cr allergy has been recognized as important ige - mediated type i hypersensitivity since 1964 .
it is associated with the development of asthma and recognized as a risk factor for emergency room admission of asthmatic patients , especially among inner city children living in low - income houses infested with cockroaches . in china , totally 25.7% of allergy patients are skin prick test ( spt ) positive to the american cr ( periplaneta americana , per a ) and 18.7% spt positive to the german cr ( blattella germanica , bla g ) .
american cr , german cr , and smoky brown cr ( periplaneta fuliginosa ) are the dominant indoor cr species which cause allergy among human population worldwide .
twenty - two immunoglobulin e ( ige ) binding components including the proteins of 23 , 28 , 35 , 38 , 40 , 49 , 72 , 78 , and 97  kda were identified as major allergens in american cr , but only per a 1 , per a 2 , per a 3 , per a 4 , per a 7 , per a 9 , and per a 10   are characterized .
for example , per a 1 is an isoallergen with 5 isoforms reported so far , that is , per a 1.0101per a 1.0105 . per a 1 is a major american cr allergen as it binds to ige in the sera of 90100% of cr allergic subjects . per a 2
. per a 5 belongs to gsts , but the allergenicity of the gst homologues in american cr has not been reported . since the gst of german cr ( bla g 5 ) is a major cockroach allergen , which induces specific ige expression in 30 to 71% of cr allergy [ 1214 ] ,
we anticipate that per a 5 is likely a major allergen of american cr and investigate the potential allergenicity of it in the present study .
two gst homologue ( per a 5 ) genes are available in the genbank ( accession : ay792949 and aev23867 ) , which provides the possibility of producing recombinant per a 5 ( rper a 5 ) in large amounts to study its role in allergic reactions .
many , but not all allergens expressed from cdna have shown a considerable ige binding reactivity that seems to be comparable to their natural counterparts . the majority of these recombinant allergens are produced in e. coli , but , unfortunately , the amount and/or reactivity is sometimes reduced when the allergen is purified and subjected to immunological and biochemical assays . to overcome some of these problems ,
the aim of the present study is to generate rper a 5 by using eukaryotic ( baculovirus - infected insect cells ) and prokaryotic ( e. coli ) expression systems and characterize its biochemical and immunologic properties .
the study protocol was approved by the ethical committee of the first affiliated hospital of nanjing medical university . written informed consent for the use of blood samples was obtained from all participants before study entry according to the declaration of helsinki .
a total of 16 allergic rhinitis patients with positive spt ( allergens were supplied by alk - abell , inc . ,
denmark ) and positive serum ige test to american cr extract ( by using immuno - cap assay ( pharmacia diagnostics ab , uppsala , sweden ) ) and 6 healthy controls ( hc ) were recruited in the study .
serum ( 4  ml ) from peripheral venous blood was collected from each patient and hc for western blot analysis .
total rna was isolated from adult female cr reared at our institute by using trizol reagent ( invitrogen , carlsbad , ca , usa ) .
the cdna was prepared by reverse transcription using a commercial rna - pcr kit according to the manufacturer 's instruction ( takara biotech co. ltd . ,
dalian , china ) . for each reaction , 1  g of total rna was reversely transcribed using oligo - d ( t ) .
the cdnas encoding per a 5 were amplified by pcr using primers based nucleotide sequence of per a 5 gene ( ay792949 and aev23867 ; forward : 5-atgaccatcgacttctacta-3 ; reverse : 5-tcacttcttggcgaggttat-3 ) .
pcr condition was 95c/5  min ( one cycle ) , 95c/1  min , 52c/1  min and 72c/1  min ( 30 cycles ) , and 72c/5  min ( one cycle ) .
the purified pcr product was cloned into apmd18-t vector ( takara biotech co. ltd . ,
dalian , china ) before being transformed into escherichia coli strain dh5. the inserts were sequenced on an abiprism 377 dna sequencer ( applied biosystems , foster , ca , usa ) .
dna sequence data were translated to amino acid sequence by show translation tool in sms software package ( http://www.bioinformatics.org/sms/ ) .
the glycosylation motifs of per a 5 were predicted by using netnglyc 1.0 server ( http://www.cbs.dtu.dk/services/netnglyc ) .
the per a 5 gene was subcloned into pfastbac1 vector ( novagen , madison , wi , usa ) using ecor i and sal i sites and the resulted construct was transformed into e. coli strain dh10bac to generate recombinant bacmid .
the recombinant bacmid was transfected into sf-9 cells by using cellfectin ( invitrogen corporation , carlsbad , usa ) and incubated in sf-900ii liquid medium ( invitrogen corporation , carlsbad , usa ) for 5 days at 27c until the cells got swollen .
a total of 500  ml of sf-9 cells were infected by p2 viruses and harvested at 72  h. the cells were lysed against 50  mm tris - hcl with 300  mm  nacl and 5% glycerol . the supernatant was loaded on ni - nta column ( genscript , nanjing , china ) , washed with running buffer containing 50  mm tris - hcl , 300  mm nacl , and 5% glycerol ( ph 8.0 ) and eluted with elution buffer containing 50  mm tris - hcl , 300  mm  nacl , 250  mm imidazole , and 5% glycerol ( ph 8.0 ) .
the eluted fractions were obtained and identified as per a 5 ( iper a 5 ) .
the purified iper a 5 was dialyzed in carbonate - bicarbonate buffer ( 0.05  m , ph 9.6 ) for further investigation .
the concentration of iper a 5 was determined by using a coomassie plus assay kit with bsa as standard ( thermo scientific pierce , rockford , il , usa ) .
the per a 5 gene was subcloned into pcold ii vector ( takara biotech co. ltd . ,
dalian , china ) using nde i and xba i sites and verified by dna sequencing .
the recombinant pcold ii - per a 5 plasmid was transformed into e. coli origami host strain .
a colony of the selected transformed e. coli origami on an overnight lb - ampicillin agar plate was inoculated into 5  ml of lb - ampicillin broth and incubated at 37c overnight . for iptg optimization , the overnight culture was added into fresh lb media in a ratio of 1  :  100 .
once cell density reached the optical density at a600  nm ( od600 ) , the cells were incubated with 0.1 , 0.5 , and 1  mm  iptg , respectively , at 15c overnight .
expression of the target protein was analyzed by sds - page . for scale - up expression , 40  ml of the culture
was inoculated into 2  l of fresh lb - ampicillin broth and incubated at 37c until od600 reached 0.6 .
iptg was added to the final concentration of 0.5  mm and the culture was incubated overnight .
the bacterial cells were harvested by centrifugation at 4,000  g at 4c for 20  min and were lysed in a lysis buffer by sonication at 20  khz , 2  min pulse - on , 3  min pulse - off .
cell debris was removed by centrifugation at 12,000  g at 4c for 20  min .
the supernatant was loaded on the nickel column ( genscript , nanjing , china ) as described above , and the eluted fractions were obtained and identified as per a 5 ( bper a 5 ) .
the purified bper a 5 was dialyzed in carbonate - bicarbonate buffer ( 0.05  m , ph 9.6 ) for further investigation .
the concentration of bper a 5 was determined by using a coomassie plus assay kit with bsa as standard .
far uv cd spectra of bper a 5 and iper a 5 were collected on a jasco j-810 spectropolarimeter ( japan spectroscopic co. , tokyo , japan ) using a 1  mm path length quartz cuvette at protein concentrations of 0.1  mg / ml . spectra were measured from 240 to 190  nm , with a 0.5  nm resolution at a scanning speed of 50  nm / min , and resulted from averaging of three scans .
results were expressed as the mean residue ellipticity ( y ) at a given wavelength .
the secondary structure content of bper a 5 and iper a 5 was calculated by using the secondary structure estimation program k2d2 .
a 96-well plate was coated with purified bper a 5 and iper a 5 at 10  g / ml in carbonate - bicarbonate buffer ( 0.05  m , ph 9.6 ) overnight at 4c , 100  l per well .
human serum samples ( 1  :  20 dilution in pbs - tween with 2% bsa ) were then added to the plates for 2  h at room temperature .
after ige binding , plates were incubated with horseradish peroxidase - labeled goat anti - human ige ( 1  :  2500 dilution ) ( kpl , inc .
, md , usa ) , and the color was developed with tetramethylbenzidine peroxidase substrate ( thermo scientific pierce , rockford , il , usa ) .
the cutoff of the elisa was calculated as the mean of the negative controls plus 2 sds .
for competitive elisa test , a 96-well plate was coated with american cockroach extract ( 10  g / ml ) in carbonate - bicarbonate buffer ( 0.05  m , ph 9.6 ) overnight at 4c .
the 1  :  20-diluted pooled serum alone or preincubated with various quantities of the crude extract , iper a 5 and bper a 5 , for 2  h was added to the plates .
crude extract of cockroaches were prepared according to the methods described previously   with few modifications .
the sample was defatted in 200  ml of ethyl ether and ethyl acetate ( 1  :  1 , by volume ) and extracted with slow overhead stirring in carbonate - bicarbonate buffer ( 0.05  m , ph 9.6 ) , containing 6  mm 2-mercaptoethanol and 1/1,000 volume of protease inhibitor cocktail ( shenggong , shanghai , china ) at 4c overnight .
the extract was then centrifuged at 10,000  g for 30  min at 4c , and the supernatant was filtered through a 0.22  m - pore - size filter ( millipore , bedford , usa ) before use .
immunoblots for detection of serum specific ige were performed with bper a 5 and iper a 5 as described previously [ 19 , 20 ] .
bper a 5 and iper a 5 ( 5  g ) were added to a sds - page ( gel concentration of 15% ) under reducing conditions and then transferred to nitrocellulose membranes .
the nitrocellulose membranes were incubated with the sera from the patients with american cr allergy ( 1  :  5 in pbs - tween with 1% bsa , 10% normal goat serum ) for 90  min . following rinsing with pbs
, the membranes were incubated with peroxidase - labeled anti - human ige monoclonal antibody .
expression of cd63 and ccr3 on basophil surface has been considered as the indicator of basophil activation [ 21 , 22 ] .
briefly , peripheral blood mononucleated cells ( pbmc ) from 20  ml blood donated by 4 healthy volunteers were separated by ficoll - paque density gradient and treated with 10  ml ls ( a solution containing 1.3  m  nacl , 0.005  m  kcl , and 0.01 lactic acid , at ph 3.9 ) for 2  min at 8c .
after neutralization with 12% tris ( ph 10.9 ) , nonspecific ige on basophils was stripped off and cells were passively sensitized with sera of the patients with american cr allergy or hc ( n = 4 , 1 in 10 dilution , 2  h at 37c ) as described previously . the cells were then challenged with various concentrations of bper a 5 and iper a 5 for 15  min at 37c . a goat anti - human ige antibody ( serotec , kidlington , uk )
san diego , ca , usa ) and anti - human cd63-fitc antibody ( invitrogen corporation , camarillo , ca , usa ) were added to cells for 15  min at 37c in the dark .
flow cytometry analysis of surface markers was performed at 488  nm on a facsaria flow cytometer ( becton dickinson , franklin lakes , nj , usa ) and analyzed by facsdiva software .
basophils were gated in the lymphocyte region of the ssc or fsc pattern , and identified as a single population of cells that stained positively for ccr3-pe antibody .
upregulation of cd63 expression was determined by an increase in fluorescence in the fl-1 channel .
responses were quantified as percentages of cd63 expressing basophils in a higher fl-1 region , which had been adjusted to contain 4% of basophils .
data are expressed as mean  sem for the indicated number of independently performed duplicated experiments . statistical significance between means
was analyzed by one - way anova or the student 's t - test utilizing the spss 13.0 version .
the cdnas encoding per a 5 were amplified by pcr using primers based on the nucleotide sequence of per a 5 gene .
it is a 645  bp gene and encoded 215 amino acids protein ( figure 1 ) .
the sequence identity of per a 5 to cdnas deposited in genbank ( accession no .
. per a 5 shows 81 , 15 , and 13% sequence similarity to german cr allergen bggstd1 , der p 8 , and bla g 5 ( figure 2 ) .
one motif ( 198nhsg201 ) was predicted to be the glycosylation motif of per a 5 .
the per a 5 gene was subcloned into pcold ii vector and transformed into e. coli origami host strain .
the optimal induction condition for per a 5 was 0.5  mm  iptg ( figure 3(a ) ) , the concentration chosen as the final condition throughout the study .
more than 6  mg bper a 5 was obtained from 2  l cell culture medium .
it showed single band with an apparent molecular weight of 25  kda ( figure 3(b ) ) .
the per a 5 encoding gene was subcloned into pfastbac1 vector and transformed into e. coli strain dh10bac to generate recombinant bacmid .
the per a 5 protein was expressed in sf-9 cells and purified by ni column .
about 16  mg of iper a 5 was obtained from 2  l cell culture medium .
the purity of the purified per a 5 was identified by sds - page , which showed major band with an apparent molecular weight of 25  kda ( figure 4 ) .
the far uv cd spectra of iper a 5 and bper a 5 showed similar curves with two minima at 220 and 209.5  nm and a large maximum at 192  nm , which represent characteristics of proteins with predominantly -helical structure ( figure 5 ) .
calculation of the secondary structure using the program k2d2 resulted in predicted 45.93 and 8.69% of -helix and -sheets in iper a 5 and 42.54 and 8.49% of -helix and -sheets in bper a 5 , respectively . in order to determine the allergenicity of per a 5
, we examined the ability of per a 5 to bind ige in the sera of american cr allergy by a direct elisa technique .
patients serum including patients 5 , 9 , 10 , and 14 showed positive ige reactivity to both bper a 5 and iper a 5 .
the results showed that 4 out of 16 ( 25% ) sera from these patients reacted to both bper a 5 and iper a 5 ( figure 6(a ) ) .
the ige reactivity of bper a 5 and iper a 5 in the sera from the per a 5 positive patients was increased by 5.0- and 7.9-fold , respectively in comparison with the sera from hc . moreover
, competitive elisa showed that bper a 5 and iper a 5 inhibited the ige reactivity to the cockroach extract by approximately 25.4 and 35.5% , respectively ( figure 6(b ) ) .
ige binding activity of per a 5 in a representative group of 3 patients and two hc was assessed by western blot and was illustrated in figure 6(c ) .
ige binding bands appeared clearer with iper a 5 than with bper a 5 . both iper a 5 and bper a 5
iper a 5 and bper a 5 at 1.0  g / ml induced approximately up to 4.5- and 3.2-fold increase in the expression of cd63 and ccr3 in cd63 and ccr3 double positive cells when incubating with passively sensitized basophils ( by sera from american cr allergy ) .
both iper a 5 and bper a 5 had no effect on the basophils sensitized by the sera from hc ( figure 7 ) .
aerosolized proteins derived from saliva , fecal material , secretions , cast skins , debris , and dead bodies of cockroaches induce ige - mediated hypersensitivity . to better understand the per a 5 mediated cr allergies and promise to improve diagnosis and treatment of cr allergies , we prepared biologically active and highly pure american cr allergen per a 5 in relatively large amount in the present study .
we have identified per a 5 as a novel american cr allergen , which is recognized by 25% of the subjects with american cr allergy .
several allergens from german and american cr have been identified and their ige cross - reactivity has been described . these include bla g 1 and per a 1 ( food intake and digestion ) ; bla g 2 and per a 2 ( inactive aspartic protease ) ; bla g 3 and per a 3 ( arylphorin - like storage protein ) ; bla g 4 and per a 4 ( male pheromone transport lipocalin ) ; and bla g 7 and per a 7 ( tropomyosin ) . among cr allergens ,
3 gsts were identified from male adults of german cr by glutathione - agarose affinity chromatography including one in which n - terminal amino acid sequence is identical to that of bla g 5 .
two ige - reactive gsts were detected from 25 ige - reactive spots by proteomic analysis , and one of them was found to be bla g 5 .
recently , a delta class gst ( bggstd1 ) , which has 15% amino acid sequence identity with bla g 5 , was purified from the german cr . however , the allergenicity and ige cross - reactivity of the gst homologues in american cr have not been reported . as a member of the delta class gsts , per a 5 shows 81% sequence similarity to bggstd1 , but only 13% similarity to bla g 5 , a sigma class gst identified from german cr . in the present study , only 25% ige reactivity to rper a 5
was observed when sera from cockroach - sensitive patients was incubated with rper a 5 , suggesting that per a 5 is not likely a major allergen in american cr
. the ige reactivity rate of per a 5 seems lower than that of bla g 5 ( 30 to 71% ) [ 1214 ] , but higher than that of bggstd1 ( 17.9% ) .
it has previously been reported that american cr extracts could not inhibit ige antibody binding to bla g 5 using an ria assay , suggesting that there was no significant ige cross - reactivity between gsts from these two species .
however , huang et al . found that gst(s ) in american cr is(are ) allergenic and is(are ) pan - allergens in cr and mites .
the finding that per a 5 reacts with specific ige of american cr allergy in the present study suggests that gst in american cr is allergenic .
we prepared rper a 5 by using two different expression systems in the present study and found that as little as 2  l of e. coli and sf-9 cell culture medium was able to produce 8 and 16  mg of highly pure rper a 5 , respectively , which is enough for functional study of per a 5 .
the e. coli system is a well - established system offering many advantages : easy handling of the bacteria cells and selection of a large variety of vectors using different promoters . among the disadvantages
, overexpressed proteins can be incorrectly folded and may require chemical refolding procedures to obtain the protein in a native , fully active , biological form . in the present study , we chose pcold ii vector and e. coli origami host strain to produce bper a 5 in a soluble form without any reconstitution process .
the eukaryotic baculovirus expression system is characterized by an extensive array of posttranslational processing , typical for higher eukaryotic cells .
the production of recombinant proteins in this system offers the advantage that secreted proteins are often glycosylated and disulphide - bonded correctly leading to a biologically active conformation .
because of the advantage of baculovirus - infected insect cell expression system , we employed it to express iper a 5 and managed to obtain substantial quantity of iper a 5 in the present study .
numerous insect allergens such as api m 1 , api m 2   from honeybee venom , sol i 3   from ant venom , dol m 5   from bald - faced hornet ( dolichovespula maculate ) , cul s 1   from the north american midge ( culicoides sonorensis ) , der f 1   from house dust mite , blo t 11   from dust mite ( blomia tropicalis ) , lep d 2   from dust mite ( lepidoglyphus destructor ) , and aed a 1 and aed a 2   from mosquito ( aedes aegypti ) have been successfully expressed in insect cells using a baculovirus expression system .
they are reported to possess similar structural and biological activities to their natural forms . regarding the immunoreactivity ,
rper a 5 produced in the two systems showed ige - binding activities towards the sera from the american cr allergic patients .
iper a 5 seems to have better binding activity than bper a 5 , implicating that the ige - binding activity of rper a 5 is likely to associate with its glycosylation sites and tertiary conformation .
one motif ( 198nhsg201 ) was predicted to be the glycosylation motif in per a 5 , but we are not sure of the numbers of -gals in it , as most of the carbohydrate epitopes are known to lead false - positive reactions except -gal .
the far uv cd spectra of recombinant iper a 5 and bper a 5 showed similar secondary structures , which consist of predominantly -helical structure .
ige - binding epitopes recognized by ige antibodies are either linear or conformational and are located on the surface of the molecule accessible to antibodies .
conformational epitope comprises amino acids that are close in space in the folded molecule , despite being noncontiguous in the amino acid sequence .
for globular inhaled allergens , conformational epitopes play a very important role in allergenicity [ 41 , 42 ] .
therefore , it seems likely that extra conformational ige - binding epitopes may exist in iper a 5 , as a result of more -helical structures in its molecular structure .
the basophil activation test we employed herein is a more advanced technique for determination of allergenicity of a given compound .
we confirm that per a 5 is an active allergen of cr as it is able to activate cr - sensitized basophils .
similar to ige - binding activity , iper a 5 seems more potent in activation of basophils compared with bper a 5 , suggesting that the basophil - activating activity of rper a 5 is likely to associate with its glycosylation sites and tertiary conformation .
the availability of recombinant allergens has increased our understanding of ige - mediated allergies and promises to improve diagnosis and treatment of these diseases . in our case ,
these observations also confirm that baculovirus - infected insect cell expression system is more suitable for the production of more active per a 5 allergen than e. coli expression system . in conclusion
, we have cloned and prepared two rper a 5 allergens by using a eukaryotic and a prokaryotic expression system .
we confirm that there is gst allergen ( per a 5 ) in american cr , though it is not a major allergen .
rper a 5 should be a useful tool for studying and understanding the role of per a 5 in cr allergy .
 glutathione s - transferase ( gst ) from various arthropods can elicit allergic reactions . in the present study , per a 5 , a gst , 
 was cloned from american cockroach ( cr ) and expressed in both baculovirus - infected insect cell ( iper a 5 ) and e. coli expression ( bper a 5 ) systems . 
 the secondary structures were predicted to be 45.93 and 8.69% of -helix -sheets in iper a 5 and 42.54 and 8.49% of -helix and -sheets in bper a 5 , respectively . 
 it is found that 4 out of 16 ( 25% ) sera from american cr allergy patients reacted to both bper a 9 and iper a 9 as assessed by elisa and western blotting analysis , confirming that per a 5 is not a major allergen of american cr . 
 induction of upregulated expression of cd63 and ccr3 on passively sensitized human basophils ( sera from american cr allergy patients ) by approximately up to 4.5- and 3.2-fold indicates that iper a 5 and bper a 5 are functionally active . 
 recombinant per a 5 ( rper a 5 ) should be a useful tool for studying and understanding the role of per a 5 in cr allergy . 
1. Introduction
2. Materials and Methods
3. Results 
4. Discussion