Patent Application
20080076124
All technical and scientific terms used herein, unless otherwise defined, have the same meanings as commonly understood by persons skilled in the art.
The term “antibody” used herein denotes intact molecules (a polypeptide or group of polypeptides) as well as fragments thereof, such as Fab, R(ab′)2, and Fv fragments, which are capable of binding the epitopes. Antibodies are produced by specialized B cells after stimulation by an antigen. Structurally, antibody consists of four subunits including two heavy chains and two light chains. The internal surface shape and charge distribution of the antibody binding domain is complementary to the features of an antigen. Thus, antibody can specifically act against the antigen in an immune response.
The term “base pair (bp)” used herein denotes nucleotides composed of a purine on one strand of DNA which can be hydrogen bonded to a pyrimidine on the other strand. Thymine (or uracil) and adenine residues are linked by two hydrogen bonds. Cytosine and guanine residues are linked by three hydrogen bonds.
The term “Basic Local Alignment Search Tool (BLAST; Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402)” used herein denotes programs for evaluation of homologies between a query sequence (amino or nucleic acid) and a test sequence. Specific BLAST programs are described as follows:
(1) BLASTN compares a nucleotide query sequence against a nucleotide sequence database;
(2) BLASTP compares an amino acid query sequence against a protein sequence database;
(3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence against a protein sequence database;
(4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames; and
(5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
The term “cDNA” used herein denotes nucleic acids that synthesized from a mRNA template using reverse transcriptase.
The term “cDNA library” used herein denotes a library composed of complementary DNAs which are reverse-transcribed from mRNAs.
The term “complement” used herein denotes a polynucleotide sequence capable of forming base pairing with another polynucleotide sequence. For example, the sequence 5′-ATGGACTTACT-3′ binds to the complementary sequence 5′-AGTAAGTCCAT-3′.
The term “deletion” used herein denotes a removal of a portion of one or more amino acid residues/nucleotides from a gene.
The term “expressed sequence tags (ESTs)” used herein denotes short (200 to 500 base pairs) nucleotide sequence that derives from either 5′ or 3′ end of a cDNA.
The term “expression vector” used herein denotes nucleic acid constructs which contain a cloning site for introducing the DNA into vector, one or more selectable markers for selecting vectors containing the DNA, an origin of replication for replicating the vector whenever the host cell divides, a terminator sequence, a polyadenylation signal, and a suitable control sequence which can effectively express the DNA in a suitable host. The suitable control sequence may include promoter, enhancer and other regulatory sequences necessary for directing polymerases to transcribe the DNA.
The term “host cell” used herein denotes a cell which is used to receive, maintain, and allow the reproduction of an expression vector comprising DNA. Host cells are transformed or transfected with suitable vectors constructed using recombinant DNA methods. The recombinant DNA introduced with the vector is replicated whenever the cell divides.
The term “insertion” or “addition” used herein denotes the addition of a portion of one or more amino acid residues/nucleotides to a gene.
The term “in silico” used herein denotes a process of using computational methods (e.g., BLAST), to analyze DNA sequences.
The term “polymerase chain reaction (PCR)” used herein denotes a method which increases the copy number of a nucleic acid sequence using a DNA polymerase and a set of primers (about 20 bp oligonucleotides complementary to each strand of DNA) under suitable conditions (successive rounds of primer annealing, strand elongation, and dissociation).
The term “polynucleotide” or “nucleic acid sequence” used herein denotes a sequence of nucleotide (guanine, cytosine, thymine or adenine) in a specific order that can be a natural or synthesized fragment of DNA or RNA. It may be single-stranded or double-stranded.
The term “protein” or “polypeptide” used herein denotes a sequence of amino acids in a specific order that can be encoded by a gene or by a recombinant DNA. It can also be chemically synthesized.
The term “RNA interference (RNAi)” used herein denotes an introduction of homologous double stranded RNA (dsRNA) into a cell to specifically inhibit the expression of a gene.
The term “reverse transcriptase-polymerase chain reaction (RT-PCR)” used herein denotes a process which transcribes mRNA to complementary DNA strand using reverse transcriptase followed by polymerase chain reaction to amplify the specific fragment of DNA sequences.
The term “transformation” used herein denotes a process describing the uptake, incorporation, and expression of exogenous DNA by prokaryotic host cells.
The term “transfection” used herein denotes a process describing the uptake, incorporation, and expression of exogenous DNA by eukaryotic host cells.
The term “variant” used herein denotes a fragment of sequence (nucleotide or amino acid) inserted or deleted by one or more nucleotides/amino acids.
According to the present invention, the polypeptides of one novel human PPEF-1-related gene variant and the nucleic acid sequences encoding the same are provided.
According to the present invention, human PPEF-1 cDNA sequence was used to query the human EST databases using BLAST program to search for PPEF-1-related gene variants. One human cDNA partial sequences (i.e., EST) similar to PPEF-1 was identified from ESTs deposited in a cDNA database constructed using a SUP-T1 (T-cell lymophoblastic lymphoma) cell line. The cDNA clone, named PPEF-1V (PPEF-1 variant), was then isolated from the SUP-T1 cDNA library and sequenced.
The full-length of the PPEF-1V cDNA is a 2130 bp clone containing a 1503 bp open reading frame (ORF) extending from nucleotides 188 to 1690, which corresponds to an encoded protein of 501 amino acid residues with a predicted molecular mass of 57.8 kDa. The initiation ATG sequence of PPEF-1V is located at nucleotides 188-190 bp. To determine the variation in sequence of PPEF-1V cDNA clone, an alignment of PPEF-1 nucleotide/amino acid sequence with PPEF-1V was performed (
In the present invention, a search of ESTs deposited in dbEST at NCBI was performed to determine the presence of PPEF-1V in silico. The result of in silico analysis showed that one EST (GenBank accession number BE546038) was found to confirm the absence of 350 bp region on PPEF-1V nucleotide sequence. Therefore, any nucleotide fragments comprising the immediately upstream and downstream sequences of the 350 bp deleted region (128-477 bp) of PPEF-1V are the “gene targets” and may be used as probes to determine the presence of PPEF-1V under high stringency conditions. An alternative approach is that any set of primers for amplifying the fragment containing the immediately upstream and downstream sequences of the 350 bp deleted region (128-477 bp) of PPEF-1V are the “gene targets” and may be used to determine the presence of the variant.
According to the present invention, the polypeptides of the human PPEF-1V and its fragments thereof may be produced via genetic engineering techniques. In this case, they are produced by appropriate host cells which have been transformed by DNAs that code for the polypeptides or fragments thereof. The nucleotide sequence encoding the polypeptide of the human PPEF-1V or their fragments thereof is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence in a suitable host. The nucleic acid sequence is inserted into the vector where the sequence will be expressed under appropriate conditions (e.g., in proper orientation and correct reading frame and with appropriate expression sequences, including an RNA polymerase binding sequence and a ribosomal binding sequence).
Any method that is known to those skilled in the art may be used to construct expression vectors containing sequences encoding the polypeptides of the human PPEF-1V and appropriate transcriptional/translational control elements. These methods may include in vitro recombinant DNA and synthetic techniques, and in vivo genetic recombinants.
A variety of expression vector/host systems may be utilized to express the polypeptide-coding sequence. These include, but not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vector; yeast transformed with yeast expression vector; insect cell systems infected with virus (e.g., baculovirus); plant cell system transformed with viral expression vector (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV); or animal cell system infected with virus (e.g., vaccina virus, adenovirus, etc.). Preferably, the host cell is a bacterium, and most preferably, the bacterium is E. coli.
Alternatively, the Polypeptides of the human PPEF-1V or fragments thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solid-phase techniques. Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Perkin-Elmer).
According to the present invention, the fragments of the polypeptides and nucleic acid sequences of the human PPEF-1V are used as immunogens, primers or probes. Preferably, the purified fragments of the human PPEF-1V are used. The fragments may be produced by enzyme digestion, chemical cleavage of isolated or purified polypeptide or nucleic acid sequences, or chemical synthesis. The fragment then may be isolated or purified. Such isolated or purified fragments of the polypeptides and nucleic acid sequences can be used as immunogens, primers or probes.
The present invention further provides the antibodies which specifically bind one or more out-surface epitopes of the polypeptides of the human PPEF-1V.
According to the present invention, immunization of mammals such as humans, rabbits, rats, mice, sheep, goats, cows, or horses with immunogens described herin is performed using procedures well known to those skilled in the art, for the purpose of obtaining antisera containing polyclonal antibodies or hybridoma lines secreting monoclonal antibodies.
Monoclonal antibodies can be prepared by standard techniques, given the teachings contained herein. Such techniques are disclosed, for example, in U.S. Pat. No. 4,271,145 and U.S. Pat. No. 4,196,265. Briefly, an animal is immunized with the immunogen. Hybridomas are prepared by fusing spleen cells from the immunized animal with myeloma cells. The fused products are screened for those producing antibodies that specifically bind to the immunogen. The positive hybridoma clones are isolated, and the monoclonal antibodies are recovered from those clones.
Immunization regimens for production of both polyclonal and monoclonal antibodies are well-known in the art. The immunogen may be injected by any route, including subcutaneous, intravenous, intraperitoneal, intradermal, intramuscular, mucosal, or a combination thereof. The immunogen may be injected in soluble form, aggregate form, attached to a physical carrier, or mixed with an adjuvant. The antisera and antibodies may be purified using column chromatography methods.
According to the present invention, antibody fragments which contain specific binding sites for the polypeptides or fragments thereof may also be generated. For example, such fragments include, but are not limited to, F(ab′)2 fragments made by pepsin digestion of the antibody molecule or Fab fragments generated by reducing the disulfide bridges of the F(ab′)2 fragments.
The subject invention also provides methods for diagnosing the diseases associated with PPEF-1V or T-cell lymphoma, more preferably, the T-cell lymphoblastic lymphoma, by utilizing the nucleic acid sequences, the polypeptide of the human PPEF-1V, or fragments thereof, and the antibodies against the polypeptides.
Many gene variants have been found to associate with diseases (Stallings-Mann et al., (1996) Proc Natl Acad Sci USA 93: 12394-9; Liu et al., (1997) Nat Genet 16:328-9; Siffert et al., (1998) Nat Genet 18: 45 to 8; Lukas et al., (2001) Cancer Res 61: 3212 to 9). Since PPEF-1V clone was isolated from T-cell lymphoblastic lymphoma cell line (SUP-T1), it is advisable that PPEF-1V serves as a marker for the diagnosis of human T-cell lymphoblastic lymphoma. Thus, the expression level of PPEF-1V relative to PPEF-1 may be a useful indicator to screen patients suspected of having T-cell lymphoma or more specifically the T-cell lymphoblastic lymphoma. This suggests that the index of relative expression level (mRNA or protein) may confer an increased susceptibility to T-cell lymphoma, more preferably, the T-cell lymphoblastic lymphoma. Fragments of PPEF-1V transcripts (mRNAs) may be detected by RT-PCR approach. Polypeptides of PPEF-1V may be determined by the binding of antibodies to these polypeptides. On the other hand, a method (RNAi; RNA interference) that is known to those skilled in the art may be used to interfere and degrade the targeted RNA using chemically synthesized double stranded short nucleic acid (about 23 nucleotides dsRNA) molecules (Montgomery et al. (1998) Proc Natl Acad Sci USA. 95:15502-7). According to the present invention, the double stranded short nucleic acid molecules containing the immediately upstream and downstream sequences of the 350 bp deleted region (128-477 bp) of PPEF-1V may be used to interfere and degrade PPEF-1V mRNA. After RNAi approach was conducted, the decreased transcripts or polypeptides may be used to determine the presence of PPEF-1V mRNA.
According to the present invention, the expression of these gene variant mRNAs may be determined by, but not limited to, RT-PCR. Using TRIZOL reagents (Life Technology), total RNA may be isolated from patient samples. Tissue samples (e.g., biopsy samples) are powdered under liquid nitrogen before homogenization. RNA purity and integrity are assessed by absorbance at 260/280 nm and by agarose gel electrophoresis. A set of primers can be designed to amplify the expected size of specific PCR fragments of PPEF-1V. For example, one of the primers is a fragment of the sequences (A fragment) or a fragment complementary to the sequences (B fragment) containing the gene targets: nucleotides 127-128 of PPEF-1V; the other is a fragment of the PPEF-1V sequences designed at any location upstream of “B fragment” or a fragment complementary to the PPEF-1V sequences designed at any location downstream of “A fragment”. Alternatively, one primer is designed at any location upstream of nucleotide 127 of PPEF-1V and the other is designed at any location complementary to a portion downstream of nucleotide 128 of PPEF-1V. The length of the PCR fragment from PPEF-1V will be 350 bp shorter than that from PPEF-1. PCR fragments are analyzed on a 1% agarose gel using five microliters (10%) of the amplified products. The intensity of the signals may be determined by using the Molecular Analyst program (version 1.4.1; Bio-Rad). Thus, the index of relative expression levels for each co-amplified PCR products may be calculated based on the signal intensities.
The RT-PCR experiment may be performed according to the manufacturer instructions (Boehringer Mannheim). A 50 μl reaction mixture containing 2 μl total RNA (0.1 μg/μl), 1 μl each primer (20 μM), 1 μl each dNTP (10 mM), 2.5 μl DTT solution (100 mM), 10 μl 5× RT-PCR buffer, 1 μl enzyme mixture, and 28.5 μl sterile distilled water may be subjected to the conditions such as reverse transcription at 60° C. for 30 minutes followed by 35 cycles of denaturation at 94° C. for 2 minutes, annealing at 60° C. for 2 minutes, and extension at 68° C. for 2 minutes. The RT-PCR analysis may be repeated twice to ensure reproducibility, for a total of three independent experiments.
The expression of gene variants can also be analyzed using Northern Blot hybridization approach. Specific fragment of the PPEF-1V may be amplified by polymerase chain reaction (PCR) using primer set designed for RT-PCR. The amplified PCR fragment may be labeled and served as a probe to hybridize the membranes containing total RNAs extracted from the samples under the conditions of 55° C. in a suitable hybridization solution for 3 hours. Blots may be washed twice in 2×SSC, 0.1% SDS at room temperature for 15 minutes each, followed by two washes in 0.1×SSC and 0.1% SDS at 65° C. for 20 minutes each. After these washes, blot may be rinsed briefly in suitable washing buffer and incubated in blocking solution for 30 minutes. Then, the blot may be incubated in suitable antibody solution for 30 minutes. Blots may be washed in washing buffer for 30 minutes and equilibrated in suitable detection buffer before detecting the signals. Alternatively, the presence of gene variants (cDNAs or PCR) can be detected using microarray approach. The cDNAs or PCR products corresponding to the nucleotide sequences of the present invention may be immobilized on a suitable substrate such as a glass slide. Hybridization can be preformed using the labeled mRNAs extracted from samples. After hybridization, nonhybridized mRNAs are removed. The relative abundance of each labeled transcript, hybridizing to a cDNA/PCR product immobilized on the microarray, can be determined by analyzing the scanned images.
According to the present invention, the presence of the polypeptide of PPEF-1V may be determined by, but not limited to, the immunoassay which uses the antibody that specifically binds to the polypeptide. The polypeptides of the gene variants may be expressed in prokaryotic cells by using suitable prokaryotic expression vectors. The cDNA fragments of PPEF-1V gene may be PCR amplified using primer set A with restriction enzyme digestion sites incorporated in the 5′ and 3′ ends, respectively. The PCR products can then be enzyme digested, purified, and inserted into the corresponding sites of prokaryotic expression vector in-frame to generate recombinant plasmids. Sequence fidelity of this recombinant DNA can be verified by sequencing. The prokaryotic recombinant plasmids may be transformed into host cells (e.g., E. coli BL21 (DE3)). Recombinant protein synthesis may be stimulated by the addition of 0.4 mM isopropylthiogalactoside (IPTG) for 3 hours. The bacterially-expressed proteins may be purified.
The polypeptide of the gene variant may be expressed in animal cells by using eukaryotic expression vectors. Cells may be maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco BRL) at 37° C. in a humidified 5% CO2 atmosphere. Before transfection, the nucleotide sequence of each of the gene variant may be amplified with PCR primers containing restriction enzyme digestion sites and ligated into the corresponding sites of eukaryotic expression vector in-frame. Sequence fidelity of this recombinant DNA can be verified by sequencing. The cells may be plated in 12-well plates one day before transfection at a density of 5×104 cells per well. Transfections may be carried out using Lipofectaminutese Plus transfection reagent according to the manufacturer's instructions (Gibco BRL). Three hours following transfection, the medium containing the complexes may be replaced with fresh medium. Forty-eight hours after incubation, the cells may be scraped into lysis buffer (0.1 M Tris HCl, pH 8.0, 0.1% Triton X-100) for purification of expressed proteins. After these proteins are purified, monoclonal antibodies against these purified proteins (PPEF-1V) may be generated using hybridoma technique according to the conventional methods (de StGroth and Scheidegger, (1980) J Immunol Methods 35:1-21; Cote et al. (1983) Proc Natl Acad Sci USA 80: 2026-30; and Kozbor et al. (1985) J Immunol Methods 81:31-42).
According to the present invention, the presence of the polypeptides of PPEF-1V in samples of T-cell lymphoblastic lymphoma may be determined by, but not limited to, Western blot analysis. Proteins extracted from samples may be separated by SDS-PAGE and transferred to suitable membranes such as polyvinylidene difluoride (PVDF) in transfer buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine, 20% methanol) with a Trans-Blot apparatus for 1 hour at 100 V (e.g., Bio-Rad). The proteins can be immunoblotted with specific antibodies. For example, membrane blotted with extracted proteins may be blocked with suitable buffers such as 3% solution of BSA or 3% solution of nonfat milk powder in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and incubated with monoclonal antibody directed against the polypeptides of gene variants. Unbound antibody is removed by washing with TBST for 5×1 minutes. Bound antibody may be detected using commercial ECL Western blotting detecting reagents.
The following examples are provided for illustration, but not for limiting the invention.
Expressed sequence tags (ESTs) generated from the large-scale PCR-based sequencing of the 5′-end of human T-cell lymphoblastic lymphoma cDNA clones were compiled and served as EST databases. Sequence comparisons against the nonredundant nucleotide and protein databases were performed using BLASTN and BLASTX programs, at the National Center for Biotechnology Information (NCBI) with a significance cutoff of p<10−10. ESTs representing putative PPEF-1V gene were identified during the course of EST generation.
One cDNA clone exhibiting EST sequence similar to the PPEF-1 gene was isolated from the T-cell lymphoblastic lymphoma cDNA library and named PPEF-1V. The inserts of these clones were subsequently excised in vivo from the λZAP Express vector using the ExAssist/XLOLR helper phage system (Stratagene). Phagemid particles were excised by coinfecting XL1-BLUE MRF′ cells with ExAssist helper phage. The excised pBluescript phagemids were used to infect E. coli XLOLR cells, which lack the amber suppressor necessary for ExAssist phage replication. Infected XLOLR cells were selected using kanamycin resistance. Resultant colonies contained the double stranded phagemid vector with the cloned cDNA insert. A single colony was grown overnight in LB-kanamycin, and DNA was purified using a Qiagen plasmid purification kit.
Phagemid DNA was sequenced using the Taq dye-deoxy terminator cycle sequencing kit for the Applied Biosystems 377 sequencing system (PerkinElmer Life Sciences). Using the primer-walking approach, full-length sequence was determined. Nucleotide and protein searches were performed using BLAST against the non-redundant database of NCBI.
The coding sequence for each cDNA clones was searched against the dbEST sequence database using the BLAST algorithm at the NCBI website. ESTs derived from each tissue were used as a source of information for transcript tissue expression analysis. Tissue distribution for each isolated cDNA clone was determined by ESTs matching to that particular sequence variants (insertions or deletions) with a significance cutoff of p<10−10.
The expression pattern of PPEF-1V was conducted in human cell lines using RT-PCR. The human cell lines were WI38 (Lung, fetus); A549 (Lung adenocarcinoma); H661 (Large cell carcinoma, lung); H520 (Squamous cell carcinoma, lung); H209 (Small cell carcinoma, lung); JHH-4 (Hepatoma); SUP-T1 (T-cell lymophoblastic lymphoma); Daudi (Burkitt's lymophoma); Ramos (Burkitt's lymophoma); RAJI (Burkitt's lymophoma); ZR75-1 (Breast carcinoma); MDA-MB-231 (Breast adenocarcinoma); Hs 578T (Breast carcinoma); G5T/VGH (Glioblastoma multiforme); TSGH9201 (Gastric carcinoma); Ca Ski (Cervix epidermoid carcinoma ); Hela S3 (Cervical epitheloid carcinoma); COLO 320 HSR (Colon adenocarcinoma); SW620 (Adenocarcinoma); TSGH8301 (Urinary bladder carcinoma); ES-2 (Ovarian carcinoma); DU145 (Brain prostate carcinoma); MIA paca-2 (Pancreatic carcinoma); CE48T/VGH (Esophagus epidermoid carcinoma); CE81T/VGH (Esophagus carcinoma well differentiated aquamous); HL-CZ (Promonocytic leukemia); Hs 181.tes (Normal testis). The purity and integrity of total RNA extracted from each of the cell lines were assessed by absorbance at 260/280 nm and by agarose gel electrophoresis. The forward and reverse primers for PPEF-1V were: 5′-CTGACACATATACTTCATGCCC-3′ and 5′-CACACAGGATCATTAGGATCTC-3′. The expected sizes of PPEF-1V PCR fragments was 265 bp.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; accession No. M33197) was used for internal control. The forward and reverse primers for GAPDH were: 5′-TGGGTGTGAACCATGAGAAG-3′ and 5′-GTGTCGCTGTTGAAGTCAGA-3′. The expected sizes of GAPDH PCR fragments was 472 bp. Briefly, a 50 μl reaction mixture containing 2 μl total RNA (0.1 μg/μl), 1 μl each primer (20 μM), 1 μl each dNTP (10 mM), 2.5 μl DTT solution (100 mM), 10 μl 5× RT-PCR buffer, 1 μl enzyme mixture and 28.5 μl sterile redistilled water were subjected to reverse transcription at 60° C. for 30 minutes followed by 35 cycles of denaturation at 94° C. for 2 minutes, annealing at 60° C. for 2 minutes, and extension at 68° C. for 2 minutes. Five microliters (10%) of the amplified products mixed with 1 μl of loading buffer were separated on a 1% horizontal agarose gel stained with ethidium bromide in 0.5× TAE buffer. The gel was electrophoresed at 100 V for 45 minutes.
All references are listed herein for the convenience of the reader. Each is incorporated by reference in its entirety.
