JP2003517299A - Sequence encoding a human neoplasia marker - Google Patents

Abstract

  (57) [Summary] The present disclosure of the present invention provides a nucleotide sequence encoding a cell surface NADH oxidase / protein disulfide-thiol exchange protein (tNOX) characteristic of neoplastic and virus-infected cells. The present disclosure also provides a recombinant DNA molecule comprising a sequence portion encoding full-length tNOX or truncated tNOX, a recombinant host cell expressing full-length tNOX or truncated tNOX, and recombinantly producing tNOX or truncated tNOX. And diagnostic methods using either the nucleotide sequence of a neoplastic cell-specific tNOX, or an antibody specific for a tNOX protein.

Description

Detailed Description of the Invention

(Cross-Reference of Related Applications) This application is US provisional application No. 60/162 filed on Nov. 1, 1999.
, 644, claim priority.

STATEMENT OF FEDERALLY SPONSORED RESEARCH This invention was made, at least in part, by funding from the NIH (National Institute of Health), and the United States Government has certain rights in this invention.

BACKGROUND OF THE INVENTION The field of the invention is that of molecular biology, and in particular to the molecular biology of neoplasia and diseased cells, in particular neoplasia and certain other diseased cells. Associated with cell surface markers for status.

[0004] Cancer and certain viruses, protozoa and parasite infections assay the above disease states because they represent a significant threat to human health and such infections result in significant economic costs. There is a long-felt need for effective, economical, and technically simple systems to do so, or to model inhibitors of the above diseases.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a recombinant plasma membrane NADH oxidase / thiol exchange protein (referred to herein as tNOX) and its coding sequence. The full-length protein has the amino acid sequence shown in SEQ ID NO: 2, and the truncated tNOX protein has the amino acid sequence shown in amino acids 220-610 of SEQ ID NO: 2. The full length sequence is nucleotides 23-185 of SEQ ID NO: 1.
2 has the specifically exemplified coding sequence, and the truncated protein has the amino acid sequence set forth at nucleotides 680-1852 of SEQ ID NO: 1. Also within the scope of the present invention are coding sequences which are synonymous with these specifically exemplified sequences. Also contemplated as within the scope of the present invention are
Sequences encoding neoplastic cell surface markers and coding sequences that hybridize under stringent conditions to the specifically exemplified full length or subsequences. Cell surface tNOX is characteristic of neoplastic states, certain viruses and other infections (eg HIV). Recombinant tNOX proteins are useful in the preparation of antigens for use in producing monoclonal antibodies or antisera for the diagnosis of cancer, other neoplastic conditions, and certain infectious disease states.

[0006] Non-naturally occurring recombinant DNA molecules comprising a portion encoding a NADH oxidase / protein disulfide-thiol exchange polypeptide are within the scope of the present invention, which portion comprises nucleotides 23 to 1852 of SEQ ID NO: 1; Nucleotides 680 to 1852 of SEQ ID NO: 1; and a nucleotide sequence selected from the group consisting of sequences that hybridize to one of the above sequences under stringent conditions, wherein the hybridizing sequence comprises Encodes a cell surface neoplastic marker protein having NADH oxidase / protein disulfide-thiol exchange activity. These recombinant DNA molecules can include a sequence that encodes a polypeptide consisting essentially of the amino acid sequence of amino acids 1-610 or amino acids 220-610 of SEQ ID NO: 2. The portion encoding the particular polypeptide may further include a translation stop codon (TGA, TAA, or TAG) immediately downstream of nucleotide 1852 of SEQ ID NO: 1. Using recombinant DNA molecules provided herein, NA in host cells (bacteria, yeast, mammals)
Also provided herein is a method of recombinantly producing a DH oxidase / protein disulfide-thiol exchange active polypeptide.

The present invention further provides a method of measuring neoplasia in a mammal, which method comprises the steps of: in a biological sample from the mammal,
NADH oxidase / protein disulfide-associated neoplastic cells compared to ribonucleic acid molecule encoding NADH oxidase associated with normal cells
Detecting the presence of ribonucleic acid encoding a thiol exchange protein,
Here, this detection step is performed using hybridization under stringent conditions or using the polymerase chain reaction, which requires a perfect match of the primer to the template, where , The hybridization probe or primer consists essentially of at least 15 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1; step; and step (a)
And correlating the results obtained with the sample of, wherein the presence of ribonucleic acid molecules in the biological sample is indicative of the presence of neoplasia. This method involves the use of a hybridization probe consisting essentially of nucleotides 680-1852 of SEQ ID NO: 1, nucleotides 23-1852, or portions thereof, wherein the neoplastic cell or viral infection. There are detectable differences in the results obtained with normal cells when compared to cells.

The present invention allows the production of antibody preparations, particularly those using recombinant tNOX or truncated tNOX or antigenic peptides derived from sequences from tNOX. The antibody preparation is a protein characterized by the amino acid sequence shown as amino acids 1 to 610 of SEQ ID NO: 2, a protein characterized by the amino acid sequence shown as amino acids 220 to 610 of SEQ ID NO: 2, or shown in SEQ ID NO: 16. It specifically binds to an antibody selected from the group consisting of proteins characterized by an amino acid sequence. These antibody preparations are useful in detecting tNOX in blood or serum from patients or animals with a neoplastic condition (eg, cancer), or in neoplastic or virally infected cells or tissues. is there.

Expression of tNOX of the invention in a host cell (eg, a mammalian host cell) results in a faster growth rate of the recombinant host cell and a significant increase in recombinant cell volume.

Northern blot analysis shows that the described cDNA is expressed in HeLa cells (human cervical carcinoma) and malignant BT-20 human breast adenocarcinoma cells. cd
The availability of NA allows rapid further testing of a variety of normal and aberrant, specificity of expression in cells and tissues.

The deduced amino acid sequence of the encoded protein is ovarian carcinoma (OVCAR-3
) It showed homology over a part of its full length to the deduced amino acid sequence of the cDNA encoding the protein detected by the K1 antibody derived from the cell line [C
hang and Pastan (1994) Int. J. Cancer 57: 9
0-97]. Although the Chang and Pastan sequences contain two errors that give rise to incorrect reading frames, the DNA of the invention is probably identical to the DNA isolated by Chang and Pastan. Based on preliminary studies with OVCAR-3 cells, MAB 12.1 used for expression screening was
It appears to not selectively react with the antigen preferentially expressed by OVCAR-3 cells, and the properties of tNOX do not correspond at all to the properties of the K1 antigen of OVCAR-3 cells.

To study the biological function of tNOX, tNOX cDNA was cloned into HindI.
II and BamHI restriction sites were used to subclone into the pcDNA3.1 expression vector. COS cells were then transfected with tNOX using calcium phosphate transfection and DMSO shock. tNOX
Overexpression was assessed based on enzyme activity analysis and Western blot analysis. Peptide antibodies against tNOX recognized expressed proteins with molecular weights of 34 and 48 kDa. Image enhanced light microscopy of tNOX-transfected cells.
The growth rate determined by t microscopy) was 2-3 times faster than that of the vector alone. Larger cell diameters resulted in a 4-5 fold increase in cell volume. The wider cell surface of the transfected cells was confirmed by electron microscopy. As expected, the transfected CO
S cells are more sensitive to tNOX inhibitors, such as capsaicin and epigallocatechin gallate (EGCg), with EC ₅₀ for growth inhibition shifting by 1-2 orders of magnitude towards lower drug concentrations. did. Therefore, tNO
X function is believed to be important in the expansion of cells and in the maintenance of uncontrolled growth of cancer cells.

DETAILED DESCRIPTION OF THE INVENTION The abbreviations used herein for amino acids are standard in the art: X or Xaa indicates an unidentified amino acid residue, but phosphorylated tyrosine, threonine. Or it can be serine, as well as any amino acid residue, including but not limited to cysteine or glycosylated amino acid residues. Abbreviations for amino acid residues as used herein are as follows: A, Ala, Alanine; V, Val, Valine; L, Leu, Leucine; I, Ile, Isoleucine; P, Pro. , Proline; F, Phe, phenylalanine; W, Trp, tryptophan; M, Met, methionine; G, G
ly, glycine; S, Ser, serine; T, Thr, threonine; C, Cys,
Cysteine; Y, Tyr, tyrosine; N, Asn, asparagine; Q, Gln,
Glutamine; D, Asp, aspartic acid; E, Glu, glutamic acid; K, L
Rs, Arg, Arginine; and H, His, Histidine.

Additional abbreviations used herein include Mes, 2- (N-morpholino) ethanesulfonic acid; DMSO, dimethyl sulfoxide; tNOX, cancer-associated and drug (capsaicin) -responsive cell surface NADH oxidase; ttN
OX, truncated tNOX; CNOX, constitutive and drug non-responsive cell surface NADH
Oxidase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; capsaicin, 8-methyl-N-vanillyl-6-nonenamide; LY181984, N- (4-methylphenylsulfonyl) -N '-(4-chlorophenyl). Urea; LY181985, N- (4-methylphenylsulfonyl) -N '-(4-phenyl) urea; EGCg, (-)-epigallocatechin gallate.

Neoplasia, as used herein, describes a disease state in a human or animal in which there are abnormally proliferating cells and / or tissues. What is the neoplastic state?
Cancers, sarcomas, tumors, leukemias, lymphomas, etc. are included, but are not limited to. The cell surface NADH oxidase / protein disulfide-thiol exchange proteins of the present invention characterize neoplastic cells, as well as neoplastic tissues and virus-infected cells (eg, human immunodeficiency virus, feline immunodeficiency virus, etc.).

A cell surface marker characteristic of diseased cells is described in US Pat. No. 5,605,81.
No. 0 (issued February 25, 1997) (incorporated herein by reference)
, And D. James Morre is described in several scientific publications, the sole and co-authors. This NADH oxidase / thiol exchange protein is found in the plasma membrane of neoplastic cells and cells infected with viruses (especially retroviruses) and protozoan parasites. This protein, called tNOX herein (t umor N ADH ox idas
e). Cell surface tNOX proteins are released in serum and urine of cancer patients, but are relatively difficult to purify. Therefore, the goal of this study is for use in recombinant production of tNOX proteins, and for the use of tNOX coding sequences or parts of coding sequences in probes and primers for the detection of tNOX transcripts or tNOX genomic sequences, To obtain a cDNA clone encoding tNOX.

Five clones were obtained by immunological screening of a HeLa cell cDNA library using a tNOX-specific monoclonal antibody. The results of the restriction digests were consistent with those from all 5 clones and with the single initial phage clone. It was confirmed by automated nucleotide sequencing that all five were different length inserts of the same DNA. The longest clone is 3.8
kb insert and 1830 bp open reading frame (SEQ ID NO: 1
23 to 1852).

The full-length cDNA resulted in an open reading frame (deduced molecular weight 70.1 kDa (Table 1)) for the deduced amino acid sequence of the 610 amino acid protein. This cDNA contains at nucleotide 20 a typical Kozac sequence (AXXATG) (Kozac, 1987) which promotes translational expression. Following the initiation methionine at nucleotide 23, F5 is followed by a sequence of 12 hydrophobic residues that act as a signal sequence for association with the membrane. Nucleotide 1
The stop codon at 853 is followed by a typical polyadenylation signal (AATAAA) at nucleotide 3625. Based on available genome information (
Bird, 1999), the tNOX cDNA contains multiple exons (at least 9) in the positive N-terminal portion of the full-length precursor (Fig. 2).

The C-terminal part of the derived amino acid sequence was proven in previous studies (M
orre et al., 1995a, 1996a; Chueh et al., 1997; del Ca.
still Oliveres et al., 1998), mature, processed 3
It corresponds to a molecular weight of 4 kDa (about 33.5 kDa derived from serum). Several possible functional motifs required for tNOX were included in this part of the tNOX protein as follows; the sequence E394-E-M-T-E contains 5 of the conserved 5 amino acids. Form a putative quinone binding site with 4 amino acids (Table 2
). The C505-XX-XX-X-C510 motif represents a potential active site for protein disulfide-thiol exchange activity based on site mutagenesis (Table 3). Also from site mutagenesis, and from C-XX-XX-XX-
Inhibition of activity by the serum against the X-C-containing peptide (LAILPACATPATCNPD) indicates a possible active site for protein disulfide-thiol exchange activity at C569-XX-XX-XX-C-575. Yes (amino acids 569-575 of SEQ ID NO: 2).

Along with E605, the sequence T590-GVGAS-S-L (amino acids 590-595 of SEQ ID NO: 2) represents 5 of the 7 conserved amino acids of known mitochondrial adenine binding proteins. Form a putative binding site for the adenine portion of NADH (Leblanc et al., 1995). The H546-VH motif, which is conserved in periplasmic copper oxidase, is
Together with is467 it forms a potential copper binding ligand. Furthermore, H54
The 6-V-H-E-G-F motif (amino acids 546 to 551 of SEQ ID NO: 2) is
It is conserved in both human and avian superoxide dismutase, and this portion provides a putative copper binding site (Shinina et al., 1996). Analysis of copper by atomic absorption spectroscopy showed that the protein purified from cancer patient's serum was 34k
It revealed 1 mole of copper per processed tNOX subunit of Da.

Possible N-glycosylation sites (NXS / T) are 138, 358, 41.
They were in 8th and 525th positions. The potential O-glycosylation site is amino acid 3
8 threonine, amino acid 71 threonine, amino acid 35 serine and amino acid 240 serine.

TNOX is a membrane associated protein. Three putative signal sequences near the N-terminus and a cleavage site were identified as involved in membrane targeting. The second signal sequence near M220 is 45.6 kD containing all of the functional motifs identified above.
yields a protein. The third potential signal sequence near M314 is 3
It produces a 4 kDa protein. In vitro translation of M220-initiated truncated tNOX cDNA using rabbit reticulocyte lysates in the presence and absence of canine pancreatic microsomal membranes is an indicator of both membrane insertion and membrane-dependent processing. It also showed no apparent change in the molecular weight of certain in vitro translation products. Truncated tNOX is encoded by nucleotides 680-1852 of SEQ ID NO: 1.

TNOX is an exogenous protein on the outer surface of the plasma membrane that does not bind lipids (Morre, 1995). tNOX is released from the membrane by incubation at pH 5 (del Castillo et al. 1998). Hydropathic plots of the derived amino acid sequence of tNOX do not predict the presence of the transmembrane domain (Figure 3).

The deduced amino acid sequence of the tNOX protein (Table 1) showed homology over the deduced amino acid sequence of the cDNA previously named APK1 antigen with part of its entire length (tNOX K357 to T610, sequence Amino acid 35 of number 2
7-610) (Chang and Pastan, 1994), so tNOX and K
The question arose whether one antigen was the same protein. APK1 antigen cDNA
The sequence was first obtained by expression cloning using the K1 antibody produced from the ovarian carcinoma cell line (OVCAR-3) as the immunogen. A portion of the tNOX cDNA was isolated by Chang and Pastan, except that the sequence contained an extra T at nucleotide 929 and one less G at nucleotide 1092 (nucleotides 83 and 247 of their sequence). It seems to be the same as the part. These differences resulted in incorrect reading frames. Two mistakes
Confirmed by Sugano et al. (2000). The monoclonal antibody used for the cDNA screen did not react with the K1 antigen expressed by OVCAR-3 cells, and the properties of tNOX did not correspond to those of the K1 antigen at all.
The lack of identity of tNOX and K1 antigen is consistent with the subsequent identification of CAK1 protein identified as a protein reactive with K1 antibody (Chang et al., 1
992; Chang and Pastan, 1994).

Neither cell surface tNOX, serum-derived tNOX, nor expressed tNOX share significant features with the K1 antigen. A high titer polyclonal antibody to recombinant tNOX was produced in the unprocessed form of tNO expressed by OVCAR cells.
X (70 kDa) and processed tNOX form (34 kDa), but in both the detergent-solubilized fraction (FIG. 4) and the unsolubilized fraction, 30 kDa (APK1 antigen) or 40kDa (CAK1)
It showed no reactivity with the part of the gel corresponding to the molecular weight of. The CAK1 protein is expressed predominantly in cell lines of mesothelial origin (Chang et al., 1992) and is anchored to the membrane by a glycosylated phosphatidylinositol (GPI) anchor. In contrast, tNOX has no GPI anchor.

E. Expression of the tNOX cDNA in E. coli was initiated with M220, a truncated 46 kDa (ttNOX), 46 kDa histidine-tagged ttNOX.
, Some forms of tN, including a truncated tNOX of 34 kDa starting at G327
This resulted in OX. E. subcloned cD expressed in E. coli
The entire sequence of NA was confirmed by resequencing. The tNOX protein was identified by reaction with a tNOX-specific monoclonal antibody (Figure 5).
The apparent molecular weight of 48 kDa ttNOX on SDS-PAGE is 46.
It was in agreement with the calculated molecular weight from the deduced amino acid sequence of kDa. The molecular weight of truncated tNOX starting at G327 was 42 kDa on SDS-PAGE. Direct amino acid sequencing revealed that the expressed protein purified from bacterial extracts matched the deduced amino acid sequence. The induced bacterial extract showed a 23-minute cycle of NADH oxidase activity (arrow in FIG. 6A). Extracts of both induced bacteria (open circles in FIGS. 6A and 7) or uninduced extracts (FIG. 6B) when measured in the presence of 1 μM capsaicin or 100 μM capsaicin showed cyclic activity. Didn't have. Addition of 1 μM anti-tumor sulfonylurea LY181984 also completely inhibited the activity.

Shown in FIG. 7 is the second unique feature of cell surface tNOX activity, which results in two activities associated with the cloned and expressed proteins: hydroquinone (NADH) oxidase and protein. The maximum rates of disulfide-thiol exchange (dithiodipyridine cleavage) alternate. As the rate of NADH oxidation decreased, the rate of DTDP cleavage increased, resulting in NADH
DTDP cleavage is maximal when oxidation is minimal. Both activities had approximately the same cycle length of 23 minutes.

Peptide antiserum directed against the C-terminus of tNOX was 48 in SDS-PAGE.
We recognized an expressed truncated protein species with a molecular weight of kDa (produced by recombinant COS-1 cells) (Figure 8). Two lower molecular weight peptides were also present. Image enhanced light microscope of ttNOX-transfected cells.
The growth rate measured by y) was about twice that of the vector alone (Fig. 9).
). The increased growth rate also reflected the increased cell size. At confluence, the average cell diameter of COS cells transfected with tNOX was approximately 30 μm, while the average cell diameter of COS cells transfected with vector alone was approximately 20 μm (FIG. 10). Larger cell diameters resulted in a 4- to 5-fold increase in cell volume. The increased cell surface of the transfected cells was confirmed by electron microscopy. tNOX and the growing phase of cell proliferation (enla
defines a close relationship of tNOX activity to the r.
Pogue et al., 2000), Proliferation of COS cells transfected with tNOX cDNA while retaining the characteristic drug responsiveness of oxidative activity was compared to cells transfected with the vector tNOX inhibitor. It showed a 10- to 100-fold greater sensitivity to (Table 3). The tNOX inhibitors include capsaicin, (−)-epigallocatechin gallate (EGCg), adriamycin, and the active antitumor sulfonylurea LY181984 (N.
-(4-methylphenylsulfonyl) -N '-(4-chlorophenyl) urea)
(Table 4) is mentioned. EC5 for growth inhibition in all four inhibitors
0 shifted by 1-2 orders of magnitude towards lower drug concentrations as a result of transfection with tNOX cDNA. LY181985 (N is an inactive antitumor sulfonylurea that differs from LY181984 at one chlorine
-(4-methylphenylsulfonyl) -N '-(4-phenyl) urea) is t
Neither the cells transfected with the NOX cDNA nor the control cells transfected with the vector alone inhibited. Similarly, the proliferative response to methotrexate (an antifolate), which is not a tNOX inhibitor, is tNOX cDN
A was not affected by transfection.

The conclusion that the recombinant T lymphocyte protein and the NOX protein isolated from the 34 kDa serum represent the same protein is based, in part, on the overall properties that define the two proteins. These properties include two distinct enzymatic activities, hydroquinone (NADH) oxidation and protein disulfide-thiol exchange (FIGS. 6 and 7), and changes in these two activities that result in a 22 minute cycle length. 7, Table 3). Moreover, the activity of both proteins responds to the same series of quinone site inhibitors and antitumor agents in situ and in solution (Tables 3 and 4). define tNOX, and tNO
It is the latter property that distinguishes X from other NOX proteins that lack drug responsiveness.

As previously demonstrated (Chueh et al., 1997; del Castill
O et al., 1998) personally folded and active NOX proteins are blocked for direct and N-terminal sequencing, and / or enzymatic or chemical cleavage. However, a direct sequence association between the monoclonal antibody antigen used in cloning and the deduced amino acid sequence for the 34 kDa processed NOX form from cell surface resulted from protein purification studies. It was An incompletely processed 38.5 kDa protein that cross-reacts with the monoclonal antibody and is converted to the 34 kDa form by digestion with proteinase K was isolated from the HeLa cell surface. 38
． The 5 kDa protein yielded a partial N-terminal sequence that matched the N-terminal sequence of the predicted amino acid sequence of tNOX shown in SEQ ID NO: 2.

Further features of NOX proteins are two activities, NADH oxidative activity and protein disulfide-thiol exchange activity, which change every 12 minutes resulting in a regular periodic pattern (temperature regulated, about 24 (It has a period that can be extended to the minute) (FIG. 7). Exactly as compared to CNOX with a period length of 22 minutes (Po
gue et al., 2000), ttNOX had a shorter period of 23 minutes. Mutant ttNOX proteins with different cysteine to alanine substitutions were transformed into E. co
expressed in li. Of course, C505A and C569A are no longer NADH
It showed no oxidase activity. The other four cysteine mutants retained NADH oxidase activity but altered cycle length (Table 3). C575A and C
For 602A, the cycle length for both NADH oxidase and protein disulfide-thiol exchange increased to 36 minutes. C510A and C
For 558A, the cycle length was 39 minutes.

Our studies have identified aberrant cellular NADH oxidase activity on the cell surface and plasma membrane of plant and animal cells. The physiological function of the oxidative part of the NOX cycle is that of hydroquinone oxidase (Kishi et al., 19
99), External NADH oxidation provides a convenient measure of enzymatic activity. The interest of these proteins depends not only on their location at the plasma membrane, but on their timing (t
Potential role as ime-keeping protein (Wang et al.,
1998), and the relationship between cyclical enzyme activity and the increasing phase of cell growth (Mor.
Re, 1998; Pogue et al., 2000). NOX protein is 2
It is unique in that it exhibits two distinct activities, hydroquinone oxidation and protein disulfide-thiol exchange. The two activities change (Morre, 1
998; Sun et al., 2000), yielding a cycle of approximately 24 minutes.

While there may be several NOX forms, the first NOX form that has been cloned and identified is the cancer-specific form designated tNOX. tNOX differs from the constitutive CNOX forms present in both cancerous and non-cancerous tissues in its sensitivity to some anti-cancer and thiol agents. Processed tN from cancer patient sera based on selection with monoclonal antibodies using the response of tNOX activity to the quinone site inhibitor capsaicin
The identity of the cloned cDNA was finally confirmed based on complete capsaicin inhibition of the activity of the protein expressed in bacteria as a guide for purification of the OX protein (Figure 6).

Monoclonal antibodies against NADH oxidase inhibited by capsaicin from the sera of cancer patients unambiguously identified a single cDNA sequence encoding the antigen. This sequence was the APK1 antigen, a sequence previously thought of as a cytoplasmic protein (Chang and Pastan, 1994). AP
The K1 antigen was considered to be the antigen recognized by the monoclonal antibody designated K1 produced from hybridoma cells derived from mice immunized with ovarian carcinoma (OVCAR-3) cells. The longest cDNA in the study by Chang and Pastan (1994) is 789-bp, which encodes a 30.5 kDa protein.
2,444-bp with the open reading frame of Chan
Although the cDNAs isolated by g and Pastan contain missing and extra bases that produce an open reading frame that differs from our open reading frame,
It was almost identical to the tNOX cDNA.

The protein reactive with the K1 antibody was first identified as CAK1 (Cha
ng et al., 1992). CAK1 is a membrane-bound protein with a molecular weight of 40 kDa, while the expressed APK1 gene product produced soluble cytoplasmic proteins (Chang and Pastan, 1994). CAK1 is expressed in ovarian cancer and mesothelioma as well as normal mesothelial cells. CAK1 appears to be a different antigen expressed in mesothelial-derived cancers, such as the epithelial-like type mesothelioma and ovarian cancer. CAK1 is a protein that is very different from tNOX. Using the monoclonal antibody K1, Chang and Pastan used CA
The 2,138-bp cDNA encoding K1 was finally isolated (Chang.
And Pastan, 1996). The cDNA had a 1,884-bp open reading frame encoding a 69 kDa protein. 69 kDa
Was processed into a 40 kDa form and the protein was named mesothelin because it was characteristic of mesothelial cells. When this cDNA is transfected into COS cells and NIH3T3 cells, the antigen is found on the cell surface and can be released by treatment with phosphatidylinositol-specific phospholipase C. tNOX is not anchored to the plasma membrane by GPI binding and is not released by treatment with phosphatidylinositol-specific phospholipase C. Mesothelin (CAK1) is associated with cell membrane through the tail of glycosylphosphatidylinositol,
Mesothelin (CAK1) is not released into the serum of cancer patients and does not appear in the conditioned medium that supports the growth of cultured cells (Chang and Pastan, 1994).
). As previously described, tNOX was derived from growth medium by growth of HeLa cells (Wilkinson et al., 1996) and from serum of cancer patients (Chue).
h., et al., 1997). Furthermore, no protein sequence homology was reported between CAK1 and tNOX.

In a previous experiment, we found that [ ³² P] NAD (H) caused tNOX
Photoaffinity labeling of proteins was successful. This indicates that the tNOX protein contains a NADH binding site. NOX activity also responds to adenine nucleotides (Morre, 1998b). A typical adenine nucleotide binding sequence motif (D or E with a downstream distant acidic amino acid residue (
G-X-G-X-X-G) (Yano et al., 1997) is T589-G-V-G.
-A-S-L (amino acids 589-595 of SEQ ID NO: 2) and E6 near the C-terminus
05 is most closely indicated. This sequence is based on Chondous cris
Sequence TG-from mitochondrial ATP synthase protein 9 from pus
Most similar to V-A-A-G-V-G (SEQ ID NO: 3) (Leblanc et al., 1).
995).

The NOX protein is an anti-tumor sulfonylurea LY181984 (Morre
, 1995c) and activity is inhibited or stimulated depending on the redox environment of the protein (Morre, 1998b). Reduced coenzyme Q
Is readily oxidized by this protein (Kishi et al., 1999), and other substances that inhibit this activity (eg, capsaicin and adriamycin) are believed to occupy the quinone site. Ubiquinone is sulfonylurea L
Protect against inhibition of Y181984 binding and activity. Therefore, tNOX
The sequence was searched for the presence of a motif that is an indicator of quinone binding, and a motif that is an indicator of binding of other molecules known to occupy sulfonylurea and quinone sites.

The site of the methionine-histidine pair has been suggested to be the quinone binding site of pyruvate oxidase by its similarity to the quinone binding protein of several chloroplast photosystem II complexes (Grabau). And Cronan, 19
86). All known urea and sulfonylurea herbicide inhibitors of Photosystem II are directed to this site (Duke, 1990). Based on these considerations, a preliminary consensus sequence for amino acids surrounding charged residues important for the sulfonylurea and quinone binding sites has been identified as AMHG (
SEQ ID NO: 4) or a closely related sequence (Table 2). Clearly, arginine could replace the key histidine. For example, cyanobacteria (S
The putative quinone binding site of the D1 protein of Y. cinechococcus is sequence E
-T-M-R-E (SEQ ID NO: 5). A sequence similar to the E-TMR-E sequence is present in the chloroplast NADH ubiquinone dehydrogenase. Serum albumin also binds to sulfonylureas, and its putative sulfonylurea binding site is also included in Table 1. We have, as a possible quinone binding site, a sequence E-, which has neither H nor R at its 4-position but still has considerable similarity to other putative quinone and / or sulfonylurea binding sites. M-T-E (amino acids 395-398 of SEQ ID NO: 2) was found. This E- as a drug binding site
The accuracy of the identification of the M-T-E sequence is supported by findings from the M396A mutant that retained NADH oxidase activity but lost its inhibition by capsaicin (Table II).

The first demonstration of the thiol exchange activity of the tNOX protein is via reduction, refolding under non-denaturing conditions, and reoxidation to form the correct secondary structure stabilized by internal disulfide bonds. , As a major criterion for restoration of activity of yeast RNase that is reduced, denatured and oxidized (scrambled) (
Morre et al., 1997c). Restoration of activity against scrambled yeast RNase was similar to that of activity catalyzed by endoplasmic reticulum protein disulfide isomerase (Freedman, 1989), but was clearly attributed to the activity of different proteins. This activity was not altered by the presence of two different antisera against protein disulfide isomerase (Morr.
e et al., 1997c). One was a mouse monoclonal antibody from StressGen Biotechnologies (SPA-891) against protein disulfide isomerase from bovine liver (cross-reactive with human, monkey, rat, mouse and hamster cell lines). The other is a self-derived peptide antibody directed against a characteristic C-XX-C motif that is common to many (but not all) members of the protein disulfide isomerase family of proteins (Sharrosh and Dixon. , 1991), not present in tNOX. The C-X-X-C motif is also present in thioredoxin reductase and related proteins, where it appears to bind bound flavins and catalyze electron transport (Russel and Model, 1998; Ohnishi
Et al., 1995). In addition to lacking C-X-X-C, tNOX does not contain bound flavin and its activity dependent upon the addition of flavin (FAD or FMN). Thus, tNOX-catalyzed protein disulfide-thiol exchange appears to be distinct from traditional disulfide isomerase or thioredoxin reductase exchanges.

The redox-active disulfide of thioredoxin reductase from the malaria parasite Plasmodium falciparum has a motif C88-XX-XX-C93 (Gilber) similar to that found in tNOX.
ger). This motif, as well as downstream of His509, has been shown to be a putative hydrogen ion donor / acceptor. A second C535-XX-XX-X-C540 motif in the same protein is critically involved in substrate coordination and / or electron transport (Gilberger et al., 1998). As shown by site-directed mutagenesis, four of the eight cysteines present in truncated tNOX can be functionally paired. Site-directed mutagenesis (Table II) was performed using C505
It is shown that the A and C569A mutations show a loss of both NADH oxidase oxidase and protein disulfide thiol exchange activity (manuscript in preparation). Therefore, these two motifs, C505-XX-XX-X-C510.
And C569-XX-XX-X-C575, alone or with downstream histidine, may function as part of the tNOX active site. tNOX was initially tested for thioredoxin reductase activity and found nothing. tNOX
Shows the sequence C505-A-S-R-L-C510 (amino acid 505 of SEQ ID NO: 2) despite the deletion of the two C-X-X-X-X-C motifs characteristic of flavin proteins. -510) or the sequence C569-T-S-D-V-E-C575.
(Amino acids 569-575 of SEQ ID NO: 2) may represent a potential protein disulfide-thiol exchange motif.

The remaining four cysteine mutations analyzed further showed a modified period length of oscillation in tNOX activity (Table II). Here, both oxidation of NADH and protein disulfide thiol exchange appear to be affected in parallel.
The length of this period increased from 23 minutes to 36 minutes for C575A and C602, where for C510A and C558A the length of this period increased to 36 minutes. The 6 amino acid motif of tNOX M588-TGVGA (588-593 of SEQ ID NO: 2) is a Drosophila melanogast.
The observation that it is shared with the er clock period protein is potentially interesting (Kliman and Hey, 1993).

Under at least certain conditions, tNOX proteins catalyze the transport of electrons and hydrogen ions to molecular oxygen. Oxygen uptake by plasma membranes prepared from HeLa cells, like other aspects of tNOX activity, is inhibited by anti-tumor sulfonylureas LY181984 with approximately the same dose response (eg Mo.
See rre et al., 1998a). Therefore, tNOX and NOX proteins are generally assumed to bind oxygen. The minimum requirement for oxygen sites is
It appears to be a metal with appropriate covalent bonding action such as hydrogen bonding (MacBe
Th et al., 2000). They are the [4Fe-4S] cluster binding site (C-X
There is no indication that a representative motif for -X-C-XX-X-C) and proline may be clustered with the distant cysteine that follows. tNOX contains a conserved copper site, which may provide the basis for oxygen binding.

E. Expression of truncated tNOX in E. coli and COS cells confirmed that the cloned cDNA did show the characteristics of the tNOX protein. All forms of tNOX, including truncated and processed forms,
It is recognized by the tNOX-specific monoclonal antibody used in expression cloning. Furthermore, the expressed protein exhibited both enzymatic activity associated with NOX protein (FIGS. 6 and 7). Overexpression of tNOX protein in COS cells stably transfected with tNOX cDNA resulted in COS
The cells were given tNOX-specific characteristics. Cells transfected with rNOX cDNA have a cell size of 1.5-1.5 compared to control cells.
It shows a 2-fold increase (3-5 fold increase in cell volume) and is sensitive to tNOX inhibitory drugs including capsaicin, (−)-epigallocatechin gallate (EGCg), adriamycin and antitumor sulfonylureas. It showed an increase of ~ 2 log order (Table II). EGCg is the major catechin responsible for the effects of green tea and green tea extracts on cancer prevention and growth of cancer in culture (Chang, 2000). As is characteristic of other NOX inhibitors, E
GCg inhibits the activity of tNOX, but has little effect on constitutive CNOX (Morre et al., 2000).

In summary, the findings discussed herein confirm the molecular cloning and expression of the tNOX protein. The bioavailability of the cDNA and the expressed protein will greatly facilitate future studies of the potential contribution of tNOX to unregulated growth and the loss of differentiated features associated with cancer.

The primary screen of a commercially available HeLa cell cDNA library is
This was done by selecting from a total of 16 150 mm plates. Five positive clones (clone 1, 2, 4, 5 and 6; clone 3 was concluded to be a false positive clone in the secondary screen) were identified, and through at least 3 rounds of screening Further purified. Due to its simplicity and speed, subsequent in vivo excision was performed rather than subcloning. Clone 1 contained the longest DNA insert having about 3900 bp and clone 5 contained the shortest DNA insert having about 2000 bp (
(Fig. 1). Several restriction endonucleases were used to determine restriction sites (
(Fig. 1). The Uni-Zap XR library used in this study was
Constructed with a double digest of coRI and XhoI. However, digestion with EcoRI or XhoI alone showed that both an internal EcoRI site and XhoI were present near the 5'end of the antisense strands of clone 1, clone 2 and clone 4. The lack of internal EcoRI and XhoI sites in both clone 5 and clone 6 indicated that the DNA inserts of these two clones were located further downstream of the shorter 3'end. In addition, all five clones contained internal BamHI and XbaI sites. Double digestion of these two enzymes in each clone produced a small (about 400 bp) DNA segment. This phenomenon verified that these sites were identical in all five clones. This restriction mapping revealed that five independent clones were identical except for DNA inserts of different lengths. Clone 1
Contains the longest DNA insert, which was selected for full-length DNA sequencing. The remaining four clones were sent in one automated sequencing. The DNA sequences of all 5 clones were examined on the GenBank looking for identities or relationships with other known genes. A computer-assisted search revealed that all five clones were similar to the DNA sequence designated as the APK1 antigen [Chang and Pastan (1994) supra]. When all five clones are compared to the nucleotide sequence of the APK1 antigen, two potential differences are observed at positions 83 and 246 of the APK1 antigen sequence. These two differences result in changes in the open reading frame and the deduced amino acid sequence.

Nucleotide sequences encoding human tNOX, recombinant human tNOX proteins and recombinant cells expressing recombinant human tNOX may be used in whole cancer ( pan cancer).
2.) In the production of recombinant tNOX for use in diagnostic protocols and as a target for new anti-cancer drugs (screening).

There may be a limited number of amino acid substitutions in the plasma membrane NADH oxidase protein without significantly affecting function, and undemonstrated plasma membrane NADH oxidase, viral or viral in neoplastic mammalian cells. It will be appreciated by those skilled in the art that the parasite-infected mammalian cell or capsaicin-responsive plant plasma membrane NADH oxidase protein may have some amino acid sequences that differ from the amino acid sequences specifically exemplified. Such naturally occurring variants are
For example, at least about 70% nucleotide sequence homology, preferably about 80%,
More preferably, under conditions suitable to detect about 90% or 95-100% sequence homology, to the exemplified coding sequence (or a portion thereof capable of specifically hybridizing to the human tNOX sequence). It can be identified by hybridizing to it. Preferably, the encoded tNOX has at least about 90% amino acid sequence identity to the exemplified tNOX amino acid sequence. In exploring sequences that are not exemplified, the characteristic plasma membrane NADH oxidase and protein thiol exchange activities and the susceptibility of these activities to inhibitors such as capsaicin can be determined by those skilled in the art to produce functional proteins. Allows you to check.

Also within the scope of the invention is an isolated nucleic acid molecule that comprises a nucleotide sequence encoding a tNOX protein. This is a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1,
Alternatively, it hybridizes to the sequence corresponding to its nucleotides 23 to 1852 under stringent conditions. A DNA molecule having at least 85% nucleotide sequence identity to a specifically exemplified tNOX coding sequence of the present invention will be highly labeled under stringent conditions using the probes disclosed herein. Identified by hybridization. Stringent conditions are eg aqueous solution (5 × SSC, 5 × Denhardt's solution, 1% sodium dodecyl sulphate) using 0.2 × SSC at room temperature, washing in 0.1% sodium dodecyl sulphate. Hybridization at a temperature between 65 ° C and 68 ° C in or about 42 ° C in 50% formamide solution. Specific exemplified tNOX of the present invention
The ability of a sequence to relate to a sequence can be readily tested as known to those of skill in the art.

As used in the present context, the percent homology or percent identity of two nucleic acid molecules can be calculated according to Karlin and Altschul (1990) Proc.
Natl. Acad. Sci. USA 87, 2264-2268 (Karli
n and Altschul (1993) Proc. Natl. Acad. Sci
． USA 90,5873-5877), modified as described in USA 90,5873-5877). An algorithm like this is Altsch
ul et al. (1990) J. Mol. Biol. 215, 402-410 NBLA
Incorporated into the ST and XBLAST programs. BLAST nucleotide searches are performed using the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the nucleotide sequences of the invention.
BLAST protein searches are performed using the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the reference polypeptide sequence. To obtain a gapped alignment for comparison purposes, Altsch
1 et al. (1997) Nucl. Acids Res. 25,3389-3402 utilized gapped BLAST. When using BLAST and BLAST programs with gaps, the respective programs (XBLAST
ST and NBLAST) default parameters. Gaps introduced to optimize alignment are treated as mismatches when calculating identity. For example, http: // www. ncbi. nlm. gov. checking ...

In the biological field, specific amino acid substitutions can be made in protein sequences without affecting the function of the protein. In general, conservative amino acids are resistant without affecting protein function. Similar amino acids can be similar in size and / or charge properties; for example, asparagine and glutamine and isoleucine and valine are both pairs of similar amino acids. The similarity between amino acid pairs has been evaluated in the art in a number of ways. For example, Dayhoff et al. [(1978): Atlas of Protein S
sequence and Structure, Volume 5, Addendum 3, Chapter 22, 3
45-352], which is incorporated herein by reference and provides a frequency table for amino acid substitutions that can be used as a measure of amino acid identity. Day
The frequency table of hoff et al. is based on a comparison of amino acid sequences for proteins with identical function from different evolutionarily different sources. This technology is tNO
Methods are provided for determining X activity, including its property of reacting with specific inhibitors (such as capsaicin, adriamycin, quassinoids).

A polynucleotide or fragment thereof, optionally aligned with another polynucleotide (with appropriate nucleotide insertions or deletions), contains about 60%, usually about 70%, and more usually, of the nucleotide bases. About 80%, preferably about 90%
, And more preferably, is substantially homologous (or substantially similar) to another polynucleotide if there is 95% -100% nucleotide sequence identity of the nucleotide bases.

Alternatively, substantial homology (or similarity) exists when a polypeptide or fragment thereof hybridizes to another polynucleotide under selective hybridization conditions. Hybridization selectivity exists under hybridization conditions that allow the target polynucleotide of interest to be distinguished from other polynucleotides. Typically, a selective high is present when there is about 55% similarity, preferably about 65%, more preferably about 75%, and most preferably about 90% similarity over about 14 nucleotide stretches. Hybridization occurs. For example, Kanehisa [(1984) Nuc.
l. Acids Res. 12: 203-213]. As noted, the lengths of homology comparisons span stretches over longer stretches, and in certain embodiments are often stretches of greater than about 17-20 nucleotides, and preferably about 36 or greater. It is a nucleotide. Hybridization of polynucleotides includes salt concentration, temperature, as well as base composition, complementary strand length, and the number of nucleotide base mismatches between the hybridizing polynucleotides, as will be readily appreciated by those skilled in the art. Or it is affected by conditions such as an organic solvent. However, the combination of parameters is much more important than the measurement of any single parameter [Wetmur and Davidson (196
8) J. Mol. Biol. 31: 349-370].

An isolated or substantially pure polypeptide is a polynucleotide that is substantially separated from other polynucleotide sequences that naturally accompany the native tNOX protein coding sequence. This term includes polynucleotide sequences that have been removed from their naturally-occurring environment, and has been recombinant or cloned DNA.
Includes isolates, chemically synthesized analogs and analogs biologically synthesized by heterologous systems.

A polynucleotide is a polypeptide when it is in its native state or engineered in a manner known to those of skill in the art, it can be transcribed, and / or translated to produce fragments of the polypeptide. Is said to code. The antisense strand of such a polynucleotide is also said to encode the sequence. The assay method as described herein below provides that active tNOX protein having an intact reaction pattern to an authentic inhibitor of tNOX is
It makes it possible to confirm that the coding sequences disclosed herein in a recombinant host cell are produced upon expression.

A nucleotide sequence is operably linked when it is placed into a functional relationship with another nucleotide sequence. For example, a promoter is operably linked to a coding sequence if it affects its transcription or expression. Generally, operably linked means that the sequences being linked are contiguous, and, where necessary to join the two protein coding sequence regions, contiguous and in reading frame. To do. However, certain genetic elements (eg, enhancers) may be operably linked, whether distant (ie, not in close proximity).

The term naturally-occurring or recombinant nucleic acid molecule refers to two other distinct segments of sequence achieved by the artificial manipulation of a segment of an isolated polypeptide by genetic engineering techniques or chemical synthesis. A polynucleotide produced by the combination of By doing so, one of ordinary skill in the art can ligate with a polynucleotide segment of the desired function to produce the desired combination of functions.

Polynucleotide probes include isolated polynucleotides attached to labels or reporter molecules and can be used to identify and isolate other tNOX protein coding sequences. Probes containing synthetic oligonucleotides or other polynucleotides can be naturally occurring or can be derived from recombinant single-stranded or double-stranded nucleic acids, or are chemically synthesized. These can be used in the polymerase chain reaction and hybridization. The polynucleotide probe can be labeled by any of the methods known in the art, such as random hexamer labeling, nick translation, or Klenow filling reaction.
Oligonucleotide or polynucleotide primers useful in PCR are readily understood and accessible to those of skill in the art using the information provided herein using methods well known in the art.

Large amounts of polynucleotide can be produced by replication in a suitable host cell. Encodes tNOX incorporated into a recombinant polynucleotide construct (typically a DNA construct) that is transducible and replicable in prokaryotic or eukaryotic cells, desirably yeast cells, and preferably Saccharomyces cerevisiae cells. Natural or synthetic DNA fragments are provided by the present invention. This construct is usually suitable for replication in a unicellular host (eg yeast or bacterium) with or without incorporation into the genome of the host cell, but multicellular eukaryotic hosts are also suitable. possible. Commonly used prokaryotic hosts include Escherichia coli strains, but other prokaryotes (eg, Bacillus subtilis or Pseudomo).
nas) may also be used. Examples of yeast suitable for the present invention include Saccharo
species of Myces and Pichia (eg, Pichia pastoris). Mammalian (eg, CHO or COS cells) or other eukaryotic host cells include filamentous fungi, plants, insects, amphibians and birds.
These factors as the ease of manipulation, the appropriately glycosylated expressed protein, the extent and control of protein expression, the easy purification of the expressed protein without cell contamination, or other factors determine the choice of host cell. obtain. Suitable vectors for use in the aforementioned host cells are well known in the art and are widely available in the laboratory and commercially.

This polynucleotide may also be used, for example, in Beaucage and Caruth.
ers [(1981) Tetra. Letts. 22: 1859-1862] by the phosphoramidite method, or by Matteuci et al. [(1981) J. Am. Chem. ． Soc. 103: 3185], and can be performed chemically on a commercially automated oligonucleotide synthesizer. Double-stranded fragments are synthesized by complementary strands and annealed the strands together under appropriate conditions, or adding the complementary strands using DNA tNOX protein with appropriate primer sequences. Either can be obtained from a chemically synthesized single-stranded product.

A DNA construct prepared for introduction into a prokaryotic or eukaryotic host cell comprises a replication system recognized by the host cell (ie, containing a DNA fragment intended for encoding the desired polypeptide). Vector), and preferably also includes transcription initiation and translation initiation regulatory sequences operably linked to the tNOX protein coding segment. As an expression system (expression vector),
Origins of replication or autonomously replicating sequences (ARS) and expression control sequences, promoters, enhancers and necessary processing information sites (eg ribosome binding sites, RNA splice sites, polyadenylation sites), transcription termination sequences, and An mRNA stabilizing sequence is included. A signal peptide may also be included at the site where a secreted polypeptide of the same or related species is appropriate. This allows the protein to cross and / or stay across the cell membrane or be secreted from the cell.

Appropriate promoters and other necessary vector sequences are chosen to be functional in the host. Examples of working combinations of cell lines and expression vectors are Sa
mbrook et al. [(1989) et al .; Ausubel et al., Ed. (199).
2) Current Protocols in Moecular Biol
Ogy, Greene Publishing and Wiley Int
erscience, New York] and Metzger et al. [(1988
) Nature 334: 31-36]. Many useful vectors for expression in bacteria, yeast, mammals, insects, plants, or other cells are well known in the art and can be found in Stratagene, New England B.
It can be obtained from iolabs, Promega, and other manufacturers and the like. In addition, the construct can be added to an amplifiable gene (eg, DHFR) such that multiple copies of the gene are made. For suitable enhancers and other expression control sequences, see also, for example, Enhancers and Eukaryo.
tic Gene Expression, Cold Spring Harb
or Press, NY (1983). Although such expression vectors are capable of autonomous replication, they may not replicate more favorably by being inserted into the host cell genome.

Expression and cloning vectors desirably include a selectable marker (ie,
Gene encoding a protein required for survival or growth of a host cell transformed with the vector). Such marker genes can be carried on separate polynucleotide sequences that can be co-introduced into the host cell, but are most often included on cloning vectors. Only those host cells into which the marker gene has been introduced will survive and / or grow under selective conditions. A typical selection gene is (
a) confers resistance to antibiotics or other toxic substances such as ampicillin, neomycin, methotrexate, etc .; (b) complements auxotrophy.
Or (c) encodes a protein that supplies important nutrients not available from complex media. The choice of the proper selectable marker depends on the host cell; suitable markers for different hosts are known in the art.

The coding and deduced amino acid sequences for tNOX are provided in Table 1. See also, for example, SEQ ID NO: 1 and SEQ ID NO: 2.

Restriction via PCR using an oligonucleotide containing the desired restriction site for cloning (not present in the coding sequence), ribosome binding site, translation initiation codon (ATG) and codon for the first amino acid of tNOX. A combination of endonuclease cleavage and site-directed mutagenesis can be used to engineer tNOX for recombinant expression. Site-directed mutagenesis strategy
For example, modified using PCR as would be readily understood by one of skill in the art,
Boone et al. [(1990) Proc. Natl. Acad. Sci. USA
87: 2800-2804].

Those of skill in the art will appreciate the exemplified t for improved expression in particular recombinant host cells.
It will be appreciated that it may be advantageous to modify the NOX coding sequence. Such modification, which can be carried out without undue experimental expense using the present disclosure incorporating readily accessible knowledge and techniques in the art, results in modified tNO.
Adapting the codon usage so that the X protein coding sequence has a codon usage substantially similar to the known usage for the target host cell. Such modification may be chemical synthesis of a coding sequence synonymous with the exemplified coding sequence, or
It can be affected by oligonucleotide site-directed mutagenesis of selected portions of the coding sequence.

Compositions and immunogenic preparations (including vaccine compositions) comprising a substantially purified recombinant tNOX virus, or an immunogenic peptide having an amino acid sequence derived therefrom and a carrier therefor, Provided by the present invention. Alternatively, the hydrophilic regions of tNOX can be identified by one of skill in the art and, if required for use in vaccines, in raising polyclonal or monoclonal antibodies specific for the exemplified tNOX, the peptide antigen is It can be synthesized and conjugated to a suitable carrier protein such as bovine serum albumin or keyhole limpet hemocyanin. An immunogenic composition is one that, when injected into a human or animal, results in the production of specific antibodies. The vaccine preparation contains an immunogenic amount of the exemplified tNOX or immunogenic fragment thereof. Such vaccines may include tNOX alone or in combination with another protein or other immunogen. "Immunogenic amount" means an amount capable of inducing the production of antibodies directed against the exemplified tNOX in the individual or animal to which the vaccine is administered.

Immunogenic carriers can be used to enhance the immunogenicity of peptides derived from tNOX or sequences thereof. Such carriers include, but are not limited to, proteins and polysaccharides, liposomes, and bacterial cells and membranes. The protein carrier may be attached to the tNOX protein or peptides derived therefrom to form fusion proteins by recombinant or synthetic means or chemical coupling. Useful carriers and means for coupling such carriers to polypeptide antigens are known to those of skill in the art.

Preferred fusion proteins effective in stimulating an immune response, especially when administered orally (eg in food or water), include fusion proteins with cholera toxin fragments, the so-called LTB fusions. These methods are
an et al. [(1990) Biochem. Soc. Trans. 18: 746-7
48] and Elson et al. [(1984) J. Immunol. 132: 273
6-2741].

The immunogenic composition and / or vaccine can be formulated by any means known in the art. These are typically prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for suspension or solution in liquid prior to injection can also be prepared. The preparation may also be emulsified or protein / peptide encapsulated in liposomes, for example.

The active immunogenic ingredient is often mixed with excipients or carriers that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, but are not limited to, water, saline, dextrose, glycerol, ethanol and the like, and combinations thereof. The concentration of immunogenic polypeptide in the injectable formulation is usually in the range of 0.2-5 mg / ml.

In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and / or adjuvants that enhance the effectiveness of the vaccine. Examples of adjuvants that may be effective include, but are not limited to: aluminum hydroxide; N-acetyl-muramyl-L-threonyl-
D-isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-
Alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP); N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2- (1'-2'-dipalmitoyl-sn- Glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP 19835A, referred to as MIP-PE)
; And RIBI. This RIBI contains three components extracted from bacteria: monophosphoryl lipid A in 2% squalene / Tween 80 emulsion, trehalose dimycolate, and cell wall skeleton (MPL + TDM + CWS). Efficacy of adjuvants can be determined by measuring the amount of antibody directed against the immunogen resulting from administration of the immunogen in a vaccine that also contains various adjuvants. Such additional formulations and modes of administration known in the art can also be used.

To tNOX as exemplified herein and / or to other tNOX proteins having a primary structure that is derived from this tNOX or is similar (> 90% identical) to the exemplified tNOX protein. The derived epitope fragment or peptide sequence may be formulated into the vaccine in neutral or salt form. Pharmaceutically acceptable salts include, but are not limited to, acid addition salts (salts formed with free amino groups of peptides). The salt is formed with an inorganic acid (eg hydrochloric acid or phosphoric acid); and an organic acid (eg acetic acid, oxalic acid, tartaric acid and maleic acid). Salts formed with free carboxyl groups also include inorganic bases (eg, sodium, potassium, ammonium, calcium, or iron hydroxide) and organic bases (eg, isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, and Procaine).

A multi-antigenic peptide having an amino acid sequence derived from tNOX which is exemplified for use in immunogenic compositions is described by Briand et al. [(1992) J. Im
munol. Methods 156: 255-265].

The immunogenic composition or vaccine is administered in a manner compatible with the dosage formulation and in such amount as is prophylactically and / or therapeutically effective. The amount administered (typically within the range of about 100-1000 μg of protein per dose, more typically,
Within the range of about 5-500 μg protein per dose),
It depends on the capacity of the individual immune system to synthesize the antibody, and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgment of the veterinarian, physician or dental technician and may be peculiar to each individual, but such determinations would be well known to those of ordinary skill in the art. It is one of the technologies. Particularly for poultry, the immunogenic composition may be administered orally from a food or water preparation containing an effective amount of protein (s) and / or peptide (s), and The sex composition may be formulated with liposomes as is known in the art.

The vaccine or other immunogenic composition may be given in a single dose or a multiple dose schedule. A multi-dose schedule with one first vaccination step
To 10 or more individual doses, which, in turn, may be followed by consecutive time intervals necessary to maintain and / or enhance the immune response (eg, 2 doses every 1-4 months, and Other medications, if required, given in consecutive doses (one or more) several months later.

Antibodies specific for plasma membrane tNOX, and shed forms in urine and serum of cancer patients and animals with neoplastic disorders can be expressed, for example, by D
It is useful as a probe for screening NA expression libraries or for detecting or diagnosing neoplastic disorders in samples of human or animal origin. Desirably, these antibodies (or secondary antibodies specific for antibodies recognizing tNOX) are either covalently or non-covalently bound,
It is labeled by linking a substance that produces a detectable signal. Suitable labels include, but are not limited to, radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent reagents, chemiluminescent reagents, magnetic particles and the like. US patents describing the use of such labels are: 3,817,837; 3,850,752; 3,939,
No. 350; No. 3,996,345; No. 4,277,437; No. 4,275.
No. 149; and No. 4,366,241. Antibodies useful in diagnostic and screening assays are prepared using a peptide antigen of sequence derived from all or part of SEQ ID NO: 2 (eg, SEQ ID NO: 16, full length protein or protein corresponding to amino acids 220-610). obtain.

All references cited herein are hereby incorporated by reference in their entirety to the extent there is no conflict with the present disclosure.

Peptide synthesis, cloning, except as described hereinafter.
Standard techniques for DNA isolation, amplification and purification, DNA ligase, DNA, tN
Standard techniques for enzymatic reactions, including OX proteins, restriction endonucleases, as well as a variety of separation techniques, are known to those of skill in the art and are routinely used.
Many standard techniques have been described by Sambrook et al. (1989) Molecular Cl.
oning, 2nd Edition, Cold Spring Harbor Laborat
ory, Plainview, New York; Maniatis et al., (19
82) Molecular Cloning, Cold Spring Har
bor Laboratory, Preview, New York; Wu (eds.) (1993) Meth. Enzymol. 218, Part 1: Wu (ed.) (19
79) Meth Enzymol. 68; Wu et al. (Eds.) (1983) Meth.
Enzymol. 100 and 101; Grossman and Moldave
(Edit) Meth. Enzymol. 65; Miller (ed.) (1972) Ex
perimeters in Molecular Genetics, Cold
Spring Harbor Laboratory, Cold Spring
g Harbor, New York, Old and Primrose (198).
1) Principles of Gene Manipulation, Un
diversity of California Press, Berkele
y; Schleif and Wensink (1982) Practical M
methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Volumes 1 and 2, IRL Pres.
s, Oxford, UK; Hames and Higgins (eds.) (1985).
Nucleic Acid Hybridization, IRL Press
, Oxford, UK; Setlow and Hollaender (1979).
Genetic Engineering: Principles and M
Methods, Volumes 1-4, Plenum Press, New York. Abbreviations and nomenclature (as used herein) are considered standard in the art and are commonly used in specialized journals such as those cited herein.

The foregoing discussion and the following examples are illustrative, but not intended to limit the invention.
Those skilled in the art will appreciate that alternative methods may be used to practice the invention.

Example 1. Materials and Bacterial Cultures Monoclonal antibody antigens were isolated as previously described [Chueh.
(1997)]. Peroxidase-conjugated goat anti-mouse IgG (Jackson
ImmunoResearch Laboratories, West Gr
ove, PA) was used to form the antigen-antibody-antibody-AP color complex. E. c
oli strains XL1-blue and E. coli. coli strain SOLR, Uni-Zap X
The R HeLa cell cDNA library, helper phage and expression vector pET11 were purchased from Stratagene (La Jolla, Ca). Luria-Bertani broth (LB broth) medium and agar were added to DIF.
Purchased from CO (Detroit, MI). A DNA marker, restriction endonuclease and plasmid DNA purification kit were added to Promega (Madiso).
n, WI). A mammalian expression system (including the expression vector pcDNA3.1) was purchased from Invitrogen (Carlsbad, CA). Unless otherwise indicated, all chemicals are Sigma Chemical Co. (S
t. Louis, MO).

RecA ^- E. E. coli host strain (XL1-blue) was first added to 100 mmL
Streak on B-tetracycline (12.5 μg / ml) agar plate, then 3
Incubated overnight at 7 ° C. One isolated colony was picked with a sterile wire loop and then plated in LB medium at 37 ° C. This plate
Cover with parafilm and place in the refrigerator at 4 ° C until the next stroke.

50 ml of LB broth was supplemented with 0.2% (v / v) maltose and 10 mM MgSO ₄ in a sterile flask. Cells are grown overnight at 37 ° C with gentle shaking. On day two, liquid cultures were centrifuged for 15 minutes in a 4,000 rpm sterile conical tube, then the cell pellet was cleared of medium. Pellets were gently resuspended in 10 mM MgSO ₄ solution in 15 ml. continue,
The cells were diluted with 10 mM MgSO ₄ to an OD ₆₀₀ of 0.5 for later use. New streak plates were used in all experiments.

Example 2. Production of Monoclonal Antibodies The antigens utilized in the production of monoclonal antibodies were isolated and characterized from pooled cancer patient sera (Chueh et al., 1997). Fractions containing an approximately 34 kDa protein with capsaicin-inhibited tNOX activity were labeled with Centricon.
(Amicon, MA) and then washed 3 times with PBS to remove excess salt. Monoclonal antibodies and hybridomas were tested using standard protocols (Sch
Mo, Purdue Cancer Center according to ook, 1987).
It was generated by nonclonal Antibody Facility. Two B
ALB / c mice were immunized with tNOX protein mixed with complete Freund's adjuvant and boosted 3 times at 3 week intervals. Hybridomas were screened by both enzyme activity assay and Western blot analysis. An antiserum-producing clone was selected with the following characteristics: ability to completely block drug-responsive NOX activity of cancer cells and serum of cancer patients, capsaicin-inhibiting NADH from serum pooled from cancer cell surfaces and cancer patients. Ability to immunoprecipitate proteins with oxidase activity, no effect on serum from healthy volunteers on NADH oxidase activity, and reactivity with HeLa cells and cancer patient serum with 34 kDa cell surface proteins.

Example 3. Isolation of cDNA Clones The HeLa Uni-Zap cDNA library was cloned into monoclonal complex water (1: 100 dilution) and peroxidase as described in [Sambrook et al. (1989) supra]. Initially screened at approximately 50,000 plaque forming units per 150 mm plate with conjugated goat anti-mouse IgG (1: 50,000 dilution). Five positive plaques were isolated from about 8 × 10 ⁵ total plaques screened and the total amount of homogeneously purified bacteriophage by at least 3 rounds of screening and selection. In vivo excision of positive phage clones with ExAssist helper phage (M13) was then performed on Un.
It was performed according to Stratagene's protocol for converting the i-Zap plasmid into pBluescript phagemid. Circular phagemid DNA
Was extracted using the Wizard Plus miniprep DNA purification kit (Promega, Madison, WI) according to the manufacturer's recommended use. ExoR
Restriction enzyme mapping using I, XhoI, and BamHI showed that all 5 clones were identical in origin. The tNOX insert was added to T3 'and T7.
Sequencing was performed using the primers. The complete nucleotide sequence of cDNA clone 1 was cloned using the gene walking technique and 10 17 bp synthetic primer (DNA
Sequencing Service, Tufts University,
Boston, MA). A search in the NCBI / GenBank database had nucleotide and deduced amino acid sequence information corresponding to the naked longest open reading frame.

Example 4 DNA Agarose Electrophoresis 1.2% agarose gel, 0.9 g agarose, 75 ml TBE buffer (10.8 g Tris, 5.5 g boric acid, and 0.93 g Na ₂ EDTA
2H ₂ O was made up to 1 liter in distilled deionized water) and heated until all the agarose gel was completely dissolved. TBE buffer was filtered before use. Ethidium bromide was added to the gel solution at a final concentration of 0.5 μg / ml solution before placing the gel solution in the mold. Immediately, the mixture was poured into a mold and the comb was in place. The gel was modeled for at least 30 minutes before electrophoresis. The comb was removed, the gel was placed in the electrophoresis system, and TBE buffer was added until the gel was covered with buffer. Marker and DNA samples were mixed with loading buffer and pipetted into separate wells. Electrophoresis was performed at 90V for about 1.5 hours. Example 5. Sequencing analysis and restriction mapping Several restriction endonucleases (EcoRI, XhoI, BamHI, X).
baI, Kpnl and SalI) were used to determine the restriction sites. Digestion was performed according to the protocol provided by Promega (Madison, WI). 11 μl H ₂ O, 2 μl 10 × reaction buffer, 2 μl BSA (
1 μg / μl), 4 μl of DNA and 1 μl of each restriction endonuclease were mixed by pipetting into Eppendorf tubes and centrifugation for a few seconds. This mixture was incubated at optimal temperature for 3-4 hours depending on the enzyme. Subsequently, agarose electrophoresis was performed after each digestion. DNA sequence
Initially analyzed by an automated sequencer using T3 and T7 primers. The complete nucleotide sequence was determined by both DNA strands. 10 gene walks
This was done by using a 17 bp synthetic primer. The nucleotide sequences of all 5 clones and the deduced amino acid sequence of clone 1 were analyzed for homology to GenBank using the BLAST and Pedro programs.

Example 6 Expression of tNOX and Histidine-Tagged tNOX Protein in Bacteria The tNOX cDNA from clone 1 was shortened (ttNOX) (M220).
As a fusion protein (ttNOX-his) in which 6 histidine residues and the amino terminus of ttNOX are fused.
Start with). expressed in E. coli. First, ttNOX
The open reading frames of DNA and nucleotides of the 3'untranslated region were amplified by PCR, digested with NdeI and BamHI, and subsequently ligated into the protein expression vector pET-11b. Label all primers
ory for Macromolecular Structure (Pur
Due University, IN).

[0087]

[Chemical 1] The forward primer was designed to incorporate 6 histidine residues at the amino terminus of the tNOX protein. The amplification carried out comprises a step of initiation at 94 ° C. for 90 seconds,
Subsequent denaturation at 94 ° C for 90 seconds, annealing at 55 ° C for 90 seconds, and 72
The extension was 90 seconds at 90 ° C. for 29 cycles.

E. E. coli [BL21 (DE3)] and transfected at 25 ° C for 16
For a period of time, they were grown in LB medium containing ampicillin (100 μg / ml) and collected. The DNA sequence of the ligation product was confirmed by DNA sequencing. Expression of all forms of tNOX was confirmed by SDS-PAGE with silver staining and immunoblotting. Immunoblot analysis used anti-tNOX monoclonal antibody. For detection, an alkaline phosphate-conjugated anti-mouse antibody was used.

Example 7. Expression of ttNOX in COS cells Transient transfection of COS cells was performed by following the manufacturer's protocol, pcDNA3.1 (Invitrogen) and Calcium Phos.
Phate Transfection Kit (Invitrogen, Ca
rlsbad, CA) was used.

[0090]

[Chemical 2] The gene product was double digested with HindIII and BamHI enzymes. The digested products were separated on an agarose gel and subjected to DNA Extract.
Extracted using Kit (Qiagen, Valencia, CA). This DNA was then ligated into the pcDNA3.1 vector containing the cytomegalovirus enhancer promoter for high level expression. Plasmid DN
For propagation of A, the ligation product was used to transform XL-1 blue competent cells using the heat pulse technique (Sambrook et al., 1989, supra). Positive clones were identified by PCR. The resulting plasmid was then used to transfect COS cells.

COS-1 cells (African monkey kidney cell line) were added to 4c per 100 mm Petri dish.
10 ⁵ cells were plated one day before transfection. 36 μl Ca
30 μg of pcDNA3.1 or pcDNA3.1-tNOX in Cl ₂ (2M) and 300 μl of sterile water was added to 300 μl of 2 × Hepe at room temperature for 30 minutes.
s buffer solution (HBS) was slowly added dropwise. The transfection mixture was then added dropwise to the cell's medium and incubated overnight at 37 ° C. After overnight exposure to DNA precipitate, cells were washed twice with PBS and 3 ml DMSO 2.5.
Added for minutes. DMSO was then removed and cells were fed with fresh medium for 2-3 days. tNOX expression was evaluated based on enzyme activity and Western blot analysis. The antibiotic G418 sulfate (Invitrogen, Carlsbad, CA) was used to select stable transfectants. COS7
0.5 m after transfection of cells with tNOX cDNA expression plasmid
G / ml G418 sulphate was added to the medium twice a week and the culture was maintained until colonies 2-3 mm in diameter were formed. A total of 3 colonies were selected and individually trypsinized and transferred to wells of 24-well plates and then to 35 mm Petri dishes. Cells were harvested at 80% confluency. Transfection was confirmed by immunoblot.

Example 8 N-Terminal Amino Acid Sequencing of Expressed tNOX For partial amino acid sequencing, recombinant E. Recombinant tNOX protein from E. coli extract was precipitated with 20% ammonium sulphate and 12% SDS-
Electrophoresed on PAGE and poly (vinylidene difluoride)
lidene difluoride)) membrane. The protein was stained with Coomassie blue and the protein band was excised and then the L
laboratory for Macromolecular Structu
Re, Purdue University for automated Edman decomposition (Appl
Sequenced by ied Biosystems, Procise 492).

Example 9 Production of Peptide Antiserum Peptide antiserum directed against the tNOX terminus (including the putative adenine binding site KQEMTGVGASLEKRW (SEQ ID NO: 16)) was used as Covance Reese.
rch Products Inc. Produced in rabbits using standard techniques by (Dever, PA). This antiserum was diluted 1: 300 before use.

Example 10. Production of polyclonal antisera Recombinant E. Recombinant truncated tNOX from E. coli extract was precipitated with 20% ammonium sulfate, the solubilized protein was separated on 12% SDS-PAGE and stained with Coomassie blue. This tNOX protein band was cut out and cut into fine pieces. Then add 0.5 ml of this protein
Was mixed with Complete Freund's Adjuvant, and injected into 2 rabbits. Three boosts of antigen in incomplete Freund's adjuvant were given every 3 weeks. This antiserum was diluted 1: 300 before use.

Example 11. RNA Isolation and Northern Analysis Total RNA was analyzed by the guanidinium method (Ausubel et al. (1992), Curren.
t Protocols in Molecular Biology, Wil
ey Interscience, New York, NY) and prepared from HeLa (or other cells). Denatured RNA is Sam
Transferred to nitrocellulose membrane for hybridization and autoradiography essentially as described in Brook et al. ((1989) supra).

MRNA is isolated from a biological sample, biopsy material, tumor tissue or others thereof and separated using gel electrophoresis. Appropriate condition is 1.2%
Agarose and 2.2 mol / liter formaldehyde. mRNA
Size is assessed relative to a marker molecule (eg, a commercially available 0.28 to 6.58 kb marker (eg, a marker from Promega Madison, WI)). Lanes containing marker molecules are stained with ethidium bromide and photographed with UV illumination. Transfer of RNA molecules from the gel to the nitrocellulose filter is accomplished as described by Maniatis et al. (1982) supra.
Blots for 2 hours at 42 ° C., 50% formaldehyde, 5 × SSPE, 2 ×
Denhardt's solution, and 0.1% sodium dodecyl sulfate (SDS
) Together with the above). Denatured radiolabel or other labeled probe nucleic acid was added directly to the prehybridization solution and the incubation continued for a further 16-24 hours. The blot is then incubated at room temperature for 20 minutes in 1 × S.
Wash with SC, 0.1% SDS, followed by three washes of 0.2 x SSC at 68 ° C for 20 minutes and 0.1% SDS at 68 ° C for 20 minutes, respectively. The labeled probe is then visualized according to the label used. If the label is radioactive, autoradiography may be used.

Samples for use in nucleic acid based diagnostics include 15-25 ml of peripheral blood sample. For biopsy or other tumor tissue samples, the tissue or biopsy sample is frozen in liquid nitrogen immediately after collection. Cells of basal tissue or blood are lysed in guanidinium thiocyanate, left at 50 ° C. for 15 minutes, then centrifuged at 3000 rpm for 5 minutes. The supernatant was layered on cesium chloride and centrifuged. The RNA pellet was lysed with diethyl pyrocarbonate. Approximately 2 mg of RNA is provided to the supplier (eg Promega
Use for cDNA synthesis using commercially available reagents according to the instructions in a). PCR
It can be carried out using commercially available reagents and primers specific for tNOX mRNA. The integrity of the RNA sample is confirmed using an inappropriate gene product, for example glyceraldehyde 3 phosphate dehydrogenase, the sequence of which is well known.

Example 12 Mutagenic Oligonucleotides and Site-Directed Mutagenesis Eight sets of oligonucleotides were tested for amino acid residues potentially involved in tNOX activity by site-directed mutagenesis according to Braman et al. (1996). Designed to replace. C505, C510, C558, C569, C575, and C
The cysteine codon corresponding to 602 was replaced with an alanine codon. The coding sequence was independently modified to replace alanine for methionine at the putative drug binding site (M396A). The coding sequence of tNOX was independently modified to replace valine for glycine at the potential adenine binding site (G592V). The oligonucleotides were as follows:

[0099]

[Chemical 3] Due to this site-directed mutagenesis, high fidelity thermostable Pfu DNA polymerase (low cycle number) and primers designed only to replicate the parental strand in a linear fashion were used The second site mutation was minimized. A double-stranded (supercoil) expression plasmid pET11tNOX (40 ng) and a mutagenic sense primer and a mutagenic antisense primer (100 ng) were added,
50 μl of reaction mixture (deoxyribonucleotides, buffer, and Pfu D
This product manufacturer's protocol (St
ratagene, La Jolla, CA). The cycling parameters were 16 cycles in total, with one cycle consisting of 95 ° C. for 30 seconds, 55 ° C. for 1 minute, and 68 ° C. for 12.8 minutes. The linear amplification product was treated with the endonuclease DpnI (10 units / μl) to remove the parent template.
Treated for 1 hour. Subsequently, an aliquot of 4 μl of this reaction mixture containing the double-nick mutated plasmid was added to supercompetent E. coli XL-1
Used for transformation of Blue cells (Stratagene). All variants were analyzed by DNA sequencing to confirm that the correct substitution was achieved.

[0100]

[Equation 1]

[0101]

[Table 1] Table 1 is the nucleotide and deduced amino acid sequence of tNOX-cDNA.
The first putative translation is at nucleotides 23-25 (ATG) at 1855-1
It ends at 857 (TAA). The putative signal peptide is underlined and the cleavage site of the signal peptide is indicated by an arrow. Putative quinone binding sequence (E394EMTE)
It is indicated by a two-dot chain line (long dash-dot dot line). The copper binding site H546VH and downstream H467 is indicated by an asterisk. Possible adenine (N
The ADH) binding sequence (T589GVGASL) is shown as a dashed line.

[0102]

[Table 2] Table 2 is a comparison of amino acid sequences in known quinone and sulfonylurea binding sites of several proteins.

[0103]

[Table 3] Table 3 shows the effect of site-directed mutagenesis of bttNOX on NADH oxidase enzyme activity, duration, and inhibition by capsaicin.

[0104]

[Table 4] Table 4 is the response of COS cells stably transfected with tNOX cDNA to target drugs.

[Sequence list]

[Brief description of drawings]

[Figure 1]   FIG. 1 summarizes the results of restriction maps of tNOX cDNA clones.

FIG. 2 schematically shows the constitution of intron-exon of the gene encoding human tNOX. Black squares in the genomic DNA map indicate exons encoding the identified proteins. The tNOX gene is located at the Xq25-26 chromosomal locus.
At least 9 exons have been identified within the available partial genomic information (Bird, 1999)

FIG. 3 is a deduced amino acid sequence of tNOX and Kyte and Doolit.
1 is a hydropathy plot prepared using the algorithm of tle (1982). One strongly hydrophobic region extending from amino acids 535-558 of SEQ ID NO: 2 was identified.

FIG. 4 shows the results of Western blot analysis of OVCAR-3 cells using antisera raised in rabbits immunized with recombinant tNOX. 12% SD
After separation on S-PAGE, the proteins were electroblotted onto nitrocellulose and with polyclonal antibody against tNOX diluted 1: 250,
Incubated overnight at 4 ° C. Detection used an alkaline phosphatase-conjugated antibody diluted 1: 5000, followed by incubation with NBT-BCIP. All fractions were prepared according to Chang and Pastan (1994). Lane 1, membrane pellet after octyl glycoside solubilization. Lane 2, supernatant after solubilization of octyl glycoside. Arrows indicate immunoreactive unprocessed tNOX (72 kDa), and processed tNOX (34 kDa).
Indicates. APK1 (29 kDa) and mesothelin (
The area of the gel corresponding to 40 kDa) lacks immunoreactive material.

5A-5C show periodic changes in the rate of oxidation of NADH (up to 5) as a function of time over 100 minutes. FIG. 5A: Enzyme source was tNOX cDNA from HeLa library in which expression of protein was induced by addition of IPTG.
Was a crude preparation from bacterial cells expressing Figure 5B: The crude preparation is identical to Figure 5A, except that expression of the cloned tNOX cDNA under the regulatory control of the lac promoter is not induced. Figure 5C: The crude preparation is the same as Figure 5A except the activity was measured as a function of time. The solid curve shows the oxidation of NADH measured as in FIG. 5A. The dashed line indicates cleavage of the dithiopyridine (DTP) substrate as a measure of protein disulfide-thiol exchange.

FIG. 6 shows overexpression of tNOX in COS cells as determined after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first two lanes (from left) are the results of Ponceau staining (lane 1, tNOX cDNA to pcDNA).
3.1 cloning into expression vector, transfection, and CO
Expressed in S; lane 2, vector without insert). The remaining lanes are t
Western blot results and detection with NOX-specific antibody (lane 3, tNOX cDNA; lane 4, vector without insert).

FIG. 7 graphically shows that the diameter of transfected COS cells is larger (approximately twice the diameter of untransfected COS cells).

FIG. 8: Periodic changes in the rate of cell expansion (proliferation) of COS cells transfected with the vector without insert (upper curve) and trans with the vector containing the tNOX cDNA insert. Compare with infected COS cells (lower curve). COS cells transfected with tNOX cDNA grow at approximately twice the rate of control cells.

FIG. 9 shows that COS cells transfected with tNOX cDNA were more sensitive to anti-cancer agents and capsaicin, a known tNOX inhibitor.

FIG. 10 demonstrates that COS cells transfected with tNOX cDNA were more sensitive to epigallocatechin gallate (EGCg), the major anti-cancer component of green tea.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) C12Q 1/68 G01N 33/566 G01N 33/53 33/574 A 33/566 C12N 15/00 ZNAA 33/574 5/00 B (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR) , OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD) , SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU. , ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MG, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA , ZW (72) Inventor Moor, Dee. James United States Indiana 47906, West Lafayette, Cherry Lane 1112 (72) Inventor Moor, Dorothy M. United States Indiana 47906, West Lafayette, Cherry Lane 1112 (72) Inventor Chue, Pinju United States Indiana 47906, West Lafayette, Arnold Drive 228-7 F Term (reference) 4B024 AA01 AA11 BA08 BA45 CA04 CA07 CA09 CA11 DA02 DA06 EA02 EA04 FA02 GA01 GA11 HA01 HA12 4B050 CC01 CC03 CC05 DD07 FF14 LL01 LL10 4B063 QA01 QA18 QA19 QQ01 QQ42 QQ52 QR08 QR42 QR55 QR62 QR82 QS25 QS34 QS36 QX02 4B065 AA26X AA90X AA90Y AB01 AB02 BA01 BA08 CA25 CA28 CA44 CA46 4H045 AA11 AA20 AA30 BA10 BA41 CA40 DA76 EA50 FA72 FA74

Claims (23)

[Claims] 1. A non-naturally occurring recombinant DNA molecule comprising a portion encoding a NADH oxidase / protein disulfide-thiol exchange polypeptide, said portion comprising nucleotides 23 to 1852 of SEQ ID NO: 1. A nucleotide sequence selected from the group consisting of nucleotides 680 to 1852 of SEQ ID NO: 1; and a sequence that hybridizes to one of these sequences under stringent conditions, wherein the hybridizes A sequence is a DNA molecule that encodes a cell surface neoplastic marker protein having NADH oxidase / protein disulfide-thiol exchange activity. 2. The non-naturally occurring recombinant DNA molecule of claim 1, wherein the polypeptide is from the group consisting of amino acids 1 to 610 of SEQ ID NO: 2; amino acids 220 to 610 of SEQ ID NO: 2. A DNA molecule consisting essentially of a selected amino acid sequence. 3. The non-naturally occurring recombinant DNA molecule of claim 2, wherein the portion encoding the polypeptide is shown in nucleotides 23 to 1852 of SEQ ID NO: 1 excluding the translation stop codon. A DNA molecule consisting essentially of a nucleotide sequence encoding the NADH oxidase / protein disulfide-thiol exchange polypeptide. 4. The non-naturally occurring recombinant DNA molecule of claim 2, wherein the portion encoding the polypeptide is set forth in nucleotides 680 to 1852 of SEQ ID NO: 1 excluding the translation stop codon. A DNA molecule consisting essentially of a nucleotide sequence encoding the NADH oxidase / protein disulfide-thiol exchange polypeptide. 5. The non-naturally occurring recombinant DNA molecule of claim 3 or 4, further comprising a translation stop codon, wherein the translation stop codon is TGA.
, TAA, or TAG, and immediately downstream of nucleotide 1852 of SEQ ID NO: 1. 6. A transformed or transfected host cell comprising a recombinant DNA molecule according to claim 1. 7. The host cell according to claim 6, which is a bacterial cell. 8. The host cell according to claim 7, wherein the bacterial cell is an Escherichia coli cell. 9. The host cell of claim 6, wherein the cell is a eukaryotic cell. 10. The host cell of claim 9, wherein the cell is a mammalian cell. 11. The host cell of claim 10, wherein the cell is a COS cell. 12. A transformed or transfected host cell comprising the recombinant DNA molecule of claim 2. 13. The host cell of claim 12, which is a bacterial cell. 14. The host cell according to claim 13, wherein the bacterial cell is an Escherichia coli cell. 15. The host cell of claim 12, wherein the cell is a eukaryotic cell. 16. The host cell of claim 15, wherein the cell is a mammalian cell. 17. The host cell of claim 16, wherein the cell is a COS cell. 18. A method for recombinantly producing a NADH oxidase / protein disulfide-thiol exchange active polypeptide in a host cell, the method comprising the steps of: a) activating the host cell within the host cell. A step of infecting or transforming with a vector containing a promoter, wherein the promoter is an amino acid sequence selected from the group consisting of amino acids 1 to 610 of SEQ ID NO: 2; amino acids 220 to 610 of SEQ ID NO: 2. With the NA
DH oxidase / protein disulfide-thiol exchange polypeptide coding sequence, or encoding NADH oxidase / protein disulfide-thiol exchange polypeptide, and nucleotides 23-1852 of SEQ ID NO: 1 or SEQ ID NO: 1 Producing a recombinant host cell operably linked to a coding sequence that hybridizes under stringent conditions to a nucleic acid molecule set forth at nucleotides 680 to 1852 of; Culturing under conditions that express the polypeptide; 19. A method of measuring neoplasia in a mammal, the method comprising:
The following steps: a) NADH associated with normal cells in a biological sample of mammalian origin.
Detecting the presence of ribonucleic acid encoding a NADH oxidase / protein disulfide-thiol exchange protein associated with neoplastic cells, as compared to a ribonucleic acid molecule encoding an oxidase, wherein the detecting step comprises: Performed using hybridization under stringent conditions, or using the polymerase chain reaction where a perfect match of the primer to the template is required, where the hybridization probe or primer is A step consisting essentially of at least 15 contiguous nucleotides of the nucleotide sequence shown in SEQ ID NO: 1; b) associating the results obtained with the sample of step (a),
Wherein the presence of the ribonucleic acid molecule in the biological sample is an indicator of the presence of neoplasia. 20. The method of claim 19, wherein the hybridization probe consists essentially of the nucleotide sequence set forth as nucleotides 680-1652 of SEQ ID NO: 1. 21. The method of claim 19, wherein the hybridization probe or primer consists essentially of the nucleotide sequence set forth as nucleotides 23-1852 of SEQ ID NO: 1. 22. A protein characterized by the amino acid sequence shown by amino acids 1 to 610 of SEQ ID NO: 2, a protein characterized by the amino acid sequence shown by amino acids 220 to 610 of SEQ ID NO: 2, and shown by SEQ ID NO: 16. An antibody preparation that specifically binds an antigen selected from the group consisting of proteins characterized by an amino acid sequence. 23. A method of measuring neoplasia in a mammal, the method comprising:
The following steps: a) detecting the presence of NADH oxidase / protein disulfide-thiol exchange protein associated with neoplastic cells in a biological sample of mammalian origin as compared to normal cells, wherein In the detection step, the protein characterized by the amino acid sequence shown in amino acids 1 to 610 of SEQ ID NO: 2; amino acids 220 to 610 of SEQ ID NO: 2
A protein characterized by the amino acid sequence shown in, or SEQ ID NO: 1
Step b) using an antibody specific for a protein characterized by the amino acid sequence shown in 6; and b) correlating the results obtained with the sample of step (a),
Wherein the presence of the protein in the biological sample is an indicator of the presence of neoplasia.