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Human Cytomegalovirus (HCMV) Antigens Identification

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  1. Metods:
  1. Study design:

The present study consists of two parts. In the first part, analysis and database search were performed to find a conserved and specific regions of HCMV genome leading to selection of immunogenic and proper region expressed in several species of CMV. The second part consists of various analyses of the selective gene such as epitope mapping, secondary and tertiary structure, antigenicity and B-cell epitope prediction, etc.

  1. Sequence availability and database:

Immunological studies that determine HCMV antigens importance in immunity, have demonstrated that the tegument phosphoprotein pp65 is the most dominant target for cellular immune responses against HCMV (Gyulai et al., 2000). Another usually recognized antigen of HCMV is pp150, that can induce specific CTLs responses (Elkington et al., 2003). Also similarity researches have shown that glycoprotein B (gB) is a principal target for neutralizing antibodies against HCMV (Pass et al., 1999; Reap et al., 2007).

In this study we selected a pp65-derived epitope (RQYDPVAALFFFDIDL), defined by specific features such as: the capability to induce immune reactions in both CD4+ and CD8+ T cells even in pharmacologically immunosuppressive individuals, high immunogenicity, wide range of HLA class I and II, ability to stimulate of producing TNF-α , INF-γ and proliferating CD4 T cells (Provenzano et al., 2009). Other information on T-cell Epitope sequences was obtained from researches by (Elkington et al., 2003) and (Jelinek et al., 2012) involving the recognition of epitopes and efficient sites.

Conformationally and linear binding sites on the glycoprotein B molecule have been mapped by Bioinformatic analysis. Linear binding sites have two conservative linear epitopes on SU segment and also two linear antibody sites on TM segment (Britt, Jarvis, Drummond, & Mach, 2005).

Accordingly, we chose two regions that efficiently stimulate the production of antibodies against HCMV.

T-cell Epitope sequences

HLA

HCMV antigens

ORF Names

Reference

RQYDPVAALFFFDIDL

 

pp65340-355

UL83

(Provenzano et al., 2009)

(Elkington et al., 2003)

(Provenzano et al., 2003)

ARNLVPMVATVQGQNLKYQEFFW

A2

pp65493-515

UL83

(Provenzano et al., 2009)

(Diamond, York, Sun, Wright, & Forman, 1997)

(Jelinek et al., 2012)

WPRERAWAL

B7/B8

pp150641-649

UL32

(Elkington et al., 2003)

Antibody-binding site

HCMV antigens

ORF Names

Reference

AD2 27-86

gB

UL55

(Beninga, Kropff, & Mach, 1995)

(Wagner et al., 1992)

(Meyer, Masuho, & Mach, 1990)

AD1 540-645

gB

UL55

(Gicklhorn, 2003)

(Ohlin, Sundqvist, Mach, Wahren, & Borrebaeck, 1993)

(Britt et al., 2005)

(Zhao et al., 2009)

The selected sequences of the antigen glycoprotein gB were obtained from Uniprot Database at http:// www.uniprot.org (Protein ID (Uniprot): P06473) and NCBI at http://www.ncbi.nlm.nih.gov (GenBank: ACL51135.1 GI:219879600). The sequences submitted to the Basic Local Alignment Search Tool (BLAST) and showed that the selected sequences were extremely conserved between various strains of CMV(???)

C:\Users\mabna\Desktop\Untitled.png

Putative conserved domain belonging to glycoproten B superfamily have been detected in full length sequence of gB

Accession no.

Description

Max score

Total score

Query coverage

E value

P06473.1

RecName: Full=Envelope glycoprotein B; Short=gB; Flags: Precursor

1896

1896

100%

0.0

AFR54557.1

envelope glycoprotein B [Human herpesvirus 5]

1891

1891

100%

0.0

AHB19702.1

envelope glycoprotein B [Human herpesvirus 5]

1881

1881

100%

0.0

  1. Chimeric construction design and Bioinformatic analysis:

The five selected segments were fused together for designing a synthetic chimeric construction. In order to correctness translation and increase expression of mRNA in the eukaryotic host up to 10 fold, the Kozak sequence (Kozak, 1989) was inserted before initiation codon. And the sequence KDEL (LYS-ASP-GLU-LEU) was added for proficient increase of the recombinant protein in Endoplasmic Reticulum (ER), at the 3' end of the synthetic chimeric gene. In addition, 6-histag inserted to its C-terminus to confirm the chimera protein expression, and according to the application of construction in future, for affinity purification (Amani, Mousavi, Rafati, & Salmanian, 2009). Arrangements of fragment junctions are shown in Fig.

In silico structural analysis of chimeric recombinant protein were defined using stand-alone software.

In order to determination of various physicochemical parameters such as molecular weight, theoretical pI, amino acid composition, total number of negatively and positively charged residues, estimated half-life, instability index and aliphatic index “protparam” (Gasteiger et al., 2005) online software at http://expasy.org/tools/protparam.html were computed (Jahangiri et al., 2011).

Another recombinant protein features such as flexibility (Bhaskaran & Ponnuswamy, 1988), hydrophilicity (Black & Mould, 1991), turns (Levitt, 1978), polarity, exposed surface and antigenic propensity (Kolaskar & Tongaonkar, 1990; Rahbar, Rasooli, Mousavi Gargari, Amani, & Fattahian, 2010) were analysis at http://web.expasy.org/protscale/ .

In order to estimate the antigenicity of the whole chimeric protein and each sequence, VaxiJen software (Doytchinova & Flower, 2007) at http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html was used (Jahangiri et al., 2012; Nazarian et al., 2012). In order to predict the immunogenicity of the whole chimeric protein, different factors such as antigenic propensities, protein complexity, hydrophobicity, hydrophilicity, surface accessibility and electrostatic potential were evaluated. These were performed using the CLCprotein Workbench 5.0.1, Protean, IEDB server and the PCE web service (http://bioserv.rpbs.jussieu.fr/PCE) (Motamedi, Amani, Shahsavandi, & Salmanian, 2013).

Antibody-binding site

Antigen index

T-cell Epitope sequences

Antigen index

AD2 27-86

0.6110

pp65 340-355

0.8969

AD1 540-645

0.4728

pp65 493-515

1.0890

pp150 641-649

1.5113

  1. Protein solubility prediction:

chimeric protein solubility was interpreted using the PROSO II server (http://mips.helmholtz-muenchen.de/prosoII) (Motamedi et al., 2013). chimeric protein (construct) solubility was evaluated using recombinant protein solubility prediction at www.biotech.ou.edu (Davis, Elisee, Newham, & Harrison, 1999; Roger G. Harrison, 2000; Wilkinson & Harrison, 1991)

  1. Secondary Structures Prediction:

The chimeric protein secondary structure evaluation was performed by GOR online software (Garnier, Gibrat, & Robson, 1995; Kloczkowski, Ting, Jernigan, & Garnier, 2002) at http://npsa-pbil.ibcp.fr/cgi-bin/secpred_gor4.pl (Jahangiri et al., 2012; Rahbar et al., 2012).

PredictProtein server (Rost, Yachdav, & Liu, 2004) was employed for sequence analysis and the protein structural and functional evaluation including low-complexity regions, regions lacking regular structure, secondary structure, solvent accessibility, transmembrane helices, coiled-coil regions, disulfide-bonds, sub-cellular localization as well as functional annotations (Nazarian et al., 2012).

  1. Tertiary Structures Prediction:

In order to identification chimeric protein 3D models I-TASSER online software (Zhang, 2009) was applied (Forghanifard, Amani, Gheybi, & Abbaszadegan, 2012).


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