Patent Application: US-58898596-A

Abstract:
the present invention provides a cdna of dock180 gene , which has a nucleotide sequence of seq id no : 1 and of which translation product binds to the proto - oncogene product crk , a recombinant vector containing this cdna , a dock180 protein expressed from the edna which has a amino acid sequence of seq id no : 2 , and an antibody to the dock180 protein . according to the present invention , it is possible to develop new diagnosing and therapeutic techniques using dock180 protein and antibody against the protein .

Description:
the cdna of the present invention can be isolated and purified from higher eukaryotes such as cells derived from human , mouse , and chicken by the application of , for example , the method of sambrook et al . ( molecular cloning , second edition , cold spring harbor laboratory , new york , 1989 ). more specifically , the cdna can be obtained by purifying mrna of dock180 protein gene from animal cell , and then synthesizing a cdna chain from the mrna by the use of reverse transcriptase . from among the thus synthesizable cdnas of dock180 protein , the nucleotide sequence of cdna derived from human cells and the amino acid sequence of the translation domain thereof are defined in sequence listing by seq id nos : 1 and 2 . then , the recombinant vector of the present invention can be prepared by inserting a fragment of the resultant cdna into known cloning vectors . ligation of the cdna fragments and the vectors may be accomplished by , for example , the above - mentioned method of sambrook et al . preferable vectors include a plasmid and a λ - phage for escherichia coli as the host , and an applicable plasmid is , for example , one derived from pbr322 . a preferable λ - phase is λ gt11 . transfer of the thus prepared recombinant vectors into the host may be conducted through infection by λ - phage as presented in the above - mentioned paper by sambrook et al . for example , a transformed cells with the recombinant vector containing the above - mentioned cdna are available by inoculating the recombinant λ gt11 into escherichia coli y1090 at 37 ° c . selection of a transformed cell may be accomplished by using a known method , depending upon the kind of the cloning vector . for example , selection of a transformed cell with λ gt11 can be performed as follows . the above - mentioned recombinant λ gt11 containing the cdna can be inoculated into escherichia coli y1090 under a temperature condition of 37 ° c ., and the e . coli cells are cultured on an ager plate containing trypton , yeast extract , nacl and ampicillin ( hereinafter abbreviated as an &# 34 ; agar medium &# 34 ;). then , a nitrocellulose membrane containing isopropyl thio - d - galactoside ( hereinafter abbreviated as &# 34 ; iptg &# 34 ;) is placed on the plate for another several hours to induce transcription from the integrated cdna . after the binding of an enzyme - labelled crk protein to the membrane ( matsuda et al ., mol . cell . biol ., 12 : 3482 - 3489 , 1992 ), an lamda phase plaques having the recombinant vector can be selected by putting a substrate for the enzyme in it . a preferable labelling enzyme is alkaline phosphatase or peroxidase . crk protein may be manufactured as a fusion protein with glutathione s - transferese ( hereinafter abbreviated as &# 34 ; gst &# 34 ;), and selection may be accomplished by using an antibody against this gst . the cdna fragment inserted into λ phage can be cleaved out and re - inserted into a plasmid vector , if necessary . an example or recombinant plasmid vector of the invention is pdock180 which is prepared by inserting the cdna of seq id no : 1 into vector plasmid pblue scriptiiks (+), for which details are presented in example 2 later . the pdock180 was transfected into the xl1 - blue strain derived from escherichia coli k12 strain , and the transformant e . coli dock180 having the pdock180 was deposited to national institute of biocience and human technology , an international depository , under budapest treaty as a deposit no . ferm bp - 5362 on jan . 12 , 1996 . then , the dock180 protein of the present invention can be exressed from the cdna of seq id no : 1 . for production of the protein , an expression vector is prepared by inserting the cdna fragment into a known expression vector . the cdna fragment can be isolated from the above - mentioned cloning vector , pdock180 . while there is no particular limitation as to the expression vector , a preferable one is pgex1 , pg5x2t or pgex3x using e . coli as the host . the expression vector containing the cdna fragment can be introduced into an escherichia coli ( for example , dh5 strain derived from escherichia coli k12 strain ) by a known method . the dock180 protein of the present invention is easily produced in a large scale by culturing the transformed cells . more specifically , a concrete example comprises the stops of culturing the transformed escherichia coli at 37 ° c . for 3 to 24 hours on l - broth containing ampicillin , bacteriolyzing through ultrasonic blending , and adsorbing this sample to a carrier such as glutathione sephalose ( made by pharmacia p - l biochemicals company ), thereby isolating and purifying the target dock180 protein . an antibody against dock180 protein is available by inoculating the thus purified dock180 protein to an animal by a conventional method . applicable animals include rabbit , mouse , goat , sheep , horse and hamster , and among others , rabbit or mouse is preferable . the thus obtained anti - dock180 protein antibody can be used , for example , for quantitative determination or separation of dock180 protein in a sample , and further , serves as a useful material for missile therapy using an antitumor agent with dock180 as a target . the cdna , the protein and the antibody of the present invention provide various genetic manipulation materials useful for development of a new cancer therapy . these materials include an antisense rna of dock180 gene , a variant protein of dock180 , and a virus vector which expresses these rna , variant protein , and anti - dock180 protein antibody in tumor cells . the present invention will be described below in further detail by means of examples . it is needless to mention that the present invention is not limited in any manner by the following examples . a cdna fragment to human dock180 protein gene was isolated and cloned as follows . an mrna of dock180 gene was isolated from human spleen and a cdna fragment was synthesized from the mrna . the cdna fragment was then integrated into λ gt11 , and a recombinant vector thereof was infected to escherichia coli y1090 , which was plated onto la agar culture medium . after six hours , a nitrocellulose membrane containing 1 mm iptg was placed on this medium , and after three hours of culture , this nitrocellulose membrane was incubated for an hour with a phosphate buffer solution ( ph : 7 . 5 ) containing 2 % skim milk and 0 . 05 % tween 20 . then , after reaction for an hour with a phosphate buffer solution containing 1 μg / ml gst - crk and 1 μg / ml anti - gst monoclonal antibody , and for an hour with 1 μg / ml alkaline phosphate - labelled anti - mouse antibody ( made by tago company ), the phage having an ability to bind to crk protein was identified by means of ap purple ( made by bio iol company ), a substrate for alkaline phosphatase . this phage was purified through three runs of plaque formation , and then dna thereof was isolated by the phenol extraction method , and cleaved with a restriction enzyme ecori . next , part of cdna of dock180 gene was prepared through electrophoresis . this cdna fragment was isotope - labelled with random oligo primer ( made by behlinger company ) and 32p - deoxycytidine triphosphate . with the use of this labelled cdna , the above - mentioned recombinant λ gt11 with cdna derived from a human spleen was screened through plaque hybridization by the method of sambrook et al . ( molecular cloning , second edition , cold spring harbor laboratory , new york , 1989 ), and further six clones of recombinant λ gt11 having dock180 protein cdna were obtained . dnas of these phages were cleaced with restriction enzyme ecori to isolate cdna of dock180 gene and subcloned into phagemid vector puc119 . a single stranded dna was purified from the thus obtained recombinant vector , and the nucleotide sequence thereof was determined by the use of an automatic nucleotide sequence reader ( made by abi company ). the identified nucleotide sequence thereof is shown in sequence listing by seq id no ; 1 , and the amino acid sequence of the anticipated translation product , by seq id no : 2 . as a result of retrieval of this amino acid sequence in the database in the genbank of the european molecular biology laboratories ( embl ), the amino terminal end of dock180 protein shares homology with fyn and yes of tyrosine kinase by more than 20 %. this domain has a structure known as sh3 , and is present in various protein groups involved in signal transduction of cell proliferation in addition to that of tyrosine kinase . however , dock180 protein was found to be different from any known proteins and to be a new signal transduction factor . from the group of recombinant puc119 obtained in example 1 , the dna fragments excluding overlapping portions were isolated and ligated each other to prepare a fragment containing all the translation domain of the cdna . the resultant cdna fragment was then inserted into the plasmid vector pblue script ii ks (+) to obtain the recombinant vector pdock180 . this pdock180 has a constitution as shown in fig1 . further , this cloning vector pdock180 was introduced into an xl1 - blue strain derived from escherichia coli k12 to obtain a transformant e . coli dock180 ( ferm bp - 5362 ). the cloning vector pdock180 obtained by example 2 was cleaved with a restriction enzyme to prepare a cdna domain of dock180 protein . the resultant cdna fragment was then inserted into plasmid pgex1 , thereby preparing expression vectors . transformant cells were prepared by introducing the expression vectors into escherichia coli dh5 . after culturing this transformant cells in 1 l l - broth containing ampicillin up to an absorbance of 0 . 6 , iptg was added to 0 . 5 mm , and culturing was continued for another three hours . after collection , the bacteria were ultrasonic - treated to remove crushed pieces of bacteria , and the supernatant was mixed with glutathione sephalose ( made by pharmacia p - l biochemicals company ). after rinsing glutathione sephalose with a phosphate buffer solution , dock180 protein was eluded with the use of a phosphate buffer solution containing 5 mm glutathione . this protein was dialyzed with a phosphate buffer solution , and then a portion thereof was analyzed with sds - polyacrylamide gel : a gst fused - dock180 protein having a purity of over 90 % was synthesized . the dock180 protein purified in example 3 was subcutaneously inoculated three times to a rabbit , together with complete freund &# 39 ; s adjuvant , and then , serum was sampled . reactivity of this serum with the purified dock180 protein was investigated by the western blotting technique : a clear reactivity was demonstrated with the dock180 protein even when diluted to about 1 , 000 . this permitted confirmation of applicability thereof as an antibody against dock180 protein . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 2 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 6519 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna to mrna ( iii ) hypothetical :( iv ) anti - sense :( v ) fragment type :( vi ) original source : spleen cell of homo sapiens ( ix ) feature :( a ) name / key : cds ( b ) location : 24 .. 5619 ( xi ) sequence description : seq id no : 1 : gcacgagcggctccggcggcgccatgacgcgctgggtgcccaccaagcgcgaggagaagt60acggcgtggctttttataactatgatgccagaggagcggatgaactttctttacagatcg120gagacactgtgcacatcttagaaacatatgaagggtggtaccgaggttacacgttacgaa180aaaagtctaagaagggtatatttcctgcttcatatattcatcttaaagaagcgatagttg240aaggaaaagggcaacatgaaacagtcatcccgggtgacctccccctcatccaggaagtca300ccacgacactccgagagtggtccaccatctggaggcagctctacgtgcaagataacaggg360agatgtttcgaagtgtgcggcacatgatctatgaccttattgaatggcgatcacaaattc420tttctggaactctgcctcaggatgaactcaaagaactgaagaagaaggtcacagccaaaa480ttgattatggaaacagaattctagatttggacctggtggttagagatgaagatgggaata540ttttggatccagaattaactagcacgattagtctcttcagagctcatgaaatagcttcta600aacaagtggaggaaaggttacaagaggaaaaatctcaaaagcagaacatagatattaaca660gacaagccaagtttgctgcaaccccttctctggccttgtttgtgaacctcaaaaatgtgg720tttgtaaaataggagaagatgctgaagtcctcatgtctctatatgaccctgtggagtcca780aattcatcagtgagaactacctggttcgctggtccagttcaggattacctaaagacatag840acagattacataatttgcgagccgtgtttactgacctcggaagcaaagacctgaaaaggg900agaaaatcagttttgtctgtcagattgttcgcgtgggtcgcatggagctgagggacaaca960acaccaggaaactgacctcggggttgcggcgaccttttggagtggctgtgatggatgtaa1020cagatataataaatggaaaagtagatgatgaagataagcagcatttcattccctttcagc1080cggtggcaggggagaatgacttccttcagactgttataaacaaagtcatcgctgccaaag1140aagtcaaccacaaggggcagggtttgtgggtaacattgaaattacttcctggagatatcc1200atcagatccgaaaagagtttccgcatttagtggacaggaccacagctgtggctcgaaaaa1260cagggtttccggagataatcatgcctggtgatgttcgaaatgatatctatgtaacattag1320ttcaaggagattttgataaaggaagcaaaacaacagcgaagaacgtggaggtcacggtgt1380ctgtgtacgatgaggatgggaaacgattagagcatgtgattttcccgggtgctggtgatg1440aagcgatttcagagtacaaatctgtgatttactaccaagtaaagcagccacgctggtttg1500agactgttaaggtggccattcccatcgaggacgttaaccgcagtcaccttcggtttacct1560tccgccacaggtcatcacaggactctaaggataaatctgagaaaatatttgcactagcat1620ttgtcaagctgatgagatacgatggtaccaccctgcgagacggagagcacgatcttatcg1680tctataaggccgaagcgaagaagctggaagatgctgccacgtacttgagtctgccctcca1740cgaaggcagagttggaagaaaagggccactcggccaccggcaagagcatgcagagccttg1800ggagctgcaccattagcaaggactccttccagatctccacgctcgtgtgctccaccaaac1860tgactcagaacgtggaccttctggggctcttgaaatggcgctccaacaccagcctgctgc1920agcagaacttgaggcagctgatgaaagtcgatggtggtgaagtagtgaagtttcttcagg1980acacgttggatgccctcttcaacatcatgatggagaactcagagagtgagacttttgaca2040cgttagtctttgatgctctggtatttatcattggactgattgctgatagaaaatttcagc2100attttaatcctgttttggaaacttacattaagaaacactttagtgcaacgttagcctaca2160cgaagttgacaaaagtgttgaagaactacgtggacggtgctgagaagccgggagtaaatg2220agcagctgtacaaagccatgaaagcgctagaatccatcttcaagttcatcgtgcgctcca2280ggatcctgttcaatcaactgtatgaaaacaagggagaggctgacttcgtggaatctttgc2340tgcagctcttcaggtccatcaatgacatgatgagcagcatgtcagaccagaccgtccggg2400tgaagggggcagcactgaaatacttaccaacgatcgtcaacgatgtgaaattggtgtttg2460atcccaaagagctcagcaaaatgtttactgaattcatcctcaatgttcccatgggcttgc2520tgaccatccagaaactctactgcttgatcgaaatcgtccacagtgacctcttcacacagc2580atgactgcagagagatcctgcttcccatgatgaccgatcagctcaagtaccatctggaga2640gacaggaggacctggaggcctgctgtcagctgctcagccacatcctggaggtgctgtaca2700ggaaggacgtggggccaacccagaggcacgtccagattatcatggagaaacttctccgga2760ccgtgaaccgaaccgtcatttccatgggacgagattctgaactcattggaaacttcgtgg2820cttgcatgacagctattttacgacaaatggaagattaccattatgcccacttgatcaaga2880cttttgggaaaatgaggactgatgtggtagatttcctaatggaaacattcatcatgttta2940agaacctcattggaaagaacgtttaccccttcgactgggtgatcatgaacatggtgcaaa3000ataaagtcttcctgcgagcaattaatcagtatgcagatatgctgaacaaaaaatttctgg3060atcaagccaactttgagctacagctgtggaacaactactttcacctggctgttgctttcc3120ttactcaagagtccctgcaactggagaatttttcaagtgccaagagagccaaaatcctta3180acaagtacggagatatgaggagacagattggctttgaaatcagagacatgtggtacaacc3240ttggtcaacacaagataaagttcattccagaaatggtgggcccaatattagaaatgacat3300taattcccgagacggagctgcgcaaagccaccatccccatcttctttgatatgatgcagt3360gtgaattccattcgacccgaagcttccaaatgtttgaaaatgagatcatcaccaagctgg3420atcatgaagtcgaaggaggcagaggagacgaacagtacaaagtgttatttgataaaatcc3480ttctggaacactgcaggaagcacaaatacctcgccaaaacaggagaaacttttgtaaaac3540tcgttgtgcgcttaatggaaaggcttttggattatagaaccatcatgcacgacgagaaca3600aagaaaaccgcatgagctgcaccgtcaatgtgctgaatttctacaaagaaattgaaagag3660aagaaatgtatataaggtatttgtacaagctctgtgacctgcacaaggagtgtgataact3720acaccgaagcggcttacaccttgcttctccatgcaaagcttcttaagtggtcggaggatg3780tgtgtgtggcccacctcacccagcgggacgggtaccaggccaccacgcagggacagctga3840aggagcagctctaccaggaaatcatccactacttcgacaaaggcaagatgtgggaggagg3900ccattgccttgggcaaggagctagccgagcagtatgagaacgaaatgtttgattatgagc3960aactcagcgaattgctgaaaaaacaggctcagttttatgaaaacatcgtcaaagtgatca4020ggcccaagcctgactattttgctgttggctactacggacaagggttccccacattcctgc4080ggggaaaagttttcatttaccgagggaaagagtatgagccccgggaagattttgaggctc4140ggctcttaactcagtttccaaacgccgagaaaatgaagacaacatctccaccaggcgacg4200atattaaaaactctcctggccagtatattcagtgcttcacagtgaagcccaaactcgatc4260tgcctcctaagtttcacaggccagtgtcagagcagattgtaagtttttacagggtgaacg4320aggtccagcgatttgaatattctcggccaatccggaagggagagaaaaacccagacaatg4380aatttgcgaatatgtggatcgagagaaccatatatacaactgcatataaattacctggaa4440ttttaaggtggtttgaggtcaagtctgttttcatggtggaaatcagccccctggagaatg4500ccatcgagaccatgcagctgacgaacgacaagatcaacagcatggtgcagcagcacctgg4560atgaccccagcctgcccatcaacccgctctccatgctcctgaacggcatcgtggacccag4620ctgtcatggggggcttcgcaaactacgaaaaggccttctttacagaccggtacctgcagg4680agcaccctgaggcccatgaaaagatcgagaagctcaaggacctgattgcttggcagattc4740cttttctggccgaagggatcagaatccatggagacaaagtcacggaggcactgaggccgt4800tccacgagaggatggaggcctgtttcaaacagctgaaggaaaaggtggagaaagagtacg4860gcgtccgaatcatgccctcaagtctggatgatagaagaggcagccgcccccggtccatgg4920tgcggtccttcacgatgccttcctcatcccgccctctgtctgtggcctctgtctcttccc4980tctcatcggacagcaccccctccagaccaggctccgacgggtttgccctggagcctctcc5040tgccaaagaaaatgcactccaggtcccaggacaagctggacaaggatgacctggagaagg5100agaagaaggacaagaagaaggaaaaaaggaacagcaaacatcaagagatatttgagaaag5160aatttaaacccaccgacatttccctgcagcagtctgaggctgtgatcctttcggaaacga5220taagtcccctgcggccccagagaccgaagagccaggtgatgaacgtcattggaagcgaaa5280ggcgcttctcggtgtccccctcgtcaccgtcctcccagcaaacaccccctccagttacac5340caagagccaagctcagcttcagcatgcagtcgagcttggagctgaacggcatgacggggg5400cggacgtggccgatgtcccaccccctctgcctctcaaaggcagcgtggcagattacggga5460atttgatggaaaaccaggacttgctgggctcgccaacacctccacctccccctccacacc5520agaggcatctgccacctccactgcccagcaaaactccgcctcctccccctccaaagacaa5580ctcgcaagcagacatcggtggactctgggatcgtgcagtgacatcgcaaggctctctgga5640aagagtgtgctgcccctccccatctccatgccctctccttctgtgtcccctgagtctgct5700gtttacctcattgggcctgtgatgttaacatttcgtgcgactgctttttcttcaaaggag5760ttcagttctcaccatggagtgagtggcctttagcgtcatggagcaaggtgggtctgggag5820gtagatatgggtccgggatgtgccatcgtagttaccagagttgggggcctctgagtgtgt5880ctggctctgagagagtctgagtcttgcccaaacattctttctttttgtgccaaatgactt5940gcatttgcaaagagctcaattgctctgagctcagccaagtaggagaggctaggccatcac6000tcttgggaagctgtgtagtgatgatgtataagaatcctcctcactgtcatgggatgttgt6060atccagcccctccttgttccagccggtggtgtgacttcgttggttgaggtgtgtctccaa6120cctacatcagaccatgaagttcaacccctccagggaagctcctgatttcccctgcataat6180tgaaaataggatattctcagctattgaacagttactaatttatggggtggaaacagcatt6240aagaatactgaatcaaatggaaaaacaaatgaatacaggaagataagtgttcgttctttt6300ctgaaaaaagagtatgtgtaccacaagagctggttttaattgggtgaattgtttttgtcc6360tcattctgtacagaaatttgtatatatgatggttcttagaacttgttttaatttttgtgg6420tccttctgtttattataataggcgtccaccaatgattatccatatgtgttcttaattttt6480aactgctggaagtgttaaaacacacacaccccggaattc6519 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 1865 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : protein ( iii ) hypothetical :( iv ) anti - sense :( v ) fragment type :( vi ) original source : spleen cell of homo sapiens ( xi ) sequence description : seq id no : 2 : 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