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Title:  Chimeric gene formed of the DNA sequences that encode the antigenic determinants of four proteins of L. infantum, useful of serologic diagnosis of canine leishmaniosis and protein obtained

United States Patent:  6,525,186

Issued:  February 25, 2003

Inventors:  Alonso Bebate; Carlos (Madrid, ES); Requena Rolania; Jose Maria (Madrid, ES); Soto Alvarez; Manuel (Madrid, ES)

Assignee:  C.B.F. Leti S.A. (Madrid, ES)

Appl. No.:  788345

Filed:  February 21, 2001

Abstract

A chimeric polypeptide encoded by a chimeric gene formed by DNA sequences that encode four antigenic determinants of L. infantum is disclosed. These antigenic determinants are obtained from rLiP2a, rLiP2b, rLiH2A and rLiPO. The protein obtained, has a molecular mass of 38 KD with an isoelectric point of 7.37. This chimeric polypeptide is useful for diagnosing, preventing and/or treating leishmaniosis in animals or humans.

DESCRIPTION OF THE INVENTION

In a first aspect, the invention relates to a chimeric gene formed by the DNA sequences that encode antigenic determinants of four proteins of L.infantum, useful for the serum diagnosis of canine Leishmaniosis.

In a further aspect, the invention relates to a protein encoded by said chimeric gene, containing one or more of the antigenic determinants of four proteins of L. infantum encoded by the chimeric gene.

The invention further relates to a diagnostic method for determining the presence of canine Leishmaniosis in a human being or an animal, in particular a dog, and/or in samples of biological fluids derived from humans or animals, such as a blood sample. In this diagnostic method, the chimeric gene of the invention or the protein encoded by it can be used. Alternatively, in the diagnostic method, a nucleic acid probe sequences specific for the chimeric gene of the invention, or a part thereof, can be used, i.e. to establish the presence of canine Leishmaniosis in a patient or a sample.

Also, in the diagnostic method, antibodies against the protein encoded by the chimeric gene of the invention, or a antigenic part thereof such as an epitope, can be used.

The invention further relates to assays or other qualitative or quantitative methods for determining the presence of canine Leishmaniosis in a human being or an animal, in particular a dog, and/or in samples of biological fluids derived from humans or animals, such as a blood sample. Such assays can use the chimeric gene of the invention, the protein encoded by it, probes specific for the chimeric gene or part thereof, and/or antibodies directed to the protein encoded by the chimeric gene of the invention, or any antigenic part thereof. Such assays can further be carried out in a manner known per se, for instance for probe-hybridization assays or immunoassays.

In a further aspect, the invention relates to diagnostic kits, at least comprising either a chimeric gene of the invention, a protein encoded by said chimeric gene, a probe specific for the chimeric gene of the invention, or an antibody directed to the protein of the invention. The kits can further contain all components for diagnostic kits and/or diagnostic assays known per se.

It should be noted that when herein, reference is made to the chimeric gene of the invention, this term also encompasses nucleic acid sequences that can hybridize with the sequence mentioned below under moderate or stringent hybridizing conditions.

In this context, heterologous hybridisation conditions can be as follows: hybridisation in 6.times.SSC (20.times.SSC per 1000 ml : 175.3 g NaCl, 107.1 g sodium citrate.5H2O, pH 7.0), 0.1% SDS, 0.05% sodium pyrophosphate, 5* Denhardt's solution (100.times.Denhardt's solution per 500 ml : 10 g Ficoll-400, 10 g polyvinyl-pyrrolidone, 10 g Bovine Serum Albumin (Pentax Fraction V)) and 20 {character pullout}g/ml denatured herring sperm DNA at 56oC. for 18-24 hrs followed by two 30 min. washes in 5.times.SSC, 0.1% SDS at 56oC. and two 30 min. washes in 2.times.SSC, 0.1% SDS at 56oC.

For instance, sequences that can hybridize with the sequence mentioned below include mutant DNA sequences which encode proteins with the same biological function as the protein encoded by the sequence mentioned hereinbelow. Such mutant sequences can comprise one or more nucleotide deletions, substitutions and/or additions to the sequence mentioned below. Preferably, the mutant sequences still have at least 50%, more preferably at least 70%, even more preferably more than 90% nucleotide homology with the sequence given hereinbelow.

The term chimeric gene as used herein also encompasses nucleic acid sequences that comprise one or more parts of the sequence mentioned hereinbelow. Preferably, such sequences comprise at least 10% more preferably at least 30%, more preferably at least 50% of the nucleotide sequence given hereinbelow. Such sequences may comprise a contiguous fragment of the sequence mentioned hereinbelow, or two or more fragments of the sequence given below that have been combined in and/or incorporated into a single DNA sequence.

It should be noted that when herein, reference is made to a protein encoded by the chimeric gene of the invention, this term also includes mutant proteins that still essentially have the same biological function. Such mutant proteins can comprise one or more amimo acid deletions, substitutions and/or additions compared to the protein encoded by the sequence mentioned below. Preferably, the mutant proteins still have at least 50%, more preferably at least 70%, even more preferably more than 90% amino acid homology with the sequence given hereinbelow.

The term protein also encompasses fragments of the protein encoded by the chimeric gene of the invention. Such fragments preferably still show the biological activity of the full protein. Preferably, such proteins comprise at least 30%, more preferably at least 50% of the amino acid sequence of the full protein. Also, two or more fragments of the full protein encoded by the chimeric gene of the invention may be combined to form a single protein.

The invention also relates to a nucleotide sequence and to a protein useful for pharmacological purposes, in particular for the prevention and/or treatment of Leishmaniosis, in particular canine Leishmaniosis, having the DNA (SEQ ID NO:11) and amino acid (SEQ ID NO:12) sequence expressed in the vector PQ31. Amino acid residues 1-29 belong to the PQ31 vector and amino acid residues 30-35 belong to both PQ31 and pMal vectors or a mutant or fragment thereof that can be used for generating a protective immune response in a human or animal against Leishmaniosis, and to a pharmaceutical composition for the prevention and treatment, in humans or animals, of Leishmaniasis, comprising this protein or a mutant or fragment thereof that can be used for generating a protective immune response in a human or animal against Leishmaniosis. This protein is derived from the insertion of gene PQV in the expression vector pQE31. Here, said chimeric gene preferably encodes a polypeptide generated with a moleuclar weight of 38 kD and an isoelectric point of 7.37.

Probes of the invention are such that they can--most preferably selectively--hybridize with the chimeric gene of the invention or part thereof, in particular under moderate as stringent hybridizing conditions, such as those mentioned above. Preferably, a probe of the invention will be essentially homologous with the nucleotide sequence of the chimeric gene of the invention or a part thereof, i.e. show a homology of more than 80%, preferably more than 90%, more preferably more than 95%.

A skilled person will be able to select suitable probes. Usually, such probes will contain at least 15 bp, preferably more than 24 bp, of the sequence given hereinbelow.

The chimeric gene formed of the DNA sequences that encode the antigenic determinants of four proteins of L. infantum, useful for serological diagnosis of canine Leishmaniosis and protein obtained, that the invention proposes, in its own right constitutes an obvious novelty within its field of application, as according to the invention, a synthetic chimeric gene is produced that as it is obtained by assembly, containing the DNA region encoding the antigenic determinants specific to the proteins LiP2a, LiP2b, LiPO and H2A, thus constructing a protein rich in antigenic determinants. The chimeric gene obtained is expressed in Escherichia coli and the product has been analysed with respect to its antigenic properties. The results confirm that this chimeric protein maintains all the antigenic determinants of the parent proteins and that it constitutes a relevant diagnostic element for canine VL, with a sensibility that oscillates between 80% to 93%, and a specificity of between 96% to 100%.

More particularly, the chimeric gene formed by the DNA sequences that encode the antigenic determinants of four proteins of L. infantum, useful for the serological diagnostic of canine Leishmaniosis and protein obtained object of the invention, is produced by means of the following stages, namely:

Construction of the chimeric gene. Methodology.

Cloning strategy.

Cloning of DNA sequences that encode antigenic determinants of the histone protein H2A.

Cloning of the sequences that encode rLiP2a-Q and rLiP2b-Q.

Cloning of the sequence rLiPO-Q.

Cloning of the chimeric gene.

Construction of the chimeric gene from the construction of intermediate products.

Cloning of epitopes specific to the L. infantum antigens.

Construction of the final product

Construction of the chimeric gene that encodes a polypeptide that contains all the selected antigenic determinants.

Evaluation of the final product.

Serums.

Purification of proteins

Electrophoresis of proteins and immuno-analysis.

Measurements by Fast-ELISA

Evaluation of the final product.

Antigenic properties.

Sensitivity and specificity of the chimeric protein CP in the serum diagnosis of canine VL.

The strategy followed by the cloning of DNA sequences that encode each one of the selected antigenic determinants is the same in all cases, and in a first step, the sequence of interest is amplified by means of a PCR and the use of specific oligonucleotides that contain targets for restriction enzymes at the extremes.

For the cloning step, the amplified product is directed by means of the appropriate restriction enzyme and it is inserted in the corresponding restriction site of the plasmid pUC18.

After sequencing the DNA, the insert is recovered and sub-cloned to the corresponding restriction site of the modified plasmid denominated pMAL-c2. The modification is made by inserting a termination codon downstream of the target HindIII in the polylinker of pMal-c2, denominating the resulting plasmid pMAL-c2*.

Regarding the cloning of the DNA sequence that encodes the antigenic determinants of the histone protein H2A, it should be pointed out that the cDNA of the clone cL71, that encodes the histone H2A of L. infantum, is used as a template for the PCR reactions, and for the DNA amplification, that encodes the N-terminal region of the histone H2A, more exactly rLiH2A-Nt-Q, the following oligonucleotides are used:

    sense 5'-
    CCTTTAGCTACTCCTCGCAGCGCCAAG-3' (SEQ ID NO: 1)
    (position 84-104 of the sequence cL71); antisense
    5'CCTGGGGGCGCCAGAGGCACCGATGCG-3' (SEQ ID NO: 2)
 

(inverse and complimentary to position 204-224 of the sequence cL71).

The sequences that are included in the oligonucleotides for the cloning and that are not present in the parent sequence cL-71 are marked in boldface type.

The amplified DNA fragment is cloned directly from the restriction site XmnI of pMAI-c2*.

The fragment is sequenced by means of the initiator #1234 ma1E and the antigenic C-terminal region of histone H2A, in particular rLiH2A-Ct-Q, is amplified with the following oligonucleotides. These are:

 

    Sense, 5'-GAATTCTCCGTAAGGCGGCCGCGCAG-3' (SEQ ID NO: 3)
    (position 276-296 of the sequence cL71).
    Antisense, 5'-GAATTCGGGCGCGCTCGGTGTCGCCTTGCC-3' (SEQ ID NO: 4)


(inverse and complimentary to the positions 456-476 of the plasmid cL71).

A triplet that encodes proline (indicated as GGG after the underlined letters) is included in the anti-sense oligonucleotide, the restriction site EccRI that is included in both oligonucleotides for cloning is indicated by underlining.

Regarding the cloning of the sequences that encode rLiP2a-Q, it should be pointed out that the regions of interest are amplified by PCR from cDNAs encoding LiP2a and LiP2b.

The oligonucleotides that are used for constructing the expression clone LiP2a-Q, are the following.

 

Sense, 5'-GTCGACCCCATGCAGTACCTCGCCGCGTAC-3'        (SEQ ID NO: 5).
Anti-sense, 5'-GTCGACGGGGCCCATGTCATCATCGGCCTC-3'   (SEQ ID NO: 6).

It should be pointed out that the Sa1I restriction sites added to the 5' extremes of the oligonucleotides have been underlined.

When constructing the expression clone LiP2b-Q, the oligonucleotides used were:

 

Sense, 5'-TCTAGACCCGCCATGTCGTCGTCTTCCTCGCC-3'     (SEQ ID NO: 7).
Anti-sense, TCTAGAGGGGCCATGTCGTCGTCGGCCTC-3'       (SEQ ID NO: 8).

At the 5' extremes of the oligonucleotides the restrictions sites are included for the enzyme XbaI (underlined), and due to the cloning needs, an additional triplet, encoding a proline residue, is included downstream of the restriction site.

Regarding the cloning of the sequence rLiPO-Q, it should be pointed out that the cloning of the DNA sequence of the C-terminal region of the protein PO of L. infantum is carried out by amplifying a clone of cDNA called L27 and the following oligonucleotides:

Sense, 5'-CTGCAGCCCGCCGCTGCCGCGCCGGCCGCC-3' (SEQ ID NO:9) (positions 1-24 of the L27 cDNA) and the initiator of the pUC18 sequence (#1211), the amplified DNA is directed by the enzymes PstI+HindIII, with later insertion into the plasmid pMAL-c2.

The resulting clone is denominated pPQI and it should be noted that the restriction site PstI is included in the nucleotide with sense (underlined sequence) and that the restriction target HindIII is present in the cDNA L27.

Regarding the cloning of the chimeric gene, it should be pointed out that the DNA sequences that encode the five antigenic determinants are assembled into a chimeric gene, and this assembly is carried out on the clone pPQI, to which the codifying regions for the antigenic regions LiP2a-Q are added sequentially in the 3' direction (naming the results of cloning pPQ2), LiP2b-Q (clone pPQ3), LiH2a-Ct-Q (clone pPQ4) and LiH2A-Nt-Q (clone pPQ5).

Finally, the insert obtained after the SacI+HindIII digestion of the final clone pPQ5 is inserted into the pQE31 expression plasmid, naming the resulting clone pPQ.

Claim 1 of 5 Claims

What is claimed is:

1. An isolated polynucleotide, comprising a recombinant cDNA encoding a chimeric polypeptide which contains at least one antigenic determinant, recognized by serum from dogs with Visceral Leishmaniosis, from each of the LiP2a, LiP2b, LiH2a and LiPO proteins from Leishmania infantum.

 


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