DE69635501T2 - cDNA of the DOCK180 gene and DOCK180 protein - Google Patents

Description

Territory of invention

The The present invention relates to a cDNA of the DOCK180 protein which to the proto-oncogene product CRK binds a recombinant vector containing this cDNA DOCK 180 protein expressed from the cDNA and an antibody against the DOCK180 protein. The cDNA of the present invention, the DOCK180 protein and the antibody are very useful for the diagnosis of tumor cells and for the development of a cancerocide Method of suppression of tumor cells.

State of technology

Of the youngest Advancement of cell biology and molecular biology is indeed remarkable. Size Efforts are being made with regard to carcinogenesis at the genetic level, and through such efforts many oncogenes and anti-oncogenes have become identified in humans. It is also known that of these Genes different proteins are expressed.

Z. B. is the CRK protein expressed by the proto-oncogene CRK is one of the key proteins affecting the proliferation of higher eukaryotic cells Control, and it is known to be in many malignant tumors regulates the proliferation of tumor cells. See for example Knudsen et al., J. Biol. Chem., 269: 32781-32787, 1994 and Feller et al., EMBO J., 13: 2341-2351, 1994, relating to proteins attached to the N-terminal SH3 domain from CRK tie.

It It has now been discovered that this CRK protein has its function exerts by binding to the DOCK180 protein. Therefore DOCK180 pulls as diagnostic indicator of tumor cells and as a target for cell-directed Therapy using various anti-tumor agents general attention on yourself.

however It was because there was no information about a gene encoding the DOCK180 protein was available, yet impossible Protein on a larger scale for the diagnosis of cancerous diseases, for the education of Carcinogenesis mechanisms or to develop a new cancer therapy to use.

Summary the invention

A The object of the present invention is to provide a cDNA of the DOCK180 gene as well as genetically engineered material that facilitates the easy manipulation of this cDNA and the expression of this protein on a large scale allowed to provide.

A Another object of the present invention is to provide a DOCK180 protein, which is expressed by said cDNA, and antibodies against to provide this protein. Subject of the present invention is a cDNA of the DOCK180 gene corresponding to those described in the Sequence Listing SEQ ID NO: 1 has defined sequence.

One Another object of the present invention is a recombinant Vector containing the cDNA of the DOCK180 gene. More precisely, the present invention the plasmid pDOCK180, in E. coli DOCK180 (FERM BP-5362), ready.

One Another object of the present invention is a DOCK180 protein with the sequence defined in the Sequence Listing as SEQ ID NO: 2 and prepared using the DOCK180 protein as an antigen antibody against DOCK180.

Summary the drawing

1 Figure 3 is a constitutional diagram of pDOCK180, which is a cloning vector of the invention.

detailed Description of the invention

The cDNA of the invention can be obtained by using z. The method of Sambrook et al. (Molecular Cloning, second edition, Cold Spring Harbor Laboratory, New York, 1989) from higher eukaryotes, e.g. B. from humans, mice and chickens cells isolated and purified. More specifically, the cDNA can be obtained by purifying the mRNA of the DOCK180 protein gene from an animal cell and then preparing a cDNA chain from the mRNA using reverse transcriptase. Among the cDNAs of the DOCK180 protein so prepared, the nucleotide sequence of the cDNA obtained from human cells and the amino acid sequence of its translation domain in the sequence listing are defined as SEQ ID NO: 1 and 2.

Of the Recombinant according to the invention Vector can then be created by adding a fragment of the resulting cDNA inserted into known cloning vectors. The Ligation of the cDNA fragment and the vectors may e.g. B. by the above-mentioned method of Sambrook et al. respectively. To preferred Vectors belong a plasmid and a λ phage for Escherichia coli as a host and a useful plasmid is e.g. One of pBR322 derived. A preferred λ phage λ is λgt11.

Of the Transfer of recombinant vectors prepared in this way into the host cells can be made by infection with λ phage, as in the above-mentioned publication by Sambrook et al. shown. For example, can a transformed cell with the recombinant vector containing the contains the above mentioned cDNA, can be obtained by recombinant λ gt11 at 37 ° C with Escherichia coli Y1090 incubated.

The Selection of a transformed cell may be through appropriate use of a known method, depending on the type of cloning vector. For example, the selection of a cell transformed with λ gt11 as follows respectively. The above mentioned recombinant λ gt11, which contains the cDNA can at a temperature of 37 ° C in Escherichia coli Y1090, and E. coli cells are cultured on an agar plate containing tryptone, yeast extract, Contains sodium chloride and ampicillin (hereafter abbreviated as "agar medium") Nitrocellulose membrane containing isopropylthio-D-galactoside (hereinafter abbreviated to "IPTG") several for another hour or so the plate spread to the transcription of the integrated trigger cDNA. After binding of an enzyme-labeled CRK protein to the membrane (Matsuda et al., Mol. Cell. Biol., 12: 3482-3489, 1992), λ-phage plaques, containing the recombinant vector can be selected by adding a substrate for the Enzyme admits. A preferred labeling enzyme is alkaline phosphatase or peroxidase. The CRK protein can be used as a fusion protein with glutathione-S-transferase (hereafter abbreviated as "GST"), and selection can be achieved by using an antibody against used this GST.

The in the λ phage pasted cDNA fragment can if necessary cut out and reinserted into a plasmid vector. An example for a recombinant according to the invention Plasmid vector represents pDOCK180, which is manufactured by the cDNA of SEQ ID NO: 1 into the vector plasmid pBluescript II KS (+) inserts, for what later in example 2 the details are presented. The pDOCK180 was in the of Transfected strain Escherichia coli K12 derived strain XL1-Blue, and the transformant contained in pDOCK180, E. coli DOCK180 at the National Institute of Bioscience and Human Technology, a international depository, as deposit No. FERM BP-5362 deposited under the Budapest Treaty.

The DOCK180 protein of the invention can then be expressed from the cDNA of SEQ ID NO: 1. For extraction the protein is an expression vector prepared by the Insert cDNA fragment into a known expression vector. The cDNA fragment can be obtained from the abovementioned cloning vector pDOCK180 be isolated. While there is no specific limitation with regard to the expression vector are pGEX1, pGEX2T or pGEX3X using E. coli as the host is preferred. The that cDNA expression fragment-containing expression vector can by a known Method in an Escherichia coli strain (for example, that of Escherichia coli K12-derived strain DH5). The DOCK180 protein of the invention is easily transformed by culturing the transformed cells produced on a large scale. More specifically, a concrete example includes the following steps: 3 to 24 hour culture of transformed Escherichia coli at 37 ° C on ampicillin-containing L-broth, bacteriolysis by sonication and absorbing the mixture to a carrier such as glutathione-Sepharose (manufactured by Pharmazia P-L Biochemicals Company), whereby the DOCK180 target protein isolated and purified.

One antibody against the DOCK180 protein is available by following one usual Method of vaccinating an animal with the thus purified DOCK180 protein. To be used animals Rabbits, mice, Goats, sheep, horses and hamsters, and among others are rabbits or mice prefers.

The thus-obtained anti-DOCK180 protein antibody may e.g. As used for the quantitative determination or separation of the DOCK180 protein in a sample, moreover, it serves as a useful Ma targeted therapy using an antitumour agent with DOCK180 as the target.

The cDNA, the protein and the antibody according to the present Invention provide various for the development of a new cancer therapy useful Materials for genetic manipulation ready. These materials include an antisense RNA of the DOCK180 gene, a protein variant from DOCK180 and a viral vector containing this RNA, protein variant and the anti-DOCK180 protein antibody expressed in tumor cells.

The The present invention will be detailed below by way of examples described. It does not need to be pointed out that the present Invention in no way limited by the following examples becomes.

example 1

One cDNA fragment corresponding to human DOCK180 protein gene, became like this isolated and cloned.

A mRNA of the DOCK180 gene was isolated from human spleen and a cDNA fragment was prepared from the mRNA. The cDNA fragment was then integrated into λgt11, and E. coli Y1090 was infected with the recombined vector and plated on LA agar culture medium. After six hours, a nitrocellulose membrane containing 1 mM IPTG was spread on this medium, and after three hours of culture, this nitrocellulose membrane was incubated for one hour with a phosphate buffer (pH 7.5) containing 2% skimmed milk and 0.05% Tween 20. incubated. After one hour of reaction with a phosphate buffer solution containing 1 μg / ml GST-CRK and 1 μg / ml of a monoclonal antibody to GST, and incubation with 1 μg / ml of an alkaline phosphatase-labeled anti-mouse antibody (produced from TAGO Company), phages capable of binding to CRK protein were identified by AP PURPLE (manufactured by BioMol Company), a substrate for alkaline phosphatase. This phage was purified by three cycles of plaque formation and its DNA was isolated by the phenol extraction method and cut with the restriction enzyme EcoRI. In the next step, part of the DOCK180 gene cDNA was recovered by electrophoresis. This cDNA fragment was radioactively labeled with a random oligomer as a primer (made by Böhringer Company) and ³² P-deoxycytidine triphosphate. Using this labeled cDNA, the recombinant λ gt11 recombinant human spleen cDNA was amplified by plaque hybridization according to the method of Sambrook et al. (Molecular Cloning, second edition, Cold Spring Harbor Laboratory, New York, 1989) and six other clones of recombinant λ gt11 containing cDNA for DOCK180 were obtained. The DNA of these phages was cut with the restriction enzyme EcoRI to isolate the cDNA of the DOCK180 gene and recloned into the phagmid vector pUC119. From the recombinant vector thus obtained, a single-stranded DNA was purified, and its nucleotide sequence was determined using an automatic nucleotide sequence reader (manufactured by ABI Company). The identified nucleotide sequence is shown in the Sequence Listing as SEQ ID NO: 1, and the amino acid sequence of the predicted translation product as SEQ ID NO: 2. As a result of the search of this amino acid sequence in the database of the Genbank of the European Molecular Biology Laboratory (EMBL), it was found that the amino-terminal end of the DOCK180 protein is more than 20% similar to the tyrosine kinases Fyn and Yes. This domain has a structure known as SH3, and besides tyrosine kinase it is present in various protein structures involved in signal transduction in cell proliferation. However, it has been found that the DOCK180 protein is different from all known proteins and represents a novel signal transduction factor.

Example 2

From the group of recombinant pUC119 vectors obtained in Example 1, DNA fragments excluding overlapping portions were isolated and ligated together to prepare a fragment containing the entire translated portion of the cDNA. The resulting cDNA fragment was then inserted into the plasmid vector pBlue Script II KS (+) to produce the recombinant vector pDOCK180. This pDOCK180 has a constitution like in 1 shown.

Then the cloning vector pDOCK180 was transformed into one derived from E. coli K12 Trunk XL1-Blue introduced, to obtain the transformant E. coli DOCK180 (FERM BP-5362).

Example 3

Of the obtained in Example 2 cloning vector pDOCK180 was with a Restriction enzyme cut to recover a cDNA piece for the DOCK180 protein. The resulting cDNA fragment was then inserted into the plasmid pGEXI inserted, whereby an expression vector was prepared. Transformants were prepared by introducing the expression vector into E. coli DH5. After culture of these transformed cells in 1 l ampicillin-containing L broth up to At an optical density of 0.6, IPTG was at a concentration of 0.5 mM, and the culture was continued for a further three hours. After harvesting, the bacteria were sonicated, around crushed pieces of bacteria to remove, and the supernatant was glutathione-Sepharose (from Pharmacia P-L Biochemicals Company made) mixed. After rinsing the glutathione-Sepharose with a phosphate buffer solution was the DOCK180 protein using a 5 mM glutathione-containing Phosphate Buffer Solution eluted. This protein was dialyzed against a phosphate buffer solution, and a part of it was then analyzed on an SDS-polyacrylamide gel: a GST-fused DOCK180 protein with over 90% purity was produced.

Example 4

With the DOCK180 protein purified in Example 3, along with complete Freund's adjuvant a rabbit was inoculated subcutaneously three times, and then serum samples taken.

The reactivity of this serum with the purified DOCK180 protein was examined by the Western blotting technique: a marked reactivity with the DOCK180 protein was demonstrated, even at dilutions of about 1,000. This allowed confirmation of its utility as an antibody to the DOCK180 protein. sequence Listing

Claims (5)

cDNA of a DOCK180 gene, the one in the Sequence Listing having SEQ ID NO: 1 defined nucleotide sequence and its Translational product binds to the proto-oncogene CRK. Recombinant vector containing the cDNA according to claim 1 contains. Plasmid pDOCK180, carried by E. coli DOCK180 (FERM BP-5362). DOCK180 protein, the one in the sequence listing SEQ ID NO: 2 has defined amino acid sequence. antibody against a DOCK180 protein according to claim 4, prepared using of the DOCK180 protein according to claim 4 as antigen.