Link:  Pharm/Biotech Resources

Title:  Mosaic protein and restriction endonuclease assisted ligation method for making the same

United States Patent:  6,960,659

Issued:  November 1, 2005

Inventors:  Khudyakov; Yury E. (Duluth, GA); Fields; Howard A. (Marietta, GA)

Assignee:  The United States of America as represented by the Department of Health and Human Services (Washington, DC)

Appl. No.:  491146

Filed:  January 25, 2000

A mosaic protein comprising a variety of immunoreactive antigenic epitopes from several genotypes of hepatitis C virus. The mosaic protein provides a sensitive and specific immunological hepatitis detection assay. A restriction enzyme assisted ligation method of making an artificial gene permits controlled construction of mosaic proteins, and allows confirmatory expression of the intermediate gene products.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions used to improve the sensitivity, the spectrum of immunoreactivity, and the specificity of antigens used as immunologic targets for detection. In preferred embodiments, the detection can be performed by enzyme immunoassay (EIA). The method, designated Restriction Endonuclease Assisted Ligation (REAL), involves the construction of an artificial gene from synthetic oligonucleotides. The compositions are synthetic proteins composed of a mosaic of broadly immunoreactive antigenic epitopes from several genotypes of, for example HCV.

REAL employs the use of the Klenow fragment of DNA Polymerase I to convert specially designed complimentary oligonucleotides into double stranded DNA fragments, which are subsequently amplified by PCR. Restriction sites were engineered into the cloning vector and used to produce complimentary overhangs for the addition of consecutive fragments. Each fragment may be cloned and expressed individually, for example in Escherichia coli to determine their immunoreactivity or may be assembled into full length product without cloning. Two consecutive fragments are subsequently ligated, amplified by PCR, cleaved with restriction endonucleases, and ligated with DNA ligase to assemble each fragment into a longer fragment in a consecutive process. By repeating this process fragments of increasing length are assembled, expressed and analyzed for immunoreactivity, and reiterated until the full length gene is assembled.

The invention provides mosaic proteins comprising a plurality of homologous antigenic peptides from different genotypes of a hepatitis virus. In particular, the invention provides mosaic proteins comprising a plurality of homologous antigenic nucleocapsid peptides from different genotypes of a hepatitis C virus. Further, the invention provides mosaic proteins comprising a plurality of homologous antigenic non-structural peptides from different genotypes of a hepatitis C virus. The mosaic proteins and genes encoding therefor can be used for immunologic detection or vaccination.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions used to improve the sensitivity, the spectrum of immunoreactivity, and the specificity of antigens used as immunologic targets for detection. In preferred embodiments, the detection can be performed by enzyme immunoassay (EIA). The method, designated Restriction Endonuclease Assisted Ligation (REAL), involves the construction of an artificial gene from synthetic oligonucleotides. The compositions are synthetic proteins composed of a mosaic of broadly immunoreactive antigenic epitopes from several genotypes of a species, for example hepatitis C virus (HCV). The mosaic protein compositions can be used for immunologic detection of, or vaccination against, the organisms from which they are derived. The invention further contemplates that the nucleic acids encoding the mosaic proteins can be used for immunologic detection of, or vaccination against, the organisms from which they are derived. Therefore, in addition to compositions and methods for detecting hepatitis, the invention provides a hepatitis vaccine comprising a mosaic protein, or a gene encoding therefor.

REAL employs the use of the Klenow fragment of DNA Polymerase I to convert specially designed complimentary oligonucleotides into double stranded DNA fragments, which are subsequently amplified by PCR. Restriction sites were engineered into the cloning vector and used to produce complimentary overhangs for the addition of consecutive fragments. Each fragment may be cloned and expressed individually, for example in Escherichia coli to determine their immunoreactivity or may be assembled into full length product without cloning. Two consecutive fragments are subsequently ligated, amplified by PCR, cleaved with restriction endonucleases, and ligated with DNA ligase to assemble each fragment into a longer fragment in a consecutive process. By repeating this process fragments of increasing length are assembled, expressed and analyzed for immunoreactivity, and reiterated until the full length gene is assembled.

In particular, the present invention provides a method of constructing an artificial gene, comprising synthesizing an initial oligonucleotide containing an initial gene segment encoding an initial gene product. The initial gene segment is flanked in the upstream direction (5′) by an upstream initial ligating sequence, a first endonuclease recognition sequence that is recognized by a first endonuclease that cleaves at the first endonuclease recognition sequence, and a second endonuclease recognition sequence which is recognized by a second endonuclease that cleaves downstream of the first endonuclease recognition sequence and within the upstream initial ligating sequence.

Additionally, the initial gene segment is flanked in the downstream direction (3′) by a downstream initial ligating sequence, a stop codon, a third endonuclease recognition sequence that is recognized by a third endonuclease that cleaves at the third endonuclease recognition sequence, and a fourth endonuclease recognition sequence which is recognized by a fourth endonuclease that cleaves upstream of the third endonuclease recognition sequence, upstream of the stop codon, and within the downstream initial ligating sequence. An example of such an initial oligonucleotide can be seen in FIG. 4, wherein "SEGMENT" designates the initial gene segment and "NNNNNN" designates the initial ligating sequence.

The method further comprises synthesizing a subsequent oligonucleotide containing a subsequent gene segment encoding a subsequent gene product. The subsequent gene segment is flanked in the upstream direction (5′) by an upstream subsequent ligating sequence, a first endonuclease recognition sequence which is recognized by the first endonuclease that cleaves at the first endonuclease recognition sequence, and a second endonuclease recognition sequence which is recognized by the second endonuclease that cleaves downstream of the first endonuclease recognition sequence and within the upstream subsequent ligating sequence.

The subsequent gene segment is flanked in the downstream direction (3′) by a downstream subsequent ligating sequence, a stop codon, a third endonuclease recognition sequence which is recognized by the third endonuclease that cleaves at the third endonuclease recognition sequence, and a fourth endonuclease recognition sequence which is recognized by the fourth endonuclease that cleaves upstream of the third endonuclease recognition sequence, upstream of the stop codon, and within the downstream subsequent ligating sequence. An example of such an subsequent oligonucleotide can be seen in FIG. 4, wherein "SEGMENT" designates the subsequent gene segment and "NNNNNN" designates the subsequent ligating sequence.

The method further comprises the step of cleaving the initial oligonucleotide with the fourth endonuclease, and cleaving the subsequent oligonucleotide with the second endonuclease. An example of this step is shown in FIGS. 6 and 7.

The method further comprises the step of ligating the initial oligonucleotide and the subsequent oligonucleotide together at the downstream initial ligating sequence of the initial oligonucleotide and the upstream subsequent ligating sequence of the subsequent oligonucleotide to form an artificial gene. An example of this step is shown in FIG. 8. The invention contemplated that additional subsequent oligonucleotides can be prepared, cleaved and ligated in a likewise fashion to make any artificial gene.

In a preferred embodiment, the invention provides the subsequent step of cleaving the artificial gene with the first and third endonucleases and inserting the remaining artificial gene into a vector previously cleaved with the first and third endonucleases. This step permits insertion of the final vector construct into a living organism, such as E. coli, and the recombinant expression of the artificial gene to produce the mosaic protein.

The invention provides the unique opportunity following the synthesizing the initial oligonucleotide step, and before the cleaving the initial oligonucleotide with the fourth endonuclease step, of confirming the operability of the initial oligonucleotide by cleaving the initial oligonucleotide with the first and third endonucleases and inserting the remaining initial oligonucleotide into a vector previously cleaved with the first and third endonucleases, and expressing the initial gene segment. An example of such an additional step is shown in FIG. 5, wherein "SEGMENT" designates the initial gene segment and "NNNNNN" designates the initial ligating sequence. This step is made possible by the inclusion of the stop codon downstream of the gene segment, which is removed by the addition of the fourth endonuclease in subsequent steps.

In preferred embodiments, the first and third endonucleases are EcoRI and BamHI, respectively. However, it will be understood that any endonucleases that cleave at the recognition sequence, can be used, with the proviso that two different endonucleases are employed. Examples of other suitable restriction endonucleases include: AflII, Alw44I, ApaI, ApaII, BclI, BglII, BspHI, BssHII, HindIII, KpnI, MluI, NarI, NcoI, PstI, SalI, or XhoI.

In preferred embodiments, the second and fourth endonucleases are BbvI and FokI, respectively. However, it will be understood that any endonuclease that cleaves downstream of the first endonuclease recognition sequence, or upstream of the third endonuclease recognition sequence, respectively, and within the ligating sequences, can be used, with the proviso that two different endonucleases are employed. Examples of other suitable restriction endonucleases that restrict the nucleic acid at a site away from the recognition site include: BspMI, HgaI, MboII, or SfaNI.

The invention provides that in preferred embodiments the initial and subsequent gene segments encode antigenic regions of a homologous protein from different genotypes of a hepatitis virus. The invention contemplates, however, that the REAL technique can be used for the construction of any mosaic or chimeric protein. In preferred embodiments, the gene segments encode antigenic regions of homologous proteins of different genotypes of a hepatitis C virus. Preferably, the gene segments encode antigenic regions of a nucleocapsid protein or a non-structural protein of different genotypes of a hepatitis C virus.

The invention further provides an artificial gene constructed by the above methods. The invention further provides a mosaic protein encoded by the artificial gene constructed by the above methods. The invention further provides a method of detecting a hepatitis infection in an individual comprising combining a serum sample from the individual with the mosaic protein made by the above methods, and detecting the presence of antibody binding to the mosaic protein, the presence of binding indicating a hepatitis infection in the individual. Preferably, an enzyme immunoassay (EIA) is performed for detection of a hepatitis infection, as is described in Examples 2 and 3. The detection of antibody binding can be facilitated by the use of detectable moieties, such as fluorescence, radioisotopes or solid substrate capture.

The invention provides mosaic proteins comprising a plurality of homologous antigenic peptides from different genotypes of a hepatitis virus. In particular, the invention provides mosaic proteins comprising a plurality of homologous antigenic nucleocapsid peptides from different genotypes of a hepatitis C virus. Further, the invention provides mosaic proteins comprising a plurality of homologous antigenic non-structural peptides from different genotypes of a hepatitis C virus. The invention also provides the gene sequences which encode for such mosaic proteins.

In one preferred embodiment, the mosaic protein can comprise a plurality of homologous antigenic nucleocapsid peptides from different genotypes of a hepatitis C virus as set forth in the amino acid sequences set forth in SEQ ID NOs:23-33, detailed in Example 2 (see Original Patent), herein. In another preferred embodiment, the mosaic protein can comprise a plurality of homologous antigenic non-structural peptides from different genotypes of a hepatitis C virus as set forth in the amino acid sequence of SEQ ID NO:52, detailed in Example 3 (see Original Patent), herein. It will be understood that certain minor or silent amino acid modifications and/or substitutions can be made in the amino acid sequences, while maintaining the antigenic functionality of the mosaic proteins. It will also be understood that certain silent or wobble nucleotide modifications and/or substitutions can be made in the gene sequences, while maintaining the ability of the gene to be ligated by the REAL technique and the antigenic functionality of the encoded mosaic proteins.
 

Claim 1 of 6 Claims

1. A nucleic acid encoding a mosaic protein comprising more than two antigenic peptides from the same domain from different genotypes of hepatitis C virus and wherein the mosaic protein comprises the amino acid sequences set forth in SEQ ID NOs:23-33.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.