTp search results were shown in FIG. 4. Outside of TgAMA-1, although the homology was less than 50%, several AMA-1 of Plasmodium species were hit as proteins having amino acid sequence homology to that of SEQ ID No. 2, which also strongly supports that the protein of SEQ ID No. 2 is Eimeria AMA-1. Up to current times, the nucleotide or amino acid sequence of Eimeria AMA-1 has not been published yet, and the sequence of this invention is the first report about full sequence data of Eimeria AMA-1.
From the amino acid sequence profile of SEQ ID No. 2, Eimeria AMA-1 includes a signal peptide at N-terminus (4th to 25th), and trans-membrane domain at the C-terminus (445th to 467th). Therefore it is guessed that this protein exists on the surface of Eimeria parasite and its C-terminus (from 468th to the end) is the cytoplasmic domain.
EXAMPLE 3
Production of Antigenic Protein of Eimeria AMA-1
3.1 Construction of an E. coli Expression Vector for Eimeria AMA-1
To express an antigenic protein of Eimeria AMA-1 as a GST fusion protein, an E. coli expression vector pGEX-EtAMA was constructed as follows (FIG. 5). To ligate a DNA encoding Eimeria AMA-1 into a pGEX-6p-3 vector (AMERSHAM #27-4599-01) containing GST gene, a DNA fragment of about 1.1-kbp was amplified by PCR using pBS-EtAMA1 as a template, and EcoAMA1 (SEQ ID No. 10) and AMA1Sal (SEQ ID No. 11) as a set of primers.
The amplified DNA fragment was cut with EcoRI and SalI, fractionated by an agarose gel electrophoresis, and purified with QIAquick Gel Extraction Kit. Plasmid pGEX-6p-3 was also cut with EcoRI and SalI, fractionated by an agarose gel electrophoresis, and purified with QIAquick Gel Extraction Kit. These recovered DNA fragments were ligated to generate pGEX-EtAMA, and transferred into competent E. coli cells of JM109 (TaKaRa Bio Inc., Japan). The resulting ampicillin-resistant transformants were randomly picked up and cultured with each 2-ml LB broth containing ampicillin (LB+amp). Each plasmid was prepared by the standard (alkaline lysis) methods, and analyzed by restriction enzyme cutting. An objective plasmid selected by restriction enzyme analysis was sequenced with two sequence primers of AMERSHAM #27-1410-01 and 27-1411-01 to confirm that Eimeria AMA-1 gene was inserted in frame into the PGEX vector.
3.2 Expression of an Antigenic Protein of Eimeria AMA-1
E. coli BL21 cells (AMERSHAM #27-1542-01) were transformed with the pGEX-EtAMA. The resulting transformants were cultured with LB+amp broth for 16 hours, inoculated into fresh 100 volumes of LB+amp broth and continues to be culture for two hours. They were cultured for more three hours after adding IPTG to a final concentration of 1 mM, harvested by centrifugation, and lysed with Laemmli sample buffer (60 mM Tris-Cl (pH6.8), 25% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.01% bromphenol blue) by boiling at 100° C. The lysate was applied to 12.5% SDS-polyacrylamide gel electrophoresis (PAGE), and the gel was stained with 0.05% Coomassie brilliant blue (CBB) solution. The expected protein of about 53-kDa was induced very well as a GST-fusion protein.
3.3 Purification of an Antigenic Protein of Eimeria AMA-1
BL21 cells transformed with pGEX-GAPDH were cultured in a 2-ml LP+amp broth for 16 hours, and inoculated into fresh 100-ml LB+amp broth and continued culture for two hours. They were cultured for more three hours after adding IIPTG to a final concentration of 1 mM, harvested and wash with Phosphate buffer saline (PBS) twice. The cell pellet was suspended with Lysis buffer (50 mM Tris-Cl (pH8.0), 150 mM NaCl, 1 mM EDTA, 0.2 mM 4-2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (Merck Ltd., Japan; product Name: Pefabloc SC), 1.5 mg/ml lysozyme), and shaken slowly for one hour and more 20 minutes followed by adding TRITON X-100, a non-ionic surfactant (octylphenoxypolyethoxyethanol), to a final concentration of 0.3%. The suspension was transferred to a tube and centrifuged at 12,000×g for 30 minutes. The pellet and supernatant were separated and applied to 12.5% SDS-PAGE. Most of fusion proteins were found in the pellet fraction.
The pellet was suspended with Laemmli sample buffer, boiled and applied to 8% SDS-PAGE. The objective fusion protein of 53-kDa was eluted and fractionated from the gel region in which the protein existed using Mini Whole Gel Eluter (Bio-Rad) with the elution buffer (60 mM Tris-Cl (pH 8.7), 25 mM boric acid). Recovered proteins in the fractions were checked by a 12.5% SDS-PAGE and CBB staining, and the fraction in which the objective protein existed and other proteins were not contaminated was selected. The antigenic protein of Eimeria AMA-1 of about 1 mg was purified from BL21 transformants cultivated with LB+amp broth of 100 ml as described herein.
EXAMPLE 4
Immune Response of Chickens Vaccinated with Antigenic Protein of Eimeria AMA-1
The antigenic protein of Eimeria AMA-1 (0.3 mg/ml) purified as described in the above Example 3 was mixed with equal volume of Freund's complete adjuvant (FCA; Sigma) to form 2 ml micelles. Ten chickens of 4 weeks old were subcutaneously immunized (sc) with each of 0.2 ml micelles, and boosted sc two times at intervals of a week with the same amount of AMA-1 protein and incomplete Freund's adjuvant (IFA; Sigma).
Ten days later after the last immunization, the immunized chickens were bled for about 2 ml each, and the peripheral blood lymphocytes and sera were prepared by the standard techniques. From five non-immunized chickens, the peripheral blood lymphocytes and sera were also prepared as negative control samples.
Antibodies in the sera against E. tenella sporozoites was checked by western blotting method. E. tenella of 1×107 sporozoites were lysed with Laemmli sample buffer and boiled for 5 min, and applied to 12.5% SDS-PAGE. After PAGE, proteins of sporozoites were blotted to a PVDF membrane (MILLIPORE; product name: Immobilon). After blotting, the membrane was dried and cut into total 12 pieces. Each piece was incubated with each serum (1:500 dilution) for 1 hour at room temperature (RT). After washing with PBS twice, the membrane was incubated with Goat anti-Chicken IgG (H+L) alkaline phosphatase conjugated (1:1,000 dilution; Bethyl, Inc. Catalog #A30-106AP) for 1 hour at RT, washed with PBS several times, and developed with the substrate solution of bromochloroindolyl phosphate/nitro blue tetrazolium (BCIP/NBT). All of 10 sera prepared from immunized chickens reacted specifically to about 60-kDa protein of sporozoites, while two negative control sera did not, although there were some proteins reacted nonspecifically. These results indicated that the antigenic protein of Eimeria AMA-1 could induce humoral immunity to chickens and the Eimeria AMA-1 of about 60-kDa is expressed in the E. tenella sporozoites.
EXAMPLE 5
Immune Responses of Chickens Injected with DNA Vaccine Containing Eimeria AMA-1 Gene
The plasmid, pNZ45/46BacpA described in Examples of U.S. Pat. No. 6,764,684 was used as the backbone plasmid vector for DNA vaccine.
To get the DNA fragment containing restriction enzyme sites before and after the ORF of Eimeria AMA-1, PCR was conducted using pBS-EtAMA1 as the template, and primer set of XbaAMA1 (SEQ ID No. 12) and AMA1endSal (SEQ ID No. 13).
The amplified DNA of 1.6 kbp was cut with XbaI and SalI, and inserted into XbaI/SalI sites of pNZ45/46BacpA (7.4 kb). The resultant plasmid p45BacEtAMA1 (9 kbp) could be used as a DNA vaccine and/or a homologous vector for construction of recombinant herpes virus of turkey.
Next, to evaluate the plasmid for DNA vaccine, Eimeria tenella challenge to immunized chickens was conducted.
Fertilized SPF eggs of Line M were purchased from NIPPON INSTITUTE FOR BIOLOGICAL SCIENCE, Japan, and were hatched. The hatched chickens were divided into three groups. Plasmid p45BacEtAMA-1 of 1 mg and 0.1 mg each of adjuvant CpG (SEQ ID No. 14) and CpG-30 (SEQ ID No. 15) were diluted with 1 ml of PBS, and inoculated subcutaneously in the leg muscle of chickens of one group by in vivo electroporation (at 50V, 20 μsec three times and more three times at reverse polarity using Model ECM830; Gentronics Inc., Dan Diego, USA) twice at 2 and 9 days of age. Chickens of other two groups were not inoculated.
Four weeks later (at 37 days of age), body weights of all chickens were measured. Chickens except one of two non-immunized group were challenged orally (into maws of chickens) with 1,000 sporulated oocysts of E. tenella Rt7 strain (Japanese field isolated). Chickens not challenged were negative control (non-immunized and non-challenged). All chickens were observed until 8 days post challenge, and set up for measure of body weight. Feces were collected from 5 to 8 days post challenge, and fecal oocysts were checked by saturated saline floating method. When oocysts in feces were detected, fecal oocysts were counted microscopically after centrifugation in chromium sulfate solution, and calculated as number per gram-feces. The results of weight gains and discharged fecal oocyst numbers of each group are shown in Table 1.
Table 1 indicates that chickens immunized with DNA vaccine of this invention were statically different from non-immunized challenge control on both of discharged fecal oocyst number and weight gain, and that DNA vaccine could induce protective immunity against Eimeria tenella challenge.










TABLE 1









Number
Evaluation











of
Discharged



Group
birds
fecal oocyst *
Weight gain (%)





Non-immunized,
12
−
35.2 ± 3.8


non-challenged


control


Non-immunized
10
++
17.9 ± 2.0


control


Immunized with DNA
10
+
30.0 ± 3.0


vaccine





* Oocyst numbers per g-feces at 6 days post challenge:


(−) indicates under detectable (103);


(+) indicates less than 106;


(++) indicates over 106.

## Claims

1. An isolated DNA molecule encoding Eimeria tenella apical membrane antigen 1 comprising of the amino acid sequence of SEQ ID No: 2.

2. A DNA molecule encoding Eimeria tenella apical membrane antigen 1 , wherein the DNA comprises of the nucleotide sequence of SEQ ID No: 1.

3. A DNA vector containing the DNA molecule as described in claim 1.

4. An isolated host cell having a DNA vector as described in claim 3.

5. A DNA vector containing a DNA molecule as described in claim 2.

6. An isolated host cell having a DNA vector as described in claim 5.