Patent Application: US-17349793-A

Abstract:
dna encoding a novel human β 2 integrin α subunit polypeptide , designated α d , is disclosed along with methods and materials for production of the same by recombinant procedures . binding molecules specific for α d are also disclosed as useful for modulating the biological activities of α d .

Description:
the present invention is illustrated by the following examples relating to the isolation of a cdna clone encoding α d from a human spleen cdna library . more particularly , example 1 illustrates the use of anti - canine α tm1 antibody in an attempt to detect a homologous human protein . example 2 details purification of canine α tm1 and n - terminal sequencing of the polypeptide to design oligonucleotide primers for pcr amplification of the canine α tm1 gene . example 3 addresses large scale purification of canine α tm1 for internal sequencing in order to design additional pcr primers . example 4 describes use of the pcr and internal sequence primers to amplify a fragment of the canine α tm1 gene . example 5 addresses cloning of the human α d - encoding cdna sequence . example 6 describes northern blot hybridization analysis of human tissue and cells for expression of α d mrna . example 7 details the construction of human α d expression plasmids and transfection of cos cells with the resulting plasmids . example 8 addresses elisa analysis of α d expression in transfected cos cells . example 9 describes facs analysis of cos cells transfected with human α d expression plasmids . example 10 addresses immunoprecipitation of cd 18 in association with α d in co - transfected cos cells . example 11 relates to production of α d - specific monoclonal antibodies . the monoclonal antibody ca11 . 8h2 [ moore , et al ., supra ] specific for canine α tm1 was tested for cross - reactivity on human peripheral blood leukocytes in an attempt to identify a human homolog of canine α tm1 . cell preparations ( typically 1 × 10 6 cells ) were incubated with undiluted hybridoma supernatant or a purified mouse igg - negative control antibody ( 10 μg / ml ) on ice in the presence of 0 . 1 % sodium azide . monoclonal antibody binding was detected by subsequent incubation with fitc - conjugated horse anti - mouse igg ( vector laboratories , burlingame , calif .) at 6 μg / ml . stained cells were fixed with 2 % w / v paraformaldehyde in phosphate buffered saline ( pbs ) and were analyzed with a facstar plus fluorescence - activated cell sorter ( becton dickinson , mountain view , calif .). typically , 10 , 000 cells were analyzed using logarithmic amplification for fluorescence intensity . the results indicated that ca11 . 8h2 did not cross - react with surface proteins expressed on human peripheral blood leukocytes , while the control cells , neoplastic canine peripheral blood lymphocytes , were essentially all positive for α tm1 . because the toonotional antibody ca11 . 8h2 specific for the canine α subunit did not cross react with a human homolog , isolation of canine α tm1 dna was deemed a necessary prerequisite to isolate a counterpart human gene if one existed . canine α tm1 was affinity purified in order to determine n - terminal amino acid sequences for oligonucleotide probe / primer design . briefly , anti - α tm1 monoclonal antibody ca11 . 8h2 was coupled to affigel 10 chromatographic resin ( biorad , hercules , calif .) and protein was isolated by specific antibody - protein interaction . antibody was conjugated to the resin , according to the biorad suggested protocol , at a concentration of approximately 5 mg antibody per ml of resin . following the conjugation reaction , excess antibody was removed and the resin blocked with three volumes of 0 . 1m ethanolamine . the resin was then washed with thirty column volumes of phosphate buffered saline ( pbs ). twenty - five grams of a single dog spleen were homogenized in 250 ml of buffer containing 0 . 32m sucrose in 25 mm tris - hcl , ph 8 . 0 , with protease inhibitors . nuclei and cellular debris were pelleted with centrifugation at 1000 g for 15 minutes . membranes were pelleted from the supernatant with centrifugation at 100 , 000 g for 30 minutes . the membrane pellet was resuspended in 200 ml lysis buffer ( 50 mm nacl , 50 mm borate , ph 8 . 0 , with 2 % np - 40 ) and incubated for 1 hour on ice . insoluble material was then pelleted by centrifugation at 100 , 000 g for 60 minutes . ten milliliters of the cleared lysate were transferred to a 15 ml polypropylene tube with 0 . 5 ml ca11 . 8h2 - conjugated affigel 10 resin described above . the tube was incubated overnight at 4 ° c . with rotation and the resin subsequently washed with 50 column volumes d - pbs . the resin was then transferred to a microfuge tube and boiled for ten minutes in 1 ml laemmli ( non - reducing ) sample buffer containing 0 . 1m tris - hcl , ph 6 . 8 , 2 % sds , 20 % glycerol and 0 . 002 % bromophenol blue . the resin was pelleted by centrifugation and discarded ; the supernatant was treated with 1 / 15 volume β - mercaptoethanol ( sigma , st . louis , mo .) and run on a 7 % polyacrylamide gel . the separated proteins were transferred to immobilon pvdf membrane ( millipore , bedford , mass .) as follows . the gels were washed once in deionized , millipore - filtered water and equilibrated for 15 - 45 minutes in 10 mm 3 -[ cyclohexylamino ]- 1 - propanesulfonic acid ( caps ) transfer buffer , ph 10 . 5 , with 10 % methanol . hnmobilon membranes were moistened with methanol , rinsed with filtered water , and equilibrated for 15 - 30 minutes in caps transfer buffer . the initial transfer was carried out using a biorad transfer apparatus at 70 volts for 3 hours . the immobilon membrane was removed after transfer and stained in filtered 0 . 1 % r250 coomassie stain for 10 minutes . membranes were destained in 50 % methanol / 10 % acetic acid three times , ten minutes each time . after desmining , the membranes were washed in filtered water and air - dried . protein bands of approximately 150 kd , 95 kd , 50 kd and 30 kd were detected . presumably the 50 kd and 30 kd bands resulted from antibody contamination . n - terminal sequencing was then attempted on both the 150 kd and 95 kd bands , but the 95 kd protein was blocked , preventing sequencing . the protein band of 150 kd was excised from the membrane and directly sequenced with an applied biosystems ( foster city , calif .) model 473a protein sequencer according to the manufacturer &# 39 ; s instructions . the resulting amino acid sequence is set in seq id no : 5 using single letter amino acid designations . the identified sequence included the fnld sequence characteristic of α subunits of the integrin family [ tamura , et al ., j . cell . biol . 111 : 1593 - 1604 ( 1990 )]. primer design and attempt to amplify canine α tm1 sequences from the n - terminal sequence information , three oligonucleotide probes were designed for hybridization : a ) &# 34 ; tommer ,&# 34 ; a fully degenerate oligonucleotide ; b ) &# 34 ; patruer ,&# 34 ; a partially degenerate oligonucleotide ; and c ) &# 34 ; guessruer ,&# 34 ; a nondegenerate oligonucleotide based on mammalian codon usage . these probes are set out below as seq id nos : 6 , 7 and 8 , respectively . nucleic acid symbols are in accordance with 37 c . f . r . § 1 . 882 for these and all other nucleotide sequences herein . based on sequencing data , no relevant clones were detected using these oligonucleotides in several low stringency hybridizations to a canine spleen / peripheral blood macrophage cdna library cloned into λzap ( stratagene , la jolla , calif .). four other oligonucleotide primers , designated 5 &# 39 ; deg , 5 &# 39 ; spec , 3 &# 39 ; deg and 3 &# 39 ; spec ( asset out in seq id nos : 9 , 10 , 11 and 12 , respectively , wherein deg indicates degenerate and spec indicates non - degenerate ) were subsequently designed based on the deduced n - terminal sequence for attempts to amplify canine α tm1 sequences by pcr from phage library dna purified from plate lysates of the stratagene library described above . the α tm1 oligonucleotide primers were paired with t3 or t7 vector sequence primers , as set out in seq id nos : 13 and 14 , respectively , which hybridize to sequences flanking the polylinker region in the bluescript phagemid found in λzap . the pcr amplification was carried out in taq buffer ( boehringer mannheim , indianapolis , ind .) containing magnesium with 150 ng of library dna , 1 μg of each primer , 200 μm dntps and 2 . 5 units taq polymerase ( boehringer mannheim ) and the products were separated by electrophoresis on a 1 % agarose gel in tris - acetate - edta ( tae ) buffer with 0 . 25 μg / ml ethidium bromide . dna was transferred to a hybond ( amersham , arlington , heights , ill .) membrane by wicking overnight in 10x sspe . after transfer , the immobilized dna was denatured with 0 . 5m naoh with 0 . 6m nacl , neutralized with 1 . 0m tris - hcl , ph 8 . 0 , in 1 . 5m nacl , and washed with 2x sspe before uv cross - linking with a stratalinker ( stratagene ) crosslinking apparatus . the membrane was incubated in prehybridization buffer ( 5x sspe , 4x denhardts , 0 . 8 % sds , 30 % formamide ) for 2 hr at 50 ° c . with agitation . oligonucleotide probes 5 &# 39 ; deg , 5 &# 39 ; spec , 3 &# 39 ; deg and 3 &# 39 ; spec ( seq id nos : 9 , 10 , 11 and 12 , respectively ) were labeled using a boehringer mannheim kinase buffer with 100 - 300 μci γp 32 - datp and 1 - 3 units of polynucleotide kinase for 1 - 3 hr at 37 ° c . unincorporated label was removed with sephadex g - 25 fine ( pharmacia , piscataway , n . j .) chromatography using 10 mm tris - hcl , ph 8 . 0 , 1 mm edta ( te ) buffer and the flow - through added directly to the prehybridization solution . membranes were probed for 16 hr at 42 ° c . with agitation and washed repeatedly , with a final stringency wash of 1x sspe / 0 . 1 % sds at 50 ° for 15 min . the blot was then exposed to kodak x - omat ar film for 1 - 4 hours at - 80 ° c . the oligonucleotides 5 &# 39 ; deg , 5 &# 39 ; spec , 3 &# 39 ; deg and 3 &# 39 ; spec only hybridized to pcr products from the reactions in which they were used as primers and failed to hybridize as expected to pcr products from the reactions in which they were not used as primers . thus , it was concluded that none of the pcr products were specific for α tm1 because no product hybridized with all of the appropriate probes . large scale affinity purification of canine α tm1 for internal sequencing in order to provide additional amino acid sequence for primer design , canine α tm1 was purified for internal sequencing . three sections of frozen spleen ( approximately 50 g each ) and frozen cells from two partial spleens from adult dogs were used to generate protein for internal sequencing . fifty grams of spleen were homogenized in 200 - 300 ml borate buffer with a waring blender . the homogenized material was diluted with 1 volume of buffer containing 4 % np - 40 , and the mixture then gently agitated for at least one hour . the resulting lysate was cleared of large debris by centrifugation at 2000 g for 20 rain , and then filtered through either a corning ( coming , n . y .) prefilter or a corning 0 . 8 micron filter . the lysate was further clarified by filtration through the corning 0 . 4 micron filter system . splenic lysate and the antibody - conjugated affigel 10 resin described in example 2 were combined at a 150 : 1 volume ratio in 100 ml aliquots and incubated overnight at 4 ° c . with rocking . the lysate was removed after centrifugation at 1000 g for 5 minutes , combined with more antibody - conjugated affigel 10 resin and incubated overnight as above . the absorbed resin aliquots were then combined and washed with 50 volumes d - pbs / 0 . 19 tween 20 and the resin transferred to a 50 ml biorad column . adsorbed protein was eluted from the resin with 3 - 5 volumes of 0 . 1m glycine ( ph 2 . 5 ); fractions of approximately 900 μl were collected and neutralized with 100 μl 1m tris buffer , ph 8 . 0 . aliquots of 15 μl were removed from each fraction and boiled in an equal volume of 2x laemmli sample buffer with 1 / 15 volume 1m dithiothreitol ( dtt ). these samples were electrophoresed on 8 9 novex ( san diego , calif .) polyacrylamide gels and visualized either by coomassie stain or by silver stain using a daiichi kit ( enprotech , natick , mass .) according to the manufacturer &# 39 ; s suggested protocol . fractions which contained the largest amounts of protein were combined and concentrated by vacuum . the remaining solution was diluted by 509 with reducing laemmli sample buffer and run on 1 . 5 mm 79 polyacrylamide gels in tris - glycine / sds buffer . protein was transferred from the gels to immobilon membrane by the procedure described in example 2 using the hoefer transfer apparatus . the protein bands corresponding to canine α d1 were excised from 10 pvdf membranes and resulted in approximately 47 μg total protein . the bands were destained in 4 ml 50 % methanol for 5 minutes , air dried and cut into 1 × 2 mm pieces . the membrane pieces were submerged in 2 ml 95 9 acetone at 4 ° c . for 30 minutes with occasional vortexing and then air dried . prior to proteolytic cleavage of the membrane bound protein , 3 mg of cyanogen bromide ( cnbr ) ( pierce , rockford , ill .) were dissolved in 1 . 25 ml 709 formic acid . this solution was then added to a tube containing the pvdf membrane pieces and the tube incubated in the dark at room temperature for 24 hours . the supernatant ( s1 ) was then removed to another tube and the membrane pieces washed with 0 . 25 ml 70 % formic acid . this supernatant ( s2 ) was removed and added to the previous supernatant ( s1 ). two milliliters of milli q water were added to the combined supernatants ( s1 and s2 ) and the solution lyophilized . the pvdf membrane pieces were dried under nitrogen and extracted again with 1 . 25 ml 60 % acetonitrile , 0 . 1 % tetrafluoroacetic acid ( tfa ) at 42 ° c . for 17 hours . this supernatant ( s3 ) was removed and the membrane pieces extracted again with 1 . 0 ml 80 % acetonitrile with 0 . 08 % tfa at 42 ° c . for 1 hour . this supernatant ( s4 ) was combined with the previous supernatants ( s1 , s2 and s3 ) and vacuum dried . the dried cnbr fragments were then dissolved in 63 μl 8m urea , 0 . 4m nh 4 hco 3 . the fragments were reduced in 5 μl 45 mm dithiothreitol ( dtt ) and subsequently incubated at 50 ° c . for 15 minutes . the solution was then cooled to room temperature and the fragments alkylated by adding 5 μl 100 mm iodoacetamide ( sigma , st . louis , mo .). following a 15 minute incubation at room temperature , the sample was diluted with 187 μl milli q water to a final urea concentration of 2 . 0m . trypsin ( worthington , freehold , n . j .) was then added at a ratio of 1 : 25 ( w : w ) of enzyme to protein and the protein digested for 24 hours at 37 ° c . digestion was terminated with addition of 30 μl tfa . the protein fragments were then separated with high performance liquid chromatography ( hplc ) on a waters 625 lc system ( millipore , milford , mass .) using a 2 . 1 × 250 mm , 5 micron vydac c - 18 column ( vydac , hesperia , calif .) equilibrated in 0 . 05 % tfa and hplc water ( buffer a ). the peptides were eluted with increasing concentration of 80 % acetonitrile in 0 . 04 % tfa ( buffer b ) with a gradient of 38 - 75 % buffer b for 65 - 95 minutes and 75 - 98 % buffer b for 95 - 105 minutes . peptides were fractionated at a flow rate of 0 . 2 ml / minute and detected at 210 nm . following fractionation , the amino acid sequence of the peptides was analyzed by automated edman degradation performed on an applied biosystems model 437a protein sequencer using the manufacturer &# 39 ; s standard cycles and the model 610a data analysis software program , version 1 . 2 . 1 . all sequencing reagents were supplied by applied biosystems . the amino acid sequences of seven of the eight internal fragments are set out below wherein &# 34 ; x &# 34 ; indicates the identity of the amino acid was not certain . one internal amino acid sequence ( set out in seq id no : 22 ) obtained was then used to design a fully degenerate oligonucleotide primer , designated p4 ( r ) as set out in seq id no : 23 . the 5 &# 39 ; portion of the canine α tm1 gene was amplified from double - stranded canine splenic cdna by pcr . one gram of frozen material from a juvenile dog spleen was ground in liquid nitrogen on dry ice and homogenized in 20 ml rna - stat 60 buffer ( tel - test b , inc , friendswood , tex .). four ml chloroform were added , and the solution extracted by centrifugation at 12 , 000 g for 15 minutes . rna was precipitated from the aqueous layer with 10 ml ethanol . poly a + rna was then selected on dynal oligo dt dynabeads ( dynal , oslo , norway ). five aliquots of 100 μg total rna were combined and diluted with an equal volume of 2x binding buffer ( 20 mm tris - hcl , ph 7 . 5 , 1 . 0m licl , 1 mm edta , 0 . 1 % sds ). rna was then incubated 5 minutes with the oligo dt dynabeads ( 1 . 0 ml or 5 mg beads for all the samples ). beads were washed with buffer containing 10 mm tris - hcl , ph 7 . 5 , 0 . 15m licl , 1 mm edta and 0 . 1 % sds , according to the manufacturer &# 39 ; s suggested protocol prior to elution of poly a + mrna with 2 mm edta , ph 7 . 5 . double - stranded edna was then generated using the eluted poly a + mrna and the boehfinger mannhelm edna synthesis kit according to the manufacturer &# 39 ; s suggested protocol . oligonucleotide primers 5 &# 39 ; deg ( seq id no : 9 ) and p4 ( r ) ( seq id no : 23 ) were employed in a standard pcr reaction using 150 ng double - stranded cdna , 500 ng of each primer , 200 μm dntps and 1 . 5 units taq polymerase ( boehringer mannheim ) in taq buffer ( boehringer mannheim ) with magnesium . the resulting products ( 1 μl of the original reaction ) were subjected to a second round of pcr with the same primers to increase product yield . this band was eluted from a 1 % agarose gel onto schleicher & amp ; schuell ( keene , n . h .) na45 paper in a buffer containing 10 mm tris - hcl , ph 8 , 1 mm edta , 1 . 5m nacl at 65 ° c ., precipitated , and ligated into the pcr tm ii vector ( invitrogen , san diego , calif .) using the ta cloning kit ( invitrogen ) and the manufacturer &# 39 ; s suggested protocol . the ligation mixture was transformed by electroporation into xl - 1 blue bacteria ( stratagene ). one clone , 2 . 7 , was determined to contain sequences corresponding to α tm1 peptide sequences which were not utilized in design of the primers . the sequence of the entire insert of clone 2 . 7 is set out in seq id no : 24 . attempts to isolate the full length canine α tm1 cdna from the stratagene library ( as described in example 2 ) were unsuccessful . approximately 1 × 10 6 phage plaques were screened by hybridization under low stringency conditions using 30 % formamide with clone 2 . 7 as a probe , but no positive clones resulted . attempts to amplify relevant sequences downstream from those represented in clone 2 . 7 using specific oligonucleotides derived from clone 2 . 7 or degenerate primers based on amino acid sequence from other peptide fragments paired with a degenerate oligonucleotide based on the conserved α subunit amino acid motif gffkr [ tamura , et al ., supra ] were also unsuccessful . to attempt the isolation of a human sequence homologous to canine α tm1 the approximately 1 kb canine α tm1 fragment from clone 2 . 7 was used as a probe . the probe was generated by pcr under conditions described in example 2 using nt2 ( as set out in seq id no : 25 ) and p4 ( r ) ( seq id no : 23 ) primers . the pcr product was purified using the qiagen ( chatsworth , ga .) quick spin kit and the manufacturer &# 39 ; s suggested protocol . the purified dna ( 200 ng ) was labeled with 200 μci α 32 pdctp using the boehringer mannheim random prime labelling kit and the manufacturer &# 39 ; s suggested protocol . unincorporated isotope was removed with sephadex g25 ( fine ) gravity chromatography . the probe was denatured with 0 . 2n naoh and neutralized with 0 . 4m tris - hcl , ph 8 . 0 , before use . colony lifts on hybond filters ( amersham ) of a human spleen cdna library in pcdna / amp ( invitrogen ) were prepared . the filters were initially denatured and neutralized as described in example 2 and subsequently incubated in a prehybridization solution ( 8 ml / filter ) with 30 % formamide at 50 ° c . with gentle agitation for 2 hours . labeled probe as described above was added to this solution and incubated with the filters for 14 hours at 42 ° c . the filters were washed twice in 2x ssc / 0 . 1 % sds at 37 ° c . and twice in 2x ssc / 0 . 1 % sds at 50 ° c . final stringency washes were 1x ssc / 0 . 1 % sds , twice at 65 ° c . ( 1x ssc is 150 mm nacl , 15 mm sodium citrate , ph 7 . 0 ). filters were exposed to kodak x - omat ar film for six hours with an intensifying screen . colonies giving signals on duplicate lifts were streaked on lb medium with magnesium ( lbm )/ carbenicillin plates and incubated overnight at 37 ° c . resulting streaked colonies were lifted with hybond filters and these filters were treated as above . the filters were hybridized under more stringent conditions with the 1 kb probe from clone 2 . 7 , labeled as previously described , in a 50 % formamide hybridization solution at 50 ° c . for 3 hours . probed filters were washed with a final stringency of 0 . 1 x ssc / 0 . 1 % sds at 65 &# 39 ; c and exposed to kodak x - omat ar film for 2 . 5 hours at - 80 ° c . with an intensifying screen . positive colonies were identified and cultured in lbm / carbenicillin medium overnight . dna from the cultures was prepared using the promega wizard miniprep kit according to the manufacturer &# 39 ; s suggested protocol and the resulting dna was sequenced . the initial screening resulted in 18 positive clones , while the secondary screening under more stringent hybridization conditions produced one positive clone which was designated 19a2 . the dna and deduced amino acid sequences of the human α d clone 19a2 are set out in seq id nos : 1 and 2 , respectively . characteristics of the human α d cdna and predicted polypeptide clone 19a2 encompasses the entire coding region for the mature protein , plus 48 bases ( 16 amino acid residues ) of the 5 &# 39 ; upstream signal sequence and 241 bases of 3 &# 39 ; untranslated sequence which do not terminate in a polyadenylation sequence . the core molecular weight of the mature protein is predicted to be around 125 kd . the extracellular domain is predicted to encompass approximately amino acid residues 17 through 1108 of seq id no : 2 . this extracellular region is contiguous with about a 20 amino acid region homologous to the human cd11 c transmembrane region ( residues 1109 through 1128 of seq id no : 2 ). the cytoplasmic domain comprises approximately 30 amino acids ( about residues 1129 through 1161 of seq id no : 2 ). the protein also contains a region ( around residues 150 through 352 ) of approximately 202 amino acids homologous to the i ( insertion ) domain common to cd11a , cd11b and cd11c [ larson and springer , supra ] and in vla - 2 [ tamura , et al ., supra ]. this region has not been demonstrated to exist in any other integrin subunits . the deduced amino acid sequence of α d shows approximately 28 % identity to that of cd11a , approximately 58 % identity to cd11b and approximately 61 % identity to cd11c . an alignment of amino acid sequences for ( cd11b seq id no : 3 ), cd11c ( seq id no : 4 ) and α d ( seq id no : 2 ) is presented in fig1 . in order to determine the relative level of expression and tissue specificity of α d , northern analysis was performed using a fragment from clone 19a2 as a probe . approximately 10 μg of total rna from each of several human tissues were loaded on a formaldehyde agarose gel in the presence of 1 μg of ethidium bromide . after electrophoresis at 100 v for 4 hr , the rna was transferred to a nitrocellulose membrane ( schleicher & amp ; schuell ) by wicking in 10x ssc overnight . the membrane was baked 1 . 5 hr at 80 ° c . under vacuum . prehybridization solution containing 50 % formamide in 3 -( n - morpholino ) propane sulfonic acid ( mops ) buffer was used to block the membrane for 3 hr at 42 ° c . a 1 . 6 kb bstxi fragment of clone 19a2 was labeled with the boehringer mannheim random prime kit according to the manufacturer &# 39 ; s instructions including both αp 32 dctp and αp 32 dttp . unincorporated label was removed on a sephadex g25 column in te buffer . the membrane was probed with 1 . 5 × 10 6 counts per ml of prehybridization buffer . the blot was then washed successively with 2x ssc / 0 . 1 % sds at room temperature , 2x ssc / 0 . 1 % sds at 42 ° c ., 2x ssc / 0 . 1 % sds at 500c , 1x ssc / 0 . 1 % sds at 50 ° c ., 0 . 5x ssc / 0 . 1 % sds at 50 ° c . and 0 . 1x ssc / 0 . 1 % sds at 50 ° c . the blot was then exposed to film for 19 hr . the autoradiogram revealed a weak signal in the approximately 5 kb range in liver , placenta , thymus and tonsil total rna . no signal was detected in kidney , brain or heart samples . the amount of rna present in the kidney lane , however , was minimal . rna from three myeloid lineage cell lines was also probed . the thp - 1 cell line , previously stimulated with pma , gave a diffuse signal in the same size range ( approximately 5 . 0 kb ), with a slightly stronger intensity than the tissue signals . rna from unstimulated and dmso - stimulated hl - 60 cells hybridized with the α d probe at the same intensity as the tissue samples , however , pma treatment seemed to increase the signal intensity . u937 cells expressed the α d message and this signal did not increase with pma stimulation . the human clone 19a2 lacks an initiating methionine codon and possibly some of the 5 &# 39 ; signal sequence . therefore , in order to generate a human expression plasmid containing 19a2 sequences , two different strategies were used . in the first , two plasmids were constructed in which signal peptide sequences derived from genes encoding either cd11b or cd11c were spliced into clone 19a2 to generate a chimeric α d sequence . in the second approach , a third plasmid was constructed in which an adenosine base was added on at position 0 in clone 19a2 to encode an initiating methionine . the three plasmids contained different regions which encoded the 5 &# 39 ; portion of the α d sequence or the chimeric α d sequence . the α d region was pcr amplified ( see conditions in example 2 ) with a specific 3 &# 39 ; primer bamrev ( set out below in seq id no : 26 ) and one of three 5 &# 39 ; primers . the three 5 &# 39 ; primers contained in sequence : ( 1 ) identical nonspecific bases at positions 1 - 6 allowing for digestion , an ecori site from positions 7 - 12 and a consensus kozak sequence from positions 13 - 18 ; ( 2 ) a portion of the cd11b ( primer er1b ) or cd11c ( primer er1c ) signal sequence , or an adenosine ( primer er1d ); and ( 3 ) an additional 15 - 17 bases specifically overlapping 5 &# 39 ; sequences from clone 19a2 to allow primer annealing . primers er1b , eric or er1d are set out in seq id nos : 27 , 28 or 29 , respectively , where the initiating methionine codon is underlined and the ecori site is double underlined . all three plasmids contained a common second α d region ( to be inserted immediately downstream from the 5 &# 39 ; region described in the previous paragraph ) including the 3 &# 39 ; end of the α d clone . the second α d region , which extended from nucleotide 625 into the xbai site in the vector 3 &# 39 ; polylinker region of clone 19a2 , was isolated by digestion of clone 19a2 with bamhi and xbai . three ligation reactions were prepared in which the 3 &# 39 ; α d bamhi / xbai fragment was ligated to one of the three 5 &# 39 ; α d ecori / bamhi fragments using boehringer mannheim ligase buffer and t4 ligase ( 1 unit per reaction ). after a 4 hour incubation at 14 ° c ., an appropriate amount of vector pcdna ( invitrogen ) digested with ecori and xbai was added to each reaction with an additional unit of ligase . reactions were allowed to continue for another 14 hours . one tenth of the reaction mixture was then transformed into competent xl - 1 blue cells . the resulting colonies were cultured and the dna isolated as in example 5 . digestion with ecori identified three clones which were positive for that restriction site , and thus , the engineered signal sequences . the clones were designated patm . b1 ( cd11b / α d , from primer er1b ), patm . c10 ( cd11c / α d , from primer er1c ) and patm . d12 ( adenosine / α d from primer er1 d ). presence of the appropriate signal sequences in each clone were verified by nucleic acid sequencing . expression from the α d plasmids discussed above was effected by cotransfection of cos cells with the individual plasmids and a cd 18 expression plasmid , prc . cd18 . as a positive control , cos cells were also co - transfected with the plasmid prc . cd18 and a cd11a expression plasmid , pdc . cd11a . cells were passaged in culture medium ( dmem / 10 % fbs / pen - strep ) into 10 cm coming tissue culture - treated petri dishes at 50 % confluency 16 hours prior to transfection . cells were removed from the plates with versene buffer ( 0 . 5 mm naedta in pbs ) without trypsin for all procedures . before transfection , the plates were washed once with serum - free dmem . fifteen micrograms of each plasmid were added to 5 ml transfection buffer ( dmem with 20 μg / ml deae - dextran and 0 . 5 mm chloroquine ) on each plate . after 1 . 5 hours incubation at 37 ° c ., the cells were shocked for 1 minute with 5 ml dmem / 10 % dmso . this dmso solution was then replaced with 10 ml / plate culture medium . resulting transfectants were analyzed by elisa and facs as described below . in order to determine if the cos cells co - transfected with cd 18 expression plasmid prc . cd 18 and an α d plasmid expressed α d on the cell surface in association with cd18 , elisas were performed using primary antibodies raised against cd18 ( e . g ., ts1 / 18 purified from atcc hb203 ). as a positive control , elisas were also performed on cells co - transfected with the cd18 expression plasmid and a cd11a expression plasmid , pdc . cd11a . the primary antibodies in this control included cd18 antibodies and anti - cd11a antibodies ( e . g ., ts1 / 22 purified from atcc hb202 ). for elisa , cells from each plate were removed with versene buffer and transferred to a single 96 - well flat - bottomed coming tissue culture plate . cells were allowed to incubate in culture media 2 days prior to assay . the plates were then washed twice with 150 μl / well d - pbs / 0 . 5 % teleost skin gelatin ( sigma ) solution . this buffer was used in all steps except during the development . all washes and incubations were performed at room temperature . the wells were blocked with gelatin solution for 1 hour . primary antibodies were diluted to 10 μg / ml in gelatin solution and 50 μl were then added to each well . triplicate wells were set up for each primary antibody . after 1 hour incubation , plates were washed 3x with 150 μl / well gelatin solution . secondary antibody ( goat anti - mouse ig / hrp - fc specific [ jackson , west grove , pa .]) at a 1 : 3500 dilution was added at 50 μl / well and plates were incubated for 1 hour . after three washes , plates were developed with 100 μl / well o - phenyldiamine ( opd ) ( sigma ) solution ( 1 mg / ml opd in citrate buffer ) and the plates were allowed to develop for 20 minutes before addition of 50 μl / well 15 % sulfuric acid . analysis of transfectants in the elisa format with anti - cd18 specific antibodies revealed no significant expression above background in cells transfected only with the plasmid encoding cd18 . cells co - transfected with plasmid containing cd11a and cd18 showed an increase in expression over background when analyzed with cd18 specific antibodies or with reagents specific for cd11a . further analysis of cells co - transfected with plasmids encoding cd18 and one of the α d expression constructs ( patm . c10 or patm . d12 ) revealed that cell surface expression of cd18 was rescued by concomitant expression of α d . the increase in detectable cd18 expression in cos cells transfected with patm . c10 or patm . d12 was comparable to that observed in co - transfected cd 11a / cd 18 positive control cells . for facs analysis , cells in petri dishes were fed with fresh culture medium the day after transfection and allowed to incubate 2 days prior to the assay . transfectant cells were removed from the plates with 3 ml versene , washed once with 5 ml facs buffer ( dmem / 2 % fbs / 0 . 2 % sodium azide ) and diluted to 500 , 000 cells / sample in 0 . 1 ml facs buffer . ten microliters of either 1 mg / ml fitc - conjugated cd18 , cd11a , cd11b specific antibodies ( becton dickinson ) or 800 μg / ml cfse - conjugated murine 23f2g ( anti - cd18 ) ( atcc hb11081 ) were added to each sample . samples were then incubated on ice for 45 minutes , washed 3 × with 5 ml / wash facs buffer and resuspended in 0 . 2 ml facs buffer . samples were processed on a becton dickinson facscan and the data analyzed using lysys ii software ( becton dickinson ). cos cells transfected with cd18 sequences only did not stain for cd18 , cd11a or cd11b . when co - transfected with cd11a / cd18 , about 15 % of the cells stained with antibodies to cd 11a or cd 18 . all cells transfected with cd18 and any α d construct resulted in no detectable staining for cd11a and cd11b . the patm . b1 , patm . c10 and patm . d12 groups stained 4 %, 13 % and 8 % positive for cd 18 , respectively . fluorescence of the positive population in the cd11a / cd18 group was 4 - fold higher than background . in comparison , the co - transfection of α d constructs with the cd18 construct produced a positive population that showed a 4 - to 7 - fold increase in fluorescence intensity over background . biotin - labeled immunoprecipitation of cd18 / α d complexes from co - transfected cos cells immunoprecipitation was attempted on cells co - transfected with cd 18 and each of the α d expression plasmids separately described in example 7 in order to determine if α d could be isolated as part of the αβ heterodimer complex characteristic of integrins . transfected cells ( 1 - 3 × 10 8 cells / group ) were removed from petri dishes with vetserie buffer and washed 3 times in 50 ml / group d - pbs . each sample was labeled with 2 mg sulpho - nhs biotin ( pierce , rockford , ill . for 15 minutes at room temperature . the reaction was quenched by washing 3 times in 50 ml / sample cold d - pbs . washed cells were resuspended in 1 ml lysis buffer ( 1 % np40 , 50 mm tris - hcl , ph 8 . 0 , 0 . 2m nacl , 2 mm ca ++ , 2 mm mg ++, and protease inhibitors ) and incubated 15 minutes on ice . insoluble material was pelleted by centrifugation at 10 , 000 g for 5 minutes , and the supernatant removed to fresh tubes . in order to remove material non - specifically reactive with mouse immunoglobulin , a pre - clearance step was initially performed . twenty - five micrograms mouse immunoglobulin ( cappel , west chester , pa .) was incubated with supernatants at 4 ° c . after 2 . 5 hr , 100 μl ( 25 μg ) rabbit anti - mouse ig conjugated sepharose ( prepared from protein a sepharose 4b and rabbit anti - mouse igg , both from zymed , san francisco , calif .) was added to each sample ; incubation was continued at 4 ° c . with rocking for 16 hours . sepharose beads were removed from the supernatants by centrifugation . after pre - clearance , the supernatants were then treated with 20 μg anti - cd18 antibody ( ts1 . 18 ) for 2 hours at 4 ° c . antibody / antigen complexes were isolated from supernatants by incubation with 100 μl / sample rabbit anti - mouse / protein a - sepharose preparation described above . beads were washed 4 times with 10 mm hepes , 0 . 2m nacl , and 1 % triton - x 100 . washed beads were pelleted and boiled for 10 minutes in 20 μl 2x laemmli sample buffer with 2 % β - mercaptoethanol . samples were centrifuged and run on an 8 % prepoured novex polyacrylamide gel ( novex ) at 100 v for 30 minutes . protein was transferred to nitrocellulose membranes ( schleicher & amp ; schuell ) in tbs - t buffer at 200 mamps for 1 hour . membranes were blocked for 2 hr with 3 % bsa in tbs - t . membranes were treated with 1 : 6000 dilution of strep - avidin horse radish peroxidase ( pod ) ( boehringer mannheim ) for 1 hour , followed by 3 washes in tbs - t . the amersham enhanced chemiluminescence kit was then used according to the manufacturer &# 39 ; s instructions to develop the blot . the membrane was exposed to hyperfilm mp ( amersham ) for 0 . 5 to 2 minutes . immunoprecipitation of cd18 complexes from cells transfected with prc . cd18 and either patm . b1 , patm . c10 or patm . d12 revealed surface expression of a heterodimeric species consisting of approximately 100 kd β chain , consistent with the predicted size of cd18 , and an α chain of approximately 150 kd , corresponding to α d . transiently transfected cells from example 7 were washed three times in dulbecco &# 39 ; s phosphate buffered saline ( d - pbs ) and injected at 5 × 10 6 cells / mouse into balb / c mice with 50 μg / mouse muramyl dipeptidase ( sigma ) in pbs . mice are injected two more times in the same fashion at two week intervals . the pre - bleed and immunized serum from the mice is screened by facs analysis as outlined in example 9 and the spleen from the mouse with the highest reactivity to cells transfected with α d / cd 18 is fused . hybridoma culture supernatants are then screened separately for lack of reactivity against cos cells transfected with cd11a / cd18 and for reactivity with cells co - transfected with an α d expression plasmid and cd18 . numerous modifications and variations in the invention as set forth in the above illustrative examples are expected to occur to those skilled in the art . consequently only such limitations as appear in the appended claims should be placed on the invention . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 29 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 3726 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( ix ) feature :( a ) name / key : cds ( b ) location : 3 .. 3485 ( xi ) sequence description : seq id no : 1 : tgaccttcggcactgtgcttcttctgagtgtcctggcttcttatcat47thrpheglythrvalleuleuleuservalleualasertyrhis15 1015ggattcaacctggatgtggaggagcctacgatcttccaggaggatgca95glypheasnleuaspvalglugluprothrilepheglngluaspala20 2530ggcggctttgggcagagcgtggtgcagttcggtggatctcgactcgtg143glyglypheglyglnservalvalglnpheglyglyserargleuval35 4045gtgggagcacccctggaggtggtggcggccaaccagacgggacggctg191valglyalaproleugluvalvalalaalaasnglnthrglyargleu50 5560tatgactgcgcagctgccaccggcatgtgccagcccatcccgctgcac239tyraspcysalaalaalathrglymetcysglnproileproleuhis6570 75atccgccctgaggccgtgaacatgtccttgggcctgaccctggcagcc287ileargproglualavalasnmetserleuglyleuthrleualaala8085 9095tccaccaacggctcccggctcctggcctgtggcccgaccctgcacaga335serthrasnglyserargleuleualacysglyprothrleuhisarg100 105110gtctgtggggagaactcatactcaaagggttcctgcctcctgctgggc383valcysglygluasnsertyrserlysglysercysleuleuleugly11512 0125tcgcgctgggagatcatccagacagtccccgacgccacgccagagtgt431serargtrpgluileileglnthrvalproaspalathrproglucys130135 140ccacatcaagagatggacatcgtcttcctgattgacggctctggaagc479prohisglnglumetaspilevalpheleuileaspglyserglyser145150 155attgaccaaaatgactttaaccagatgaagggctttgtccaagctgtc527ileaspglnasnasppheasnglnmetlysglyphevalglnalaval160165170 175atgggccagtttgagggcactgacaccctgtttgcactgatgcagtac575metglyglnphegluglythraspthrleuphealaleumetglntyr180185 190tcaaacctcctgaagatccacttcaccttcacccaattccggaccagc623serasnleuleulysilehisphethrphethrglnpheargthrser195200 205ccgagccagcagagcctggtggatcccatcgtccaactgaaaggcctg671proserglnglnserleuvalaspproilevalglnleulysglyleu210215220acgttcacggccacgggcatcctgacagtggtgacacagctatttcat719thrphethralathrglyileleuthrvalvalthrglnleuphehis225230235cataaga atggggcccgaaaaagtgccaagaagatcctcattgtcatc767hislysasnglyalaarglysseralalyslysileleuilevalile240245250255aca gatgggcagaagtacaaagaccccctggaatacagtgatgtcatc815thraspglyglnlystyrlysaspproleuglutyrseraspvalile260265270ccc caggcagagaaggctggcatcatccgctacgctatcggggtggga863proglnalaglulysalaglyileileargtyralaileglyvalgly275280285cacgc tttccagggacccactgccaggcaggagctgaataccatcagc911hisalapheglnglyprothralaargglngluleuasnthrileser290295300tcagcgcctc cgcaggaccacgtgttcaaggtggacaactttgcagcc959seralaproproglnasphisvalphelysvalaspasnphealaala305310315cttggcagcatccagaag cagctgcaggagaagatctatgcagttgag1007leuglyserileglnlysglnleuglnglulysiletyralavalglu320325330335ggaacccagtccagg gcaagcagctccttccagcacgagatgtcccaa1055glythrglnserargalaserserserpheglnhisglumetsergln340345350gaaggcttcagcac agccctcacaatggatggcctcttcctgggggct1103gluglypheserthralaleuthrmetaspglyleupheleuglyala355360365gtggggagctttagct ggtctggaggtgccttcctgtatcccccaaat1151valglyserphesertrpserglyglyalapheleutyrproproasn370375380atgagccccaccttcatcaac atgtctcaggagaatgtggacatgagg1199metserprothrpheileasnmetserglngluasnvalaspmetarg385390395gactcttacctgggttactccaccgagcta gccctgtggaagggggta1247aspsertyrleuglytyrserthrgluleualaleutrplysglyval400405410415cagaacctggtcctgggggccccccg ctaccagcataccgggaaggct1295glnasnleuvalleuglyalaproargtyrglnhisthrglylysala420425430gtcatcttcacccaggtgtccaggc aatggaggaagaaggccgaagtc1343valilephethrglnvalserargglntrparglyslysalagluval435440445acagggacgcagatcggctcctacttc ggggcctccctctgctccgtg1391thrglythrglnileglysertyrpheglyalaserleucysserval450455460gatgtggacagcgatggcagcaccgacctgatc ctcattggggccccc1439aspvalaspseraspglyserthraspleuileleuileglyalapro465470475cattactatgagcagacccgagggggccaggtgtccgtgtg tcccttg1487histyrtyrgluglnthrargglyglyglnvalservalcysproleu480485490495cctagggggcagagggtgcagtggcagtgtgacgctg ttctccgtggt1535proargglyglnargvalglntrpglncysaspalavalleuarggly500505510gagcagggccacccctggggccgctttggggcagcc ctgacagtgttg1583gluglnglyhisprotrpglyargpheglyalaalaleuthrvalleu515520525ggggatgtgaatgaggacaagctgatagacgtggccatt ggggccccg1631glyaspvalasngluasplysleuileaspvalalaileglyalapro530535540ggagagcaggagaaccggggtgctgtctacctgtttcacggagc ctca1679glygluglngluasnargglyalavaltyrleuphehisglyalaser545550555gaatccggcatcagcccctcccacagccagcggattgccagctcccag1 727gluserglyileserproserhisserglnargilealasersergln560565570575ctctcccccaggctgcagtattttgggcaggcgctgagtgggggtcag 1775leuserproargleuglntyrpheglyglnalaleuserglyglygln580585590gacctcacccaggatggactgatggacctggccgtgggggcccggggc 1823aspleuthrglnaspglyleumetaspleualavalglyalaarggly595600605caggtgctcctgctcaggagtctgccggtgctgaaagtgggggtggcc 1871glnvalleuleuleuargserleuprovalleulysvalglyvalala610615620atgagattcagccctgtggaggtggccaaggctgtgtaccggtgctgg1919 metargpheserprovalgluvalalalysalavaltyrargcystrp625630635gaagagaagcccagtgccctggaagctggggacgccaccgtctgtctc1967glugluly sproseralaleuglualaglyaspalathrvalcysleu640645650655accatccagaaaagctcactggaccagctaggtgacatccaaagctct2015thri leglnlysserserleuaspglnleuglyaspileglnserser660665670gtcaggtttgatctggcactggacccaggtcgtctgacttctcgtgcc2063val argpheaspleualaleuaspproglyargleuthrserargala675680685attttcaatgaaaccaagaaccccactttgactcgaagaaaaaccctg2111ilephe asngluthrlysasnprothrleuthrargarglysthrleu690695700ggactggggattcactgtgaaaccctgaagctgcttttgccagattgt2159glyleuglyil ehiscysgluthrleulysleuleuleuproaspcys705710715gtggaggatgtggtgagccccatcattctgcacctcaacttctcactg2207valgluaspvalvalserp roileileleuhisleuasnpheserleu720725730735gtgagagagcccatcccctccccccagaacctgcgtcctgtgctggcc2255valarggluproile proserproglnasnleuargprovalleuala740745750gtgggctcacaagacctcttcactgcttctctccccttcgagaagaac2303valglyserglnasp leuphethralaserleupropheglulysasn755760765tgtgggcaagatggcctctgtgaaggggacctgggtgtcaccctcagc2351cysglyglnaspglyle ucysgluglyaspleuglyvalthrleuser770775780ttctcaggcctgcagaccctgaccgtggggagctccctggagctcaac2399pheserglyleuglnthrleut hrvalglyserserleugluleuasn785790795gtgattgtgactgtgtggaacgcaggtgaggattcctacggaaccgtg2447valilevalthrvaltrpasnalaglyglu aspsertyrglythrval800805810815gtcagcctctactatccagcagggctgtcgcaccgacgggtgtcagga2495valserleutyrtyrproalaglyleu serhisargargvalsergly820825830gcccagaagcagccccatcagagtgccctgcgcctggcatgtgagaca2543alaglnlysglnprohisglnseral aleuargleualacysgluthr835840845gtgcccactgaggatgagggcctaagaagcagccgctgcagtgtcaac2591valprothrgluaspgluglyleuargs erserargcysservalasn850855860caccccatcttccatgagggctctaacggcaccttcatagtcacattc2639hisproilephehisgluglyserasnglythr pheilevalthrphe865870875gatgtctcctacaaggccaccctgggagacaggatgcttatgagggcc2687aspvalsertyrlysalathrleuglyaspargmetleumet argala880885890895agtgcaagcagtgagaacaataaggcttcaagcagcaaggccaccttc2735seralasersergluasnasnlysalaserserserly salathrphe900905910cagctggagctcccggtgaagtatgcagtctacaccatgatcagcagg2783glnleugluleuprovallystyralavaltyrthrm etileserarg915920925caggaagaatccaccaagtacttcaactttgcaacctccgatgagaag2831glnglugluserthrlystyrpheasnphealathrser aspglulys930935940aaaatgaaagaggctgagcatcgataccgtgtgaataacctcagccag2879lysmetlysglualagluhisargtyrargvalasnasnleuser gln945950955cgagatctggccatcagcattaacttctgggttcctgtcctgctgaac2927argaspleualaileserileasnphetrpvalprovalleuleuasn960 965970975ggggtggctgtgtgggatgtggtcatggaggccccatctcagagtctc2975glyvalalavaltrpaspvalvalmetglualaproserglnserleu 980985990ccctgtgtttcagagagaaaacctccccagcattctgacttcctgacc3023procysvalsergluarglysproproglnhisserasppheleuthr 99510001005cagatttcaagaagtcccatgctggactgctccattgctgactgcctg3071glnileserargserprometleuaspcysserilealaaspcysleu 101010151020cagttccgctgtgacgtcccctccttcagcgtccaggaggagctggat3119glnpheargcysaspvalproserpheservalglnglugluleuasp102 510301035ttcaccctgaagggcaatctcagtttcggctgggtccgcgagacattg3167phethrleulysglyasnleuserpheglytrpvalarggluthrleu1040 104510501055cagaagaaggtgttggtcgtgagtgtggctgaaattacgttcgacaca3215glnlyslysvalleuvalvalservalalagluilethrpheaspthr 106010651070tccgtgtactcccagcttccaggacaggaggcatttatgagagctcag3263servaltyrserglnleuproglyglnglualaphemetargalagln 107510801085atggagatggtgctagaagaagacgaggtctacaatgccattcccatc3311metglumetvalleuglugluaspgluvaltyrasnalaileproile109 010951100atcatgggcagctctgtgggggctctgctactgctggcgctcatcaca3359ilemetglyserservalglyalaleuleuleuleualaleuilethr1105 11101115gccacactgtacaagcttggcttcttcaaacgccactacaaggaaatg3407alathrleutyrlysleuglyphephelysarghistyrlysglumet11201125 11301135ctggaggacaagcctgaagacactgccacattcagtggggacgatttc3455leugluasplysprogluaspthralathrpheserglyaspaspphe1140 11451150agctgtgtggccccaaatgtgcctttgtcctaataatccactttcctgtt3505sercysvalalaproasnvalproleuser11551160tatc tctaccactgtgggctggacttgcttgcaaccataaatcaacttacatggaaacaa3565cttctgcatagatctgcactggcctaagcaacctaccaggtgctaagcaccttctcggag3625agatagagattgtaatgtttttacatatctgtccatctttttcagcaatg acccactttt3685tacagaagcaggcatggtgccagcataaattttcatatgct3726 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 1161 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 2 : thrpheglythrvalleuleuleuservalleualasertyrhisgly151015pheasnleuaspvalglugluprothrilepheglngluaspalagly 202530glypheglyglnservalvalglnpheglyglyserargleuvalval354045glyalaproleugluvalva lalaalaasnglnthrglyargleutyr505560aspcysalaalaalathrglymetcysglnproileproleuhisile657075 80argproglualavalasnmetserleuglyleuthrleualaalaser859095thrasnglyserargleuleualacysglyprothrleuh isargval100105110cysglygluasnsertyrserlysglysercysleuleuleuglyser115120125argtrpglu ileileglnthrvalproaspalathrproglucyspro130135140hisglnglumetaspilevalpheleuileaspglyserglyserile145150 155160aspglnasnasppheasnglnmetlysglyphevalglnalavalmet165170175glyglnphegluglythraspthrleuph ealaleumetglntyrser180185190asnleuleulysilehisphethrphethrglnpheargthrserpro19520020 5serglnglnserleuvalaspproilevalglnleulysglyleuthr210215220phethralathrglyileleuthrvalvalthrglnleuphehishis225 230235240lysasnglyalaarglysseralalyslysileleuilevalilethr245250255aspglyglnlystyrlys aspproleuglutyrseraspvalilepro260265270glnalaglulysalaglyileileargtyralaileglyvalglyhis275280 285alapheglnglyprothralaargglngluleuasnthrileserser290295300alaproproglnasphisvalphelysvalaspasnphealaalaleu3 05310315320glyserileglnlysglnleuglnglulysiletyralavalglugly325330335thrgln serargalaserserserpheglnhisglumetserglnglu340345350glypheserthralaleuthrmetaspglyleupheleuglyalaval355 360365glyserphesertrpserglyglyalapheleutyrproproasnmet370375380serprothrpheileasnmetserglngluasnvalasp metargasp385390395400sertyrleuglytyrserthrgluleualaleutrplysglyvalgln405410 415asnleuvalleuglyalaproargtyrglnhisthrglylysalaval420425430ilephethrglnvalserargglntrparglyslysalagluvalthr 435440445glythrglnileglysertyrpheglyalaserleucysservalasp450455460valaspseraspglyserthraspleu ileleuileglyalaprohis465470475480tyrtyrgluglnthrargglyglyglnvalservalcysproleupro485490 495argglyglnargvalglntrpglncysaspalavalleuargglyglu500505510glnglyhisprotrpglyargpheglyalaalaleuthrval leugly515520525aspvalasngluasplysleuileaspvalalaileglyalaprogly530535540gluglngluasnargg lyalavaltyrleuphehisglyalaserglu545550555560serglyileserproserhisserglnargilealaserserglnleu565 570575serproargleuglntyrpheglyglnalaleuserglyglyglnasp580585590leuthrglnaspglyleumetaspleuala valglyalaargglygln595600605valleuleuleuargserleuprovalleulysvalglyvalalamet610615620argph eserprovalgluvalalalysalavaltyrargcystrpglu625630635640glulysproseralaleuglualaglyaspalathrvalcysleuthr 645650655ileglnlysserserleuaspglnleuglyaspileglnserserval660665670argpheaspleualaleua spproglyargleuthrserargalaile675680685pheasngluthrlysasnprothrleuthrargarglysthrleugly690695 700leuglyilehiscysgluthrleulysleuleuleuproaspcysval705710715720gluaspvalvalserproileileleuhisleuasnpheserleu val725730735arggluproileproserproglnasnleuargprovalleualaval740745750glysergl naspleuphethralaserleupropheglulysasncys755760765glyglnaspglyleucysgluglyaspleuglyvalthrleuserphe77077 5780serglyleuglnthrleuthrvalglyserserleugluleuasnval785790795800ilevalthrvaltrpasnalaglygluaspsert yrglythrvalval805810815serleutyrtyrproalaglyleuserhisargargvalserglyala8208258 30glnlysglnprohisglnseralaleuargleualacysgluthrval835840845prothrgluaspgluglyleuargserserargcysservalasnhis850 855860proilephehisgluglyserasnglythrpheilevalthrpheasp865870875880valsertyrlysalathrleugl yaspargmetleumetargalaser885890895alasersergluasnasnlysalaserserserlysalathrphegln900905 910leugluleuprovallystyralavaltyrthrmetileserarggln915920925glugluserthrlystyrpheasnphealathrseraspglulysl ys930935940metlysglualagluhisargtyrargvalasnasnleuserglnarg945950955960aspleualaile serileasnphetrpvalprovalleuleuasngly965970975valalavaltrpaspvalvalmetglualaproserglnserleupro980 985990cysvalsergluarglysproproglnhisserasppheleuthrgln99510001005ileserargserprometleuaspcysserilea laaspcysleugln101010151020pheargcysaspvalproserpheservalglnglugluleuaspphe1025103010351040thrleulysglyasnleuserpheglytrpvalarggluthrleugln104510501055lyslysvalleuvalvalservalalagluilethrpheaspthrser 106010651070valtyrserglnleuproglyglnglualaphemetargalaglnmet107510801085glumetvalleugluglua spgluvaltyrasnalaileproileile109010951100metglyserservalglyalaleuleuleuleualaleuilethrala110511101115 1120thrleutyrlysleuglyphephelysarghistyrlysglumetleu112511301135gluasplysprogluaspthralathrpheserglya spasppheser114011451150cysvalalaproasnvalprolysser11551160 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 1153 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 3 : metalaleuargvalleuleuleuthralaleuthrleucyshisgly1510 15pheasnleuaspthrgluasnalametthrpheglngluasnalaarg202530glypheglyglnservalvalglnleuglnglyserargva 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euaspleualaleualaproglyargleuserpro675680685argalailepheglngluthrlysasnargserleuserargvalarg690 695700valleuglyleulysalahiscysgluasnpheasnleuleuleupro705710715720sercysvalgluaspse rvalileproileileleuargleuasnphe725730735thrleuvalglylysproleuleualapheargasnleuargpromet740 745750leualaalaleualaglnargtyrphethralaserleupropheglu755760765lysasncysglyalaasp hisilecysglnaspasnleuglyileser770775780pheserpheproglyleulysserleuleuvalglyserasnleuglu785790 795800leuasnalagluvalmetvaltrpasnaspglygluaspsertyrgly805810815thrthrilethrphe serhisproalaglyleusertyrargtyrval820825830alagluglyglnlysglnglyglnleuargserleuhisleuthrcys835 840845cysseralaprovalglyserglnglythrtrpserthrsercysarg850855860ileasnhisleuilepheargglyg lyalaglnilethrpheleuala865870875880thrpheaspvalserprolysalavalglyleuaspargleuleuleu885 890895ilealaasnvalsersergluasnasnileproargthrserlysthr900905910ilepheglnleuglule uprovallystyralavaltyrilevalval915920925serserhisgluglnphethrlystyrleuasnphesergluserglu930 935940glulysgluserhisvalalamethisargtyrglnvalasnasnleu945950955960glyglnargaspleuproval serileasnphetrpvalprovalglu965970975leuasnglnglualavaltrpmetaspvalgluvalserhisprogln980 985990asnproserleuargcysserserglulysilealaproproalaser99510001005asppheleualahisilegln lysasnprovalleuaspcysserile101010151020alaglycysleuargpheargcysaspvalproserpheservalgln10251030 10351040glugluleuaspphethrleulysglyasnleuserpheglytrpval104510501055argglnileleuglnl yslysvalservalvalservalalagluile106010651070ilepheaspthrservaltyrserglnleuproglyglnglualaphe1075 10801085metargalaglnthrilethrvalleuglulystyrlysvalhisasn109010951100proileproleuilevalglyser serileglyglyleuleuleuleu1105111011151120alaleuilethralavalleutyrlysvalglyphephelysarggln112 511301135tyrlysglumetmetgluglualaasnglyglnilealaprogluasn114011451150glythrglnthrp roserproproserglulys11551160 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 12 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 5 : pheasnleuaspva lglugluprometvalphegln1510 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 35 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 6 : ttyaayytggaygtngargarccnatggtnttyca35 ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 36 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 7 : ttcaacctggacgtggaggagcccatggtgttccaa36 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 36 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 8 : ttcaacctggacgtngaasancccatggtcttccaa36 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 23 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 9 : ttyaayytngaygtngargarcc23 ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 10 : ttyaayytggacgtngaaga20 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 17 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 11 : tgraanaccatnggytc17 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 18 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 12 : ttggaagaccatnggytc18 ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 17 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( x i ) sequence description : seq id no : 13 : attaaccctcactaaag17 ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 17 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 14 : aatacgactcactatag17 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 11 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 15 : valpheglngluxaaglyalaglypheglygln1510 ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 14 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 16 : leutyraspxaavalalaalathrglyleuxaaglnproile1510 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 12 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 17 : proleuglutyrxaaaspvalileproglnalaglu1510 ( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 10 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 18 : pheglngluglypheserxaavalleuxaa1510 ( 2 ) information for seq id no : 19 :( i ) sequence characteristics :( a ) length : 14 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 19 : thrserprothrpheilexaametserglngluasnvalasp1510 ( 2 ) information for seq id no : 20 :( i ) sequence characteristics :( a ) length : 17 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 20 : leuvalvalglyalaproleugluvalvalalavalxaaglnthrgly151015arg ( 2 ) information for seq id no : 21 :( i ) sequence characteristics :( a ) length : 9 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 21 : leuaspxaalysproxaaaspthrala15 ( 2 ) information for seq id no : 22 :( i ) sequence characteristics : ( a ) length : 7 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 22 : pheglygluglnpheserglu15 ( 2 ) information for seq id no : 23 :( i ) sequence characteristics :( a ) length : 21 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 23 : raanccytcytgraaactytc21 ( 2 ) information for seq id no : 24 :( i ) sequence characteristics :( a ) length : 1006 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 24 : ttcaacctggacgtggaggagcccatggtgttcaagaggatggagctggctttggacaga60gcgtggcccagcttggcggatctagactcgtggtgggagccc ccctggaggtggtggcgg120tcaaccaaacaggaaggttgtatgactgtgtggctgccactggccttgtcaacccatacc180cctgcacacacccccagatgctgtgaacatgtccctgggtctgtccctgtcagccgccgc240cagtcgcccctggctgctgg cctgtggcccaaccatgcacagagcctgtggggagaatat300gtatgcagaaggcttttgcctcctgttggactcccatctgcagaccatttggacagtacc360tgctgccctaccagagtgtccaagtcaagagatggacattgtcttcctgattgatggttc42 0tggcagtatgagcaaagtgactttaaacaaatgaaggatttgtgagagctgtgatgggac480agtttgagggcacccaaaccctgttctcactgatacagtatcccacctccctgaagatcc540acttcaccttcacgcaattccagagcagctggaaccctctga gcctggtggatcccattg600tccaactggacggcctgacatatacagccacgggcatccggaaagtggtggaggaactgt660ttcatagtaagaatggggcccgtaaaagtgccaagaagatcctcattgtcatcacagatg720gcaaaaatacaaagaccccc tggagtacgaggacgtatccccaggcagagagagcggatc780atccgctatgccattggggtgggagatgctttctggaaacccagtgccaagcaggagctg840gacaacattggctcagagccggctcaggaccatgtgttcagggtggacaactttgcagca90 0ctcagcagcatccaggagcagctgcaggagaagatctttgcactcgaaggaacccagtcg960acgacaagtagctctttccaacatgagatgttccaagaagggttca1006 ( 2 ) information for seq id no : 25 :( i ) sequence characteristics :( a ) length : 17 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 25 : gtnttycargargaygg17 ( 2 ) information for seq id no : 26 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 26 : ccactgtcaggatgcccgtg20 ( 2 ) information for seq id no : 27 :( i ) sequence characteristics :( a ) length : 42 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 27 : agttacgaattcgccaccatggctctacgggtgcttcttctg42 ( 2 ) information for seq id no : 28 :( i ) sequence characteristics :( a ) length : 42 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 28 : agttacgaattcgccaccatgactcggactgtgcttcttctg42 ( 2 ) information for seq id no : 29 :( i ) sequence characteristics :( a ) length : 36 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( xi ) sequence description : seq id no : 29 : agttacgaattcgccaccatgaccttcggcactgtg36