cloning and expression of ama r 1, as a novel …...cloning and expression of ama r 1, as a novel...

Research ArticleCloning and Expression of Ama r 1 as a Novel Allergen ofAmaranthus retroflexus Pollen

Payam Morakabati1 Mohammad-Ali Assarehzadegan12 Gholam Reza Khosravi1

Bahareh Akbari1 and Fatemeh Dousti1

1Department of Immunology Faculty of Medicine Ahvaz Jundishapur University of Medical Sciences Ahvaz 1478736714 Iran2Department of Immunology School of Medicine Iran University of Medical Sciences Tehran Iran

Correspondence should be addressed to Mohammad-Ali Assarehzadegan assarehmagmailcom

Received 6 August 2015 Accepted 28 December 2015

Academic Editor Desiderio Passali

Copyright copy 2016 PayamMorakabati et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Sensitisation to Amaranthus retroflexus pollen is very common in tropical and subtropical countries In this study we aimed toproduce a recombinant allergenic Ole e 1-like protein from the pollen of this weed To predict cross-reactivity of this allergen (Amar 1) with other members of the Ole e 1-like protein family the nucleotide sequence homology of the Ama r 1 was investigated Theexpression of Ama r 1 in Escherichia coli was performed by using a pET-21b(+) vector The IgE-binding potential of recombinantAma r 1 (rAma r 1) was evaluated by immunodetection and inhibition assays using 26 patientsrsquo sera sensitised to A retroflexuspollenThe coding sequence of the Ama r 1 cDNA indicated an open reading frame of 507 bp encoding for 168 amino acid residueswhich belonged to the Ole e 1-like protein family Of the 26 serum samples 10 (3846) had significant specific IgE levels for rAmar 1 Immunodetection and inhibition assays revealed that the purified rAma r 1 might be the same as that in the crude extract Amar 1 the second allergen from the A retroflexus pollen was identified as a member of the family of Ole e 1-like protein

1 Introduction

Pollen from Amaranthus retroflexus (redroot pigweed) awell-known species of the Amaranthaceae family which isfound throughout the world is an important trigger ofrespiratory allergies in different regions with temperate anddry climates such as Saudi Arabia Kuwait India Iran thewestern United States Australia and the Mediterranean area[1ndash4] Allergy to A retroflexus pollen one of the most com-mon sources of respiratory allergies among Iranian allergypatients has also been well defined [1 5] This annual weedis abundant in open fields and in farmlands or grasslandsThe flowering season of this plant is from around August toOctober [6]

Immunochemical characterisation of the pollen extractof A retroflexus revealed several components ranging from10 kDa to 85 kDa [6] Furthermore based on the studies ofsera of patients with respiratory allergies the proteins ofaround 10 15 18 39 45 and 85 kDa have been reported asIgE-binding proteins from A retroflexus pollen using allergic

patientsrsquo sera [6 7] The first allergen from A retroflexuspollen (Ama r 2) was identified as a member of the profilinfamily [8] To the best of our knowledge despite a highrate of sensitisation to pollens from Amaranthus species indifferent areas of theworld [1 4 5 9 10] few studies about themolecular characterisation of A retroflexus pollen allergenshave been conducted to date

In this study we introduced Ama r 1 as the second aller-gen fromA retroflexus pollen which is amember of the Ole e1-like protein family The prototypic member of this family isthe major olive pollen allergen Ole e 1 [11] Several allergensfrom the Ole e 1-like protein family have been identifiedpreviously in other plants such as Chenopodium album (Chea 1) [12] Salsola kali (Sal k 5) [13] Fraxinus excelsior (Fra e 1)[14] Ligustrum vulgare (Lig v 1) [15] and Syringa vulgaris (Syrv 1) [16] In the present study we aimed to produce Ama r 1in Escherichia coli and then determined the homology of itsprotein sequence that was determined by comparing it withthe most common allergenic Ole e 1-like proteins

Hindawi Publishing CorporationJournal of AllergyVolume 2016 Article ID 4092817 8 pageshttpdxdoiorg10115520164092817

2 Journal of Allergy

2 Materials and Methods

21 Protein Extraction from A retroflexus Pollen Flowers ofA retroflexus were accumulated from August to October inAhvaz city in the southwest of Iran Gathering of pollenmaterials and handling were performed by trained pollencollectors Floral parts other than pollenwere separated usingthe sieves with different sizes (100 200 and 300 meshes)successively [17]

The final fine powder was subjected to a purity checkfor pollen content using a microscope Pollen materials withmore than 96 pollen and less than 4 of the other parts ofthe same plant were gathered for protein extraction Pollenswere defatted using repeated changes of diethyl ether Forprotein extraction two grams of pollen was mixed with12mL phosphate-buffered saline (PBS) 001M (pH 74) bycontinuous stirring for 16 h at 4∘C The supernatant wasseparated by centrifugation at 13000timesg for 20min andfiltered and the supernatant collected [18]

The protein content of the extract was evaluated usingBradfordrsquos method [19] Finally the extract was freeze-driedand stored at minus20∘C for later use in the present study

22 Patientsrsquo Sera and Skin Prick Test (SPT) In this study weused sera from 26 patients from Ahvaz city southwest IranThe patients were 12 men and 14 women (mean age 2988 plusmn688 years age range 20ndash41 years) with respiratory allergiesand seasonal rhinitis who had positive skin prick test (SPT)results forA retroflexus pollen extract Eight subjects withoutallergies who presented with negative SPTs and no specificIgE to the A retroflexus pollen extract were assigned asnegative controls All patients and control subjects gave ustheir written informed consent to participate Serum samplesof the subjects were prepared and then immediately stored atminus20∘C until used

23 Total IgE and Specific Indirect Enzyme-Linked Immunosor-bent Assays (ELISA) Total serum IgE levels were evaluatedin serum samples in duplicate using a commercially avail-able ELISA kit according to the manufacturerrsquos instructions(Euroimmun Lubeck Germany) To evaluate the levels ofspecific IgE to A retroflexus pollen proteins in patients withrespiratory allergies an indirect ELISA was devised In briefwells of an ELISA microplate (Nunc AS Roskilde Den-mark) were coated with 100 120583Lwell of A retroflexus pollenextract [10 120583gwell in coating buffer (15mMNa

2CO3 35mM

NaHCO3 pH 96)] at 4∘C for overnight After blocking with

a solution composed of 150 120583L of phosphate buffered saline(PBS) and 2 bovine serum albumin (BSA) solution for1 hour at 37∘C the plates were incubated with 100 120583L ofpatientsrsquo sera for 3 hours at room temperature Each wellwas then incubated for 2 hours at room temperature with a1 500 dilution of biotinylated goat anti-human IgE antibody(Nordic-MUbio Susteren Netherlands) in 1 PBSThe wellswere then washed five times with PBS containing 005Tween-20 (T-PBS) to eliminate unbound anti-IgE There-after 100 120583L of horseradish peroxidase- (HRP-) conjugatedstreptavidin (Bio-Rad Laboratories Hercules CA USA) wasplaced in each well and each well was then incubated for

1 hour at room temperature After five washes with T-PBSeach well received 100 120583L of tetramethylbenzidine substratesolution (Sigma-Aldrich St LouisMOUSA) that was placedin eachwell and the plate was incubated at room temperaturefor 20min before the reaction was stopped by addition of100 120583L of 2M HCl Subsequently the absorbance in eachwell was measured at 450 nm using an ELISA plate readerAll results were expressed as optical density (OD) units AnOD value four times higher than the average values of threedeterminations of a pooled sera from negative controls (iegt015 OD units) was considered to be positive

24 Subcloning of Ama r 1 Allergen cDNA and DNA Sequen-cing Total RNA was extracted from 100mg of A retroflexuspollen by using the Chomczynski and Sacchi method[20] The first strand of cDNA was synthesised using theRevertAidTM First Strand cDNA Synthesis Kit (Thermo Sci-entific Waltham MA USA) according to the manufac-turerrsquos instructions The primers used for cDNA amplifi-cation were designed according to the known nucleotidesequence for reported allergens from the Ole e 1-like proteinfamily that have a high degree of amino acid sequenceidentity [12ndash14 21ndash23] These primers include the sense 51015840-ATGGGGAAGTGTCAAGCTGT-31015840 and the antisense 51015840-TTAATTAGCTTTAACATCATAAAGATCC-31015840 The ampli-fied fragment was ligated into a PTZ57RT TA cloning vectorusing the InsTAcloneTM PCR Cloning Kit (Thermo Scien-tific Waltham MA USA) according to the manufacturerrsquosinstructions E coli TOP10 cells (Invitrogen Carlsbad CAUSA) were transformed with the ligation products using themanufacturerrsquos protocol A recombinant plasmidwas selectedbywhiteblue screening and then purified from the gel using aplasmid extraction kit (GeNet Bio Chungnam Korea) DNAsequence analysis was performed using the dideoxy methodat the Bioneer Inc (Daejeon Korea)

The obtained sequence was submitted to the GenBankdatabase of the National Center for Biotechnology Informa-tion (httpwwwncbinlmnihgov) under accession num-ber KR870437

25 Construction of Prokaryote Expression Vector and Pro-duction of Recombinant Ama r 1 (rAma r 1) To create apET-21b(+) expression plasmid (Novagen Gibbstown NJUSA) restriction sites of Not I and Xho I restriction enzymesites were introduced at the 51015840- and 31015840-ends of the Ama r1 cDNA by the polymerase chain reaction (PCR) methodusing two specific primers as follows the sense primer 51015840-TCCgcggccgcATGGGGAAGTGTCAAGCTGT-31015840 (Not Irestriction site is in lowercase) and the antisense primer 51015840-CCctcgagTTAATTAGCTTTAACATCATAAAGATCC-31015840(Xho I restriction site is in lowercase) After PCR amplifi-cation the resulting product was digested with Not I andXho I restriction enzymes according to the manufacturerrsquosprotocol (Thermo Scientific Waltham MA USA) The puri-fied digested PCR product was ligated into the digested pET-21b(+) plasmid with the same enzymes Correct constructswere transformed into competent E coli BL21 (DE3) cells(Novagen Gibbstown NJ USA)

Journal of Allergy 3

A clone of recombinant plasmid pET-21b(+)Ama r 1 wasinoculated into 2mL of lysogeny broth (LB) medium con-taining 100 120583gmL of ampicillin and incubated at 37∘CExpression of the recombinant protein was induced byadding isopropyl 120573-d-thiogalactopyranoside (IPTG) to afinal concentration of 05mM [18] Afterwards the cells wereharvested by centrifugation (3500timesg for 15min at 4∘C)resuspended in lysis buffer (50mM Tris-HCl pH 68 15mMimidazole 100mM NaCl 10 glycerol and 05 Triton X-100) and then disrupted by sonication Purification of rAmar 1 was performedwithNi-nitrilotriacetic acid (NTA) agarose(Invitrogen Carlsbad CA USA) from the soluble phase oflysate according to the manufacturerrsquos instructions

26 Determination of Specific IgE Levels to rAma r 1 In orderto assess the serum IgE levels to the purified rAma r 1 an

indirect ELISAwas developed as described above except thatthe wells of the ELISA microplate were coated with 100 120583Lwell of the purified rAma r 1 at a concentration of 2 120583gmLin coating buffer (15mM Na

2CO3and 35mM NaHCO

3 pH

96) overnight at 4∘CThe results were expressed in OD unitsBased on the mean value of two normal sera OD

450greater

than three times the median values of negatives controls wasconsidered to be positive

27 ELISA Inhibition Assays for rAma r 1 ELISA inhibitionwas performed as described above except for a pooled serum(1 2 volvol) from patients allergic to A retroflexus (patients1 2 4 6 and 8) which was preincubated for overnight at 4∘Cwith either 1000 100 10 1 01 or 001 120583g of rAma r 1 as aninhibitor or with BSA as a negative control The inhibitionpercentage was calculated using the following relationship

Inhibition = (OD of sample without inhibitor minusOD of sample with inhibitorOD of sample without inhibitor

) times 100 (1)

28 IgE-Immunoblotting and IgE-Immunoblotting Inhibitionfor rAma r 1 Proteins from A retroflexus pollen extract andE coli lysate and purified rAma r 1 were analysed by sodiumdodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) using 125 acrylamide separation gels and underreducing conditions according to the method of Laemmli[24] The molecular masses of protein bands were estimatedwith Image Lab analysis software (Bio-Rad LaboratoriesHercules CA USA) and compared with protein markers ofknown molecular weights (Amersham low molecular weightcalibration kit for SDS electrophoresis GE Healthcare LittleChalfont UK) Separated protein bands from the elec-trophoresis of A retroflexus pollen were electrotransferred topolyvinylidene difluoride (PVDF) membranes (GE Health-care Little Chalfont UK) as described elsewhere [18] Inbrief after blocking andwashingmembranes were incubatedwith a serum pool or individual sera from patients with Aretroflexus allergy or with control sera (1 5 dilutions) for3 hours Biotinylated goat anti-human IgE (Nordic-MUbioSusteren Netherlands) (1 1000 volvol in PBS) was added tothe blotted membrane strips and incubated for 2 hours atroom temperature The unbound antibodies were removedfrom blots by washing with T-PBS and incubated at1 10000 volvol in T-PBS-HRP-linked streptavidin (Sigma-Aldrich St Louis Mo USA) for 1 hour at room temperatureAfter several washes with T-PBS strips were incubated usingthe SuperSignal West Pico Chemiluminescent Substrate Kit(Thermo Scientific Waltham MA USA) for 5 minutesand proteins were then visualised by chemiluminescenceusing the ChemiDoc XRS+ System (Bio-Rad LaboratoriesHercules CA USA)

To study cross-inhibition among natural and recombi-nant Ama r 1 a mixture of 100 120583L of pooled serum (1 5 volvol) was incubated with natural A retroflexus pollen extract(65 120583gmL as an inhibitor) rAma r 1 (10 120583gmL as an inhib-itor) or BSA (as a negative control) overnight at 4∘C with

shaking Preincubated sera were used to assess the reactivityof a PVDF membrane blotted with natural A retroflexuspollen extract and rAma r 1

3 Results

31 Measurement of Total and Specific IgE The mean totalIgE serum in the subjects was determined as 25633 IUmLIn patients reactive to Ama r 1 the mean of total IgE was18380 IUmL (Table 1) Sera from 26 allergic patients wereassessed for specific IgE binding to proteins from Aretroflexus pollen extract All of these patients had sig-nificantly elevated specific IgE levels to the extract of Aretroflexus pollen (mean OD

450= 147 plusmn 050 range 079ndash

221) The mean OD450

for specific IgE in patients reactive torAma r 1 was 095 plusmn 016 (range 078ndash123) (Table 1)

32 Nucleotide and Protein Sequence Analysis of Ama r 1 Thesequence analysis of Ama r 1 indicated an open reading frameof 507 bp coding for 168 amino acid residues with a predictedmolecular mass of 18379 kDa and a calculated isoelectricpoint (pI) of 470 We compared the deduced amino acidsequence of Ama r 1 with other allergenic plant-derived Olee 1-like proteins in the protein database (Figure 1) and wedetected a high level of sequence identity (93) that wasdetected between Ama r 1 and Che a1 (Table 2)

33 SDS-PAGE and IgE-Binding Components of A retroflexusPollen Extract The reducing SDS-PAGE separation of thepollen extract showed several resolved protein bands in theA retroflexus pollen extract with molecular weights ran-ging from approximately 10 to 85 kDa (Figure 2) IgE-bindingreactivity of the separated protein bands from the electropho-resis of the A retroflexus pollen extract was assessed by con-ducting immunoblotting experiments The results revealedthat several IgE-reactive bands range from about 15 to 85 kDa


Table 1 Clinical attribute skin prick test responses and specific IgE values of patients reactive to recombinant Ama r 1

Patients number Age (years)gender1 Symptoms2 Total IgE (IUmL) A retroflexus pollen extract rAma r 1-specific IgESkin prick test3 Specific IgE4

1 38M A R 152 8 180 0952 32F A R L 185 12 210 1233 21F A R 162 8 098 0804 38F A R L 224 12 189 1105 23M A R L 132 10 110 0846 29F A R L 166 12 195 0987 41M A R 175 9 092 0818 32F A R L 305 15 221 1159 22M A R 159 11 112 08710 35F A L 178 10 097 0781M male F female2A allergic rhinitis L lung symptoms (breathlessness tight chest cough and wheeze) R rhinoconjunctivitis3Themean wheal areas are displayed in mm2 Histamine diphosphate (10mgmL)mdashpositive control Glycerinmdashnegative control4Determined in specific ELISA as OD (optical density) at 450 nm

MAKCQAVFLLVGALCVLSLDDVAKAPVSQFHIQGLVYCDTCRIQFMTRISTIMEGATVKLECRNITAGTQ

AGNAENHKVM

GAGNAENHKVMV

---------------------KGGGHNLYVKMIVMSQLE

------------------------PQARIELEFIPSRQDGENSI

------------------MEPQPQARIKLEFITSRQDKENDV

-------------------EPQPPQARIELEFIPGIRQKDGEHKI

-------------------EPQPYQARIELEFIPGRQKDGENKV

TFKAEAVTDKVGQYSIPVNGDFEDDICEIELVKSFNSECSEVSHSVYAKQSAKVSLTSNNGEASDIRSAN

HQ

LMTKDHQVGQIPNSEATVN

TEVGSRAELMLIERHKEFTISSRKDDIPVEGWVPLFMNTV---TTTV

TEIGYRGELMFERHKNEFTLSGRKDNIPIEGWVPLFINTV---TTTI

TEIGYRAELMLEHKNEFTLSSRKDEIPIEGWVPLFINTV---TTTI

TEVGYKAELNMLIERHKNEFTISSRKDDIPTEGWVPLFVNTV---TTTI

ALGFMRKEPLEECPEVLKE-LDLYDVKAN----

K-----

K-----

LKAP-MPGSVTQN

PFKAPKQFNK-GMPPDM-----

PFKAPQAQYNK-GMPPNM-----

PFKVPKQFNK-GMPPNM-----

PLKVPKQFNK-GMPPNM-----

Ama r 1

10 20 30 40 50 60 70

7070704946525151

80 90 100 110 120 130 140

140140140119

119118118

168168168151140146145145

113

150 160 170

Che a 1Cro s 1Sal k 5Ole e 1Fra e 1Syr v 1Lig v 1

Ama r 1Che a 1Cro s 1Sal k 5Ole e 1Fra e 1Syr v 1Lig v 1


Figure 1 Comparison of the A retroflexusOle e 1-like protein (Ama r 1) amino acid sequence with allergenic Ole e 1-like protein from otherplants Chenopodium album (Che a 1 G8LGR01) Crocus sativus (Cro s 1 XP0041436351) Salsola kali (Sal k 5 ADK228421) Olea europaea(Ole e 1 P199632) Fraxinus excelsior (Fra e 1 AAQ835881) Syringa vulgaris (Syr v 1 S43243) and Ligustrum vulgare (Lig v 1 O820152) Theamino acid sequence identity and the similarity ofAma r 1 (KR870437) to othermembers of theOle e 1-like family are shown inTable 2The topline indicates the location of secondary structures that are created by PSIPRED protein sequence analysis (httpbioinfcsuclacukpsipred)The cylinder arrows and black line correspond to alpha-helices beta-strands and coil structure respectively


Table 2 Percentage of similarity and identity between Ama r 1 andselected allergenic Ole e 1-like proteins

Allergenslowast GenBank accession number Ama r 1 Similarity Identity

Che a 1 G8LGR01 95 93Cro s 1 AAX937501 95 91Sal k 5 ADK228421 88 70Ole e 1 ABP586351 61 43Fra e 1 AAQ835881 62 42Lig v 1 O820152 61 40Syr v 1 S43243 60 42lowastChe a 1 (C album) Cro s 1 (C sativus) Sal k 5 (S kali) Ole e 1 (O europaea)Fra e 1 (F excelsior) Syr v 1 (S vulgaris) and Lig v 1 (L vulgare)

970

660

450

300

201

144

(kDa) MW 21

Figure 2 SDS-PAGE and immunoreactivity of A retroflexus pollenextract Lane MW molecular weight marker (GE Healthcare LittleChalfont UK) lane 1 Coomassie Brilliant Blue-stained SDS-PAGEof the crude extract of A retroflexus pollen (125 acrylamide gel)and lane 2 immunoblotting ofA retroflexus pollen extractThe stripwas first blottedwithA retroflexuspollen extract and then incubatedwith pooled sera of patients allergic to A retroflexus (patients 1 2 46 and 8) and detected for IgE reactive protein bands Natural Amar 1 is indicated by white arrow

34 Expression and Purification of Ama r 1 Protein ApET-21b(+)Ama r 1 clone was constructed and confirmed by diges-tion with Not I and Xho I restriction enzymes This recom-binant plasmid was expressed in E coli strain BL21 (DE3)pLysS as a fusion protein with a His

6-tag in the C-terminus

rAma r 1 was present in a soluble form in the supernatantwhere it was further purified by Ni2+ affinity chromatog-raphy to yield purified protein The purified rAma r 1 wasquantified by using Bradfordrsquos protein assay which showedthat approximately 17mg of recombinant protein had beenpurified from 1 L of the bacterial expression medium SDS-PAGE revealed that the apparent molecular weight of thefusion proteinwas about 19 kDa (Figure 3)The allergenicOle

e 1-like protein from A retroflexus pollen as a new allergenwas designated Ama r 1 by the WHOInternational Unionof Immunological Societies (IUIS) Allergen NomenclatureSubcommittee (httpwwwallergenorg)

35 Specific IgE ELISA of rAma r 1 The levels of specific IgEto the purified rAma r 1 were determined using 26 individualpatientsrsquo sera Of the 26 patients 10 (3846) had significantspecific IgE levels to rAma r 1 (Table 1) Serum samples fromthe patients allergic to A retroflexus pollen were furthertested for IgE reactivity to rAma r 1 by immunoblottingassays The results showed that the recombinant form ofAma r 1 was reactive with 10 individualsrsquo sera These resultswere consistent with those obtained by specific IgE ELISA(Table 1)

36 In Vitro Inhibition Assays ELISA inhibition experimentswere performed to evaluate the IgE-binding capacity of thepurified rAma r 1 compared with its natural counterpart inA retroflexus pollen extract The ELISA inhibition resultsrevealed a dose-dependent inhibition of the IgE directedtowards rAma r 1 in patientsrsquo sera positive to A retroflexusPreincubation of pooled serawith 1mgmLof rAma r 1 andAretroflexus pollen extract showed significant inhibition (86and 80 resp) of IgE binding to rAma r 1 inmicroplate wells(Figure 4)

Immunoblot inhibition assays indicated that preincuba-tion of serum samples with rAma r 1 almost completelyinhibited the IgE binding to a protein band with an apparentmolecular weight of 19 kDa (Figure 5 line 3) Altogether invitro inhibition assays showed a similar IgE reactivity forrAma r 1 and its natural counterpart in A retroflexus pollenextract In addition the results indicated that preincubationof serum samples with native crude extract of A retroflexuspollen completely inhibited the IgE binding to natural Amar 1 counterparts in A retroflexus pollen extract and otherreactive proteins (Figure 5 line 2) However preincubationof the pooled sera with BSA did not affect the IgE reactivityto rAma r 1 (Figure 5 line 1)

4 Discussion

A retroflexus is a weed broadly distributed across wastelandsand farms in various climates and it produces such a largequantity of pollen that it has become one of the most aller-genic weeds in different countries throughout the world [1ndash5 10] In this study the cloning and production of the secondallergen of the A retroflexus pollen is reported This allergenwas shown to be a member of Ole e 1-like protein familyand in accordance with the IUIS Allergen NomenclatureSubcommittee was designated as Ama r 1 Several allergensfrom this family such as Sal k 5 Che a 1 Cro s 1 Pla l 1 Syr v 1Lig v 1 and Fra e 1 have been recognised in previous studies[12ndash14 21ndash23]

The open reading frame of Ama r 1 contained a sequenceencoding an 1837 kDa protein related to the molecular spec-ifications of a known plant Ole e 1-like protein family [13 1421] Until now several members of Ole e 1-like protein aller-gens from different plant sources have been reported with


970

660

450

300

201

144 rAma r 1

(kDa) MW 2 31

(a)

NTC

rAma r 1

1 2 3 4 5

6 7 8 9 10

(b)

Figure 3 SDS-PAGE and immunoreactivity of recombinant Ama r 1 (rAma r 1) (a) Lane MW molecular weight marker (GE HealthcareLittle Chalfont UK) lane 1 Coomassie Brilliant Blue-stained SDS-PAGEof soluble fraction of cell culture (IPTG-induced pET-21b(+) withoutinsert) lane 2 rAma r 1 (IPTG-induced pET-21b(+)Aca f 1) in soluble fraction and lane 3 purified rAma r 1 (as an approximately 19 kDarecombinant protein) with Ni-NTA affinity chromatography on 125 acrylamide gel (b) IgE immunoblot of purified rAma r 1 using sera ofpatients with respiratory allergies Lanes 1ndash10 probed with sera from patients with positive for rAma r 1 lane NTC negative control

13

29

50

6575

86

10

32

48

60

70

80

1 1 15 22 28 240

10

20

30

40

50

60

70

80

90

100

Inhi

bitio

n (

)

001 01 1 10 100 1000Concentration (120583gmL)

Recombinant Ama r 1A retroflexus pollen extract

BSA

Figure 4 ELISA inhibition with A retroflexus pollen extract andrAca f 1 Inhibition of IgE binding to rAca f 1 by ELISA using Aretroflexus pollen extract and rAma r 1 Control experiments wereperformed with BSA

various molecular weights such as 1708ndash1762 kDa in twomembers of the Amaranthaceae family (Che a 1 Sal k 5)20 kDa in C sativus pollen (Cro s 1) and 17ndash20 kDa (gly-cosylated and nonglycosylated) in Plantago lanceolata (Pla l1) [13 22 25] These relative disparities in molecular weightmay be due to differences in some amino acid residues levelsof glycosylation or molecular weight measurement methods

970660

450

300

201

144

(kDa) MW 2 31

Figure 5 Immunoblotting inhibition assays Lane MW molecularweight marker (GE Healthcare UK) lane 1 A retroflexus proteinstrip incubated with pooled sera without inhibitor (negative con-trol) lane 2 A retroflexus protein strip incubated with pooled seracontaining 65 120583g of A retroflexus pollen extract as an inhibitor(positive control) and lane 3 A retroflexus protein strip incubatedwith pooled sera containing 10120583g purified rAma r 1 as an inhibitor

MWs methods Ama r 1 like Che a 1 Cro s 1 and Sal k 5 hasa conserved sequence for potential N-linked glycosylation inthe same position of the polypeptide chain (Asn-IleLeu-Thr-Ala) which is actually engaged by a glycan in these proteins


Immunoblotting assays of A retroflexus pollen extractusing pooled sera from the patients also revealed an IgE-binding protein band with an estimated molecular weight of19 kDa (Figure 1) The IgE-binding capability of the purifiedrAma r 1 to sera from patients with A retroflexus allergieswas evaluated by specific ELISA and immunoblotting assaysto confirm that rAma r 1 was correctly folded and bound toIgE as the natural counterpart in A retroflexus extract Thepurified rAma r 1 was recognised in 10 patients allergic to theA retroflexus pollen extract (1026 384)

The results of immunoblotting assays for natural Amar 1 with a molecular weight of 19 kDa were consistent withthose obtained for rAma r 1 A nearly complete inhibitionof IgE-binding to natural Ama r 1 was also obtained afterpreincubation of pooled sera with purified rAma r 1 It seemsthat rAma r 1 is composed of IgE epitopes similar to those ofits natural counterpart

Cross-reactivity between A retroflexus pollen compo-nents and other allergenic members of the Amaranthaceaefamily (S kali C album and Kochia scoparia) and someunrelated allergenic plants such as Acacia farnesiana andProsopis juliflora has been described previously [6 7 17]The present study was conducted to detect the amino acidsequence homology of Ole e 1-like proteins from allergenicregional plants The results of amino acid sequence identityanalysis indicated that Ama r 1 protein has a great degreeof identity with the selected allergenic Ole e 1-like proteinfamily from the most common allergenic regional plantsparticularly C album (Che a 1) C sativus (Cro s 1) and Skali (Sal k 5) (93 91 and 70 resp) Identification of theAma r 1 sequence will warrant further studies on the basisof in vitro assays to investigate the molecular basis of cross-reactivity among these important pollen allergens

5 Conclusion

In conclusion in this study we investigated a new allergenfrom A retroflexus pollen Ama r 1 with a detectably specificIgE in 384of patients allergic toA retroflexus pollen Ama r1 was identified as a member of the Ole e 1-like protein familyIn addition the results demonstrate that rAma r 1 expressedin E coli has immunoreactivity similar to that of the naturalform of the allergen Analysis of the amino acid sequencesof Ama r 1 and several allergenic members of the Ole e1-like protein family from other plants also indicated thatcross-reactivity between plants belongs to unrelated familieswhichmay be predicted by the degree of amino acid sequenceidentity of potential conformational epitopes

Concerning the more prevalent of sensitisation to Aretroflexus pollen and the abundance of it in different coun-tries throughout the world identification and production ofthe recombinant forms of common allergens of this pollenmay lead to the exploration of new guidelines for diagnostictherapeutic and preventive purposes

Disclosure

This paper is issued from the thesis of Mr P Morakabati

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

Financial support was provided by Ahvaz Jundishapur Uni-versity of Medical Sciences (Grant no U-93179)

References

[1] M-A Assarehzadegan A Shakurnia and A Amini ldquoThemostcommon aeroallergens in a tropical region in SouthwesternIranrdquo World Allergy Organization Journal vol 6 no 1 article7 2013

[2] D Ghosh P Chakraborty J Gupta et al ldquoAssociations betweenpollen counts pollutants and asthma-related hospital admis-sions in a high-density indian metropolisrdquo Journal of Asthmavol 49 no 8 pp 792ndash799 2012

[3] K Min M Yoshida R Miike and E Tam ldquoAeroallergen sen-sitivity in Hawaii association with asthma and increased prev-alence of sensitivity to indoor allergens Since 1996rdquo HawaiiJournal of Medicine amp Public Health vol 73 no 9 supplement1 pp 9ndash12 2014

[4] M Villalba R Barderas S Mas C Colas E Batanero andR Rodrıguez ldquoAmaranthaceae pollens review of an emergingallergy in the mediterranean areardquo Journal of InvestigationalAllergology and Clinical Immunology vol 24 no 6 pp 371ndash3812014

[5] M-A Assarehzadegan A H Shakurnia and A Amini ldquoSen-sitization to common aeroallergens among asthmatic patientsin a tropical region affected by dust stormrdquo Journal of MedicalSciences vol 13 no 7 pp 592ndash597 2013

[6] M Tehrani M Sankian M A Assarehzadegan R Falak FJabbari and A Varasteh ldquoImmunochemical characterization ofAmaranthus retroflexus pollen extract extensive cross-reactiveallergenic components among the four species of Amaran-thaceaeChenopodiaceaerdquo Iranian Journal of Allergy Asthmaand Immunology vol 9 no 2 pp 87ndash95 2010

[7] P A Wurtzen H S Nelson H Lowenstein and H IpsenldquoCharacterization of chenopodiales (Amaranthus retroflexusChenopodium album Kochia scoparia Salsola pestifer) pollenallergensrdquo Allergy vol 50 no 6 pp 489ndash497 1995

[8] M Tehrani M Sankian M A Assarehzadegan et al ldquoIdenti-fication of a new allergen from Amaranthus retroflexus pollenAma r 2rdquo Allergology International vol 60 no 3 pp 309ndash3162011

[9] S M Hasnain A R Al-Frayh J L Subiza et al ldquoSensitizationto indigenous pollen and molds and other outdoor and indoorallergens in allergic patients from Saudi Arabia United ArabEmirates and Sudanrdquo World Allergy Organization Journal vol5 no 6 pp 59ndash65 2012

[10] A B Singh and P Dahiya ldquoAntigenic and allergenic propertiesof Amaranthus spinosus pollenmdasha commonly growing weed inIndiardquo Annals of Agriculture and Environmental Medicine vol9 no 2 pp 147ndash151 2002

[11] J de Dios Alche M Mrsquorani-Alaoui A J Castro and M IRodrıguez-Garcıa ldquoOle e 1 the major allergen from olive (Oleaeuropaea L) pollen increases its expression and is released tothe culturemedium during in vitro germinationrdquo Plant and CellPhysiology vol 45 no 9 pp 1149ndash1157 2004


[12] R Barderas M Villalba M Lombardero and R RodrıguezldquoIdentification and characterization of Che a 1 allergen fromChenopodium album pollenrdquo International Archives of Allergyand Immunology vol 127 no 1 pp 47ndash54 2002

[13] L Castro S Mas R Barderas et al ldquoSal k 5 a member ofthe widespread ole e 1-like protein family is a new allergen ofrussian thistle (Salsola kali) pollenrdquo International Archives ofAllergy and Immunology vol 163 no 2 pp 142ndash153 2014

[14] R Barderas A Purohit R Rodrıguez G Pauli andM VillalbaldquoIsolation of the main allergen Fra e 1 from ash (Fraxinusexcelsior) pollen comparison of the natural and recombinantformsrdquo Annals of Allergy Asthma amp Immunology vol 96 no 4pp 557ndash563 2006

[15] E Batanero M A Gonzalez De La Pena M Villalba R IMonsalve M Martin-Esteban and R Rodriguez ldquoIsolationcDNA cloning and expression of Lig v 1 themajor allergen fromprivet pollenrdquo Clinical and Experimental Allergy vol 26 no 12pp 1401ndash1410 1996

[16] E Gonzalez M Villalba and R Rodrıguez ldquoImmunologicalandmolecular characterization of themajor allergens from lilacand privet pollens overproduced in Pichia pastorisrdquoClinical andExperimental Allergy vol 31 no 2 pp 313ndash321 2001

[17] M H Shamsbiranvand A Khodadadi M-A AssarehzadeganS H Borci and A Amini ldquoImmunochemical characterizationof acacia pollen allergens and evaluation of cross-reactivitypattern with the common allergenic pollensrdquo Journal of Allergyvol 2014 Article ID 409056 7 pages 2014

[18] F Zarinhadideh A Amini M A Assarehzadegan S H BorsiN Sepahi and H Ali-Sadeghi ldquoImmunochemical andmolecu-lar characterization of allergenic profilin (Koc s 2) from Kochiascoparia pollenrdquo Journal of the Korean Society for AppliedBiological Chemistry vol 58 no 3 pp 443ndash451 2015

[19] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[20] P Chomczynski and N Sacchi ldquoSingle-step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroformextractionrdquo Analytical Biochemistry vol 162 no 1 pp 156ndash1591987

[21] J A Asturias M C Arilla N Gomez-Bayon J MartınezA Martınez and R Palacios ldquoCloning and expression of thepanallergen profilin and the major allergen (Ole e 1) from olivetree pollenrdquo Journal of Allergy andClinical Immunology vol 100no 3 pp 365ndash372 1997

[22] B N Calabozo A Dıaz-Perales G Salcedo D Barber and FPolo ldquoCloning and expression of biologically active Plantagolanceolata pollen allergen Pla l 1 in the yeast Pichia pastorisrdquoBiochemical Journal vol 372 no 3 pp 889ndash896 2003

[23] A-R Varasteh M Moghadam F Vahedi T Kermani and MSankian ldquoCloning and expression of the allergenCro s 2 profilinfrom saffron (Crocus sativus)rdquoAllergology International vol 58no 3 pp 429ndash435 2009

[24] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[25] R Barderas M Villalba and R Rodrıguez ldquoChe a 1 recombi-nant expression purification and correspondence to the naturalformrdquo International Archives of Allergy and Immunology vol135 no 4 pp 284ndash292 2004


2 Materials and Methods

21 Protein Extraction from A retroflexus Pollen Flowers ofA retroflexus were accumulated from August to October inAhvaz city in the southwest of Iran Gathering of pollenmaterials and handling were performed by trained pollencollectors Floral parts other than pollenwere separated usingthe sieves with different sizes (100 200 and 300 meshes)successively [17]

The final fine powder was subjected to a purity checkfor pollen content using a microscope Pollen materials withmore than 96 pollen and less than 4 of the other parts ofthe same plant were gathered for protein extraction Pollenswere defatted using repeated changes of diethyl ether Forprotein extraction two grams of pollen was mixed with12mL phosphate-buffered saline (PBS) 001M (pH 74) bycontinuous stirring for 16 h at 4∘C The supernatant wasseparated by centrifugation at 13000timesg for 20min andfiltered and the supernatant collected [18]

The protein content of the extract was evaluated usingBradfordrsquos method [19] Finally the extract was freeze-driedand stored at minus20∘C for later use in the present study

22 Patientsrsquo Sera and Skin Prick Test (SPT) In this study weused sera from 26 patients from Ahvaz city southwest IranThe patients were 12 men and 14 women (mean age 2988 plusmn688 years age range 20ndash41 years) with respiratory allergiesand seasonal rhinitis who had positive skin prick test (SPT)results forA retroflexus pollen extract Eight subjects withoutallergies who presented with negative SPTs and no specificIgE to the A retroflexus pollen extract were assigned asnegative controls All patients and control subjects gave ustheir written informed consent to participate Serum samplesof the subjects were prepared and then immediately stored atminus20∘C until used

23 Total IgE and Specific Indirect Enzyme-Linked Immunosor-bent Assays (ELISA) Total serum IgE levels were evaluatedin serum samples in duplicate using a commercially avail-able ELISA kit according to the manufacturerrsquos instructions(Euroimmun Lubeck Germany) To evaluate the levels ofspecific IgE to A retroflexus pollen proteins in patients withrespiratory allergies an indirect ELISA was devised In briefwells of an ELISA microplate (Nunc AS Roskilde Den-mark) were coated with 100 120583Lwell of A retroflexus pollenextract [10 120583gwell in coating buffer (15mMNa

2CO3 35mM

NaHCO3 pH 96)] at 4∘C for overnight After blocking with

a solution composed of 150 120583L of phosphate buffered saline(PBS) and 2 bovine serum albumin (BSA) solution for1 hour at 37∘C the plates were incubated with 100 120583L ofpatientsrsquo sera for 3 hours at room temperature Each wellwas then incubated for 2 hours at room temperature with a1 500 dilution of biotinylated goat anti-human IgE antibody(Nordic-MUbio Susteren Netherlands) in 1 PBSThe wellswere then washed five times with PBS containing 005Tween-20 (T-PBS) to eliminate unbound anti-IgE There-after 100 120583L of horseradish peroxidase- (HRP-) conjugatedstreptavidin (Bio-Rad Laboratories Hercules CA USA) wasplaced in each well and each well was then incubated for

1 hour at room temperature After five washes with T-PBSeach well received 100 120583L of tetramethylbenzidine substratesolution (Sigma-Aldrich St LouisMOUSA) that was placedin eachwell and the plate was incubated at room temperaturefor 20min before the reaction was stopped by addition of100 120583L of 2M HCl Subsequently the absorbance in eachwell was measured at 450 nm using an ELISA plate readerAll results were expressed as optical density (OD) units AnOD value four times higher than the average values of threedeterminations of a pooled sera from negative controls (iegt015 OD units) was considered to be positive

24 Subcloning of Ama r 1 Allergen cDNA and DNA Sequen-cing Total RNA was extracted from 100mg of A retroflexuspollen by using the Chomczynski and Sacchi method[20] The first strand of cDNA was synthesised using theRevertAidTM First Strand cDNA Synthesis Kit (Thermo Sci-entific Waltham MA USA) according to the manufac-turerrsquos instructions The primers used for cDNA amplifi-cation were designed according to the known nucleotidesequence for reported allergens from the Ole e 1-like proteinfamily that have a high degree of amino acid sequenceidentity [12ndash14 21ndash23] These primers include the sense 51015840-ATGGGGAAGTGTCAAGCTGT-31015840 and the antisense 51015840-TTAATTAGCTTTAACATCATAAAGATCC-31015840 The ampli-fied fragment was ligated into a PTZ57RT TA cloning vectorusing the InsTAcloneTM PCR Cloning Kit (Thermo Scien-tific Waltham MA USA) according to the manufacturerrsquosinstructions E coli TOP10 cells (Invitrogen Carlsbad CAUSA) were transformed with the ligation products using themanufacturerrsquos protocol A recombinant plasmidwas selectedbywhiteblue screening and then purified from the gel using aplasmid extraction kit (GeNet Bio Chungnam Korea) DNAsequence analysis was performed using the dideoxy methodat the Bioneer Inc (Daejeon Korea)

The obtained sequence was submitted to the GenBankdatabase of the National Center for Biotechnology Informa-tion (httpwwwncbinlmnihgov) under accession num-ber KR870437

25 Construction of Prokaryote Expression Vector and Pro-duction of Recombinant Ama r 1 (rAma r 1) To create apET-21b(+) expression plasmid (Novagen Gibbstown NJUSA) restriction sites of Not I and Xho I restriction enzymesites were introduced at the 51015840- and 31015840-ends of the Ama r1 cDNA by the polymerase chain reaction (PCR) methodusing two specific primers as follows the sense primer 51015840-TCCgcggccgcATGGGGAAGTGTCAAGCTGT-31015840 (Not Irestriction site is in lowercase) and the antisense primer 51015840-CCctcgagTTAATTAGCTTTAACATCATAAAGATCC-31015840(Xho I restriction site is in lowercase) After PCR amplifi-cation the resulting product was digested with Not I andXho I restriction enzymes according to the manufacturerrsquosprotocol (Thermo Scientific Waltham MA USA) The puri-fied digested PCR product was ligated into the digested pET-21b(+) plasmid with the same enzymes Correct constructswere transformed into competent E coli BL21 (DE3) cells(Novagen Gibbstown NJ USA)





2CO3and 35mM NaHCO

3 pH


450greater




) times 100 (1)




3 Results



221) The mean OD450









AGNAENHKVM

GAGNAENHKVMV







HQ

LMTKDHQVGQIPNSEATVN






K-----

K-----

LKAP-MPGSVTQN





Ama r 1

10 20 30 40 50 60 70

7070704946525151

80 90 100 110 120 130 140

140140140119

119118118

168168168151140146145145

113

150 160 170









970

660

450

300

201

144

(kDa) MW 21









4 Discussion




970

660

450

300

201

144 rAma r 1

(kDa) MW 2 31

(a)

NTC

rAma r 1

1 2 3 4 5

6 7 8 9 10

(b)


13

29

50

6575

86

10

32

48

60

70

80

1 1 15 22 28 240

10

20

30

40

50

60

70

80

90

100

Inhi

bitio

n (

)



BSA



970660

450

300

201

144

(kDa) MW 2 31







5 Conclusion



Disclosure




Acknowledgment


References































2CO3and 35mM NaHCO

3 pH


450greater




) times 100 (1)




3 Results



221) The mean OD450









AGNAENHKVM

GAGNAENHKVMV







HQ

LMTKDHQVGQIPNSEATVN






K-----

K-----

LKAP-MPGSVTQN





Ama r 1

10 20 30 40 50 60 70

7070704946525151

80 90 100 110 120 130 140

140140140119

119118118

168168168151140146145145

113

150 160 170









970

660

450

300

201

144

(kDa) MW 21









4 Discussion




970

660

450

300

201

144 rAma r 1

(kDa) MW 2 31

(a)

NTC

rAma r 1

1 2 3 4 5

6 7 8 9 10

(b)


13

29

50

6575

86

10

32

48

60

70

80

1 1 15 22 28 240

10

20

30

40

50

60

70

80

90

100

Inhi

bitio

n (

)



BSA



970660

450

300

201

144

(kDa) MW 2 31







5 Conclusion



Disclosure




Acknowledgment


References
































AGNAENHKVM

GAGNAENHKVMV







HQ

LMTKDHQVGQIPNSEATVN






K-----

K-----

LKAP-MPGSVTQN





Ama r 1

10 20 30 40 50 60 70

7070704946525151

80 90 100 110 120 130 140

140140140119

119118118

168168168151140146145145

113

150 160 170









970

660

450

300

201

144

(kDa) MW 21









4 Discussion




970

660

450

300

201

144 rAma r 1

(kDa) MW 2 31

(a)

NTC

rAma r 1

1 2 3 4 5

6 7 8 9 10

(b)


13

29

50

6575

86

10

32

48

60

70

80

1 1 15 22 28 240

10

20

30

40

50

60

70

80

90

100

Inhi

bitio

n (

)



BSA



970660

450

300

201

144

(kDa) MW 2 31







5 Conclusion



Disclosure




Acknowledgment


References































970

660

450

300

201

144

(kDa) MW 21









4 Discussion




970

660

450

300

201

144 rAma r 1

(kDa) MW 2 31

(a)

NTC

rAma r 1

1 2 3 4 5

6 7 8 9 10

(b)


13

29

50

6575

86

10

32

48

60

70

80

1 1 15 22 28 240

10

20

30

40

50

60

70

80

90

100

Inhi

bitio

n (

)



BSA



970660

450

300

201

144

(kDa) MW 2 31







5 Conclusion



Disclosure




Acknowledgment


References




























970

660

450

300

201

144 rAma r 1

(kDa) MW 2 31

(a)

NTC

rAma r 1

1 2 3 4 5

6 7 8 9 10

(b)


13

29

50

6575

86

10

32

48

60

70

80

1 1 15 22 28 240

10

20

30

40

50

60

70

80

90

100

Inhi

bitio

n (

)



BSA



970660

450

300

201

144

(kDa) MW 2 31







5 Conclusion



Disclosure




Acknowledgment


References































5 Conclusion



Disclosure




Acknowledgment


References



























cloning and expression of ama r 1, as a novel …...cloning and expression of ama r 1, as a novel...

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