2.1- Sequence Retrieval and phylogenetic analysis The sequence of

2.1- Sequence Retrieval and phylogenetic analysis

The sequence of Iran fusion protein gene NDV (accession
number KJ176996.1) were obtain from nucleotide database NCBI (https://www.ncbi.nlm.nih.gov/nuccore/KJ176996.1). To find similar sequences with our sequence we used
nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) & the
phylogenetic tree were draw by phylogeny online
tools phylogeny from results of BLAST. Finally Sequence alignment done by Bio-Edit software (version, in order to find conserved
regions between Iran F protein and other NDV F proteins. We also evaluated
percent identity matrix by using clustal W version 12.1.

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2.2- Codon Optimization

The codons of the fusion protein (F) codons from NDV were
optimized by the GenScript service (http://www.genscript.com/gene_synthesis.html). based on the codon usage table of Pichia Pastoris for
the back translation of the sequence. Some
parameters that are critical to the efficiency of gene expression including
codon adaptation index (CAI), frequency of optimal codon (FOP) and GC content adjustment
were optimized. Moreover structure such as stem-loop structures which have on
the translation process a negative effect, were eliminated.


2.4- Protein
Structures Prediction

The Phyre2
(http://www.sbg.bio.ic.ac.uk/~phyre2/) server used to obtain the secondary structure of fusion
protein NDV. Secondary structure prediction with used of alpha helix, beta
sheet or randomcoil. The prediction of 3D models protein structure were performed
by Swiss model servers (https://swissmodel.expasy.org/interactive) and SPDB (version 4.10) used for the visualization of 3D


2.5- Prediction of Post-Translational Modification

Three dimensional structural quality of the fusion protein was performed by the Swiss-PDB Viewer software were
used for energy minimization. Certain post-translational modifications which
may occur in the

eukaryote protein sequences such as NetNGly, NetOGly
and YingOYang were predicted at http://www.cbs.dtu.dk.


2.3- prediction of epitopes
and antigenicity cites

The amino acid sequence was analyzed for both continuous
(linear) and discontinuous (Conformational) epithets use different B and T
cell’s epitopes predication algorithm. BacPerd and Immune Epitope Data Base
(IEDB) servers (http://www.iedb.org) use predicted these
epitopes. In the IEDB server, the prediction is performed on the based
algorithms that include (a) Chou and Fasman beta-turn (b) Emini surface
accessibility (c) Karplus and Schulz flexibility and (d) Parker hydrophilicity. Moreover, the prediction of T-cell epitopes was performed based on MHC-I by
using tools at IEDB server. The predictions were carried out with default
threshold values of the programs to avoid variation.