Cloning, expression, and analysis of the group 2 allergen from Dermatophagoides farinae from China

To obtain the recombinant group 2 allergen product of Dermatophagoides farinae (Der f 2), the Der f 2 gene was synthesized by RT-PCR. The full-length cDNA comprised 441 nucleotides and was 99.3% identical to the reference sequence (GenBank AB195580). The cDNA was bound to vector pET28a to construct plasmid pET28a(+)-Der f 2, which was transformed into E. coli BL21 and induced by IPTG. SDS-PAGE showed a specific band of about 14kDa in the whole cell lysate. As estimated by chromatography, about 3.86 mg of the recombinant product was obtained, which conjugated with serum IgE from asthmatic children. The protein had a signal peptide of 17 amino acids. Its secondary structure comprised an alpha helix (19.86%), an extended strand (30.82%), and a random coil (49.32%). The subcellular localization of this allergen was predicted to be at mitochondria. Furthermore, its function was shown to be associated with an MD-2-related lipid-recognition (ML) domain. The results of this study provide a solid foundation for large-scale production of the allergen for clinical diagnosis and treatment of allergic disorders.


INTRODUCTION
Dust mites have been recognized to be associated with the rising incidence of asthma and other allergic diseases (Milián andDíaz 2004, Nadchatram 2005).At present, mite extract is used in clinical diagnosis and treatment of allergic diseases, especially in developing countries.However, the extract is a complex mixture of allergens, non-allergic or toxic proteins, and some enzymes, which have been reported to be associated with flushing, swelling, induration, necrosis and other local reaction, shock, laryngeal edema, bronchospasm, urticaria, angioedema, generalized erythema, and other systemic side effects (Mauro et al. 2006, Zhang et al. 2009).Over the past 10 years, some groups of mite allergens have been cloned, sequenced, expressed and purified.
The three-dimensional structures of some allergens were determined, and B and T-cell epitopes were mapped (Jeong et al. 2006, Mothes et al. 2006).The recombinant allergens were produced in bacteria, yeasts and insect cell expression systems, and have been demonstrated to have good IgE binding activity similar to their natural counterparts.They have also shown good effects in skin prick tests and other allergen diagnosis methods (Jeong et al. 2006, Mothes et al. 2006).These recombinant allergens are intended to replace natural allergens and to allow the development of new immunotherapy projects, including the use of hypoallergens, allergens coupled to IgE suppressive adjuvants, and peptide-based therapies (Dinakar andPortnoy 2004, Linhart andValenta 2005).
Polymorphisms have been reported in dust mite allergens.Therefore, recombinant allergens should be produced according to thorough study of the preval-An Acad Bras Cienc (2010) 82 (4) 942 YU-BAO CUI, YING ZHOU, WEIHONG SHI, GUIFANG MA, LI YANG and YUNGANG WANG ent allergens in different regions (Linhart and Valenta 2005).In our previous study, we obtained the gene encoding the group 2 allergen from Dermatophagoides farinae (Der f 2).An additional region of 87 bp (from 77 to 163 bp) was present in our strain that was absent from the reference sequence (GenBank AB195580) (Cui et al. 2007).We attempted to express this gene in Escherichia coli (E.coli) with no success.We, then, isolated total RNA from Dermatophagoides farinae again, and synthesized the cDNA encoding Der f 2 by RT-PCR.We obtained a Der f 2 gene that was identical in length to the reference sequence, and successfully expressed it in E. coli.

PATIENT SERA
Five asthmatic children were involved in this study.They were demonstrated to be allergic to dust mites by the skin prick test with Dermatophagoides farinae extract provided by the Department of Parasitology, Medical College of Fudan University.Blood (5 ml) was extracted from each child and centrifuged for serum.We mixed five sera samples and treated the mixture as the positive control in western-blotting.On the other hand, umbilical serum samples were taken from five children whose mothers had no atopic history, centrifuged, mixed, and used as negative controls.
5 μl of pET28a (+)-Der f 2 plasmid were purified using the MiniBEST Plasmid Purification Kit Ver.2.0 (TaKa-Ra Biotech Co. Led, Code No. DV801A) and transformed into 100 μl of E. coli BL21 (DE3, Stratagene, USA).E. coli BL21 carrying pET28a (+)-Der f 2 was grown on LB plates containing 50 μg/ml of kanamycin at 37 • C overnight.A single colony was inoculated into 2 ml LB plates at 37 • C overnight, 100 μl of which was taken and added into a glass tube containing 5 ml LB fluid containing kanamycin and, then, cultured at 37 • C. 100 mM of isopropyl-β-D-thiogalactopyranoside (IPTG, 50 μl, final 1 mM) was added to induce the lac promoter.The E. coli cells were harvested by centrifugation, and PBS Buffer (200 μl/tube) was added in.After centrifugation and resuspension, ultrasonic disruption was used until the obtention of a transparent fluid, 50 μl of which was taken as the whole cell lysate.The lysate was centrifuged and the supernatant and pellet were regarded as the soluble and insoluble protein samples, respectively.The whole cell lysate (10 μl), soluble and insoluble protein samples were taken individually, and 2.5 μl of 5× SDS sample buffer were added into each of them.The samples were heated for 10 min at 95 • C and subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 12.5% of polyacrylamide gel and CBB-R250 staining.
ISOLATION, PURIFICATION, AND RENATURATION OF RECOMBINANT RDER F 2 PROTEINS E. coli BL21 carrying pET28a (+)-Der f 2 was grown on LB plates containing 50 μg/ml of kanamycin at 37 • C overnight.A single colony inoculated into 5 ml of LB fluid with shaking at 250 rpm overnight at 37 • C was treated as the seed fluid for fermentation.50 ml of the seed fluid were inoculated into 2000 ml LB fluid containing 50 μg/ml of kanamycin at 30 • C shaking at 250 rpm for fermentation.100 mM IPTG were added until the final concentration reached 1 mmol/l.The induced cells were collected, suspended in PBS (pH 7.4), and broken by ultrasonic waves in an ice bath until the fluid became transparent.This fluid with broken cells was centrifuged at 7000 rpm at 4 • C for 15 min to collect rough inclusion bodies, which were then washed in PBS buffer containing 2 mol/l of urea for the purified product.The purified inclusion bodies were resuspended in PBS buffer (pH 8.0) containing 8 mol/L of urea and 5 mmol/l of DTT, stirred slightly at 4 • C overnight, and centrifuged at 1000 rpm for 10 min.The supernatant was filtered by microfiltration membrane with a pore size of 0.22 μm.This protein product was further purified by nickel-affinity chromatography at a speed of 4.0 ml/min, eluted with 25, 50, 75, 100, 125, and 150 mmol/L of imidazole solution at a speed of 6 ml/min.The elution peak at each stage was collected and determined for molecular weight and purity by SDS-PAGE.All of the elution fractions were freeze-dried for protein power.Lastly, the product was re-natured by gradient dialysis with a renaturation solution containing 2 mol/l to 0.2 mol/L of urea, and 0.2 mol/l of EDTA continuously.

WESTERN BLOTTING
For western blotting analysis, proteins were transferred to a PVDF membrane (TIANGEN, Biotech Co. Led, Beijing, China) and incubated with primary antibodies kept in the sera from asthmatic children allergic to dust mites at 4 • C overnight.They were, then, incubated with a secondary antibody (Horse anti-human IgE, AP conjugated, ZYMED Laboratories, USA) for 1 hr at room temperature, and the proteins were visualized using 3 ml of BCIP/NBT (Roche, Switzerland).

NUCLEOTIDE SEQUENCE, INFERRED AMINO ACID SEQUENCE, STRUCTURAL AND FUNCTION ANALYSIS, ALIGNMENT AND PHYLOGENY
Sequences were edited to remove the vector sequence and the extra restriction sites.The open reading fragment (ORF) was obtained using the ORF finder in the NCBI (National Center for Biotechnology Information) website.The amino acid sequence of Der f 2 was deduced using Translate Tools, and its physiochemical properties were predicted by ProtParam Tools in ExPaSy web server.The signal peptide of Der f 2 was analyzed by the SignalP 3.0 server.The functional site was searched for in the InterPro database in EBI (European Bioinformatics Institute) website.The subcellular localization for Der f 2 was predicted by CELLO v.2.5, and its secondary structure was analyzed by GOR4.0 software.The published sequences that matched our sequenced cDNA were obtained from GenBank using BLAST.The published sequences of ticks and mites were chosen for alignment using ClustalW2 at the EBI website, and computed for similarity by VECTOR NTI 9.0 software (IBI, New Haven, CT, USA).A phylogenetic tree was constructed with maximum parsimony methods in Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0 software.

CLONING AND SEQUENCING OF CDNA ENCODING DER F 2
Total RNA was isolated from adult mites, and Der f 2 cDNA fragments were amplified by RT-PCR, obtaining a 441 bp product (Fig. 1A).The recovered PCR product was cloned into vector pMD19-T and transformed into E. coli competent cells (JM109) and positive clones.The product was confirmed by restriction enzyme digestion and automatic DNA sequencing (Fig. 1B, the vector sequence and the added restriction sites were removed from the sequencing results).The sequencing results showed that our Der f 2 cDNA is 99.3% identical to the previously published sequence (GenBank AB195580).There is one mutation at position 328 (G>A) resulting in amino acid mutation from Aspartate to Asparagine, and the other two mutations at positions 382 (G>A) and 384 (T>C) resulting in amino acid mutation from Valine to Isoleucine.Using the ORF Finder, a complete ORF was found within the Der f 2 cDNA; its length is 441 bp from the start codon ATG to the stop codon TGA (Fig. 1B).
EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEIN DER F 2 The cDNA encoding Der f 2 allergen was excised from recombinant plasmid pMD19-T-Der f 2, sub-cloned into expression vector of pET28a (+) and confirmed by restriction analysis (Fig. 2A).E. coli BL21 was transformed with plasmid pET28a (+)-Der f 2, and protein expression was induced with IPTG.We observed a single band of about 14 kDa on SDS-PAGE, which agreed with the predicted molecular weight of 14.076 kDa for the Der f 2 protein (Fig. 2B).E. coli BL21 carrying pET28a (+)-Der f 2 was recovered from a 2000 ml of fermentation solution, collected, ultrasonically disrupted, and centrifuged.The precipitate was washed, dissolved and filtered.The supernatant was purified by nickel-affinity chromatography and eluted with imidazole solution, and the pure protein presented in 25, 50, 75, 100, and 125 mmol/l of elution fluid was shown by SDS-PAGE (Fig. 2C).These elution fractions were collected and freeze-dried, and 3.86 mg of protein power was obtained.The recombinant protein can bind the IgE antibody in asthmatic children by western-blotting (Fig. 2D), which demonstrated that the expression product has good allergenicity.To identify the physiochemical properties of the recombinant protein, the sequenced result was translated into an amino acid sequence, which should encode a protein comprising 146 amino acids with a 17 amino acid signal peptide analyzed by SignalP 3.0 software.After the removal of the signal peptide sequence, the mature allergen comprised 129 amino acids residues with a relative molecular weight of 14.076, an isoelectric point of 6.46, a molar extinction coefficient of 8970, and an instability index of 21.31.The complete pro-protein was computed for a Grand average of hydropathicity (GRAVY) An Acad Bras Cienc (2010) 82 (4) (Broad); Lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a; Lane 2, the supernatant of cells containing pET28a; Lane 3, the pellet of cells containing pET28a; Lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a (+)-Der f 2; Lane 5, the supernatant of cells containing pET28a (+)-Der f 2; Lane 6, the pellet of cells containing pET28a (+)-Der f 2. Arrows indicate the rDer f 2 band.(C) SDS-PAGE analysis of purified rDer f 2 protein from E. coli BL21 cells.Lane M: TaKaRa Protein Marker (Broad); Lane 1, 2, 3, 4, 5, 6, eluted fraction with 25, 50, 75, 100, 125 and 150 mmol/L imidazole elution buffer, respectively.(D) Western blotting analysis of rDer f 2 in E. coli BL21 cells.Lane 1, the reaction result between rDer f 2 and negative serum; Lane 2, the reaction result between rDer f 2 and positive serum; Lane M 1 , Precision Plus Protein Standards; Lane M 2 , perfect protein marker. of 0.105 by ProtParam Tools, which indicated that it should be hydrophilic.With regard to Der f 2 secondary structure, GOR4 predicted that 19.86% (29 aa) of the protein were in alpha helices, 30.82% (45 aa) in extended strands, and 49.32% (72 aa) in random coils (Fig. 3).Using CELLO v.2.5, the subcellular localization of Der f 2 from China was concluded to be mitochondrial (Table I).A MD-2-related lipid-recognition (ML) domain from residue 22 to 141 was found in the Der f 2 pro-protein by InterProScan.

DISCUSSION
More than 30 groups of allergens were found in mite extracts, including enzymes, enzyme inhibitors, and structural proteins, which were named according to the order in which they were discovered.Group 2 allergens are regarded as a major cause of mite allergy (Thomas et al. 2002(Thomas et al. , 2004)).In this study, we obtained a Der f 2 cDNA fragment with 99.3% identity with the reference sequence, by conventional RT-PCR.When the plasmid pET28a (+)-Der f 2 was transformed into E. coli BL21, the obvious bands of about 14 kDa were observed in the whole cell lysate and the pellet of cells on SDS-PAGE, which agreed with the predicted result and suggest that the prokaryotic expression system for Der f 2 was successfully developed, as well as the product presented in the inclusion bodies.The expression vector PET28a(+), with a total length of 5369bp, expressed the recombinant protein containing the six-histidine tail, which bound to the nickel resin and could be eluted with a chelating agent after other proteins were removed with the appropriate buffers.The single specific reaction band of about 14 kDa was present in the 25, 50, 75, 100, and 125 mmol/l of imidazole elution fractions on SDS-PAGE.Furthermore, the purified recombinant allergen could bind to IgE in sera from asthmatic children, which showed that the recombinant protein had good allergenicity and was a good foundation for the production of genetic allergens.The cDNA sequence for Der f 2 was translated  into an amino acid sequence, and the signal peptide sequence was deduced at position 1 to 17.After the removal of the signal peptide, the mature protein was predicted to be a hydrophilic protein of 129 residues and 14.076 kDa of molecular weight.Interestingly, the subcellular location for this protein was predicted to be mitochondrial according to its N-peptide, physicochemical and neighboring sequence compositions, by CELLO2.5.Additionally, the group 2 allergen was predicted to have a MD-2-related lipid-recognition (ML) domain, which is associated with lipid recognition, particularly with the recognition of pathogen-related products.The ML domain has an immunoglobulin-like betasandwich fold similar to that of E-set Ig domains.Many studies have been published concerning house dust mites, which belong to the family Pyroglyphidae, especially Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Euroglyphus maynei.Other mite species, referred to as "storage mites" because of their consumption of stored food, are also considered allergenic, although their study is more limited (Fernández-Caldas et al. 2007).We used the deduced amino acid sequence of Der f 2 from China to search the protein databases at NCBI using BLASTp.Similar amino acid sequences of other mite species were identified, including D. siboney, D. pteronyssinus and Euroglyphus maynei listed in Pyroglyphidae; Lepidoglyphus destructor and Blomia tropicalis in Glycyphagidae; Suidasia medanensis, Aleuroglyphus ovatus, Tyrophagus putrescentiae and Acarus siro in Acaridae.Besides Dermatophagoides spp.other mite-species, especially Blomia tropicalis due to its abundance in tropical and subtropical regions, were previously considered to be important from economic and sanitary perspectives, but are now being recognized as important contributors to the allergen content in house dust.They can cause occupational respiratory allergies in farmers and other occupationally exposed individuals (Nadchatram 2005).Many of these allergens have shown sequence homology and biological functional similarity to those described in Dermatophagoides spp.The amino acid sequence similarity between the group 2 allergen of D. farinae and that of D. pteronyssinus, D. siboney and E. maynei were calculated at 88%, 96%, and 83%, respectively, in this paper.In the phylogenetic tree constructed by Mega 4.0, D. farinae, D. pteronyssinus, D. siboney and E. maynei were clustered with 100% of bootstrap values, which agreed with the present taxonomy.D. farinae and D. siboney were clustered with 59% of bootstrap values, and D. pteronyssinus and Euroglyphus maynei with 63%.Generally, if the bootstrap value is below 70%, the relationship in the phylogenetic tree will be out of accord with the real taxonomy.In our previous report (Cui et al. 2008), Der p1 shared more than 87% identity in an amino acid sequence with Eur m1, but only 80% with Der f 1. D. pteronyssinus was evolutionarily closer to E. maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same genus (Dermatophagoides) in the phylogenetic tree constructed for the group 1 allergens from different mite-species.Therefore, the relationship among the common mite-species should be investigated using much more molecular data.

ACKNOWLEDGMENTS
This work was supported by National Sciences Foundation of China (NSFC 30060166), and Health Department of Jiangsu Province in China (Grant Number: Z200914 and J200907).

Fig. 1 -
Fig. 1 -Cloning and sequencing of cDNA encoding Der f 2. (A) Amplification of the cDNA encoding for Der f 2 protein by RT-PCR.Total RNA was isolated from mites using RNA isolator and subjected to RT-PCR as described in "Materials and Methods".The PCR products were separated on a 1% agarose gel containing ethidium bromide.Lane M, DNA Marker DL2, 000; Lane 1, PCR product (441bp).(B) Sequencing results for the recombinant plasmid pMD19-T-Der f 2. Being compared with the reference sequence (GenBank AB195580), there are three mutations at positions 328 (G>A), 382 (G>A) and 384 (T>C), which were marked with an arrow.