JPH04502101A - Full-length cDNA encoding human laminin binding protein - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] name of invention The full-length invention described herein encodes a human laminin binding protein. is recognized by the National Institutes of Health under Grant Number POI CA44704, PO Done in the course of research under ICA22427 and ROI 0M38318. Ta.

Cross-reference of related applications This application is filed in U.S. Patent Application No. 07/238.9 on August 31, 1988. This is a continuation-in-part application of No. 55.

Technical field FIELD OF THE INVENTION This invention relates generally to human laminin binding proteins, and in particular to human colon cancer. Full-length cDNA encoding human laminin-binding protein derived from 1”ull length cDNA) and their molecular cloning. .

Background technology Colon cancer is one of the leading causes of cancer death in humans. in the US In 1988, more than 140,000 new cases of colon cancer were expected. There is. More than 70,000 of these patients will die from the disease. cormorant. Current diagnostic techniques are insufficient to ensure effective surgical control. During this period, less than half of the patients are detected. In particular, patients with poorly differentiated colon cancer Detection and monitoring are difficult for patients who do. This seems like Malignancy is often a useful marker for highly and moderately differentiated colon cancer. This is because it is generally rare to express the carcinoembryonic antigen (CEA). be.

Conversion of normal colon epithelial cells to tumor state and differentiation of colon cancer cells at the molecular level However, very little is known about it.

Over 100 human colon cancer-derived cell lines have been established. These cell lines are human Despite sharing common characteristics of colonic epithelial origin, the origin of their differentiation, degree of tumorigenesis, tumorigenicity in nude mice, histology of induced tumors, and metastatic potential. power, and the transformed phenotype in culture can be quite different. those is a potentially rich source for elucidating new markers.

Laminin is involved in a wide variety of biological processes. Most associated with colon cancer, La Minin leads to cell adhesion, morphogenesis, division, differentiation and translocation. Laminin is It is a major component of the basement membrane. Laminin consists of A chain (Mr 400,000), B Three chains: L chain (Mr210,000) and B2 chain (Mr200,000) It is a large glycoprotein with a cruciform structure consisting of polypeptide chains. This is normal and mediates tumor cell adhesion to the basement membrane.

Proteolytic fragments of laminin from the B1 chain and pentaminin that possess cell-adhesive activity Iy-3e r-A rg has been identified in the peptide Try-11e- . Iwamoto et al., 5science, 238.1132-1134 (1987 ), this pentapeptide inhibited melanoma metastasis in mice. Many of the effects of salt laminin have been reported to appear to be 87 KDa. This has been shown to be mediated through laminin receptors, cell surface glycoproteins. It is.

U.S. Patent 4. issued to Liotta et al. on January 21, 1986. 565.789 discloses the isolation and characterization of certain aspects of laminin receptors. There is.

Wewer et al., Proc, Nat, Acad, Sci, (US A), 83.7137-7141 (198B) is the human laminin receptor 2 In addition to the nucleotide and hypothetical amino acid sequences of the H5 epitope, the laminin receptor Discloses the isolation of cDNA.

In recent years, two additional laminin receptors have been identified, 18KDa and 110KDa molecules. (Kleinman et al., Proc.

Nat, Aead, Sci, (USA), pp. 1282-1288, vol. 85 ( 1900), and 5aalheiser et al., Proc. Nat. Acad. Sci, (LISA), pp. 6457-6461 (1987).

Summary of the invention The present invention provides a complete cell-derived protein derived from human colon cancer cells containing substantially the sequence shown in FIG. The objective is the human laminin binding protein cDNA sequence. This also applies to Starting from nucleotide number 162 and ending at nucleotide number 829 as shown in Figure 3 It relates to a subsequence that substantially corresponds to an array.

In another aspect, the invention provides a construct encoding a human laminin binding protein. It also relates to replacement clones.

In another aspect, the invention provides a laminar sequence encoded by any sequence. The above-mentioned cDNA encodes a laminin-binding protein. , relating to substantially isolated DNA.

Also, methods for detecting the presence of cancer in a patient and methods for detecting the presence of cancer in a patient. Concerning reagents for extraction.

Brief description of the drawing Figure 1 shows RNA from surgically obtained human colon cancer and adjacent normal epithelium. and 32p-labeled cDNA inserts from three plasmids - High Diagram showing hybridization. Lanes 1.3.5 are from normal human colonic epithelium. Ane 2.4.6 used total RNA from colon cancer. Lane 1.2 is 8-2V, Lane 3.4 hybridizes with 3-4-D, and lane 5.6 hybridizes with 1-4E. Ru. Arrows indicate the main hybridized mRNAs, each 1.2 kb , 1.7 kb and 0.7 kb.

Figure 2 shows RNA from 20 human colon cancer cell lines and 8-2v plasmid. 32p mark? Plot with lcDNA insert-hybridization Diagram showing the option. Lane 1 is from HT29, 2 is from CX-1, 3 is RCA? 4 is from CCL222, 5 is from CCL220.1, 6 is from CCL22 8 from HCT116b, 8 from HTB39, 9 from clone A , 10 is from clone D, J] is from) UP] 01, 12 is from CCL2 29, 13 from c+y, 14 from Mo5er, 15 from CCL23 3, 16 from CCL224, 17 from CCL237, 18 from CL R from I87, 19 from CCL227, and 20 from CCL234 NA was used. The arrow indicates the 1.2 kb major hybridized mRNA. vinegar.

Figure 3 shows the full length cDNA insert from the J-9λgtlO phage. Figure showing the nucleotide sequence and deduced amino acid sequence. 8-2v insert Substantially all sequences of (nucleotide numbers) located in the sequences shown herein Nos. 162 to 829). Sequences of interest are underlined.

FIG. 4 shows the predicted amino acid sequence shown in FIG. 3. Bathy Puot Chyt iropatt+y plot).

Figures 5 and 6 show human esophagus, neck, duodenum, lung, prostate cancer and human cancer. Different types showing significant levels of expression of 1.2 Kb mRNA in melanoma cell lines Figure 3 shows a Northern plot of total RNA isolated from human cancer cell lines.

Figure 7 shows in vitro translational production of laminin-binding genes from rabbit reticulocytes. The substance binds to the laminin Sepharose column, and the laminin-binding protein in The molecular weight of the vitro-translated product was 45 kd protein for SDS PAGE. A diagram showing how to move.

Deposit report 1. A recombinant clone named J~9 was created in 1988 under the Budapest Treaty. Published in American Type Culture Collection (ATCC) on August 30th. Deposited and received ATCC accession number 40489.

A recombinant clone named 2,8-2V was produced in 1988 under the Butabest Treaty. Deposited with ATCC+ on August 30th and received ATCC reception number 40490.

Unless otherwise defined, technical or scientific terms used herein are defined as It has the same meaning as commonly understood by one of ordinary skill in the relevant art. child The publications mentioned herein are incorporated herein by reference.

The term “derived from” refers to proteins with the same sequence or mild substitutions or additions. or code for a functional equivalent that has been removed.

The term "substantially purified" typically refers to the association in a particular genome. Synthesized without other cellular components or naturally occurring isolated means isolated.

A cDNA library was constructed from a human colon cancer cell line.

These libraries hybridize with mRNAs that are more abundant in colon cancer. Screening was performed for specific clones. Recombination described below By hybridization with the cDNA insert in clone 8-2■ Thus, 1.2 kb mRNA was identified. This mRNA is found in human colon cancer. It was approximately nine times more abundant than in the adjacent normal colonic epithelium. This 1.2 kb mR The NA cDNA is the original, full-length human genome containing substantially the sequence shown in Figure 3. The cDNA sequence of human laminin-binding protein derived from human colon cancer cells is thought to be be exposed.

More than one nucleotide triblet can code for the same amino acid , it is understood that changes can occur in the nucleotide sequence due to the presence of degenerate code. has been done. Therefore, there may be changes in the nucleotide sequence shown in Figure 3. This is also within the scope of this invention.

8-2 ■ Identification of plasmid. are more abundant in human colon cancer than in their normal counterparts. In order to explore the cellular mRNAs that exist in Using poly(A) + mRNA isolated from the cell strain, clone A, plasmid A cDNA library was constructed in pBR322. First, the screening is 1 ．． 500 plasmids were introduced into the highly differentiated human colon cancer cell line CX-1 and other cells. by hybridizing with cDNA generated from clone A mRNA. I did it. Most plasmids showed equal or greater CX- c from clone A than from CX-1. Ten plasmids were studied because they hybridized more with DNA. did. Next, the cDNA inserts of these plasmids were plotted on an RNA gel. Normal human colonic epithelial tissue and colon from the same patient by hybridization. It was used to compare mRNA levels between cancers. One of the plasmids, 8-2V, is adjacent to 1 to 2 kb mRNA, which is more abundant in cancer cells than in normal epithelium, It bred (Fig. 1). Comparing equal amounts of total RNA from cancer cells and normal tissue did. Analysis by densitometry shows that the level of this mRNA is higher in cancer cells than in normal epithelium. It was suggested that it was about 9 times higher than the previous year. For comparison, the same pair of RNAs were prepared from two different species. was hybridized with the cDNA insert from the plasmid. Plasmid 1 -4E is a 0.7 kb protein that is present in much larger amounts in normal colonic epithelium than in cancer cells. hybridized with mRNA. In contrast, plasmid 3-4W is between the two It hybridized with 1 and 7 kb mRNA, which are equally abundant. these The results showed that 1 and 2 kb m hybridized with the 8-2■ cDNA insert. RNA encodes a polypeptide useful in distinguishing colon cancer cells from normal colon epithelium He suggested that he had the ability to do so.

Plot of total RNA from various cell lines and 8-2V cDNA insert. 1, which hybridizes with 8-2■ present in hybridization colon cancer cells; Increased levels of 2 kb mRNA, especially that by poorly differentiated cells. To investigate whether expression is a general phenomenon, we tested 20 human colon cancer cell lines (described above). ) was plotted and hybridized between equal amounts of total RNA and 32p-labeled probe. Tested by Figure 2 shows a highly differentiated, mucin-producing rectal adenoma cell line. All human colon cancer cell lines tested, with the exception of CCL234, which has essentially this It has been shown to express significant levels of mRNA. is hardly differentiated All cell lines (CCL220.l, HCTl16b, clone A and MI PIOI) has a high expression level and is expressed only at a lower level are highly differentiated (CCL233 and GLY, both are high CEA producers) However, there is a tendency between the level of this 1.2 kb mRNA and the degree of differentiation. The correlation was not clear. Based on equal total RNA, this The level of 1.2 kb mRNA is in the same range as the colon cancer surgical tissue shown in Figure 1. It was surrounded.

Nucleotide sequence of 8-2V cDNA insert 8-2■ Plasmid cDNA The insert begins at nucleotide number 162 in FIG. It had eeg bp substantially corresponding to the sequence ending with number 829. D.N. A computer search of the A sequence database revealed that 8-2■ contains approximately 349 nucleotides. It was revealed that it contains an array of codes. This sequence is similar to the human sequence reported by Webb et al. Contains a partial cDAN sequence of laminin receptor. 2H5 monoclonal antibody , binds to laminin receptors in immunoplots and is active in EL ISA. reacts with adult laminin receptors and directs laminin to either the cell membrane or the plasma membrane interfering with binding and blocking cell attachment to the laminin-rich amniotic basement membrane. It has been known. The epitope or deduced amino acid sequence recognized by this antibody exists in Furthermore, the predicted polypeptide inhibits laminin-mediated cell adhesion 20 amino acid sequences recognized by polyclonal antibodies (Pro-T hr-Glu-Asp-Trp-8er-A I a-G I n-Pro-A l a-Th r-G I u-As p-T rp-8er-A I a- A　a-o　ro- Thr-Ala). Considering this, hybridization with 8-2V cDNA It is also very likely that the 1 to 2 kB mRNA product binding to laminin It seems like something that could happen.

1 for molecular cloning and hybridizing with the 8-2V cDNA insert; cDNA sequence corresponding to 2kb mRNA Next, 8-2V 32p-labeled c DNA inserts were made from mRNA of the human colon cancer cell line ex-i. λgtl.

Cloning of cDNA with longer inserts from a cDNA library It was used for One of the cDNAs, J-9, had an insert of approximately 1 kb.

The cDNA sequence of this insert is essentially the same as shown in FIG.

The 8-2V cDNA sequence from the Clone A cell line is completely within this cDNA sequence. It is located in Therefore, the antigen recognized by the 2H5 monoclonal antibody A polyclonal protein that blocks laminin binding to the sexual domain and laminin receptors. The C-terminal 20-amino acids recognized by null antibodies come from three different sources, human with identical nucleotide sequences from clonal venous endothelial cells, clone A and CX-1. are doing. Starting from the first ATG, as shown by computer-aided analysis, It is clear that the oven reading frame (ORF) ends with Mari TAA. became. This ORF is preceded by an in-frame stop codon at position -18 and an There is an ANN immediately before the first ATG. Signals for polyadenosine phosphorylation There is also a file array.

Deduced amino acid sequence This ORF encodes a 32.817 Da polypeptide of approximately 295 amino acid residues. can be decoded. How many deduced amino acid sequences are substantially as shown in Figure 3? revealed some interesting characteristics. At the N-terminus, many cell surface proteins A leading signal sequence for entering the endoplasmic reticulum, which has been reported for proteinaceous cells, was discovered. It has not been. Bitorobacy plot shows that 2/3 of the N-terminus is It has a hydrophobic fragment that can function as a membrane sequence or confer membrane anchoring. N series Consensus on Asn-XAA-(Ser/Thr) regarding glycosylation There is no such array. Simply two Cy separated by 14 amino acid residues There is only s. After amino acid residue 225, arginine or lysine has not been found. Therefore, trypsin completely destroys this polypeptide. When degraded, it is a 70-amino acid, strongly acidic polypeptide with no positively charged residues. should be the C-terminal domain (13 asparagine and glutamine residues). be.

The normal sequence was noted previously (Wewer, U.M., Liotta.

L, A,, Jaye, M,, Ricca, G, A,, Kanohan, %/, N6 , CIays+++ith, A.

Pl., Rao, C.N., Wirth, P., Coligan, J.E., A. lbrachtsen, R., Mudryj, M., and 5obel. M.E. (198B) Proc, Nat, Acad, Sci.

83.7] 37-7141) 2 Thr-Glu-Asp-Trp-8e r-A! a-X-Pro repeats 2B4-271 and 273-280 (★ ) Fig. 3); Two AI a-A I a-A I a-X-X-A l a 91-96 and 21B-221 (A) Figure 3); three Lys-G L u-G I u repetition 11-13.212-214 and 224-2 26), two of which are Lys-G I u-G l u-Gln- X-X-X-Glu (212-219) and Lys-G l u-G I Present in u-X-G l n-X-Glu (224-230); two As p(G:Iu)-X-X-X-C; Iu(Asp)-X-Tyr-X-Tyr- Lys(Arg)-X-X-X-Asp(Glu) repeats 31-44 and 196-209 (:) Figure 3); One symmetrical arrangement Leu-Met-Tr Contains p-Trp-Met-Leu (173-1711i (o) Figure 3) .

Hybridized with the cDNA insert of the recombinant clone named 8-2v. Increased levels of l', 2 kb mRNA are found in mostly undifferentiated and and a highly differentiated human colon cancer cell line. Currently, mostly differentiated Markers for human colon cancer cells that are not present are needed. this is this This is because these cells hardly express carcinoembryonic antigen. laminin binding tan The 1- and 2-kb mRNA encoding proteins are based on interaction with laminin. , has no effect on both poorly differentiated and highly differentiated Intestinal cancer cells can be distinguished from normal colon epithelial cells.

Figures 5 and 6 show human esophagus, neck, duodenum, lung, prostate cancer and human cancer. The expression of 1.2 Kb mRNA in melanoma cell lines was found to be at a significant level. It shows.

Figure 5 shows a Northern plot of total RNA isolated from various human cancer cell lines. . Total RNA (15ug/lane) was added to 1.0% agarose/formaldehyde gel. separated for laminin-binding protein, transferred to nitrocellulose filter, and laminin-bound protein. 32p-labeled cDNA insert from protein-encoding clone 8-2v It was explored using. Lanes 1 to 6 are RNA isolated from the following cancer cell lines. including. 1 is CE81T, 2 is CE48T, 3 is HID (1, 2 and 3 are Hi esophageal cancer cells), 4 is HTB34MS751.5 is HTB35SiHa, 6 is HeLa (4, 5 and 6 are human cervical cancer cells). The head of the arrow is a hybrid The 12 kb RNA that was raised is shown.

FIG. 6 also shows a Northern plot of total RNA isolated from various human cancer cell lines. shows. Total RNA (15ug/lane) in 1.0% agarose/formalde Separate for hydrochloride, transfer to nitrocellulose filter, and laminin binding protein. P-labeled cDNA insert from clone 8-2■ encoding protein Searched using. Lanes 1 to 6 are RNA isolated from cancer cell methods below. including. 1 is T24.2 is EJ (1 and 2 are human bladder adenomas), 3 is ax-1, 4 is CCI, 218111iDr (3 and 4 are human colon adenoma), 5 is human ten Diodenal adenoma 1ITB40HuTu80.6 is CCL18A549.7 is 0 AT4.8 is Ca! u (6, 7 and 8 are human lung cancer cells), 9 is A2058 (human melanoma), 10 is CRL1435PC-3 (human prostatic adenoma) . Arrow heads indicate hybridized species of 1.2 kb RNA.

At the protein level, Figure 7 shows that the laminin-binding protein gene is The in vitro translation product is a protein that binds to the laminin Sepharose column. Therefore, it appears as a protein of 45k (j) in SDS PAGE. It shows. In Figure 7, the in vitro analysis of the lamini:/binding protein gene Toro translation products were obtained from rabbit reticulocyte lysate.

Molecules of laminin-binding protein in vitro translation products, as shown in lane 2. The amount migrated on 5DSPAGE as a 45 kd protein. lane] for the in vitro translation of this particular batch of rabbit reticulocyte lysate!・ ・Tsuku ground contrast. This in vitro translation product was converted to laminin Passed through loin affinity column. 50+++M after washing longer) of the 45 kd band using 0.5 M NaCΩ buffered at pH 8.0. Protein was eluted (lane 3).

Laminin receptors bind laminin, which facilitates invasion and subsequent translocation. It has been suggested that the tumor cells are deeply involved in the initial interaction of tumor cells with the vascular basement membrane. It is. Increased levels of this mRNA in colon cancer cells compared to normal epithelial cells. The expression of laminin receptors in cancer cells tends to increase when compared to This would be consistent with prior observations. highly malignant and poorly differentiated cancers, including colon cancer Human cancer cells that do not appear to be highly differentiated by immunoperoxidase staining. They have also been shown to express high levels of cytoplasmic laminin receptors. moreover , a laminin-derived pentapeptide identified by the laminin receptor, Inhibition of melanoma metastasis in model systems has been shown. laminin and its It is possible that interactions mediated between receptors prevent metastasis of colon cancer.

The size of this mRNA is similar to that of the laminin receptor previously described by Uewa et al. ．． 7kb significantly smaller than mRNA.

The ORF in the cDNA described here was described for one laminin. A polypeptide of 32.817 Da, much smaller than the 87 kDa protein. It will only satisfy the following.

However, all reported partial cDNA sequences of laminin receptors are listed here. Since it is included in the ORF that is listed, the product of 1.2 kb mRNA is either that or 67 If it is not a kDa laminin receptor, it is probably a laminin binding protein. . The product of this 1.2kb mRNA must be laminin-binding protein. This means that the epitope coding region of the 67 KDa laminin receptor is located here. This is strongly supported by the fact that all are intervening in the reported sequences. this epi Tope is a Moatsu clonal antibody that blocks the binding of laminin to its receptor. , 2I (5). Furthermore, 1. Laminin-mediated cell adhesion C-terminal 20-amino acid synthetic peptides identified by polyclonal antibodies that block A tide is also present at the C-terminus of this sequence.

Three types of laminin receptors with different molecular populations have already been reported (Kle inman et al., Proe, Nati, Aead, Sci, (USA), 1282 - p. 1288, vol. 85 (1,988) and Smalheiser et al., Proc. .

Natl, Acad, Sci, (tJsA), pp. 6457-6461, vol. 84 (1987)), we wonder whether all receptors have a common sequence eutectic. It is of interest. Plotinoλ hybridization of the RNA gel shown here. Most of the sections show a prominent node of 1.2 kb, but some of the larger size There are a few nodes. Are these bands associated with other laminin receptors? Whether this is the case requires further investigation. Furthermore, 8-2~ of human esophageal cancer i11 cell 'A 5.5 kb mRNA that strongly hybridizes with cDNA has been identified. .

The deduced amino acid sequence in Figure 3 showed several interesting features. Nine “- at the end In many cell surface proteins, the ability to enter the endoplasmic reticulum has been reported. No leading signal sequence has been found. Apparent membrane similar to other membrane proteins Inside and outside (trans+++embrane)'? +R region or amphipathic α -There are no helices. This amino acid sequence is one of the laminin receptors. If so, it may be connected to the membrane by another route. Bite Lobby Sea Pu According to Lott, the N-terminal two-thirds of the molecule functions as a signal sequence or contains a hydrophobic fragment that will confer membrane anchoring. For example, give an association with a membrane As part of the 19-amino acid fragment, the highly hydrophobic sequence Leu-Le u-Leu-Ala-A la-Arg-A 1a-118-va l-A 1 a-11e exists. Other potential membrane-anchored fragments have many hydrophobic residues The N-terminal 15-30 residues. However, laminin receptors are responsible for membrane anchoring. It is also possible to use listic acid or valmitic acid. N series connecting tube added There is no consensus sequence Asn-X-3er (Thr). Potential 〇There are 24 Thr and 14 Ser for series connection addition.

3114 Oyobi 19B-209th position (7) Asp(Glu)-X-X-X-G lu(Asp)-X-Tyr-X-Tyr-Lys (A rg)-X-X-X -Asp (GIu) is a notable repetition. By two cysteines If we consider the 40 amino acid stretch as a connecting domain, then 2 Each of the two adjacent domains will have one of this repeat sequence. These two Each of the adjacent domains contains another repeat, Ala-Ala-Ala-X-X- There is Ala-X(-)-Thr.

The C-terminus after residue 225 has 13 asparagine and glutamine residues. and contains a 70-amino acid fragment without lysine or arginine residues. this Trypsin-resistant, strongly acidic domains characterize laminin-binding proteins . This 70-amino acid domain contains five Asp(Glu)-Trp-8er There are only repeats of (Thr), two of which are almost vertical repeats Thr-Gl Present in u-Asp-Try-8er-^1a-X-Pro.

In another aspect, the invention provides reagents and methods for detecting cancer cells in a patient. Regarding. This method involves using a labeled cDNA probe having one of the cDNA sequences described above. exposing the probe to a tissue or body fluid sample from the patient, and Including monitoring reactions for hybridization. hybrida The ization selection technique is described by Maniatjs et al., Molecular Clon. ing, ^Laboratory Manual, Co1d Spring Harbor Laboratory (1982).

Useful labels include radionuclides such as 32p, 3H1 or I40, enzymes, fluorescent Hybridization agents such as photophores, chemiluminescent compounds, chromophores or chromophores This is followed by the detectable component. These labels are commonly known to those skilled in the art. The probe can be attached directly or indirectly using various techniques. Ma Additionally, identifying probes using specific labeled antibodies is also within the scope of technology in this field. It is within the range.

The following examples illustrate the invention.

From the Department of Surgery at New England Deaconess Hospital , surgical specimens of early human colon cancer cells and adjacent normal colon tissue were obtained. surgery Immediately after, normal colonic epithelium is separated from muscle and connective tissue under a dissecting microscope and drained of liquid. Placed in nitrogen (1verse, P, L, Matal, E, and Hin es, R.N. (1987) BioTechnique 5.521, 52 3) o Human colon cancer cell line Clone A, which is a subclone of DLD-1 and clone D beam, from Dexter (Dupont), MIPIOI is N, ZalTlcheck (Mallory Institute )), CX-1 is S, Bernal (Danner-Farber Cancer Institute CD RCA 5 from ana-Farber Caneer In5 titute HCT118b, Gly and Mo5er are M, Brattain (pay Baylor College of Medicine (Baylor College of Medicine) from) lT29, CL187, CCL22[1,1, CCL222, CC' L224, CCL227, CCL228, CCL229, CCL233, CCL 234, CCL237 and HTB39 are American Type Culture Co. I got it from Rection. These cell lines are all well-characterized and well-documented. and is of human colon origin. All cells were incubated with 10% fetal bovine serum (H A　Bioproducts) and 1% NutridomaNS　(Boeh 50% Dulbecco's modified Eagle's medium (GIBCO) supplemented with and 50% PPMI 1640 MEDIIJM (GIBCO), 5 % CO2 and 100% humidity at 37°C.

Preparation of RNA Total cellular RNA from cultured cells was collected by Cox, R.A. (1986). Methods Enzym, 12.120-129) and Stroman et C3trohtaan, R, C6, Mo5s, P, S, Eastwood , J.M., and 5pector, D. (1977) Ce1l 10.285 -273). Total cellular RNA from surgical specimens was Lublein et al. (Chirgwfn, J.M., Pryzbyla, A.E. , MacDormd, R.J., and Rutter, W.J. (1979). Prepared according to Biochemistry 1g, 5294-5299> . Prior to extraction with guanidine isothiocyanate, the Both normal and fti tumor tissue were ground into sand-like particles.

Construction of 98R322 clone A cDNA library Ports from clone A cells (A)” RNA was prepared by Haff et al. , (197B) Biochem, 15.4110-4115) described It was purified by using poly(U)-Sephadex column twice as follows. . AMV inversion of the first cDNA strand using oligo-dT as a primer By Molecular Genetics Re5sources The second strand was synthesized by Wickens et al. , G, N, and Sch i mke, l? , T. (1978) J.B.I.o. DNA polymerase according to I, Chem, 263.2483-2495) Prepared using I. cDNA was treated with 81 nuclease and tailed with dCTP. and fractionated using a Blo-Ge1 A-50 column. Greater than 400 bp Only the dC-tailed cDNA (dC-tailed cDNA) was pooled, and P Annealed to StI-digested poly(G)-coccyx pBR322 plasmid. Daggert (Dagert, M., and Ehrlich, S.D., (1979) Gen E. coli HBIO1 was transformed according to 6.23-28). this The complexity of the library is approximately 5 x 104 clones/;tgc　DN　A It was.

Screening of plasmid cDNA libraries Individual transformed E. coli Transfer the colonies to a master agar plate and two containing tetracytalin. (ManjaLjs, T, Frjts ch, E, F.

and 5arQbrook, J. (1982) Molecular Clo ning: A Laboratory Manual (Cold Spr. ing Harbor Laboratory, C3)l, NY)).

These colonies were arranged in a grid pattern. on all three plates A line was drawn at the same location on each colony.

Colonies on the nitrocellulose filter were lysed with alkali and fixed. Black Produced from poly(A) RNA of A or CX-1 [, 32p-labeled] The first cDNA strand obtained was used for colony hybridization. Total 1, 500 colonies were screened.

RNA gel plots - extracted from hybridized tissues and cultured cells. The total RNA (15 ug) was run on an agarose gel containing formaldehyde. Seed (Seed, B. (1982) Genet, Eng. 4.9l-102) according to GeneSc, reenPIus (DuPont ). Recombinant plasmids were isolated by alkaline lysis and CsCΩ gradient centrifugation. Prepared (Maniatis, T., Frltsch, E.F., and Sam brookl.

(1982) Mo1ecular Cloning: A Laborato ry Manual (ColdSpring Harbor Laborat ory, C8H, NY)) o Purify the plasmid DNA and digest it with PstI. Disassembled. After electrophoresis, the cDNA insert was placed in a LID/X filter series. Elute with Concentrated by ethanol precipitation and suspended in TE buffer (Maniatis, T., Frltsch, E. P., & Sambrookl, (1982). Mo1ecular Cloning: A Laboratory Ma nual (Cold Spring Harbor Laboratory, C8H, NY: l), -1-tuk translation processing 32p-labeling Plot hybridization of cDNA inserts on GeneScrcenPIus used for ization. For concentration measurement reading, LKB UltroSean The test was carried out using a laser densitometer (CLKB).

Construction of CX-1 cDNA λgtlo phage library Human colon cancer cell line By extracting CX-1 using guanidinium thione-1. NA was isolated. Poly(A)-RNA was conjugated with oligo(dT)-cellulose affix. selection using a nitty column.

(a) First strand synthesis, synthesis of first strand cDNA was performed using 50mM)Lis-)KCf7, pH 8, 3, 50rnM KCf), 6 a+M MgCf) 7.10 　mM　D　Tr, 0.5　mM　dN　TP, oligo(clT) 12-1 85D11g/ml, Poly(A)” RNA 1 (]111g RNA 100 a/ml, and chicken reverse transcriptase]00t) μ/ml (volume 20 The test was carried out at 42° CF for 1.5 hours at 0uρ). Labeled with radioactive nucleotides In order to minimize the frequent double knocking of the synthesized cDNA, the main reaction of first strand synthesis is Not radioactively labeled. The label is composed of the same components as above and a combination of the same components as above. Uptake containing 1071ci of co-dcTP (specific activity: 3000Ci/μmol) This was done in a separate tube only to monitor the reaction. This reaction was performed using SDS and EDTA at maximum concentration f), 49o and 10mM, respectively. It was terminated by adding so as to jFW product is f, ff-no and chloroform/'isoamyl alcohol (247'l), - Perform chromatography on a 5 ml column of 50 cephadex and import The rare triphosphate nucleotides were removed. Pool the cDNA fraction of the void volume. 1. Concentrate by ethanol precipitation; Incorporated into ethanol precipitated cDNA Based on the radioactivity obtained, the first strand c per g of added poly(A)'' RNA] Approximately 0.1 μ of DNA was produced.

(b) Second strand synthesis: Dissolve the cDNApeL knot from the first strand reaction in water, The second strand synthesis is performed using the first strand c DN A O,5-1,Ol! g, 20mM tri Su-HC41%, pH 7.5, 100nM)l KCf, 5mM MgCΩ 2.10 [11M DTT, 10mM (N)] 4) 2804.50j ! g/m, 9B SA (nuclease free), 150gMB-\AD)t /rnl RN aseH150μ/- E. coli DNA ligase, 40MMd NTP, 20μCiC”P] dCTP (specific activity: 3000Ci/μmol ), and a reaction containing 250μ/lIlρT4 DNA polymerase (volume 40 0 μg). Incubate overnight at 15°C, 0.49°SD Termination was performed using S and lOmMEDTA. Unincorporated triphosphate nuc Generation of double-stranded cDNA from leotide was performed using Sephadex G-50 as described above. Achieved by column chromatography. cjs from poly(A)” RNA The approximate yield of -cDNA was 1b-209o.

(C) fill-in) and methylation reaction: λgt1.0 vector Cloning of the full-length cDNA insert into the EcoRI site of In the case of a blunt-end (btu-endsd) EeoRI linker, to generate blunt-ended cDNA, and by methylation, internal Eco of cDNA is generated. Is there a need to protect R1 Zyte? The fill-in reaction is performed using 30n+M) Lis-acetate. Yu-to, pH 7,8, Go mM K-acetate, 10 mM MgCΩ2 , 0.2mM DTT, 0.1mg/ml BSA, 0.1. z/ ml RN asc A, 1-00μ/m1RNase H, 50MM B-NAD, 250Il/ml EcoRI DNA ligase, 30[11 Reaction containing 1λ, 1dNTP and 500μ/m 1T4 DNA polymerase (in vitro) The test was carried out at 37° C. for 30 minutes at a temperature of 80 μρ). Ethanol precipitation of cDNA Collected by 1, followed by 50IIM Tris KCf, ptl 8.0.5 mM EDTA, 5 mM DTT.

0.1mM Na-acetate, 50MMS-adenosylmethionine, and and 1000 μ/ml EcoRI methylase in a buffer containing Chilling was carried out at 37°C for 30 minutes. The second cDNA was recovered by ethanol precipitation. 1 followed by 50n+M) Squirrel KCf, po8. o, 5mM EDTA, 5 　mM　DTT, 0. ], ]mM\a-Acedate 50MMS-Adenosy Contains methionine and 1000 μ/’ml EcoRI methylase. Methylation was carried out in buffer at 37°C for 1 hour.

(d) Linking EcoR1 linker to cDNA: 9 parts EcoR to cDNA To generate the I site, the 5-phosphorylated EeoRI linker of the cDNA fraction Connect the blunt end of (0,5/'ml) to (50 m M!・Squirrel・) KCf, pH 7,6, 101TI101TI! 2.20mM In D T T ), i nMM ATP, T2ON A Q　O:L=・7・1・Continue overnight at 15°C.

(e) [ITJ: Ecol? ! Ren f3 Degradation of cDNA: The above ligation product was digested using tEcoRI DNAase. Degradation [7,c　D　NA external EcoRI site was exposed.

This decomposition reaction was carried out using standard EeoRI buffer (100mM l~Lis I [Ω, pt cDN in (7, 5, 100 nM Ja (I!, 5 mM MgCΩ2)) Ao, 50-100 units of EcoRI eledonuclease per 1-0,5 ■ store. Incubate at 37°C for 4-8 hours, then at 68°C for 15 hours. The EcoRI enzyme was inactivated by heating.

(f) EcoRI73-degraded cDNA by CL, -4B column chromatography. Cleaved DNA size fraction: Using CL-4B-Sepharose column, EcoR Purify I-degraded cDNA and select desired size for cDNA cloning did. 20 The purpose of column purification is to (i) effectively convert cDNA into vector DNA; Excess polymeric linker and/or degradable linker (usually 200 7, (ii) The second step is to fractionate cDNA by size before cloning into a specific vector. It is. Prepare CL-4B-200 sepharose resin in a 5-pipette and add it to the column. Buffer solution (101IIM Tris-KCf, pH 8, 0,600mM\aCΩ,] -mMEDTA.

and 0. Equilibration was carried out with I% Sarkosyl. column buffer Add EeoRI-decomposition c D NA test +4 to solution 0.3-, and add 100μ of 20 ρ fractions were collected. The radioactivity of each fraction was examined and the size was determined using 2.196 agarons. [-2] λ gtto vector with all phases (vessel 1c DNA live Let's build a rally for our 1st target, c D N greater than 5001) Select A, 2, and pool. Then, the recovered cDNA was concentrated by ethanol precipitation. and a small amount of 10mM Tris IIcΩ, pH 7.5/l IIIM E D Dissolved in TA buffer.

(g) Selection (also λgt of cDNA inner) ligation of 0 vector ＼: cDNA Cloning of the A library into the λgt, IO hector was performed using EcoRl-degradation. , Ujilam-purified and Zuizu-selected cDNA A insert was pre-cleaved with EC and oRI. and 5°-Yue phosphorylated λgtlO arm.

This ligation reaction (10ALi) was performed using vector DNA, 0.1-0.5t! g insa Standard ligation of host cDNA, lmM ATP, and 1-10 units of T4 ligase. contained in the application buffer. Incubate overnight at 15°C.

Screening of Phage cDNA Libraries 32 from Plasmid 8-2v Screening of CX-1λgtlO library with p-labeled cDNA inserts (Hanjatis, T., Frltsch, E.F., and Sa Mbrook, J. (1982) Hotecular Cloning: A Laboratory Manual (Co!d Spring) I arbor Laboratory, CSH5NY)) o approx. 10,00 Screening for 0 plaques was performed.

DNA sequencing M13 dideoxyoligonucleotide chain termination method (San ger, F., N1cklen, S., and Coullson, A. R.

(1977) Proc, Nat, Acad, Sci, 74.5483-54 87) was used.

M13 universal primer or specially synthesized 17- Using base oligonucleotides, both the 8-2v and '-9 inserts were sequentially Arranged.

#1”CTGATGTCAGTGTTATA”’#22”TGGCCA GTATTCCTGGA23' (complementary strand) #3647CTCCT; AGAAGGCA663#469' GAGTCCATTCACCCTGA68 ’ (Complementary strand) M13 is Ne Kei Engla Biolab) and Sequenase Sea Fencing Kit (Sequenase sequencing kit) is United 0 Ste. Biochemical (Llnjted 5tates Bjohemjca 1). Synthetic oligonucleotides were prepared by E. Reinhertz (E. Re1nherz) Research Institute (Danner 77-Barr Cancer Research Institute) Courtesy of P, H, 5ayer.

Computer-assisted sequence analysis is available at Molecular Biology Computer Research Resources/ Nonah-Farber Cancer Institute/X-Bird School of Public Health (Harvard 5c) It was carried out in the hall of 'Public Health).

8-2V B-4W +-4E 1234567891oll 121314151617181920FIG, 2 eJJ h:5 t-1CCJc? llI (Jc ua <w <yQ = Evacuation b3 22 Shiba = 6 foot soldiers 8IJ-1 (J> 1-II -I Cl cheer (ri, -1 (JIJ CJO riff (≧[-IlaoP4<> u-+ hm LIJ: (Jk-?Φ Q-C: l> aa hh a< a> Beto Q mountain (J-Q mouth ha,?> CJc l-1-1hf6 <飄 0α Q-〇Post West 286 shi = Kure ε 8; j　( )　CJ＞　? al　(joI　EsQ　ll-11-+　E-1Φ　OCmu3 No. 38 $ Chi = Yodo ni 8th foot ε se = Koushiba FIG, 6 FIG.7 international search report PCT/US 89103595 international search report US 8903595 SA　31014

Claims (10)

[Claims] 1. A full-length human tumor derived from human colon cancer cells containing substantially the sequence shown in FIG. Laminin binding protein cDNA sequence. 2. In FIG. 3, starting at nucleotide number 162 and starting at nucleotide number 82 of the cDNA sequence according to claim 1 which substantially corresponds to the sequence ending in 9. Subarray. 3. In Figure 3, starting with nucleotide number 1 and ending with nucleotide number 43. A subsequence of a cDNA sequence according to claim 1 which substantially corresponds to a sequence according to claim 1. 4. Recombinant clone named J-9 with ATCC accession number 40489. 5. Recombinant clone named 8-2V with ATCC accession number 40490 . 6. encoded by the sequence according to claim 1, 2 or 3. laminin-binding protein. 7. substantially encoding the laminin binding protein according to claim 6. Purified DNA sequence. 8. A method of detecting the presence of cancer cells in a patient comprising: a. Claim Paragraph 1 , provides a labeled cDNA probe having the cDNA sequence described in Section 2 or Section 3. supply, b. exposing the probe to a tissue or body fluid sample from a patient; and c. comprising monitoring the reaction of step b for hybridization. How to do it. 9. 9. The method according to claim 8, wherein the cancer cells are colon cancer cells. 10. A reagent for detecting the presence of cancer cells in a patient, comprising: Labeled cDNA probe having the cDNA sequence according to item 1, item 2 or item 3 Reagents containing.