Molecular cloning, expression and first antigenic characterization of human astrovirus VP26 structural protein and a C-terminal deleted form

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Comparative Immunology, Microbiology and Infectious Diseases 33 (2010) 1–14 www.elsevier.com/locate/cimid

Molecular cloning, expression and first antigenic characterization of human astrovirus VP26 structural protein and a C-terminal deleted form Enrique Royuela a,b,d,*, Alicia Sa´nchez-Fauquier a,c a

Centro Nacional de Microbiologı´a (CNM), Instituto de Salud Carlos III (ISCIII), 28220 Madrid, Spain b Unidad de Aislamiento y Deteccio´n de Virus, Servicio de Microbiologı´a Diagno´stica, Spain c Seccio´n de Virus Productores de Gastroenteritis, Servicio de Virologı´a, Spain d Centro de Investigaciones Biome´dicas en Red (CIBER) en Epidemiologı´a y Salud Pu´blica, Spain Accepted 12 July 2008

Abstract The open reading frame 2 (ORF2) of human astrovirus (HAstV) encodes the structural VP26 protein that seems to be the main antigenic viral protein. However, its functional role remains unclear. Bioinformatic predictions revealed that VP29 and VP26 proteins could be involved in virus–cell interaction. In this study, we describe for the first time the cloning and expression in Escherichia coli (E. coli) of a recombinant VP26 (rVP26) protein and a VP26 C-terminal truncated form (VP26DC), followed by purification by NTA-Ni2+ agarose affinity chromatography. Protein expression and purification were evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) and Western blot (WB). Then, the purified proteins were evaluated for antigenic properties in Abbreviations: HAstV, human astrovirus; ORF, open reading frame; HAstV-2, human astrovirus serotype 2; E. coli, Escherichia coli; rVP26, VP26 recombinant protein; VP26DC, VP26 C-terminal truncated protein; SDSPAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; WB, western blotting; ELISA, enzyme linked immunosorbent assay; nMAb, neutralizing monoclonal antibody; PAb, polyclonal antibody; ATCC, American Type Collection Culture; LB, Luria-Bertoni medium; CDC, centers for disease control and prevention; LLCMK2, Rhesus monkey kidney cell line; R, arginine; RT, retrotranscription; PCR, polymerase chain reaction; IPTG, isopropyl b-D-thiogalactoside; OD, optical density; PBS, phosphate buffer saline; PBST, phosphate buffer saline tween-20; OPD, O-phenylenediamine; bp, base pairs; kb, kilo bases; kDa, kilo daltons * Corresponding author at: Unidad de Aislamiento y Detección de Virus, Servicio de Microbiología Diagnóstica, Centro Nacional de Microbiología (CNM), Instituto de Salud Carlos III (ISCIII), Carretera MajadahondaPozuelo, Km 2, 28220 Majadahonda, Madrid, Spain. Tel.: +34 918223682; fax: +34 915097919. E-mail address: [email protected] (E. Royuela). 0147-9571/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2008.07.010

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enzyme linked immunosorbent assay (ELISA) using a polyclonal antibody (PAb) and a neutralizing monoclonal antibody (nMAb) named PL2, both of them directed to HAstV. The results presented herein indicate that the C-terminal end of the VP26 protein is essential to maintain the neutralizing epitope recognized by nMAb PL2 and that the N-terminus of VP26 protein may contain antigenic lineal-epitopes recognized by PAb. Thus, these recombinant proteins can be ideal tools for further antigenic, biochemical, structural and functional VP26 protein characterization, in order to evaluate its potential role in immunodiagnosis and vaccine studies. # 2008 Elsevier Ltd. All rights reserved.

Résumé Le cadre de lecture ouvert (ORF2) de l’astrovirus humain (AstVH) codifie la protéine structurale VP26 qui semble être la principale protéine antigénique virale. Par contre, son rôle fonctionnel n’est pas clair. Les prédictions bioinformatiques montrent que les protéines VP29 et VP26 pourraient être impliquées dans l’interaction virus cellule. Dans cet étude, on décrit pour la première fois le clonage et l’expression à Escherichia coli (E. coli) d’une forme recombinante (rVP26) et d’une autre tronquée (VP26DC) de la protéine VP26, suivi de l’épuration par chromatographie d ‘affinité en Ni2+ - NTA agarose. L’ expression des protéines et sa épuration ont été évaluées par SDS-PAGE et Western blot (WB). Après, les propiétés antigéniques des protéines épurées ont été évaluées par ELISA en employant un anticorps polyclonal (PAb) et un autre monoclonal neutralissant (nMAb) appellé PL2, les deux dirigés vers HAstV. Les résultats qu’on présente ici indiquent que le côté C terminal de la protéine VP26 est essentiel pour la maintenance de l’epitope neutralisant reconnue par PAb. Dans le cas du côté N terminal de la même protéine, il pourrait contenir des epitopes linéaires antigéniques reconnus par PAb. Ainsi, ces protéines recombinantes pourraient être des outils idéaux pour d’autres caractérisations antigéniques, biochimiques, structurales et fonctionnelles de la protéine VP2 qui évaluent leur rôle potentiel dans l’immunodiagnostic et les études de vaccins. # 2008 Elsevier Ltd. All rights reserved. Keywords: Human astrovirus (HAstV); VP26; Structural protein; Recombinant protein; Molecular cloning; Expression; purification; Antigenic characterization; Enzyme linked immunosorbent assay (ELISA); Western blot (WB); Immunodiagnosis; VaccineMots cle´s : Astrovirus humain (AstVH) ; VP26 ; Protéine recombinante, Clonage moléculaire ; Expression ; Caractérisation antigénique ; Essai d’un immunosorbent lié aux enzymes (ELISA) ; Western blot (WB) ; Immunodiagnostic ; Vaccine

1. Introduction Astroviruses are nonenveloped, icosahedral RNA viral particles with radiating spikes around their surface [1]. Human astrovirus (HAstV) are detected as a common cause of viral acute gastroenteritis worldwide [2–4]. The genome of HAstV is composed of an approximately 7-kilobases (kb) single-stranded positive-sense RNA molecule, organized in three ORFs (Fig. 1). The structural proteins are encoded by the open reading frame 2 (ORF2), located in the C-terminus, that is not solely encoded by genomic RNA but also by a 2.4 kb subgenomic RNA produced during viral infection [5,6]. VP32, VP29 and VP26 (32, 29 and 26-kilodaltons (kDa), respectively) are the mature structural proteins in HAstV serotype 2 (HAstV-2) particles [7], directly obtained from an 87 kDa capsid precursor

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Fig. 1. Genome organization of HAstV. A proteolytic scheme of structural proteins encoded by the ORF2 from HAstV-2 is represented. The NH2-terminal processing sites of VP29 and VP26 proteins and the cleaved products from p87 polyprotein precursor are indicated.

(p87) [8]. While research on molecular epidemiology of HAstV has advanced over the years, relatively little is known concerning the structural, functional and immunogenic role of the structural proteins. It has been reported that the capsid precursor encoded by ORF2 contains neutralizing and immunoreactive epitopes [7,9–12]. Furthermore, the implication of the viral capsid in the pathogenicity of HAstV by increasing the epithelial barrier permeability has been recently described [13]. In agreement with the above-mentioned data, bioinformatic predictions of the ORF2 aminoacid sequence revealed that the Nterminal end of capsid precursor (corresponding to the VP32 domain) may have a function in viral assembly, while, the C-terminus (corresponding to VP29 and VP26 proteins) could be the cell receptor-interaction domain [14]. Since the VP26 protein is a major structural viral component of the HAstV and it seems to be the main antigenic protein [7], details about its antigenicity could enhance our understanding of pathogenesis and immunogenesis. Methodology for cloning and expression of recombinant proteins in Escherichia coli (E. coli) has been widely used to obtain both structural [15–17] and non-structural proteins [18–20] from several organisms in order to characterize their biological properties. In our study we have cloned, for the first time, the gene encoding VP26 protein into pQE32 expression vector to obtain large amounts of recombinant VP26 (rVP26) protein in E. coli. This allowed us to analyze its antigenic features in the absence of any other viral structural proteins. Moreover, to analyze the biological significance and viral antigenicity of the C-terminus of VP26, which is shared with VP29 structural protein, we generated a Cterminus deletion form of VP26 protein (VP26DC). The VP26 sequence was deleted at the 30 end of VP26 gene by antisense primer utilizing polymerase chain reaction (PCR). After cloning, expressed proteins were purified and primary antigenicity characterized, showing an importance of VP26 C-terminal domain in maintenance of conformational epitope and the implication of the N-terminal end in antigenic recognition.

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2. Materials and methods 2.1. Biochemical reagents The restriction enzymes BamHI and HindIII were purchased from Fermentas (Fermentas Life Sciences, Fermentas Inc., Hanover, USA). T4 DNA Ligase was purchased from New England Biolabs (New England Biolabs, New England Biolabs Inc., MA, USA). NTA-Ni2+ agarose, polypropylene columns as well as the pQE32 plasmid were purchased from Qiagen (Qiagen GmbH, Hilden, Germany). The Dye Terminator Cycle Sequencing kit was purchased from Applied Biosystems (Applied Biosystems, Foster City, USA). 2.2. Culture media and bacteria E. coli DH5a cells were provided by American Type Collection Culture (ATCC) (Reference 53868). This bacterial strain was cultured on Luria-Bertoni (LB) medium (10 g/l tryptone, 5 g/l yeast extract and 10 g/l NaCl) at 37 8C for 24 h. The transformants were selected on LB-agar plates containing ampicillin (100 mg/ml) at 37 8C for 24 h. E. coli DH5a cells were used for propagation of pQE32 plasmid and recombinant constructions. E. coli M15 bacterial strain was purchased from Qiagen (Qiagen GmbH, Hilden, Germany) and it was cultured on LB medium at 37 8C for 24 h. The transformants were selected on LB-agar plates containing both ampicillin (100 mg/ml) and kanamycin (25 mg/ ml) at 37 8C for 24 h. E. coli M15 strain was chosen as the host for gene expression. Both strains were conserved in LB medium including 20% glycerol at 80 8C. 2.3. Virus and cell lines HAstV-2 was provided by Centers for Disease Control and Prevention (CDC, Atlanta, USA). HAstV-2 was propagated in the presence of 10 mg/ml of trypsin (Sigma, Sigma– Aldrich Chemie GmbH, Munich, Germany) in a Rhesus monkey kidney cell line (LLCMK2 cells) as previously described [21,22]. LLCMK2 cells were provided by ATCC (Reference CCL 7.1). 2.4. Antibodies A polyclonal antibody (PAb) directed to HAstV was used in enzyme linked immunosorbent assay (ELISA) and Western blot (WB) for detecting rVP26, VP26DC and HAstV particles. The protocol for generation of the PAb and its characterization have been described elsewhere [7,8]. The use of neutralizing monoclonal antibody (nMAb) PL2 in ELISA assays to characterize the antigenic properties of rVP26 and VP26DC proteins has also been described elsewhere [7,8]. The anti-mouse-horseradish peroxidase (HRPO) conjugated (anti-mouse-HRPO) and anti-rabbit-HRPO conjugated secondary antibodies were purchased from GE Healthcare

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(GE Healthcare Limited, Buckinghamshire, United Kingdom) and they were applied following the manufacturer’s instructions. 2.5. RNA extraction and construction of VP26 expression vectors Genomic RNA from HAstV-2 was extracted using a MagNA Pure LC instrument (Roche, F. Hoffmann-La Roche, Basel, Switzerland) with a MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche, F. Hoffmann-La Roche, Basel, Switzerland), following the manufacturer’s instructions. Extracted RNA was stored at 20 8C before use. The sequence of subgenomic RNA from HAstV-2 was used as template to design specific oligonucleotides for amplification of complete VP26 (rVP26 protein) and 30 end truncated VP26 (VP26DC protein) genes. The subgenomic RNA sequence was retrieved from Genbank (accession number L06802). Primers designed for cloning were evaluated for possible primer-dimers formations and/ or self-complementary formations using PrimerSelect software (DNASTAR, Inc., Madison, WI, USA). The theoretical primer melting temperatures were calculated using the Oligo Calculator programme (http://www.pitt.edu/rsup/OligoCalc.html). The pQE32 plasmid was used for cloning VP26 gene and VP26 30 deleted form fused to a sequence encoding a 6-histidines tag. The expressed recombinant proteins were purified using NTA-Ni2+ agarose affinity chromatography for further protein characterization. VP26 complete gene encodes aminoacids from Arginine(R)394 to R648 of ORF2. The cloning techniques were applied following the literature methods [23]. The amplification of VP26 gene was performed using extracted genomic RNA from HAstV-2 as template and the primers FVP26: 50 -GAGAGAGAGGATCCCGTACCACACCGGTAACAAC-30 , containing BamHI site, and RVP26: 50 -GAGAGAGAAAGCTTTCTGGGCAATGTGGTGTTAC-30 , containing HindIII site. Retrotranscription (RT) polymerase chain reaction assay was carried out with Qiagen One Step RT-PCR kit (Qiagen GmbH, Hilden, Germany) according to manufacturer’s instructions. A reaction mix total volume of 50 ml consisted of 5 ml of buffer 5, 10 mM of dNTP mix, 10 pmol of each primer and 2 ml of enzyme mix. The RT step was carried out for 45 min at 50 8C, followed by denaturation for 2 min at 95 8C and 40 cycles of PCR, denaturation for 1 min at 95 8C, annealing for 1 min at 59 8C and extension for 1 min at 72 8C; and a final extension step for 7min at 72 8C. The VP26DC gene encodes aminoacids from R394 to R583 of ORF2. The amplification of the deleted VP26 gene was performed using genomic RNA from HAstV-2 as template and the primers FVP26: 50 -GAGAGAGAGGATCCCGTACCACACCGGTAACAAC-30 , containing BamHI site, and RVP26DC 50 -GAGAGAGAAAGCTTTTTGAATTGCTTCATGAAG-30 , containing HindIII site. The RT-PCR assay and programme was carried out following the protocol described above. This deleted gene encodes a VP26 protein lacking its C-terminal domain. Five microliters of each PCR amplification product were analyzed by 2% agarose gel electrophoresis in the presence of ethidium bromide and visualized under ultraviolet light. The RT-PCR products were digested with BamHI and HindIII restriction enzymes and inserted into the corresponding BamHI/HindIII cloning site of the digested pQE32 vector to form pQE32-VP26 and pQE32-VP26DC recombinant constructions. Both bacterial E. coli M15 and DH5a strains were transformed by heat shock with the recombinant

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plasmids, then, the selected transformants were screened by enzyme digestion assay excising the VP26 and VP26DC inserts from pQE32-VP26 or pQE32-VP26DC constructions, respectively. Finally, the selected bacterial transformants were further screened using PCR techniques for amplifying VP26 or VP26DC genes. 2.6. Automatic sequencing The nucleotide sequence of the cloned fragment was confirmed with the Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, USA) and ABI Prism 3700 DNA sequencer (Applied Biosystems, Foster City, USA) by the Genomic Unit at National Center of Microbiology (from ISCIII). Then, the sequences were corrected and assembled using DNASTAR’s Seqman Genome Assembler to corroborate the correct nucleotide sequence. 2.7. Culture conditions and affinity chromatography purification E. coli M15 transformed cells were grown in 50 ml of LB medium containing ampicillin (100 mg/ml) and kanamycin (25 mg/ml) at 37 8C for 24 h. An aliquot of 5 ml of culture was inoculated into 500 ml of LB with both antibiotics and grown to an OD of 0.8 read at a wavelength of 600 nm before induction. Protein expression was inducted by the addition of 0.5, 1 and 2 mM of isopropyl b-D-thiogalactoside (IPTG) and they were expressed for 1, 2 and 4 h. Then, E. coli M15 cells were harvested by centrifugation at 4000  g for 30 min at 4 8C. The bacterial pellet was disrupted by sonication in Lysis Buffer (20 mM Tris–HCl, 0.5 M NaCl, 20 mM imidazole, lysozyme (10 mg/ml) and 10 mg of protease inhibitor cocktail, pH 8 (Sigma, Sigma–Aldrich Chemie GmbH, Munich, Germany)), followed by centrifugation at 14 000  g for 1 h at 4 8C. The recombinant proteins were purified from the supernatant. The purification of His-VP26 and His-VP26DC proteins was carried out by chromatography on Ni2+-NTA agarose resin. 500 ml of Ni2+-NTA agarose were equilibrated with 5 ml of Lysis Buffer and then, incubated with supernatant. The mixture was applied to a polypropylene column, equilibrated with 5 ml of Lysis Buffer. The polypropylene column containing the mixture was washed extensively with Wash buffer (20 mM Tris–HCl, 0.5 M NaCl and 40 mM imidazole, pH 8) to remove the impurities bounded unspecifically to NTA-Ni2+ agarose. The recombinant proteins were collected after addition of 5 ml of Elution buffer (20 mM Tris–HCl, 0.5 M NaCl, 250 mM imidazole, pH 8) and stored at 80 8C before use. 2.8. Protein expression analysis and quantification Analytical sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli protocol [24] in gels containing 12% acrylamide (Bio-Rad, Bio-Rad Laboratories SA, CA, USA). Gels were stained with Coomassie brilliant blue R-250 (Bio-Rad, Bio-Rad Laboratories SA, CA, USA) and destained using a solution containing 40% (v/v) methanol and 7% (v/v) acetic acid. Protein concentration was determined by Lowry protocol [25] using bovine seroalbumine (BSA) (Sigma, Sigma–Aldrich Chemie GmbH, Munich, Germany) as template to draw a standard curve.

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2.9. Western blotting analysis An immune-specific detection was performed following the WB protocol described in literature [26]. Briefly, fractions collected during the protein expression were separated by SDS-PAGE using 12% acrylamide and transferred to a nitrocellulose membrane (GE Healthcare, Chalfont St. Giles, United Kingdom) at 200 mA for 2 h. Then, the membrane was blocked with 5% non-fat dried milk in phosphate buffer saline (PBS) for 1 h at room temperature with shaking. Protein detection with PAb was made at 1:5000 dilution in PBS at room temperature for 1 h. Then, the membrane was washed with 0.05% Tween-20 (BioRad, Bio-Rad Laboratories SA, CA, USA) in PBS three times for 15 min, followed by incubation of IgG anti-rabbit-HRPO (GE Healthcare, Chalfont St. Giles, United Kingdom) at room temperature for 1 h. After three washes, bound antibodies were visualized using Western Blotting Luminol Reagent (Santa Cruz Biotechnology Inc. Private Company, CA, USA) according to manufacturer’s guidelines. 2.10. Detecting ELISA In order to characterize rVP26 and VP26DC antigenic properties, 96-well plates (Corning, Corning Costar Corp., MA, USA) were coated, in duplicate, with 10 mg/well of each purified protein in PBS and incubated for 1 h at 37 8C. After blocking with 5% non-fat milk in PBS, wells were washed with PBS containing 0.05% (v/v) Tween-20 (PBST) (Bio-Rad, Bio-Rad Laboratories SA, CA, USA) and later incubated at 37 8C for 1 h with the corresponding antibodies. The plate was washed again with PBST and then, the corresponding secondary antibodies were added and incubated for 1 h at 37 8C. The peroxidase reaction was visualized by using O-phenylenediamine (OPD) (Sigma, Sigma–Aldrich Chemie GmbH, Munich, Germany) as substrate after incubation for 10 min at room temperature. The reaction was stopped by adding 1N H2SO4 and absorbance was read at 492 nm. The cut-off value was established using the mean absorbance of negative samples. To compare the results obtained with recombinant proteins versus the pattern obtained with HAstV, 10 mg of HAstV-2 per well were analyzed using the same protocol. Ten mg of BSA per well were also analyzed as negative control.

3. Results 3.1. Cloning of VP26 and VP26DC genes The sequences of VP26 and VP26DC genes were amplified by RT-PCR using extracted genomic RNA from HAstV-2 as a template. Then, they were examined by 2% agarose gel electrophoresis for the presence of the VP26 and VP26DC products. Two bands of 775 base pairs (bp) (Fig. 2A) and 582 bp, respectively (Fig. 2B) were visualized, as expected. After enzymatic restriction with BamHI and HindIII, both cDNA sequences were inserted into the multiple-cloning site of the digested pQE32 expression plasmid. The recombinant plasmids were digested with BamHI and HindIII and, then analyzed on a 2% agarose gel electrophoresis to verify the insertions of both genes (VP26 and VP26DC) in

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Fig. 2. Electrophoretic analysis of VP26 DNA fragments. (A) Analysis of VP26 cDNA sequence amplified by RTPCR. Arrowhead indicates the VP26 cDNA with the expected size (775 bp). (B) Analysis of VP26DC sequence. Black arrow indicates the VP26DC cDNA with the expected size (582 bp). (C) Enzyme restriction assay with restriction enzymes BamHI and HindIII of pQE32-VP26 vector and (D) pQE32- VP26DC construction. Lanes ND show the non-digested plasmids. Lanes D show the digested plasmids with its expected sizes released from constructed DNA-vectors. DNA molecular size markers, in base pairs, are indicated on the left side of the picture.

the expression vectors. The result showed the released 775 bp (Fig. 2C) and 582 bp fragments (Fig. 2D). The constructed plasmids were named pQE32-VP26 and pQE32-VP26DC. Automatic sequencing and sequence analysis using Seqman software showed the correct in-frame VP26 sequence in both pQE32-VP26 and pQE32- VP26DC plasmids. 3.2. Expression and purification of rVP26 and VP26DC recombinant proteins As there are no reported post-translational modifications in native VP26 protein, we can express the recombinant VP26 protein in a prokaryotic system. The pQE32-VP26 and pQE32-VP26DC recombinant plasmids were transformed in competent E. coli M15. An induction of the transformed bacteria with IPTG 1 mM at 37 8C for 2 h resulted in optimal high expression of rVP26 and VP26DC proteins. As expected, after expression analysis by SDS-PAGE, two proteins of approximately 26 kDa (Fig. 3A) and 22 kDa (Fig. 3B) were observed in the supernatant of cell lysates. The expressed N-terminal His-tagged recombinant proteins were purified (in a single step) under experimental procedures. The optimal concentration of imidazole for lysis and washing buffers were 20 and 40 mM, respectively. After washing, no apparent contaminants remained in NTA-Ni2+ agarose. Electrophoretic mobility of HAstV purified particles, rVP26 and VP26DC proteins were analyzed by SDS-PAGE (Fig. 3C), in order to determine the correct MW of the recombinant proteins. This analysis showed that rVP26 protein had the same MW as the native VP26 protein present in the virion. The electrophoretic mobility of VP26DC was lower, as expected, corresponding to the predicted 22-kDa MW. After purification, protein concentration was by Lowry reaction, obtaining an average yield of 10 mg of purified rVP26 protein from 1 l of liquid culture, while the average yield of purified VP26DC was 6 mg from 1 l of bacterial culture. 3.3. WB verification of rVP26 and VP26DC expressed in E. coli The C-terminal domain of ORF2 polyprotein (corresponding to VP26 protein) can induce antibodies against immunodominant and neutralizing epitopes in HAstV [7,10,11].

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Fig. 3. Analysis of recombinant proteins expression by SDS-PAGE. (A) Expression of rVP26 protein (arrowhead). (B) Expression of VP26DC protein (black arrow). Lane MW shows the molecular weight markers (in kDa). Lane 1 shows soluble extracts before induction with IPTG. Lane 2 shows soluble extracts after induction with IPTG (1 mM at 37 8C for 2 h). Lanes 3 and 4 show the impurities that bound unspecifically to Ni2+-NTA agarose. Lane 5 shows the purified His-tagged protein. (C) SDS-PAGE to compare the electrophoretic mobility of rVP26 and VP26DC Vs purified HAstV particles. Lane 1 shows purified VP26DC (black arrow). Lane 2 shows purified rVP26 protein (head arrow), Lane 3 shows HAstV particles (structural proteins VP32, VP29 and VP26 are indicated on the right).

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Fig. 4. Immunodetection by western blotting using PAb. (A) HAstV purified particles (positive control) (lane 1) and rVP26 protein (lane 2). Arrowhead shows the MW of VP26. (B) Detection of VP26DC (lane 1, black arrow) and rVP26 (lane 2, arrowhead).

To demonstrate that the recombinant 26-kDa protein, observed after induction of transformed E. coli M15, contains the same aminoacid sequence as the native VP26 protein from HAstV, a WB technique using the PAb, which recognizes all HAstV structural proteins (Fig. 4A) was developed. HAstV particles were also analyzed to compare the results obtained with rVP26 protein. These results showed that the PAb was able to detect the three structural proteins that compose the mature virion in HAstV particles. The rVP26 was also detected by PAb as a 26-kDa protein, showing a similar immune-detection pattern as the native VP26 protein present in the virus. Furthermore, the recombinant VP26 C-terminal deleted form was also analyzed in WB for antigenic recognition under denaturing conditions using PAb (Fig. 4B). This assay included a sample of rVP26 protein as a positive control. The assay showed the presence of a 22-kDa protein, indicating that the N-terminal aminoacid sequence of VP26 is involved in antigenic recognition of lineal-epitopes recognized by PAb under denaturing conditions. 3.4. First antigenic characterization approach To analyze the possible humoral immune response produced by the native VP26 protein during viral infection, purified rVP26 and VP26DC proteins were tested by ELISA using nMAb PL2 and PAb directed to HAstV (Fig. 5). The results of the detecting ELISA showed that the purified rVP26 was recognized by nMAb PL2. Moreover, rVP26 protein was also detected by PAb, which recognizes non-conformational epitopes present in all the HAstV structural proteins. The results obtained for VP26DC protein by ELISA showed that this purified protein was not recognized by nMAb PL2. Nonetheless, PAb was able to recognize the VP26DC protein at a similar level as HAstV particles and rVP26 protein, indicating that the linear epitope within VP26DC recognized by PAb, under non-denaturing conditions, is the same in both the native VP26 and the rVP26 proteins. The analysis of HAstV showed that it was recognized by the nMAb PL2 and PAb, as previously described [7]. The lack of significative antigenic reactivity in the negative control (BSA) validated the assay. The antigenic pattern shown by rVP26 was very similar to the one obtained with purified HAstV-2 particles. This pattern showed that nMAb PL2 and PAb were able to detect both rVP26 and HAstV-2. However, only PAb was able to recognize VP26DC, showing a different qualitative pattern compared to rVP26 and HAstV-2 particles.

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Fig. 5. Antigenic analysis of recombinant proteins and HAstV particles by ELISA using nMAb PL2 and PAb. BSA was used as a negative control following the same protocol. The Y axis represents the absorbance values at 492 nm. The analyzed samples are represented in the X axis.

4. Discussion Little is known about biochemical features, cell location, antigenic epitopes or structural characteristics of the structural proteins of HAstV. Generally, the structural viral proteins have a variety of functions during the infective cycle, which might be closely related to its structure. For that reason, the understanding of the role of each structural protein depends on the study at a molecular level. The functional analysis of some recombinant proteins from several organisms has been possible by cloning, expression and purification based technology [15–20]. The antigenic structure of the VP26 protein has not been completely described though it has been reported that some antibodies recognizing the capsid proteins of HAstV have antigenic and/or neutralizing properties [7,11,12,27,28]. Computational predictions suggested that the region corresponding to VP26 protein could be the receptor-cell binding domain [14]. In this work, the VP26 and VP26 C-terminal truncated genes, were cloned into pQE32 vector and expressed in E. coli M15 cells. As there are no reported post-translational modifications in native VP26 protein, we could express the recombinant VP26 protein in a prokaryotic system that allowed us to obtain large-scale protein production. The monitoring of experimental protocol to optimize protein expression was made by SDSPAGE. This analysis allowed us to optimize the IPTG concentration, the time of protein expression and the imidazole concentration for lysis, wash and elution buffers. The VP26 protein expressed in E. coli was purified by Histidines Bind column chromatography. It must be noted that the rVP26 protein characterized in this study did not differ in apparent MW from native VP26 protein. Besides, some antigenic properties are similar to those of the VP26 protein in the virus particles. Consequently, these data indicate that VP26 is a good candidate for further characterization which may allow obtaining a potential model for VP26 native role. Moreover, the apparent MW of VP26DC protein analyzed by SDS-PAGE and WB was the expected 22 kDa.

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The antigenic patterns of the recombinant proteins obtained by WB, using PAb, as well as by ELISA using PAb and nMAb PL2, showed that both antibodies reacted with conformational rVP26 epitope and with purified virus particles, however they do not reacted with VP26DC. These results strongly suggest that the purified rVP26 protein seems to be structurally and antigenically very similar to native VP26 protein. The absorbance values obtained by ELISA assay to detect HAstV-2 purified particles using nMAb PL2 were higher than those obtained with rVP26. This was also expected because the complete viral particles contain more epitopes recognized by PL2 than rVP26 protein alone. Moreover, the fact that nMAb PL2 recognizes rVP26 protein, indicates that this structural component would be located on the surface of the virion, exposing epitopes, according to previous data suggesting that it could be the spike protein [1,14]. Thus, these results indicate that native VP26 protein should play an important role in generating neutralizing immune response in mice and, therefore, should have a similar implication in humans. These results also suggest that VP26 protein would be the most antigenic structural protein of HAstV and they agree with the data previously reported for HAstV [7] and with the proposed role of VP26 in viral–cell recognition [14]. It is important to notice that we have demonstrated that the protein sequence spanning aminoacids 394–648, included into ORF2, contains the minimal information necessary to produce the VP26 protein. Thus, these results allow us to establish the proteolytic sites within the tryptic map in R394 and R648, confirming the previously reported data [7]. It is of interest to note that when the amount of VP26 protein was very large (more than 0.1 mg/ml), the protein precipitated in Elution buffer, causing the loss of the ability to bind nMAb PL2 in ELISA (data not shown), probably because this MAb PL2 recognizes conformational epitopes that were not accessible. However, it was detected in WB by PAb, since this technique denatures the precipitated complex and allows the recognition of nonconformational epitopes by PAb [7]. In addition, here we describe the effect of the absence of the VP26 C-terminal end in antigenic recognition. On one hand, the VP26DC analysis revealed that PAb was able to detect this protein in ELISA, indicating that the VP26 N-terminal aminoacid sequence is involved in the antigenic recognition. On the other hand, PAb was able to detect VP26DC by WB (under denaturing conditions), suggesting that PAb recognizes non-conformational epitopes located in the N-terminal end of VP26. The lack of nMAb PL2 reactivity against VP26DC protein suggests two possible conclusions. First, the epitope involved in neutralization is on the deleted portion. Second, this epitope could be a conformational epitope but it has not been correctly reorganized. In conclusion, this is the first report on cloning, expression and purification of the VP26 protein and a VP26 C-terminus deleted form from HAstV. As it has been described for diagnosis of other pathogens [29–32], the results obtained in this study make this cloning, expression and purification system a suitable method to obtain large amounts of highly purified rVP26 for further characterization to evaluate its utility for developing new detection methods and vaccines. The rVP26 protein could be more useful than complete viral particles, which are commonly used in immunoenzyme techniques as the coating antigen in commercially available ELISA kits used to detect antibodies. Conventional propagation and purification of the complete HAstV is time consuming and labor-intensive. Expressed rVP26 protein is

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fast, easy, economical and efficient to detect antibodies against HAstV in ELISA. Furthermore, this recombinant protein, can surely be used to understand structural, functional and immunological characteristics of VP26 from HAstV and for X-ray crystallographic analysis as it was reported for other recombinant proteins [33,34]. Thus, it also offers us an important tool to know biological features about the role of one of the major capsid proteins of HAstV.

Acknowledgments This work was supported by a grant from the Instituto de Salud Carlos III (ISCIII) of Spain. We are grateful to Unidad de Geno´mica at CNM (ISCIII), for carrying out the nucleotide sequencing. We thank the staff members from Unidad de Aislamiento y Deteccio´n de Virus at CNM (ISCIII) for helpful discussions, especially to Mar Mosquera for her critical reading of the manuscript. We also thank to Ana Ba´rcena for her French Résumé and Dra. Ana Falco´n for her unending support.

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