human 
 cytomegalovirus ( hcmv ) is one of the prevalent opportunistic pathogen that causes a wide spectrum of clinical manifestations ( 1 ) .
this virus is regarded as the main morbidity and mortality cause in immune suppressed patients ( ie ; organ transplant recipients , human immunodeficiency virus ( hiv ) patient ) ( 2 ) . newborns with congenital infection
have a mortality rate of about 5 percent , and neurologic morbidity occurs in 50 to 60 percent of survivors ( 3 ) . in spite of the fact that cmv is a widespread pathogenin the human population , prevention of its disease is rather difficult .
although several vaccine approaches have been developed , none have yet been put into routine clinical practice .
the cell - mediated immune response is necessary in the recovery of infections ( 4 ) .
the best way to protect females before the onset of puberty is a cmv vaccine that stimulates cellular and humoral immune response .
this would reduce the risk of congenital cmv infection in the fetus and newborn ( 5 ) . in previous studies ,
many peptides had been reported that had been used as vaccine against cmv . the most abundant tegument protein ( pp65 protein )
is a major target of the cytotoxic t - cell ( 6 ) . besides , the most abundant envelope protein ( the glycoprotein b , gb ) is a major target of the humoral immunity ( 1 , 3 ) .
it seems that the development of a suitable vaccine for cmv , synchronously using epitopes specific for the immune dominant protein derived from these proteins , are the best choice .
a major limitation for using vaccines to induce protective immune responses against cmv infection is related to achieving efficient uptake and processing antigen - presenting cells ( apcs ) .
one way to overcome this problem is the use of the fc - fusion peptides in which one or more antigenic peptides are fused into the fc domain of immunoglobulin ( ig ) .
fusion of peptides to fc domain can increase harvesting and delivering them to the t lymphocyte , which is critical for protection against cmv .
the most effective method of antigen uptake in apcs such as macrophages and dendritic cells ( dcs ) is receptor - mediated uptake .
for this reason , dcs have several types of fc receptors ( fcr ) that bind the fc domain of igg molecules ( 7 ) .
therefore , to facilitate the binding of peptide to apc , fc receptor was added to desired fusion peptide . in recent decade , pichia pastoris was used as a useful expression system in vaccine development ( 8) . in this study , a cocktail peptide vaccine using pp65 , and gbwas designed against cytomegalovirus infection .
cloning and expression of this cocktail peptide vaccine fused with fc domain of mouse igg2a in p. pastoris was reported .
these epitopes were gb431 - 450 , gb471 - 490 and gb571 - 590 ( 9 ) for gb protein ( gen bank access number gi : 138192 ) and pp65361 - 376 ( 10 ) , pp340 - 355 ( 11 ) , and pp65417 - 427 ( 12 ) for pp65 protein ( gen bank access number gi : 130714 ) .
finally , the fc fragment of mouse igg2a ( gen bank access number gi : 51835 ) ( 7 ) was fused to c - terminal of cocktail epitopes .
sequences optimization and colon simulating in p. pastoris were checked by dna 2.0 , genscript and genius softwares .
ppicza expression vector carries -factor signal sequence ( -mf ) to drive fusion peptide secretion .
besides , this vector contains the zeocin resistance gene , shble , which is used for selection of escherichia coli and p. pastoris recombinants .
also , a c - terminal polyhistidine ( 6xhis ) tag was added to c - myc in order for fusion peptide to be detected by dot blot assay .
this strain was cultured at 37 c in lb ( luria broth ) medium supplied with ampicillin .
ppicza was used as shuttle vector for expression of fusion peptide , and the p. pastorisgs115 strain ( invitrogen , usa ) was used as an expression host .
the yeast cells were grown at 30 c on ypd ( yeast extract peptone dextrose ) medium ( 1% yeast extract , 2%bac to - peptone 2% glucose ) and ypds ( yeast extract peptone dextrose medium with sorbitol ) ( 1% yeast extract , 2% peptone , 2% glucose , 1 m sorbitol ) supplemented with 100 g / ml zeocin when necessary .
inclusion of sorbitol in ypd plates stabilizes electroporated cells as they appear to be somewhat osmotically sensitive .
restriction enzymes used for cloning were purchased from promega and used in accordance with manufacturer s recommendations . in order to amplify this synthetic fusion sequence ,
the presence of desired segment was confirmed by double digestion with related restriction enzymes and sequencing .
after minipreparation of pgh containing fusion sequence and double digestion , gel extraction and purification of desired segment were carried out by commercial kit according to manufacturer s recommendations ( 5 prime , germany ) .
this segment was cloned into ppicza shuttle vector under control of aox1 promoter , and the resulting vectors were called ppicza - cmv .
the construction of vector was confirmed by restriction digestion , pcr with aox1 and -factor primers , which amplified entire recombinant fusion peptide sequence .
pcr was performed with the primers 3aox1 5-gcaaatggcattctgacatcc-3  , and -factor , 5-tactattgccagcattgctgc-3  .
temperature profile was one cycle in 94 c for 3 min followed by 30 cycles in 94 c for 1 min , 55 c for 1 min , and 72 c for 2.30 min and finally 72c for 1 min .
stable integration into the aoxi ( aldehyde oxidase 1 ) of p. pastoris was achieved after linearization of the 5 g ppicza - cmv peptide expression vectors with saci .
after colony selection on ypds media supplemented with 100 g / ml zeocin , the recombinant p. pastoris cells were subjected to bmgy ( buffered glycerol - complex medium ) medium for fusion peptide expression . in this step , a total of 10 colonies were obtained and subsequently screened for methanol utilization phenotype . to remove inhibitory effect of other carbon sources ,
the recombinant p. pastoris strain gs115 was cultured in bmgy medium ( containing 1% yeast extract , 2% peptone , 100 mm potassium phosphate ph 6 , 1.34% yeast nitrogen base , 4 x 10 - 5% biotin and 1% glycerol ) .
after acquiring bmgy turbidity to od600 = 1.5 , the cells were harvested through centrifuge . in order to induct aox1 ,
the product in question was transferred into the bmmy medium ( the same as bmgy medium but with 0.5% methanol instead of 1% glycerol ) . in this system ,
the inducer was 0.5% methanol , which was added into the medium from day 2 up to day 7 . to confirm the expression of pp65-gb - fc
sds - page was performed in a 10% ( w / v ) polyacrylamide gel . as a control ,
these epitopes were gb431 - 450 , gb471 - 490 and gb571 - 590 ( 9 ) for gb protein ( gen bank access number gi : 138192 ) and pp65361 - 376 ( 10 ) , pp340 - 355 ( 11 ) , and pp65417 - 427 ( 12 ) for pp65 protein ( gen bank access number gi : 130714 ) .
finally , the fc fragment of mouse igg2a ( gen bank access number gi : 51835 ) ( 7 ) was fused to c - terminal of cocktail epitopes .
sequences optimization and colon simulating in p. pastoris were checked by dna 2.0 , genscript and genius softwares .
ppicza expression vector carries -factor signal sequence ( -mf ) to drive fusion peptide secretion .
besides , this vector contains the zeocin resistance gene , shble , which is used for selection of escherichia coli and p. pastoris recombinants .
also , a c - terminal polyhistidine ( 6xhis ) tag was added to c - myc in order for fusion peptide to be detected by dot blot assay .
this strain was cultured at 37 c in lb ( luria broth ) medium supplied with ampicillin .
ppicza was used as shuttle vector for expression of fusion peptide , and the p. pastorisgs115 strain ( invitrogen , usa ) was used as an expression host .
the yeast cells were grown at 30 c on ypd ( yeast extract peptone dextrose ) medium ( 1% yeast extract , 2%bac to - peptone 2% glucose ) and ypds ( yeast extract peptone dextrose medium with sorbitol ) ( 1% yeast extract , 2% peptone , 2% glucose , 1 m sorbitol ) supplemented with 100 g / ml zeocin when necessary .
inclusion of sorbitol in ypd plates stabilizes electroporated cells as they appear to be somewhat osmotically sensitive .
restriction enzymes used for cloning were purchased from promega and used in accordance with manufacturer s recommendations .
in order to amplify this synthetic fusion sequence , pgh vector was transformed into jm109 e. coli strain using calcium chloride method .
the presence of desired segment was confirmed by double digestion with related restriction enzymes and sequencing .
after minipreparation of pgh containing fusion sequence and double digestion , gel extraction and purification of desired segment were carried out by commercial kit according to manufacturer s recommendations ( 5 prime , germany ) .
this segment was cloned into ppicza shuttle vector under control of aox1 promoter , and the resulting vectors were called ppicza - cmv .
the construction of vector was confirmed by restriction digestion , pcr with aox1 and -factor primers , which amplified entire recombinant fusion peptide sequence .
pcr was performed with the primers 3aox1 5-gcaaatggcattctgacatcc-3  , and -factor , 5-tactattgccagcattgctgc-3  .
temperature profile was one cycle in 94 c for 3 min followed by 30 cycles in 94 c for 1 min , 55 c for 1 min , and 72 c for 2.30 min and finally 72c for 1 min .
stable integration into the aoxi ( aldehyde oxidase 1 ) of p. pastoris was achieved after linearization of the 5 g ppicza - cmv peptide expression vectors with saci .
after colony selection on ypds media supplemented with 100 g / ml zeocin , the recombinant p. pastoris cells were subjected to bmgy ( buffered glycerol - complex medium ) medium for fusion peptide expression . in this step ,
a total of 10 colonies were obtained and subsequently screened for methanol utilization phenotype . to remove inhibitory effect of other carbon sources ,
the recombinant p. pastoris strain gs115 was cultured in bmgy medium ( containing 1% yeast extract , 2% peptone , 100 mm potassium phosphate ph 6 , 1.34% yeast nitrogen base , 4 x 10 - 5% biotin and 1% glycerol ) .
after acquiring bmgy turbidity to od600 = 1.5 , the cells were harvested through centrifuge . in order to induct aox1 ,
the product in question was transferred into the bmmy medium ( the same as bmgy medium but with 0.5% methanol instead of 1% glycerol ) . in this system ,
the inducer was 0.5% methanol , which was added into the medium from day 2 up to day 7 . to confirm the expression of pp65-gb - fc
sds - page was performed in a 10% ( w / v ) polyacrylamide gel . as a control ,
in attempting to obtain secretion of pp65-gb - fc fusion peptide at high levels in the culture medium , a cod on - optimized -mf along with the ppicza based vector was employed . in this paper ,
the aoxi promoter sequence was used to allow integration of the ppicza - cmv expression vector via homologous recombination , as well as a cod on - optimized -factor from saccharomyces cerevisiae to drive the secretion of fusion peptide ( 13 ) . in this study ,
p. pastoris strain gs115 was used as the host and the ppicza was used as an expression vector .
pp65-gb - fc segments were cloned in xhoi / xbai site of mcs ( multiple cloning site ) from ppicza ( figure 1 ) .
schematic diagram for cloning of modified pp65-gb - fc segments in ppicza ( ppicza - pp65-gb - fc ) amino acid sequences of final recombinant protein pcr ( figure 3 ) and digestion ( figure 4 ) showed that pp65-gb - fc segments were ligated with ppicza expression vector .
the pcr and restriction digestion of the recombinant plasmid ppicza - pp65-gb - fc produced a fragment with the expected length of ~1300 bp ( figure 3 ) .
dna fragments with the expected length of the target gene ( 1290 bp ) were produced in the transformed yeast clones .
line1 : 1 kb dna ladder , line 2 - 4 : 1290 bp pcr product , line 5 : negative control double restriction digestion analysis of ppicza - cmv with xhoi / xbai restriction enzymes .
in attempting to obtain secretion of pp65-gb - fc fusion peptide at high levels in the culture medium , a cod on - optimized -mf along with the ppicza based vector was employed . in this paper ,
the aoxi promoter sequence was used to allow integration of the ppicza - cmv expression vector via homologous recombination , as well as a cod on - optimized -factor from saccharomyces cerevisiae to drive the secretion of fusion peptide ( 13 ) . in this study ,
p. pastoris strain gs115 was used as the host and the ppicza was used as an expression vector .
pp65-gb - fc segments were cloned in xhoi / xbai site of mcs ( multiple cloning site ) from ppicza ( figure 1 ) .
schematic diagram for cloning of modified pp65-gb - fc segments in ppicza ( ppicza - pp65-gb - fc ) amino acid sequences of final recombinant protein
pcr ( figure 3 ) and digestion ( figure 4 ) showed that pp65-gb - fc segments were ligated with ppicza expression vector .
the pcr and restriction digestion of the recombinant plasmid ppicza - pp65-gb - fc produced a fragment with the expected length of ~1300 bp ( figure 3 ) .
dna fragments with the expected length of the target gene ( 1290 bp ) were produced in the transformed yeast clones .
line1 : 1 kb dna ladder , line 2 - 4 : 1290 bp pcr product , line 5 : negative control double restriction digestion analysis of ppicza - cmv with xhoi / xbai restriction enzymes .
line1 : 1 kb dna ladder , line 2 - 4 digested recombinant plasmid . line 5 : undigested plasmid
digestion with saci generates a fragment , which can integrate by homologous recombination into the aoxi region .
cells of p. pastoris strain gs115 were transformed with linear ( saci - digested ) form of the expression vector , ppicza - cmv .
after expression of ppicza - cmv in induction media , supernatant of the media was studied by dot blot and 10% sds - page . in dot
blot , we used antibody against c - myc epitopes in terminal segment of cassette gene ( figure 5 ) . by using the anti - cmy antibody
, it was demonstrated that a fusion peptide was present in the supernatant during the cultivation course , while no detection was observed in the control extracts . in sds - page ,
coomassie blue staining showed that six of the ten induced clones produced a fusion peptide , which was not present in the control strain and had the expected molecular weight ( ~50 kda ) for pp65-gb - fc .
fusion peptide was present in the supernatant during total course of cultivation ( 7 days ) expression of the cytomegalovirus recombinant fusion protein .
( a ) the analysis of the p. pastorisgs115/ppicza - cmv cells by 10% sds - page stained with coomassieblue revealed a protein band profile with a molecular weight of ~50 kda .
line 1 : ppicza without fusion peptide , line 2 : p. pastoris without vector .
( b)the expression of the recombinant pp65-gb : fc2a protein was analyzed by immunoblot using anti mouse igg - hrp
nowadays , development of effective , cheap and safe vaccines is one of the major challenges in medical science . in order to design effective peptide vaccines ,
identification of proper viral antigens is important for inducing protective immune response ( 14 ) .
there is little data about using p. pastoris for hcmv peptide vaccine . in this research
, it was shown that p. pastoris yeast can be successfully used as an expression system for generating cmv fusion peptide .
p. pastoris performs some posttranslational modifications such as glycosylation and refolding on heterologous eukaryotic proteins ( 13 ) .
this glycosylation can affect the stability and activation of recombinant protein that is used for studies of some aspects of recombinant protein functions ( 15 ) .
all peptides in our research were immunogenic and have been used in previous studies , separately . in this respect ,
p. pastoris cells are attractive tools to use as antigen carrier , which subsequently induces specific ctl ( cytotoxic t cell ) immune responses against viral antigens .
a recent study looks into improving the immunogenicity of the peptides based on the fast development of multi - epitopes vaccines , which showed immunological activity against bacteria and virus ( 16 ) . in this study
, we choose eight epitopes on two immunologic proteins of hcmv . in this study , emphasize was put on p. pastoris as one of the best characterized yeasts , which is also becoming increasingly important in the field of eukaryotic cell biology and gene technology . based on the metabolization of methanol ,
p. pastoris strains were divided into three distinct phenotypes : the first phenotype is wild - type , methanol utilization plus ( mut+ ) that is characterized by the presence of the alcohol oxidase 1 gene ( aox1 ) , which is responsible for 85% of the methanol utilization ; the second is methanol utilization slow phenotype ( muts ) , characterized by the absence of aox1 gene and presence of the aox2 gene , which is about 97% homologous to aox1 but much less expressed ; and the third is methanol utilization minus phenotype ( mut ) , where both aox genes are absent . among these strains ,
the mut+ strains have a greater growth rate on methanol as the sole carbon source .
expression of the cmv cocktail peptide was efficiently achieved for the first time through an easier production schedule than when an aox1inducible promoter is employed ( 17 ) . in this research
, we utilized an expression vector , ppicza , which has the aox1 gene promoter to drive peptide expression , which can be induced by adding methanol to the growth medium . in this vector ,
the native signal peptide was replaced with that of the s. cerevisiae-factor , allowing secretion of the peptide into the extracellular medium ( 18 - 20 ) .
we have shown that fusion cmv peptide can be expressed in a yeast system by dot blot and sds - page assays .
currently , there is a growing need for cmv vaccines that are capable of eliciting cell - mediated and humoral immunity .
previously , the expression of dna encoding pp52 , as a non - fusion protein , in p. pastoris had shown good level of intracellular pp52 , which has good reactivity with human sera ( 21 ) .
according to recent studies , pp65 protein recognized by cd8 t cells from the majority of cmv - infected individuals , and evidence from preclinical and clinical studies indicates that this protein is principal target of protective cellular and humoral immune response ( 17 , 22 , 23 ) . in this context
, it has been shown that pp65495 - 503 cytotoxic cd8 t - cell epitope can stimulate pp65 t - cells in healthy volunteers ( 24 ) . in this research
recently , a subunit vaccine based on cmv glycoprotein b ( gb ) formulated with mf59 adjuvant successfully terminated phase ii clinical trials ( 25 ) . also we selected three immunodominant and mhc class i restricted epitopes in gb protein and fused by -ggggs- linkers . in order to activate antigen - specific t - cells ,
antigens must be taken up by antigen presenting cells ( apcs ) , and must be consequently presented by mhc class i and ii proteins .
dendritic cells , dcs ) including fc fusion protein will improve availability of protein antigen ( 10 , 14 ) .
previous studies demonstrated that targeting of immunogenic peptide to fc on apcs can increaseits selectively uptake and induce effective protective immune response both in vitro and in vivo .
these studies indicated that fc domain of immunoglobulin ( specially igg ) to form fusion with peptide ( such as pp65 and gb from cmv in this research ) can increase the immunogenicity of fc - fused antigens and could be considered as a delivery system ( 26 , 27 ) .
recently , it was shown that fcrn - targeted mucosal immunization produced durable memory immune responses .
immunological memory is exemplified by increased levels of effector t and b cells and , functionally , by the ability to respond faster and more vigorously to a second encounter with the vaccine antigens ( 7 ) . to the best of our knowledge ,
this is the first time that the expression of hcmv - pp65-gb - fc protein was achieved in p. pastoris .
in this study , we have shown that the pp65-gb - fc can be produced at appropriate levels by p. pastoris .
current study provides a starting point for future attempt to immunological studies on this vaccine .
we believe that secretion of hcmv pp65-gb - fc in the culture media could help downstream processing .
although further experiments are needed to determine immunogenicity of this cocktail peptide , the data outlined here underline that this vaccine could be a promising candidate for the development of a novel peptide vaccine .
 objective(s):human cytomegalovirus ( hcmv ) remains a major morbidity and mortality cause in immuno suppressed patients . 
 therefore , significant effort has been made towards the development of a vaccine . in this study , 
 the expression of the pp65 and gb fusion peptides and fc domain of mouse igg2a as a novel delivery system for selective uptake of antigens by antigen - presenting cells ( apcs ) in pichia pastoris yeast system were studied.materials and method : in this study , four immune dominant sequences in pp65 protein and 3 immuno dominant sequences in gb protein were selected according to literature review . 
 peptide linker -ggggs- was used for construction of fusion peptide . 
 this fusion peptide was cloned in the ppicza expression vector and transfected into p. pastoris host cells.results:dot blot and sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) techniques showed that a high level of pp65-gb - fc fusion peptide was expressed.conclusion:this cmv pp65-gb - fc fusion peptide could be a promising candidate for the development of a novel peptide vaccine . 
Introduction
Materials and Methods
Epitopes sequences
Strains and media
Preparation of fusion peptide sequence
Construction of pPICZA-CMV
Electroporation of yeasts
Results
Bioinformatics analysis
Construction and confirmation of pp65-gB-Fc
Expression of pPICZA-CMV in Pichia pastoris
Discussion
Conclusion
Conflict of interests