WO2004078980A1 - Nucleic acid and method of examining canceration using the nucleic acid - Google Patents

Abstract

A nucleic acid for examining canceration which is hybridizable with the base sequence represented by SEQ ID NO:1 or a base sequence complementary thereto under stringent conditions. A method of examining canceration which typically comprises: (a) using a nucleic acid hybridizable with the base sequence represented by SEQ ID NO:1 or a base sequence complementary thereto under stringent conditions and measuring the transcriptional level of the nucleic acid in a biological sample; and (b) judging whether or not the transcriptional level of the nucleic acid in the biological sample is significantly lower than that of a normal biological sample employed as a control.

Description

 Specification .

 Nucleic acid and canceration assay method using the nucleic acid

 Technical field

 The present invention relates to a novel nucleic acid, a nucleic acid for canceration assay, a method for assaying canceration of a biological sample based on a difference in the expression level of the nucleic acid in a biological sample, and a novel glycosyltransferase and a novel method. The present invention relates to a nucleic acid encoding the same.

 Background art

 In recent years, the functions of sugar chains and complex carbohydrates in vivo have been attracting attention. For example, mucopolysaccharides contained in connective tissues such as cartilage, blood vessel walls and tendons, glycoproteins determining blood types, and glycolipids involved in the functioning of the nervous system are known. Identifying enzymes that function to synthesize these sugar chains is a very important clue in analyzing the physiological activities brought about by various sugar chains and their distribution.

 For example, N-acetyl-D-galactosamine residues (hereinafter also referred to as “GalNAc”) and D-glucuronic acid (hereinafter also referred to as “GulUA”) in sugars are glycosylated in the extracellular matrix. It is a component of Saminodalican. Therefore, enzymes that transfer sugar residues (for example, see Non-Patent Document 1) are extremely important tools for analyzing the functions of sugar chains that work in various tissues in a living body.

 By the way, sugar chain synthesis is known to change very well in canceration, and is known to correlate with cancer metastasis and malignancy (Non-Patent Documents 2 to 4). In addition, comprehensive research such as these, such as the analysis of expression profiles in various tissues, is also actively aimed at elucidating the mechanism of canceration, and the mechanism of canceration may be related to the expression level of specific genes. It has been debated. As is well known, as a cancer diagnostic assay, an assay for a cancer marker in a body fluid, identification of a gene product or the like which is an indicator of canceration, and the like have already been performed. For example, cancer markers include probes and antibodies that detect or quantify such gene products.

Non-patent document 1

 Kitagawa H. et al., J. Biol. Chem. 2001, Oct 19; 276 (42): 38721-6 Non-Patent Document 2

Kobata A., Eur. J. Biochem. 15, 209 (2), 483-501, 1992 Non-patent document 3

 Santer U. V. et al., Cancer Res., Sep, 44 (9), 3730-5, 1984.

Non-patent document 4

 Taniguchi N., Biochim. Biophys. Acta., 1455 (2-3), 287-300, 1999 As mentioned above, the function of sugar chains in vivo has attracted attention. The analysis of synthesis is not sufficiently advanced. One reason is that the mechanism of sugar chain synthesis and the localization of sugar synthesis in vivo have not been sufficiently analyzed. In analyzing the mechanism of sugar chain synthesis, it is necessary to analyze sugar chain synthases, especially glycosyltransferases, and analyze what kind of sugar chains are produced using the enzymes. There is a growing demand for finding new glycosyltransferases and analyzing their functions.

 On the other hand, since sugar chain synthesis may change with canceration, identification of such enzymes involved in sugar chain synthesis is expected to provide useful indicators for cancer diagnosis. In particular, by using the amino acid sequence of such an enzyme or a nucleic acid encoding the same as an index, it is possible to provide a cancer marker prior to elucidation of its specific function. For example, nucleic acid identification can be performed on a DNA microarray, or it can be quantified by amplifying it in a small amount by PCR.

 In view of the above-mentioned problems, a first object of the present invention is to provide a cancer marker nucleic acid or polypeptide whose expression level changes significantly with canceration, or a nucleic acid or antibody for canceration assay targeting the same, and An object of the present invention is to provide a canceration assay method and the like using them.

 A second object of the present invention is to provide a novel polypeptide derived from human having glycosyltransferase activity, a nucleic acid encoding the same, and the discovery of a polypeptide that is noted as an indicator of canceration. An object of the present invention is to provide a transformant for expressing a nucleic acid, a method for producing the enzyme protein using the transformant, and the like.

 BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows each amino acid sequence aligned between the L4 protein of the present invention and chondroitin synthase.

 Summary of the invention

In order to find a novel enzyme protein having glycosyltransferase activity, the present inventors We have been conducting intensive research on various nucleic acid sequences found as sequences using queries. As one of the research results, we succeeded in cloning a nucleic acid sequence encoding a novel structural gene, and determined its nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 3). It was named. In the following, the nucleic acid relating to the nucleotide sequence of SEQ ID NO: 1 is also described as "Shi 4 gene" or "L4 nucleic acid", etc. Describe. As a result of detailed studies on L4, it was found that the expression level of L4 nucleic acid is significantly lower in cancerous tissues than in healthy tissues, and that the polypeptide encoded by the nucleic acid has a glycosyltransferase activity. The present invention has been completed. That is, the present invention relates to a nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary thereto.

 Further, the nucleic acid of the present invention is a nucleic acid consisting of at least a dozen or more consecutive nucleotide sequences in the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary thereto, preferably SEQ ID NO:

A nucleic acid comprising the nucleotide sequence described in 1 or a nucleotide sequence complementary thereto. In particular, the nucleic acids of the invention can be probes or primers. An example of a suitable primer of the present invention is a primer consisting of the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5 or a nucleotide sequence complementary thereto. Thus, the nucleic acid of the present invention can be a cancer marker. The present invention also relates to a method of assaying a biological sample for canceration. That is, the canceration assay method according to the present invention comprises:

 (a) using the nucleic acid according to any one of claims 1 to 6, measuring a transcription level of the nucleic acid in a biological sample; and

 (b) determining whether the measured value is significantly lower than the measured value for a healthy biological sample;

including.

 In one embodiment of the canceration assay method of the present invention, in the step (a), whether the measured value of the biological sample is 1 Z2 or less as compared with the measured value of a healthy biological sample And determining whether or not.

In another embodiment of the canceration assay method of the present invention, in the step (a), any one of the nucleic acids is used as a labeled probe, and the labeled probe is a stringent probe. Contacting the sample with a biological sample under hybridization conditions, and measuring the transcription level based on the signal from the labeled probe hybridized there. In another embodiment of the canceration assay method of the present invention, in the step (a), a nucleic acid amplification treatment is performed on the nucleic acid contained in the biological sample using any of the primers, And measuring the amount of the nucleic acid amplified by the primer.

 Another embodiment of the canceration assay method of the present invention is a method for assaying the effectiveness of a treatment for cancer therapy, which comprises using any one of the nucleic acids described above for an organism that has been treated for cancer therapy. Measuring the level of transcription for the nucleic acid in the sample, and determining whether the measured value is significantly lower than before or without the treatment.

 In the canceration assay of the present invention, the typical biological sample is a colon-derived sample. In another aspect of the present invention, a novel polypeptide having a glycosyltransferase activity, a novel nucleic acid encoding the same, and the like are provided based on the discovery of the nucleic acid.

 According to the studies by the present inventors, the L4 protein is presumed to be a glycosyltransferase because it has a transmembrane region and a predetermined motif sequence on the N-terminal side, which is a characteristic of glycosyltransferase in its amino acid sequence. Was done. Providing such novel enzyme proteins contributes to satisfying these diverse needs in the art.

 That is, the present invention relates to a nucleic acid encoding a polypeptide having glycosyltransferase activity, comprising the following nucleotide sequences (a) to (d):

 (a) the full-length nucleotide sequence of SEQ ID NO: 1;

 (b) the nucleotide sequence of SEQ ID NO: 1 at nucleotide numbers 73 to 107;

 (c) the base sequence according to base numbers 106 to 107 of SEQ ID NO: 1; or

 (d) the nucleotide sequence of SEQ ID NO: 1 as represented by nucleotide numbers 151 to 107;

The present invention also relates to a nucleic acid having any one of the above.

 One embodiment of the nucleic acid encoding the polypeptide of the present invention is a nucleic acid encoding a polypeptide having glycosyltransferase activity, wherein the amino acid sequence of the following (a) to (d): (a) SEQ ID NO: 3 full-length amino acid sequences;

 (b) the amino acid sequence of SEQ ID NO: 3 as defined in amino acid numbers 25-349;

 (c) the amino acid sequence of amino acid number 36-349 of SEQ ID NO: 3; or

(d) the amino acid sequence of SEQ ID NO: 3 as set forth in amino acid numbers 51-349; A nucleic acid encoding a polypeptide having any one of the above. One preferred embodiment is a nucleic acid encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3. A preferred type of nucleic acid is DNA.

 The present invention also relates to a vector containing any of the above nucleic acids, and a transformant containing the vector.

 The polypeptide of the present invention is a polypeptide having glycosyltransferase activity, and has the following amino acid sequence (a) to (d):

 (a) the full-length amino acid sequence of SEQ ID NO: 3;

 (b) the amino acid sequence of amino acid number 25-349 of SEQ ID NO: 3;

 (c) the amino acid sequence of amino acid numbers 36 to 349 of SEQ ID NO: 3; or (d) the amino acid sequence of amino acid numbers 51 to 349 of SEQ ID NO: 3. A polypeptide having the formula: One preferred embodiment is a polypeptide comprising the amino acid sequence of SEQ ID NO: 3.

 Further, the present invention provides an antibody against the polypeptide.

 Detailed description of the invention

 Hereinafter, the present invention will be described in detail by embodiments of the present invention.

 (1) Nucleic acid of the present invention for assaying for canceration

 As described above, the present inventors have found that the expression level of a nucleic acid having the nucleotide sequence of SEQ ID NO: 1 is significantly reduced in cancerous tissues. Therefore, a nucleic acid having the nucleotide sequence of SEQ ID NO: 1 or a sequence complementary thereto is attracting attention as a cancer marker useful for cancer diagnosis in a biological sample containing a transcript. In this respect, there is provided a cancer marker nucleic acid capable of hybridizing to a nucleic acid defined by the nucleotide sequence of SEQ ID NO: 1 under stringent conditions.

One embodiment of such a cancer marker nucleic acid is a primer or a probe targeting the nucleic acid in a biological sample and having a base sequence selected from the base sequence of SEQ ID NO: 1. In particular, the nucleotide sequence of SEQ ID NO: 1 is derived from mRNA encoding a structural gene, and includes the entire open reading frame (0RF) of the gene. Is found to be the full length of SEQ ID NO: 1 or most of it. From this point of view, the primer or probe according to the present invention has SEQ ID NO: 1 It can be a nucleic acid having a desired partial sequence selected from the following base sequences and thus being able to specifically hybridize to the selected base sequence in the nucleic acid.

 A typical primer or probe is a natural DNA fragment derived from a nucleic acid having the nucleotide sequence of SEQ ID NO: 1, a DNA fragment synthesized to have the nucleotide sequence of SEQ ID NO: 1, or a complementary strand thereof.

 In addition, the target nucleic acid in a biological sample can be detected and / or quantified using a primer or a probe as described above, as described later. Since the sequence on the genome can also be a target, the nucleic acid of the present invention may be used as an antisense primer for medical research or gene therapy.

Probe of the present invention

 A preferred embodiment of the nucleic acid of the present invention is a probe targeting a nucleic acid having the nucleotide sequence of SEQ ID NO: 1 or a complementary strand thereof. The probe according to the present invention has at least ten or more nucleotides selected from the nucleotide sequence of SEQ ID NO: 1, preferably at least 15 nucleotides, preferably at least 17 nucleotides, more preferably at least 20 nucleotides or a complementary strand thereof, Alternatively, it includes the full length of the 0RF region (base numbers 1 to 1047), that is, cDNA of 1407 bases or its complementary strand.

 In the case of an oligonucleotide probe, it should be understood that the nucleic acid of the present invention can specifically hybridize to the target nucleic acid under stringent conditions as long as it has more than ten bases, especially as many as 15 bases. It is. That is, a person skilled in the art can select an appropriate partial sequence of at least 15 to 20 nucleotides from the nucleotide sequence of SEQ ID NO: 1 according to various known strategies for designing oligonucleotide probes. In addition, the amino acid sequence information of SEQ ID NO: 3 is useful for selecting a unique sequence that is deemed appropriate as a probe.

 In the case of a cDNA probe, for example, a probe as a reagent or a diagnostic agent for medical research is generally difficult to handle with a large molecular weight, and from this viewpoint, the probe of the present invention for medical research is represented by SEQ ID NO: 1. 50 to 500 bases, more preferably 60 to 300 bases selected from the base sequence of

Stringent conditions described herein are defined as moderate or high stringency. Hybridizing under stringent conditions. Specifically, moderate stringent conditions can be easily determined by those skilled in the art, for example, based on the length of the DNA. The basic conditions are set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual W> Edition, Vol. 1, ι. 42-7, 45 Cold Spring Harbor Laboratory Press, 2001, and for ditrocellulose filters. 5X SSC, 0.5% SDS, pre-wash solution of 1.0 mM EDTA (pH 8.0), about 50% formamide at about 40-50 ° C, 2X SSC-6X SSC (or Hybridization conditions for other similar hybridization solutions, such as Stark's solution, in about 50% honoleamide at about 42 ° C, and at about 60 ° C, 0,5 X SSC, including the use of 0.1% SDS wash conditions. Highly stringent conditions can also be readily determined by those skilled in the art, for example, based on the length of the DNA. In general, such conditions include hybridization and Z or washing at higher temperatures and lower Z or salt concentrations than moderately stringent conditions, e.g., hybridization conditions as described above, and approximately 68 ° C, defined as 0.2 X SSC, with 0.1% SDS wash. One skilled in the art will recognize that the temperature and wash solution salt concentration can be adjusted as necessary according to factors such as the length of the probe. As described above, those skilled in the art can select based on the common general knowledge of various probe design methods and hybridization conditions known in the art, and empirical rules that can be obtained through commonly used experimental means. Moderate or moderately stringent conditions appropriate for the probe used can be easily found and implemented.

 In addition, depending on the selected base length and the hybridization conditions to be employed, the relatively short oligonucleotide probe may have one or several nucleotides of the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence. Even if there is a mismatch of one or two bases, especially one or two bases, it can function as a probe. In addition, the relatively long cDNA probe functions as a probe even if there is a mismatch of 50% or less, preferably 20% or less with the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence. obtain.

The probe of the present invention designed as described above is labeled with a fluorescent label, a radioactive label, a biotin label, or the like in order to detect or confirm a hybrid with the target sequence. Can be used as a labeled probe.

 For example, the labeled probe of the present invention can be used for confirming or quantifying a PCR amplification product of the cancer marker nucleic acid. In this case, it is preferable to use a probe that targets a base sequence in a region located between a pair of primer sequences used for the PCR. One example of such a probe is an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 (corresponding to the complementary strand of nucleotides 244-274 in SEQ ID NO: 1) (see Example 2). ). The probe of the present invention may be incorporated in a diagnostic DNA probe kit or the like, or may be fixed on a chip such as a DNA microarray.

Primer of the present invention

 Another preferred embodiment of the nucleic acid of the present invention is an oligonucleotide primer. In producing the oligonucleotide primer according to the present invention, two regions may be selected from the ΔRF region of the base sequence of SEQ ID NO: 1 so as to satisfy the following conditions.

 1) the length of each region is several tens of bases or more, particularly 15 or more bases, preferably 17 or more bases, more preferably 20 or more bases, and 50 or less bases;

2) The ratio of G + C in each region is 40 to 70%;

 In practice, it may be produced as a single-stranded DNA having the same nucleotide sequence as the two regions selected as described above or a nucleotide sequence complementary thereto, or may lose the binding specificity to these nucleotide sequences. A modified single-stranded DNA may be produced. The primer of the present invention preferably has a sequence completely complementary to the partial sequence in the ORF region of SEQ ID NO: 1, but may have a mismatch of 1 or 2 bases.

 Examples of the pair of primers according to the present invention include an oligonucleotide consisting of the base sequence of SEQ ID NO: 4 (corresponding to base numbers 194-213 in SEQ ID NO: 1), and a base sequence of SEQ ID NO: 5 (sequence (Corresponding to the complementary strand of base Nos. 3225-344 in No. 1). The above-mentioned probe that can be used for confirming or quantifying the amplification product thereby is, for example, a labeled oligonucleotide probe consisting of the base sequence of SEQ ID NO: 6 described above.

(2) Canceration assay method according to the present invention As described above, the expression level of L4 nucleic acid in a biological sample in which cancer is caused, that is, the transcription level of the gene from the genome to mRNA is significantly lower than that of the healthy biological sample. Was found. From this viewpoint, a method for assaying canceration using the transcription level of the nucleic acid in a biological sample as an index is provided. That is, since the nucleotide sequence information of the nucleic acid was disclosed in the present specification, those skilled in the art can appropriately prepare probes or primers based on the present disclosure as described in detail above, whereby the nucleic acid Transcript levels can be detected and examined.

 In a specific embodiment of the canceration assay method according to the present invention, a transcript extracted from a biological sample or a nucleic acid library derived therefrom is used as a test sample to measure the amount of the nucleic acid using the probe or primer of the present invention. And determining whether the measured value is significantly below the value for the control healthy biological sample. Here, when the value of the test biological sample is significantly lower than the value of the healthy biological sample, for example, the biological sample is determined to be cancerous or highly malignant.

 In the canceration assay according to the present invention, the value for a healthy biological sample serving as a control may be a measured value for a non-cancerous site in the same tissue of the same patient, or may be a known value obtained from a healthy biological sample. A value generalized based on the above data, for example, an average normal value may be used.

 In this assay, whether the measured value of a test sample is significantly lower or lower than that of a healthy sample depends on the precision (positive rate) required for the test and the degree of aggression to be judged. Accordingly, the judgment can be made based on criteria set.

 For example, according to an assay example (Example 2) by the present inventors, if the transcript level to be determined to be in a cancerous state is a measurement value of 1/2 or less of a healthy sample, about 80% is obtained. Determined to be positive. If the condition is set at 3-5 (0.6 times) or less, a positive rate of 100% can be achieved. Therefore, according to this assay method, a method including determining whether the measured value of a test sample is 1/2 or less of a healthy sample, preferably 3Z5 or less, etc. Is provided.

Further, for example, for the purpose of detecting a tissue with a high degree of malignancy, the above-mentioned difference that is determined to be positive is set to be larger, or the purpose is to comprehensively detect a test sample having a sign or possibility of canceration. , Such as setting the difference that is considered positive to be smaller, The criterion can be arbitrarily set according to the purpose.

Hybridization assay

 Embodiments of the canceration assay according to the present invention include, for example, a Southern blot, a Northern blot, a dot blot, and a co-needle knee hybridization method using a probe obtained from the nucleic acid of the present invention. Methods using various hybridization assays well known to those skilled in the art are included. If further amplification or quantification of the detection signal is required, an assay that combines them with an immunoassay may be used.

 According to a typical hybridization assay, a test nucleic acid or an amplified product thereof extracted from a biological sample is immobilized, hybridized with a labeled probe under stringent conditions, washed, and bound to a solid phase. The labeled label is measured.

 Extraction and purification of the transcript from the biological sample can be performed by applying any method known to those skilled in the art. ·

Assay by nucleic acid amplification

A preferred embodiment of the canceration assay according to the present invention also includes an assay using a nucleic acid amplification reaction performed using a primer selected from the nucleotide sequence of the nucleic acid of the present invention. Here, nucleic acid amplification reactions include, for example, the polymerase chain reaction (PCR) [Saiki RK, et al., Science, 230, 1350-1354 (1985)], the Lighes chain reaction (LCR) [Wu D. Υ ·, et al. al., Genomics, 4, 560-569 (1989); Barringer KJ, et al., Gene, 89, 117-122 (1990); Barany F., Proc. Natl. Acad. Sci. USA, 88, 189-. 193 (1991)] and transcription-based amplification [Kwoh DY, et al., Proc. Natl. Acad. Sci. USA, 86, 1173-1177 (1989)], etc., and strand displacement. Reaction (SDA) [Walker GT, et al., Proc. Natl. Acad. Sci. USA, 89, 392-396 (1992); Walker GT, et al., Nuc. Acids Res., 20, 1691-1696 ( 1992)], self-retaining sequence replication (3SR) [Guatelli JC, Proc. Natl. Acad. Sci. USA, 87, 1874-1878 (1990)], and the QjS replicase system [Resirdi et al., BioTechnology 6, p. 1197-1202 (1988)]. Further, a nucleic acid sequence-based amplification (Nucleic Acid Sequence Based Amplification: NASBA) reaction described in European Patent No. 0525882, which is a competitive amplification of a target nucleic acid and a mutant sequence, can also be used. Preferably This is a PCR method. Generally, a nucleic acid amplification method itself such as PCR is well known in the art, and a reagent kit and an apparatus therefor are commercially available, so that it can be easily performed.

 Specific embodiments of the assay method of the present invention utilizing a nucleic acid amplification reaction are as follows. The target nucleic acid in the transcript to be assayed is amplified by PCR using a pair of primers located at both ends of a predetermined region selected from the base sequence. In this step, if even a small amount of the nucleic acid is present in the sample, it becomes と な り and the nucleic acid region between the primer pairs is replicated and amplified one after another. By repeating a predetermined number of cycles in the PCR, the type III nucleic acid is amplified to a desired concentration. Under the same amplification reaction conditions, an amplification product proportional to the amount of the target nucleic acid present in the sample can be obtained. Then, it is possible to confirm whether the amplification product is the target nucleic acid and quantify it by using the above-mentioned probe or the like targeting the amplification region. In addition, the nucleic acid in a healthy tissue can be measured in the same manner. The measured value of the nucleic acid amount is compared to test for the presence or degree of canceration as described above. It should be noted that nucleic acid of a gene that is widely and commonly present in the same tissue, such as nucleic acid encoding dariceraldehyde-3-phosphate dehydrogenase (GAPDH) or i3-actin, is used as a control to eliminate individual differences. Good.

 The nucleic acid sample to be subjected to the PCR method may be an entire mRNA extracted from a biological sample such as a test tissue or a cell, or an entire cDNA reverse-transcribed from the mRNA. When increasing the mRNA, the NASBA method (3SR method, TMA method) using the above-described primer pair may be employed. Since the NASBA method itself is well known and a kit therefor is commercially available, it can be easily carried out using the primer pair of the present invention.

 Detection or quantification of the above amplification products can be performed by electrophoresis of the reaction solution after amplification and staining the bands with ethidium bromide, etc., or immobilization of the amplification products after electrophoresis on a solid phase such as a nylon membrane. It can be carried out by hybridizing a labeled probe that specifically hybridizes with the test nucleic acid (for example, the labeled probe of SEQ ID NO: 6), washing, and detecting the label.

Suitable PCR methods for this assay include quantitative PCR, particularly RT-PCR for kinetic analysis and quantitative real-time PCR. Especially mRNA The quantitative real-time RT-PCR method for libraries is suitable from the viewpoint that the measurement target can be directly purified from a biological sample and directly reflects the transcription level. The quantification is not limited to the quantitative PCR method, and other known DNA quantification methods such as Northern blot, dot blot, and DNA microarray using the above-described probe can be applied to the PCR product.

 It is also possible to quantify the amount of target nucleic acid in a sample by performing quantitative RT-PCR using quencher-fluorescent dye and reporter fluorescent dye. In particular, kits for quantitative RT-PCR are also commercially available, and can be easily performed. Furthermore, it is also possible to semiquantify the target nucleic acid based on the intensity of the electrophoresis band.

Test method for cancer treatment effect

 Another form of the canceration assay according to the present invention includes an assay for judging the effect of a treatment intended for a cancer curing effect. The test object includes, for example, in vitro cancer cells and cancer tissues derived from a cancer patient or an animal model for experimental carcinogenesis. Such treatments include all treatments such as administration of anticancer drugs and radiation therapy.

 According to the assay for cancer therapeutic effect according to the present invention, the transcript level of a target nucleic acid in a biological sample before or after treatment is compared with that of the biological sample to which the treatment has been applied, and the treatment does not cause cancer. It will be possible to determine what effect it has on malignancy. The treatment is effective as a cancer treatment if the transcription level is increased due to the treatment, or if the decrease in the transcription level is significantly suppressed even in a situation where it increases with canceration. Can be evaluated. Variation in transcription levels after treatment may be followed over time after treatment, as well as in comparison to untreated tissue.

 The cancer therapeutic effect assay according to the present invention includes, for example, whether or not the anticancer drug candidate substance is effective on cancerous tissue, whether or not resistance has been formed to the anticancer drug being administered to the cancer patient, and whether or not the experimental model animal has a lesion. Judgment as to whether it is effective for an organization or the like is included. The test tissues of the experimental model animals include not only in vitro but also in vivo or ex vivo samples.

In this specification, the term "measured value of transcription level" or "expression level" of a nucleic acid refers to the amount of the nucleic acid present in a certain amount of a transcript derived from a biological sample, that is, the concentration of the nucleic acid. Is shown. In addition, the assay of the present invention makes it possible to compare the measured values. When nucleic acids are amplified by PCR or the like for quantification, or when signals from probe labels are amplified, relative comparison of the amplified values is possible. It is. Therefore, the "measured value of nucleic acid" can be grasped as the amount after amplification or the signal level after amplification.

 As used herein, the term "target nucleic acid" or "the relevant nucleic acid" includes not only in vivo or in vitro but also all types of nucleic acids obtained by converting mRNA into type II. . In this specification, the term “base sequence” includes its complementary sequence unless otherwise specified.

 In the present specification, the term "biological sample" refers to an organ, a tissue and a cell, and an organ, a tissue and a cell derived from an experimental animal, but is preferably a tissue, and specifically, an esophagus, a stomach, and the like. Examples include the kidney, liver, kidney, duodenum, small intestine, large intestine, rectum, and colon. The large intestine, the rectum and the colon are preferred, and the colon is more preferred. Also,

 As used herein, the term “measurement” or “assay” includes any of detection, amplification, quantification, and semi-quantitation. In particular, the assay method according to the present invention relates to an assay for canceration of a biological sample as described above, and can be applied to cancer diagnosis and treatment in medical treatment. Here, the term “assay for canceration” includes an assay for whether a biological sample is carcinogenic and an assay for whether the malignancy is high. As used herein, the term "cancer" typically includes malignant tumors in general and disease states caused by the malignant tumors. Therefore, the assay according to the present invention is not particularly limited, but includes esophageal cancer, stomach cancer, spleen cancer, liver cancer, kidney cancer, duodenal cancer, small intestine cancer, large intestine cancer, rectal cancer, and colon cancer. Preferably, they are colorectal cancer, rectal cancer, and colon cancer, and are preferably used for colorectal cancer assay.

 (3) Nucleic Acid of the Present Invention Coding Polypeptide Having Glycosyltransferase Activity The nucleic acid of the present invention can be prepared, for example, by the following method.

Using a part of the base sequence shown in SEQ ID NO: 1, hybridization and nucleic acid amplification reaction were carried out from the cDNA library using a basic method of genetic engineering such as nucleic acid amplification reaction according to a conventional method. The L4 nucleic acids of the invention can be cloned. For example, if the sequences of SEQ ID NOs: 4 and 5 are used as primers, A DNA fragment of about lkbp is obtained as a product. This can be isolated, for example, by agarose gel electrophoresis to separate DNA fragments by molecular weight and sieving, and cut out a specific band. Details of the nucleic acid amplification reaction are as described above. The present inventors expressed the L4 protein encoded by the L4 nucleic acid isolated as described above, isolated and purified the L4 protein, and confirmed that it had biological activity. In this regard, the present invention provides nucleic acids encoding novel full length polypeptides or fragments thereof.

 First, focusing on the fact that the amino acid sequence of the isolated polynucleotide having biological activity has been identified, the nucleic acids of the present invention have the same amino acids that degenerate to the amino acid sequence of the L4 protein. Includes a finite number of any nucleic acid that can encode the sequence. Therefore, the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary thereto is one embodiment of the nucleic acid encoding the polypeptide having the biological activity.

 The nucleic acids of the present invention also include both single-stranded and double-stranded DNA, and their RNA complements. DNA includes, for example, naturally occurring DNA, recombinant DNA, chemically bonded DNA, DNA amplified by PCR, and combinations thereof. However, DNA is preferable from the viewpoint that it is stable at the time of preparing a vector or a transformant. Second, the isolated L4 nucleic acid is predicted to have a hydrophobic transmembrane domain at its end according to its deduced amino acid sequence (SEQ ID NO: 3). By preparing a region of a nucleotide sequence encoding a peptide, the nucleic acid encoding a solubilized form of the polypeptide can also be obtained. In fact, the present inventors have found that, for example, by expressing a nucleic acid consisting of the nucleotide sequence of base numbers 73 to 107 in SEQ ID NO: 1, the 25th, 36th, or 51 A soluble protein having the N-terminal of each amino acid was produced (see Example 3). Thus, incomplete length nucleic acids encoding polypeptides having the biological activity of the L4 protein are also within the scope of the invention.

 Third, those skilled in the art can obtain a nucleic acid equivalent to the sequence of SEQ ID NO: 1 by preparing a nucleic acid having a nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 1. In identifying the scope of such homologous nucleic acids of the invention, the SEQ ID NO:

When an identity search is performed for the nucleic acid sequence described in 1, the nucleic acid sequence is found to be the most homologous It has 18% identity to the nucleic acid sequence of the highest known chondroitin synthase (Non-Patent Document 1). From this viewpoint, the novel nucleic acid sequence encoding the homologous protein of the present invention is typically the entire base sequence in SEQ ID NO: 1, preferably base numbers 73 to 107, 106 to 107. Or more than 18% identity, more preferably at least 20% identity, to a partial nucleotide sequence consisting of the nucleotide sequences of 151 to 10047, or a nucleotide sequence complementary thereto. Preferably, it can be defined as having at least 30% identity.

 The above percent identity used to define homologous nucleic acids can be determined by visual inspection and mathematical calculation. Alternatively, the identity percent identity of two nucleic acid sequences is described in Devereux et al., Nucl. Acids Res. 12: 387, 1984, and the GAP computer program available from the University of Wisconsin Genetics Computer Group (UWGCG). It can be determined by comparing sequence information using version 6.0. Preferred default parameters for the GAP program include: (1) a single 'unary' comparison matrix for nucleotides (including values of 1 for identical and 0 for non-identical), and Schwartz and Dayhoff III: Modification, Atlas of Protein weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14: 6745, 1986, as described in sequence and Structure, pp. 353-358, National Biomedical Research Foundation, 1979; (2) For each gap 3.0 penalty and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for terminal gaps. Other sequences comparison programs used by those skilled in the art can also be used. Fourth, other nucleic acids homologous as the structural gene of the present invention typically include the full-length nucleotide sequence in SEQ ID NO: 1, preferably nucleotides 73 to 107, 106 to 107, Or a polypeptide that hybridizes under stringent conditions to nucleotides consisting of the respective partial nucleotide sequences of 151 to 1074 or nucleotide sequences complementary thereto and encodes a polypeptide having L4 protein activity. Nucleic acids are also included. The stringent conditions used for defining such homologous nucleic acids are as described above.

By using the L4 nucleic acid of the present invention as described above, medical research or gene therapy can be performed. Not only can it produce probes and antisense primers for therapeutic purposes, but also can express the desired L4 enzyme protein.

 (4) Vector and transformant of the present invention

 According to the present invention, there is provided a recombinant vector comprising an L4 nucleic acid. Methods for incorporating a DNA fragment of the nucleic acid into a vector such as a plasmid are described in, for example, Sambrook, J. et al., Molecular Straining, A Laboratory Manual (3rd edition), Cold spring Harbor Laboratory, 1.1 (2001). The methods described above are exemplified. For convenience, a commercially available ligation kit (for example, Takara Shuzo) may be used.

 The recombinant vector (for example, recombinant plasmid) obtained as described above is introduced into a host cell (for example, Escherichia coli DH5a, TBI, LE392, or XL-LE392 or XL-lBlue). Methods for introducing a plasmid into a host cell include the calcium chloride method or the calcium chloride method described in Sambrook, J. et al., Molecular Cloning, A Laboratory Manual (3rd edition), Cold spring Harbor Laboratory, 16.1 (2001). Examples include the rubidium chloride method, the electoral portation method, the electoral injection method, a method using a chemical treatment such as PEG, and a method using a gene gun.

 A usable vector can be simply prepared by ligating a desired gene to a recombination vector (for example, plasmid DNA or the like) available in the art by a conventional method. Specific examples of the vector used include, but are not limited to, plasmids derived from Escherichia coli such as pD0NR201, pBluescript, pUC18, pUC19, and pBR322.

 Those skilled in the art can appropriately select the restriction end so as to be compatible with the expression vector. Those skilled in the art can appropriately select an expression vector suitable for the host cell in which the enzyme of the present invention is to be expressed, and the gene is involved in gene expression so that the nucleic acid can be expressed in the target host cell. It is preferable that regions (promoter region, enhancer region, operator region, etc.) are appropriately arranged and constructed so that the nucleic acid is appropriately expressed.

The type of expression vector is not particularly limited as long as it has a function of expressing a desired gene and producing a desired protein in various prokaryotic and eukaryotic or eukaryotic host cells. Examples of the expression vector for E. coli include, but are not limited to, pQE-30, pQE-60, pMAL-C2, pMAL_p2, and pSE420. Examples of yeast expression vectors include pYES2 (Saccharomyces), pPIC3.5K, pPIC9K, and pA0815. (Above Pichia genus), insect expression vectors and LT pFastBac, pBacPAK8 / 9, pBK283, pVL1392, pBlueBac4.5, and the like. For the construction of the expression vector, a Gateway system (Invitrogen) which does not require restriction and ligation may be used. The Gateway system is a system that uses site-specific recombination that enables cloning while maintaining the orientation of the PCR product and that enables subcloning into an expression vector in which a DNA fragment has been appropriately modified. Specifically, an entry clone was created from the PCR product and the donor vector using BP clonase, a site-specific recombination enzyme, and then recombined with this clone and another recombination enzyme, LR clonase. By transferring the pCR product into a possible destination vector, an expression clone corresponding to the expression system is prepared. One of the features is that if an entry clone is created first, the laborious subcloning step of working with restriction enzyme ゃ ligase is unnecessary.

 By incorporating the expression vector containing the L4 nucleic acid of the present invention into a host cell, a transformant for producing the polypeptide of the present invention can be obtained. The host cell for obtaining the transformant may be generally a eukaryotic cell (eg, a mammalian cell, a yeast, an insect cell) or a prokaryotic cell (eg, Escherichia coli, Bacillus subtilis). In addition, cultured cells derived from human (eg, HeLa, 293T, SH—SY5Y) ゝ mouse (eg, Neuro2a, NIH3T3) and the like may be used. All of these host cells are known and are commercially available (eg, Dainippon Pharmaceutical Co., Ltd.) or available from public research institutions (eg, RIKEN Cellbank). Alternatively, embryos, organs, tissues or non-human individuals can be used.

By the way, it is considered that the L4 nucleic acid of the present invention can express an enzyme protein having a property close to that of a natural biomolecule (for example, a sugar chain added form) by using a eukaryotic cell as a host cell. From this viewpoint, it is preferable to select eukaryotic cells, particularly mammalian cells, as host cells. Specific mammalian cells include those derived from mouse, African frog, rat, hamster, monkey or human. And a cultured cell line established from those cells. Escherichia coli, yeast or insect cells as host cells include, for example, Escherichia coli (DH5a, M15, JM109, BL21, etc.), yeast (INVScl (Saccharomyces), GS115, KM71 (Pichia), etc.) And insect cells (Sf21, BmN4, silkworm larvae, etc.) and the like.

 In general, an expression vector can be prepared by linking at least a promoter, an initiation codon, a gene encoding a desired protein, a termination codon, and a terminator region to an appropriate replicable unit in a continuous and circular manner. it can. At this time, if necessary, an appropriate DNA fragment (for example, a linker, another restriction enzyme site, etc.) can be used by a conventional method such as digestion with a restriction enzyme or ligation using T4 DNA ligase. When a bacterium, particularly Escherichia coli, is used as a host cell, an expression vector generally comprises at least a promoter / operator region, an initiation codon, a gene encoding a desired protein, a stop codon, a terminator, and a replicable unit. . When yeast, plant cells, animal cells, or insect cells are used as host cells, the expression vector generally includes at least a promoter, initiation codon, a gene encoding a desired protein, a stop codon, and a terminator. Is preferred. Further, it may appropriately contain a DNA encoding a signal peptide, an enhancer sequence, 5 ′ and 3 ′ untranslated regions of a desired gene, a selectable marker region or a replicable unit.

 A replicable unit refers to DNA capable of replicating its entire DNA sequence in a host cell, and is composed of natural plasmids, artificially modified plasmids (prepared from natural plasmids). Plasmid) and synthetic plasmid. Suitable plasmids include the plasmid pQE30, pET or pCAL or an artificially modified product thereof (a DNA fragment obtained by treating pQE30, pET or pCAL with an appropriate restriction enzyme) in E. coli. In yeast, plasmid pYES2 or pPIC9K is used, and in insect cells, plasmid pBacPA 8/9 is used.

A preferred initiation codon of the vector of the present invention is methionine codon (ATG). Examples of the stop codon include common stop codons (eg, TAG, TGA, TAA, etc.). Also, enhancer array, terminator As the sequence, for example, those commonly used by those skilled in the art such as those derived from SV40 can be used.

 As the selection marker, a commonly used marker can be used by a conventional method. Examples thereof include tetracycline, ampicillin, or an antibiotic resistance gene such as dynammycin or neomycin, hygromycin, or spectinomycin.

 Introduction (also referred to as transformation or transfection) of the expression vector according to the present invention into a host cell can be performed using a conventionally known method. In the case of bacteria (E. coli, Bacillus subtilis, etc.), for example, the method of Cohen et al. [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method [Mol. Gen. Genet., 168, 111] (1979)] に よ っ て According to the competency method [J. Mol. Biol., 56, 209 (1971)], in the case of Saccharomyces cerevisiae, for example, the method of Hinnen et al. [Proc. Natl. Acad. Sci. USA, 75, 1927 (1978)] and the lithium method [JB Bacteriol., 153, 163 (1983)]. In the case of plant cells, for example, by the leaf disk method [Science, 227, 129 (1985)] and by the electroporation method [Nature, 319, 791 (1986)]. In the case of animal cells, for example, the method of Graham [Virology, 52] , 456 (1973)], and insect cells can be transformed by, for example, the method of Summer et al. [Mol. Cell Biol., 3, 2156-2165 (1983)].

 (5) The protein of the present invention

 According to the studies by the present inventors, the amino acid sequence of SEQ ID NO: 3 was estimated based on the nucleotide sequence of SEQ ID NO: 1 (see SEQ ID NO: 2), and the deduced amino acid sequence of the L4 protein was It has a certain homology with the amino acid sequence. For example, in the amino acid sequence of L4, homology is found with the amino acid sequence near the N-terminal of known chondroitin synthase (hChSy). It has been reported that this chondroitin synthase has both jS 1,3GluUA transfer activity and β1, GalNAc transfer activity (Non-Patent Document 1).

FIG. 1 shows an alignment near the N-terminal side between the L4 protein and the amino acid sequence of the chondroitin synthase. As shown in the figure, L4 protein, like chondroitin synthase, has a hydrophobic transmembrane domain at the N-terminal and a DXD motif. Have. Since these are characteristics widely observed in glycosyltransferases, it was concluded that the amino acid sequence of the L4 protein was that of a polypeptide having glycosyltransferase activity. Thus, the L4 protein can be expressed by incorporating the L4 nucleic acid having the nucleic acid sequence of SEQ ID NO: 1 into an expression vector, as exemplified in the Examples below, and thus has a glycosyltransferase activity. It can be isolated and purified as an enzyme protein.

 First, in view of the above, a typical embodiment of the protein of the present invention is an isolated glycosyltransferase protein consisting of the deduced amino acid sequence of SEQ ID NO: 3.

Donor substrate specificity:

 Examples of the donor substrate of the L4 enzyme protein include sugar nucleotides having a sugar to be transferred, such as a form of peridine diphosphate-sugar such as UDP-GalNAc or UDP_GluUA, adenosine diphosphate monosaccharide, guanosine It may be diphosphate monosaccharide or cytidine diphosphate monosaccharide.

Receptor substrate specificity:

 The acceptor substrate for the L4 enzyme protein may include, for example, a sulfated daricosaminodalican composed of GalNAc or GluUA, but may include other glycoproteins, glycolipids, oligosaccharides, or polysaccharides. Is also good.

 As described above, the L4 enzyme protein of the present invention is useful for sugar chain synthesis or modification in mucopolysaccharide, glycoprotein, glycolipid, oligosaccharide, polysaccharide and the like.

 Second, the disclosure of the amino acid sequence of SEQ ID NO: 3 representative of the primary structure of the L4 enzyme protein in the present specification allows production by a well-known genetic engineering technique in the art based on the amino acid sequence. All the proteins obtained (hereinafter also referred to as “mutated proteins” or “modified proteins”) are provided.

That is, the protein of the present invention is not limited to the protein consisting of the amino acid sequence of SEQ ID NO: 3 deduced from the nucleotide sequence of the cloned nucleic acid according to the common general knowledge in the art, and is described below. Thus, for example, a protein consisting of an incomplete-length polypeptide in which the amino acid sequence at the N-terminal or the like is partially deleted, or a protein homologous to the amino acid sequence, It is also intended to include proteins having properties. First, the enzyme protein of the present invention may preferably have an amino acid sequence from amino acid number 25 to SEQ ID NO: 3 in SEQ ID NO: 3 as obtained in Examples described later.

 Third, generally, in a protein having a physiological activity such as an enzyme, one or more amino acids in the amino acid sequence are substituted or deleted, and one or more amino acids are substituted in the amino acid sequence. It is well known that the physiological activity can be maintained even when a plurality of amino acids are inserted or added. In addition, one of the naturally-occurring proteins may be one of them due to differences in the varieties of the species producing them, mutations in genes due to differences in ecotype, or the presence of similar isozymes. It is also known that there exists a mutant protein having a plurality of amino acid mutations. From this viewpoint, in the protein of the present invention, one or more amino acids are substituted or deleted in the amino acid sequence shown in SEQ ID NO: 1, or one or more amino acids are added to the amino acid sequence. Mutant proteins having an inserted or added amino acid sequence and having the above-mentioned glycosyltransferase activity under predetermined enzyme reaction conditions are also included. Further, as the modified protein, the amino acid sequence shown in SEQ ID NO: 1 or one or several amino acids in the sequence are substituted or deleted, or one or several amino acids are inserted or added in the amino acid sequence. Those having the defined amino acid sequence are particularly preferred.

 In the above, “plurality” is preferably 1 to 200, more preferably 1 to 100, still more preferably 1 to 50, and most preferably 1 to 20. Generally, when amino acids are substituted by site-specific mutation, the number of amino acids that can be substituted is preferably 1 to 10 so that the activity of the original protein is maintained. It is.

Fourth, the modified proteins of the present invention include modified proteins obtained by substitution of amino acids having similar properties. That is, in general, substitution of amino acids having similar properties (for example, substitution of one hydrophobic amino acid with another hydrophobic amino acid, substitution of one hydrophilic amino acid with another hydrophilic amino acid, and one acidic amino acid with another acidic amino acid) Amino acid substitution or substitution of one basic amino acid with another basic amino acid) to produce a recombinant protein having a desired mutation. The methods are well known to those skilled in the art, and the modified protein thus obtained often has properties similar to the original protein. From this viewpoint, the modified protein in which such amino acid substitution is performed can be included in the present invention.

 In addition, the modified protein of the present invention is a glycoprotein having a sugar chain bound to the polypeptide as long as it has the amino acid sequence as described above and has an intrinsic enzyme activity for the target enzyme. You may.

 Fifth, in identifying the range of the homologous protein of the present invention, the amino acid sequence described in SEQ ID NO: 3 of the present invention was identified by GENET YX (Genetics). It has 18% identity to a known homologous chondroitin synthase (Non-Patent Document 1). From this viewpoint, the novel amino acid sequence as the homologous protein of the present invention has an identity of more than 18%, more preferably at least 20%, particularly preferably at least 20%, to the amino acid sequence shown in SEQ ID NO: 1. Defined to have 30% identity.

GENET YX is a genetic information processing software for nucleic acid analysis and protein analysis, and is capable of normal signal homology analysis and multi-alignment analysis, as well as signal peptide prediction, promoter site prediction, and secondary structure prediction. is there. In addition, the homology analysis program used in this specification is based on the L1pman-Pear'son method (Lipman, D.J. & Pearson, WR, which is frequently used as a high-speed and high-sensitivity method). Science, 277, 1435—144 1 (1 98 5)). As used herein, the percent identity may be determined, for example, by the BLAST program described in Altschul et al. (Nucl. Acids. Res., 25. 3389-3402 (1997)), or Pearson et al. Sci. USA, 2444-2448 (1988)), and can be determined by comparing the sequence information with FASTA. The program can be used on the Internet from the website of Nationa1CenterforBiotechnologyInformation (NCBI) or DNA Data Bank of Japan (DDB J). The various conditions (parameters) for the identity search by each program are described in detail on the same site, and some settings may be applied. Although it is possible to change it, search is usually performed using the default value. It should be noted that other sequence comparison programs used by those skilled in the art can also be used.

 Sixth, an antibody against the isolated protein of the present invention can be prepared by administering the isolated protein to an animal as an immunogen as described below. The enzyme can be measured and quantified by immunoassay using such an antibody. Therefore, the present invention is also useful for producing such an immunogen. From this point of view, the protein of the present invention also includes polypeptide fragments, mutants, fusion proteins and the like of the protein, which contain an antigenic determinant or an epitope for inducing antibody formation.

 (6) Isolation and purification of the protein of the present invention

 The protein of the present invention can be isolated and purified by the following methods.

 In recent years, as a genetic engineering technique, a technique has been established in which a transformant is cultured and grown, and a target substance is isolated and purified from the culture or grown product. The L4 protein of the present invention can also be expressed (produced), for example, by culturing a transformant containing an expression vector into which the L4 nucleic acid of the present invention has been incorporated in a nutrient medium.

 The nutrient medium for the transformant preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, sucrose, methanol and the like. Examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn chip liquor, peptone, casein, meat extract, soybean meal, potato extract and the like. If desired, other nutrients (eg, inorganic salts (eg, sodium chloride, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.)) May be included. The culturing is performed by a method known in the art. Culture conditions, for example, temperature, pH of the medium, and culture time are appropriately selected so that the protein of the present invention is produced in large quantities.

The protein of the present invention can be obtained from the culture or grown product as described below. That is, when the target protein accumulates in the host cell, The host cells are collected by an operation such as centrifugation or filtration, and the host cells are collected in an appropriate buffer (for example, Tris buffer, phosphate buffer, HEPES buffer, MES buffer, etc. at a concentration of about 100 to 100 mM). (The pH varies depending on the buffer used, but the pH is preferably in the range of 5.0 to 9.0.) After the cells are suspended, the cells are disrupted by a method suitable for the host cells to be used, and the host is centrifuged. Get the contents of the cells. On the other hand, when the target protein is secreted outside the host cell, the host cell and the medium are separated by operations such as centrifugation and filtration to obtain a culture filtrate. The host cell disruption solution or the culture filtrate can be used for isolation and purification of the protein as it is or after ammonium sulfate precipitation and dialysis.

 Methods for isolating and purifying the target protein include the following methods. That is, when a tag such as 6X histidine, GST, or maltose binding protein is attached to the protein, a method using affinity chromatography that is generally suitable for each tag can be used. On the other hand, when the protein according to the present invention is produced without such a tag, for example, a method by ion exchange chromatography can be mentioned. In addition, a method combining gel filtration, hydrophobic chromatography, isoelectric focusing, or the like may be used.

 It is also preferable to construct an expression vector that facilitates isolation and purification. In particular, isolation and purification are easy if an expression vector is constructed so as to express in the form of a fusion protein of a polypeptide having enzyme activity and a labeled peptide, and the enzyme protein is prepared by genetic engineering. It is. Examples of the above-mentioned discriminating peptide include, when the enzyme of the present invention is prepared by genetic recombination, expression of the discriminating peptide and a polypeptide having an enzymatic activity as a fusion protein, whereby the transformant is obtained. It is a peptide having a function of facilitating secretion / separation / purification or detection of the enzyme of the present invention from a grown product.

Such discriminating peptides include, for example, signal peptides (15-30 amino acids present at the N-terminus of many proteins and functioning in cells for protein selection in the intracellular membrane permeation mechanism). Peptides consisting of residues: for example, 0mpA, 0mpT, Dsb, etc., protein kinase, protein A (constituting components of S. aureus cell wall) Protein with a molecular weight of about 42,000 per minute), daltathione S-transferase, His tag (a sequence in which 6 to 10 histidine residues are arranged), myc tag (a 13 amino acid sequence derived from the cMyc protein), FLAG peptide ( Analytical marker consisting of 8 amino acid residues), T7 tag (composed of the first 11 amino acid residues of genelO protein), S tag (composed of 15 amino acid residues derived from Tengen RNaseA), HSV tag, pelB (E. coli) Outer membrane protein pelB 22 amino acid sequence), HA tag (consisting of 10 amino acid residues derived from hemadaltun), Trx tag (thioredoxin sequence), CBP tag (calmodulin binding peptide), CBD tag (cellulose binding) Domain), CBR tag (collagen binding domain), j3 -lac / blu (jS lactamase), iS-gal galactosidase), luc (lucifera one), HP-Thio (His-patch Redoxin), HSP (heat shock peptide), Lny (laminin γ peptide), Fn (fibronectin partial peptide), GFP (green fluorescent peptide), YFP (yellow fluorescent peptide), CFP (cyan fluorescent peptide), BFP (blue Peptides such as fluorescent peptide), DsRed, DsRed2 (red fluorescent peptide), MBP (maltose binding peptide), LacZ (latatose operator), IgG (immunoglobulin G), avidin, protein G, etc. It is possible to use even the discriminating peptide of the above.

Among them, signal peptide, protein kinase, protein A, glutathione S-transferase, His tag, myc tag, FLAG peptide, T7 tag, S tag, HSV tag, pelB or HA tag force It is preferable because expression and purification of such an enzyme becomes easier, and it is particularly preferable to obtain the enzyme as a fusion protein with FLAG peptide because it is extremely excellent in handling. The FLAG peptide is highly antigenic and provides a peptide to which a specific monoclonal antibody reversibly binds, allowing for rapid access and easy purification of the expressed recombinant protein. The murine hybrid dorma, referred to as 4E11, has been disclosed in the presence of certain divalent metal cations, as described in US Pat. No. 5,011,912, which is incorporated herein by reference. Produces a monoclonal antibody that binds to the FLAG peptide. The 4E11 hybridoma cell line has been deposited with the American Type 'Collection' under the accession number HB 9259. Monoclonal antibodies that bind to FLAG peptide Eastman Kodak Co., Scientific Imaging Systems Division ^ Available from New Haven, CT.

 As a basic vector that can be expressed in mammalian cells and can obtain the enzyme protein of the present invention as a fusion protein with the above-mentioned FLAG peptide, for example, pFLAG-CMV-1 (Sigma) is available. Examples of vectors that can be expressed in insect cells include, but are not limited to, pFBIF (a vector in which a region encoding a FLAG peptide is incorporated into pFastBac (Invitrogen): see Examples described later). . Those skilled in the art can select an appropriate basic vector based on the host cell used for expression of the enzyme, restriction enzymes, identification peptides, and the like.

 (7) an antibody that recognizes the protein of the present invention

 The present invention provides antibodies that are immunoreactive with the L4 protein. Such antibodies can specifically bind to the enzyme protein via the antigen-binding site of the antibody (as opposed to non-specific binding). Specifically, a polypeptide having the amino acid sequence of SEQ ID NO: 3 or a fragment thereof, a mutant protein thereof or a fusion protein thereof is used as an immunogen for producing an antibody which is immunoreactive with each. It is possible.

 More specifically, proteins, fragments, mutants, fusion proteins and the like contain antigenic determinants or epitopes that elicit antibody formation, but these antigenic determinants or epitopes may be linear or higher order. The structure (intermittent) may be used. In addition, the antigenic determinant or epitope can be identified by any method known in the art. Therefore, the present invention also relates to the antigenic epitope of the L4 enzyme protein. Such epitopes are useful for generating antibodies, particularly monoclonal antibodies, as described in more detail below.

The epitopes of the invention can be used in Atsey and as research reagents for purifying antibodies that specifically bind from substances such as polyclonal serum or supernatants from cultured hybridomas. Such epitopes or variants thereof can be produced using techniques known in the art, such as solid phase synthesis, chemical or enzymatic cleavage of proteins, or using recombinant DNA techniques. Antibodies of all aspects are induced by the proteins of the present invention. The protein As long as all or part of the polypeptide or epitope is isolated, both polyclonal antibodies and monoclonal antibodies can be prepared using conventional techniques. See, for example, Kennet et al. (Supervised), Monoclonal Antibodies, Hybridomas-'A New Dimensions in Biological Analyzes, Plenum Press, New York, 1980.

 According to the present invention, there is also provided a hybridoma cell line that produces a monoclonal antibody specific to the L4 enzyme protein. These hybridomas can be produced and identified by conventional techniques. One method for producing such a hybridoma cell line is to immunize an animal with the enzyme protein of the present invention, collect spleen cells from the immunized animal, fuse the spleen cells with a myeloma cell line, Thereby producing hybridoma cells and identifying a hybridoma cell line that produces a monoclonal antibody that binds to the enzyme. Monoclonal antibodies can be recovered by conventional techniques.

 The monoclonal antibodies of the present invention include chimeric antibodies, for example, humanized forms of murine monoclonal antibodies. Such humanized antibodies have the advantage of being administered to humans to reduce immunogenicity.

According to the present invention, there is also provided an antigen-binding fragment of the above antibody. Examples of antigen-binding fragments that can be produced by conventional techniques include, but are not limited to, £ 113 and (ab ') ₂ fragments. Antibody fragments and derivatives that can be produced by genetic engineering techniques are also provided.

 The antibody of the present invention can be used in an assay for detecting the presence of the L4 enzyme protein of the present invention or a polypeptide fragment thereof both in vitro and in vivo. The antibodies of the present invention can also be used for purifying L4 enzyme proteins or polypeptide fragments thereof by immunoaffinity chromatography.

Further, the antibodies of the present invention may be provided as blocking antibodies capable of blocking the binding of the glycosyltransferase protein to a binding partner, eg, a receptor substrate, and such binding may result in the biological activity of the enzyme. Can be inhibited. These blocking antibodies can express receptor substrates and inhibit the binding of the protein to certain cells. Identification can be made using any suitable Atsey method, such as testing antibodies for force.

 Blocking antibodies can also be identified in assays relating to their ability to inhibit the biological effects resulting from the enzyme protein binding to the binding partner of the target cell. Such antibodies can be used in in vitro methods or administered in vivo to inhibit a biological activity mediated by the entity that produced the antibody. Thus, according to the present invention, there can be provided an antibody for treating a disorder caused or exacerbated by a direct or indirect interaction between an L4 enzyme protein and a binding partner. Such therapy will involve administering to the mammal an in vivo amount of a blocking antibody effective to inhibit the binding partner-mediated biological activity. Generally, monoclonal antibodies are preferred for use in such therapies, and in one embodiment, antigen-binding antibody fragments are used.

 Hereinafter, the present invention will be described more specifically with reference to examples.

 Example

Example 1: Cloning of L4 nucleic acid

 The present inventors have found a novel structural gene through BLAST and PSI-BLAST searches using a known gene sequence called LARGE as a query, and named it L4. The open reading frame (0RF) predicted from the nucleic acid sequence of L4 is 1047 bp (SEQ ID NOS: 1 and 2) and 349 residues in amino acid sequence (SEQ ID NOs: 2 and 3). Example 2: Expression level of L4 nucleic acid in human colorectal cancer tissue and canceration assay

 Using a quantitative real-time PCR method, the expression level of the L4 gene was compared between human colorectal cancer tissue and normal colorectal cancer tissue of the same patient.

The quantitative real-time PCR method is a method of combining a fluorescently labeled probe in addition to a sense primer and an antisense primer in PCR. As the probe is amplified by PCR, the fluorescent label on the probe comes off and shows fluorescence. Since this fluorescence intensity increases in correlation with the amplification of the target gene, L4 nucleic acid is quantified using this as an index. RNA from normal colon cancer tissue of the same patient as human colon cancer tissue was extracted with the RNeasy Mini Kit (Qiagen) and oligo (dT) using Super-Script First-Strand Synthesis System (Invitrogen) Single-stranded DNA was obtained by the method. This extracted DNA Using the 5 'primer (SEQ ID NO: 4), 3' primer (SEQ ID NO: 5) and TaqMan probe (SEQ ID NO: 6) as template 定量, quantitative real-time with ABI PRISM 7700 (Applied Biosystems Japan) PCR was performed. The PCR conditions were as follows: reaction at 50 ° C for 2 minutes and 95 ° C for 10 minutes, followed by 95 ° C for 15 seconds and 60 minutes. C 1 minute was repeated 50 times. The measured value obtained by using the fluorescence intensity as an index was divided by j8-actin, which was quantified using an Applied Biosystems Japan kit as an internal standard gene, to correct inter-individual variability. A comparison was made between measurements of cancerous tissue and those of normal colorectal cancer tissue of the same patient.

 The results (Table 1) revealed that the amount of L4 nucleic acid expressed in colon cancerous tissues (ie, the transcription level in cancerous tissues) was significantly lower than that in normal tissues. Also, when compared by patient, the expression level of normal tissues was lower than that of normal tissues in the cancerous tissues of all patients, and the degree of the decrease was about 1 Z10 or less, which was 1/1/1 compared to the average value. 5 In addition, for example, if the expression level is reduced to one half of that in normal tissues, the positive rate is 83%.

 table 1 '

 Patient number Normal tissue Cancerous tissue

 1 0.034 0.023

 2 0.020 0.001

 3 0.017 0.003

 4 0.036 0.002

 5 0.117 0.011

 6 0.012 0.009

 7 0.019 0.002

 8 0.010 0.002

 9 0.203 0.005

 10 0.272 0.005

 11 0.026 0.003

 12 0.059 0.007

Average 0.069 0.006 Example 3: Expression of isolated L4 nucleic acid in mammalian cells

Introduction of L4 nucleic acid into mammalian cell expression vector

 To examine the activity of L4, L4 was expressed in mammalian cells by the following method. That is, the L4 gene was incorporated into pENTR (manufactured by Invitrogen) of the pENTR / D-TOPO Cloning Kit (manufactured by Invitrogen), and the mammalian cell expression vector pcDNA3.2-DEST (Invitrogen) and pFLAG -0RF of the relevant gene was introduced into CMV3 (manufactured by SIGMA).

 ① Preparation of entry clone

 Using a cDNA obtained from human skeletal muscle tissue (Clontech) as type III, a PCR reaction was performed using a primer 5 (L4_fullNkoz: SEQ ID NO: 7) and a 3 ′ primer (L4-fullC: SEQ ID NO: 8). Thus, the desired DNA fragment was obtained. The PCR method was carried out under the conditions of 98 ° C for 10 seconds, 54 ° C for 30 seconds, and 72 ° C for 2 minutes 30 times. Then, the PCR product was purified by MiniElute PCR Purification Kit (manufactured by QIAGEN) and isolated by a conventional method.

 This PCR product was incorporated into pENTR / D (manufactured by Invitrogen) by a T0P0 isomerase reaction to prepare an “entry clone”. The reaction was performed by incubating the desired purified DNA fragment solution 21, pENTR / D-TOP0 solution 0.51 (70 ng), and reaction buffer 0.5 μl at 25 ° C for 5 minutes. Thereafter, 2 μl of the reaction solution was mixed with 100/1 of a competent cell (Escherichia coli 0-10, manufactured by Invitrogen), and after heat shock, inoculated on an LB plate containing kanamycin and cultured. The next day, a colony was picked, and an E. coli clone retaining the target DNA fragment was confirmed by direct PCR. To further ensure the DNA sequence by sequencing, the plasmid DNA (pENTR-L4A) was extracted and purified.

 ② Preparation of expression plasmid

 The above-mentioned entry clone has attL which is a recombination site when lambda phage is cut out of E. coli on both sides of the insertion site, and LR clonase (lambda phage recombinant enzymes Int, IHF, Xis By mixing the mixture with a destination vector, the insertion site is transferred to the destination vector, and an expression clone is produced. The specific steps are as follows.

First, 1 μl of the entry clone plasmid solution and 1 μl of pcDNA3.2-DEST solution (75 ng), 2 μl of LR reaction buffer, 4 μl of TE, and 2 μl of LR clonase mix for 1 hour at 25 ° C, add proteinase K solution, incubate at 37 ° C for 10 minutes to terminate the reaction. (This recombination reaction produces pcDNA3.2-L4A). pcDNA3.2 inserts the target gene under the control of the CMV promoter and expresses the inserted gene in mammalian cells.

 (3) Preparation of soluble protein expression plasmid

 p FLAG-CMVS plasmid vector (manufactured by SIGMA) expresses a transgene by a CMV promoter that works in mammalian cells, and adds a FLAG peptide sequence (SEQ ID NO: 9) to the N-terminal part of the inserted gene product for purification. This is a plasmid vector. Since the L4 gene product is expected to be a membrane-bound protein with a hydrophobic region near the N-terminus, a soluble enzyme protein excluding the water-soluble region was used to facilitate purification of the enzyme protein. The gene is modified to express it, incorporated into the expression plasmid vector PFLAG-CMV3, transfected into mammalian cells, and expressed to obtain a soluble enzyme protein.

 The removal of the gene region encoding the hydrophobic region of the gene product was carried out by amplifying by PCR the partial sequence with a total chain length of 0RF obtained by entry and cloning.

The three types of 5'-terminal primers (L4_LformKpn77F; SEQ ID NO: 10 and L4_MformKpnllOF; SEQ ID NO: 11 and L4_NformKpnl55F; SEQ ID NO: 12) are lysine codons 1 to 73 and 106 aspartic acid codon of cDNA, respectively. Amplify the cDNA partial sequence encoding a peptide having an isoleucine codon at position 151 N-terminal. As a primer at the 3 ′ end, a sequence derived from a plasmid vector (XBM13R; SEQ ID NO: 13) contained in the entry clone pENTR-L4A plasmid was used. The PCR method was performed under the conditions of repeating 94 ° C for 30 seconds, 54 ° C for 30 seconds, and 72 ° C for 1 minute 20 times. The amplification product was purified using MiniElute PCR Purification Kit (QIAGEN). The above-mentioned modified gene was incorporated into the expression vector using restriction enzyme recognition sequences set at both ends of the PCR primer. Purification The PCR product was digested with the restriction enzymes Kpnl and Xbal at 37 ° C for 2 hours, and then purified using the MiniElute PCR Purification Kit. Similarly, the pFLAG-CMV3 plasmid vector was digested with Kpnl / Xbal and purified. The ligation of the plasmid vector and the insertion gene was performed using a ligation kit ver. 1 (TAKARA). Mix 0.5 μL of the imported gene fragment solution, 0.5 μL of the plasmid solution, and 4 μL of the LIGAI-YEONGYON Kit A solution 4 μL. The reaction was performed at C for 30 minutes. Thereafter, the reaction solution 2 _mu 1 Combi competent cells were mixed with (shed E. coli DH5, Invitrogen) 100 il, after Hitosho click method, was inoculated and cultured in LB play Bok containing ampicillin phosphorus. The next day, a colony was picked and an E. coli clone retaining the target DNA fragment was confirmed by direct PCR. After confirming the DNA sequence by sequencing for further assurance, three plasmid DNAs (pFLAG-L4, pFLAG_L4M, and pFLAG-L4N) were extracted and purified.

Introduction and expression of expression plasmid into cultured mammalian cells

After confirming that the L4 gene was inserted into the expression plasmid obtained by the above method, this plasmid was introduced into cultured mammalian cells Cosl and 293T. That is, 4 × 10 ⁶ Cosl cells or 293T cells were placed in a 150 mm Petri dish in 20 ml of DMEM medium (manufactured by Gibco Co., Ltd., Low's glucose medium was used for Cosl cells, and Hydalcos medium was used for 293T cells. 10% inactivated bovine serum was used), and the cells were cultured for 1 晚. After co-washing with the medium, 0pti_MEM (manufactured by Gibco, 10 ml) containing 200 of Lipofectamine 2000 and 60 g of plasmid DNA was added, and the cells were cultured at ³⁷ ° C. and 5% CO _{2 for} 48 hours.

 After the culture, the cells were collected by a conventional method using trypsin, and the cells and the culture supernatant were frozen and preserved, and used for enzyme activity measurement.

Resin purification of L4

NaN ₃ (0.05%), NaCl (150 mM), CaCl ₂ (2 mM), anti-Ml resin (Sigma) (100 μ 1) are mixed with 10 ml of FLAG-L4 supernatant of the above mammalian cell culture supernatant And stirred overnight at 4 ° C. The next day, centrifuge (3000 rpm, 5 minutes, 4 ° C), collect the pellet, add 9001 of 2 raM CaCl ₂ · TBS, centrifuge again (2000 rpm, 5 minutes, 4 ° C), and remove the pellet to 200 μl. Was suspended in 1 mM CaCL · TBS to prepare a sample for activity measurement (L4 enzyme solution). A part of this was subjected to Western blotting using anti-FLAG M2-peroxidase (manufactured by SIGMA) for electrophoresis by SDS-PAGE, and the expression of the desired L4 protein was confirmed. As a result, multiple bands (post-translational modification of sugar chains, etc.) This was probably due to the difference), and the expression was confirmed.

Example 4: Analysis of sugar chain structure in cultured mammalian cells

 In the cultured mammalian cells into which the L4 gene has been introduced as described in Example 3, the introduction of the L4 gene causes a change in the sugar chain structure in the cells or in the cell surface. By analyzing this change, the L4 gene product can be identified and the enzyme activity can be estimated.

Example 5: Measurement of enzyme activity

 In order to examine the specific enzymatic activity of the L4 protein, a conventional method known in the art can be used. For example, by incorporating a donor substrate group or an acceptor substrate group involved in glycosaminodalican synthesis into a reaction system using the L4 enzyme solution prepared as described in Example 3 above. The substrate specificity can be screened, and by comparing the reaction results when the type of buffer used, pH conditions, and the type of coexisting metal ions, etc., are compared, the optimal buffer and optimal pH can be determined. , Required metal ions, etc. can also be specified.

Claims

The scope of the claims

1. A nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary thereto.

 2. The nucleic acid according to claim 1, consisting of at least a dozen or more consecutive base sequences in the base sequence set forth in SEQ ID NO: 1 or a base sequence complementary thereto.

 3. The nucleic acid according to claim 2, consisting of the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary thereto.

 4. The nucleic acid according to any one of claims 1 to 3, which is a probe or a primer.

 5. The primer according to claim 4, consisting of the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or a nucleotide sequence complementary thereto.

 6. The nucleic acid according to any one of claims 1 to 5, which is a cancer marker.

 7. A method for assaying canceration of a biological sample,

 (a) using the nucleic acid according to any one of claims 1 to 6 to measure a transcription level of the nucleic acid in a biological sample; and

 (b) determining whether the measured value is significantly lower than the measured value for a healthy biological sample;

A method that includes

 8. The method according to claim 7, wherein the step (a) includes determining whether a measured value of the biological sample is 1Z2 or less as compared with a measured value of a healthy biological sample. the method of.

 9. In the step (a), the nucleic acid according to any one of claims 1 to 6 is used as a labeling probe, and the labeled probe is contacted with a biological sample under stringent hybridization conditions. The method according to claim 7 or 8, comprising measuring the transcription level based on a signal from the hybridized labeled probe.

10. In the step (a), the nucleic acid contained in the biological sample is subjected to a nucleic acid amplification treatment using the primer according to claim 4 or 5, and 9. The method according to claim 7 or 8, comprising measuring the amount of the nucleic acid amplified by the immobilizer.

 1 1. A method for assaying the effectiveness of a treatment for cancer therapy, comprising using the nucleic acid according to any one of claims 1 to 6 in a biological sample that has been treated for cancer treatment. Measuring the level of transcription for the nucleic acid and determining whether the measured value is significantly greater than before or without treatment.

 12. The method according to any one of claims 7 to 11, wherein the biological sample is a sample derived from the large intestine.

 1 3. A nucleic acid encoding a polypeptide having glycosyltransferase activity, comprising the following nucleotide sequences (a) to (d):

 (a) the full-length nucleotide sequence of SEQ ID NO: 1;

 (b) the nucleotide sequence of SEQ ID NO: 1 from nucleotide numbers 73 to 1074;

 (c) the nucleotide sequence of nucleotide numbers 106 to 1074 of SEQ ID NO: 1; or

 (d) the nucleotide sequence of SEQ ID NO: 1 as represented by nucleotide numbers 151 to 1047;

A nucleic acid having any one of the above.

 1 4. A nucleic acid encoding a polypeptide having glycosyltransferase activity, comprising the following amino acid sequence (a) to (d):

 (a) the full-length amino acid sequence of SEQ ID NO: 3;

 (b) the amino acid sequence of SEQ ID NO: 3 according to amino acid numbers 25 to 349;

 (c) the amino acid sequence of SEQ ID NO: 3 as described in amino acid numbers 36 to 349; or (d) the amino acid sequence of SEQ ID NO. 3 as described in amino acid numbers 51 to 349; A nucleic acid encoding a polypeptide having one or more of the following:

 15. The nucleic acid according to any one of claims 13 to 14, which is a DNA.

1 6. A vector comprising the nucleic acid according to any one of claims 13 to 15.

1 7. A transformant containing the vector according to claim 16.

 1 8. A polypeptide having glycosyltransferase activity, comprising the following amino acid sequence (a) to (d):

 (a) the full-length amino acid sequence of SEQ ID NO: 3;

(b) the amino acid sequence of SEQ ID NO: 3 according to amino acid numbers 25 to 349; (c) the amino acid sequence of amino acid numbers 36 to 349 of SEQ ID NO: 3; or (d) the amino acid sequence of amino acid numbers 51 to 349 of SEQ ID NO: 3; Polypeptide having one.

 19. An antibody against the polypeptide according to claim 18.