clostridium perfringens , which was previously known as clostridium welchii , is a gram - positive , anaerobic , non - motile rod - shaped bacterium and is able to form environmentally resistant spores ( 1 ) .
this ubiquitous microorganism could be found in a variety of habitats such as soil , water and lower mammals and human intestine .
the bacterium can be found widely all over the world due to its genomic variation .
it produces numerous toxins which may cause various diseases such as diarrhea , necrotizing bowel disease and gas gangrene in humans as well as diarrhea , necrotic enteritis and fatal enterotoxemia in animals .
the bacterium produces 17 toxins from which epsilon is the major one ( 2 ) . c. perfringens has a circular chromosome of approximately 3.6 mbp with a low g+c content of approximately 25% ( 3 , 4 ) .
its genome includes extra chromosomal genetic elements in the form of plasmids and phages encoding mobile genes that vary in size and composition ( 5 , 6 ) . c. perfringens is a model for genetic engineering studies due to its striking physiological features ( 7 ) and several studies on the cloning and expression of its toxin genes
epsilon toxin is the third most potent clostridial toxin after botulinum and tetanus neurotoxins ( 8) and is active in a wide range of animal species and plays a central role in enterotoxemia of sheep and lambs ( 9 ) . in 1992
, epsilon toxin gene of c. perfringens type d had been cloned and expressed in e. coli .
studies have shown that the etx gene has 987bp and is located between base nucleotide 188 ( start codon ) and the stop codon at position 1174 ( 10 ) .
type d epsilon toxin gene of c. perfringens has been cloned and expressed and its n terminal 20 amino acids sequence has been determined ( 11 ) . the recombinant toxin has been strongly recognized by anti - native epsilon antibodies .
the antibodies against this recombinant toxin recognized the native epsilon toxin and neutralized its action in mice models ( 12 ) .
the recombinant type b and the native type d toxins have similar molecular weights and isoelectric points and both of them react with the specific monoclonal antibodies . in 2010 , etx gene was cloned and the potency of e. coli expressed recombinant epsilon toxoid was evaluated through administration to the goats , sheep and cattle .
numerous items can modify the current manufacturing procedure of clostridial vaccines and their efficiency but the biological process is often economically permanent .
thus , heterologous expression systems are good alternative tools for production of these toxins . in the present study , expression of epsilon toxin in e. coli rosetta(de3 )
c. perfringens type d vaccine strain ( cwd cn409 ) , e. coli top10f and rosetta ( de3 ) ( 14 ) strain were purchased from razi institute .
c. perfringens type d vaccine strain was cultured anaerobically in the liver extract medium using anoxomat chambers ( mart microbiology , netherlands ) at 37 c , ph 7.5 for 18 hours .
primers were designed for amplification of epsilon toxin encoding gene from c. perfringens genomic dna .
complete nucleotide sequence of etx gene was retrieved from the gene deposited in genbank under accession number hq179546 .
etx gene was amplified using pfu dna polymerase and specific forward ( 5-aatcatatgaaaaaaaatcttgtaaaaagt-3 ) and reverse primers ( 5-aatctcgagttttattcctggtgccttaat-3 ) including ndei and xhoi restriction sites , respectively .
linearized pjet1.2/blunt vector was ligated with amplified etx gene in order to develop the recombinant cloning vector , pjet. screening of recombinant e. coli top10 clones was done via colony pcr using universal sequencing primers .
identity of recombinant cloning vector , pjet , was confirmed thorough restriction digestion with ndei and xhoi enzymes ( fermentas ) .
sequencing analysis of the purified recombinant vector was also performed and blast and phylogenetic analysis was accomplished .
the expression vector , pet22b ( + ) , was digested with ndei and xhoi and ligated with etx gene digested from the cloning vector .
recombinant e. coli cells were inoculated into lb broth medium and incubated at 37 c to reach 0.60.7 od 

 600 

 .
final 0.5 mm iptg concentration was used to induce the expression of recombinant protein and bacterial incubation was continued for 18 hours . to optimize the expression of recombinant protein , different concentrations of iptg ( 0.5 , 1 and 1.5 mm ) ,
different incubation temperatures ( 25 , 31 and 37 c ) and times ( 3 , 6 and 18 h. ) were examined .
protein expression was analyzed by sds - page . to purify the recombinant epsilon protein which had a c - terminal his - tag ,
the bacterial pellet was re - suspended in lysis buffer and the cells were lysed by sonication on ice .
the cell lysate was centrifuged at 13 , 680( g ) and the clarified supernatant was loaded on ni - nta resin at the flow rate of 1 ml / min .
the column was washed with 5 volumes of washing buffer and finally the protein was eluted by adding elution buffer ( 17 ) .
c. perfringens type d vaccine strain ( cwd cn409 ) , e. coli top10f and rosetta ( de3 ) ( 14 ) strain were purchased from razi institute .
c. perfringens type d vaccine strain was cultured anaerobically in the liver extract medium using anoxomat chambers ( mart microbiology , netherlands ) at 37 c , ph 7.5 for 18 hours .
primers were designed for amplification of epsilon toxin encoding gene from c. perfringens genomic dna .
complete nucleotide sequence of etx gene was retrieved from the gene deposited in genbank under accession number hq179546 .
etx gene was amplified using pfu dna polymerase and specific forward ( 5-aatcatatgaaaaaaaatcttgtaaaaagt-3 ) and reverse primers ( 5-aatctcgagttttattcctggtgccttaat-3 ) including ndei and xhoi restriction sites , respectively .
linearized pjet1.2/blunt vector was ligated with amplified etx gene in order to develop the recombinant cloning vector , pjet. screening of recombinant e. coli top10 clones was done via colony pcr using universal sequencing primers .
identity of recombinant cloning vector , pjet , was confirmed thorough restriction digestion with ndei and xhoi enzymes ( fermentas ) .
sequencing analysis of the purified recombinant vector was also performed and blast and phylogenetic analysis was accomplished .
the expression vector , pet22b ( + ) , was digested with ndei and xhoi and ligated with etx gene digested from the cloning vector .
recombinant e. coli cells were inoculated into lb broth medium and incubated at 37 c to reach 0.60.7 od 

 600 

 .
final 0.5 mm iptg concentration was used to induce the expression of recombinant protein and bacterial incubation was continued for 18 hours . to optimize the expression of recombinant protein , different concentrations of iptg ( 0.5 , 1 and 1.5 mm ) ,
different incubation temperatures ( 25 , 31 and 37 c ) and times ( 3 , 6 and 18 h. ) were examined .
to purify the recombinant epsilon protein which had a c - terminal his - tag , the bacterial pellet was re - suspended in lysis buffer and the cells were lysed by sonication on ice .
the cell lysate was centrifuged at 13 , 680( g ) and the clarified supernatant was loaded on ni - nta resin at the flow rate of 1 ml / min .
the column was washed with 5 volumes of washing buffer and finally the protein was eluted by adding elution buffer ( 17 ) .
genomic dna of c. perfringens type d was extracted and used for pcr amplification of etx gene ( approximately 927 bp ) ( fig .
1 ) . colony pcr on top10 colonies containing the recombinant pjet cloning vector using etx forward and reverse primers confirmed the presence of a 927 bp dna fragment via 1% agarose gel electrophoresis ( fig .
2 ) . identity confirmation was done through restriction digestion with ndei and xhoi ( fig .
pcr on recombinant e. coli top10fcolonies containing pjet. lanes 111 : pcr products ( 927 bp ) ; lane 6:100 bpdna ladder .
lane1 : 100 bpdna ladder ; lane 2 : undigested vector ; lane 3 : digested pjet. double digested etx gene was purified using agarose gel extraction kit according to the manufacturer and its sequence was confirmed through sequencing .
subcloning of the gene into pet22b ( + ) expression vector was done and the recombinant vector was transformed into e. coli rosetta ( de3 ) strain .
iptg induction of protein expression was tested through sds - page analysis showing the expression of recombinant protein 3 h after induction ( fig .
the effect of different temperatures and incubation times , after iptg induction , on protein expression was significant ( figs . 6 and 7 ) .
lane 1 : protein molecular weight marker ; lane 2 : negative control ( e. coli rosettade3 strain containing empty pet22b+ ) ; lane 3 & 4 : recombinant e. coli strain before and after 3 h induction ; arrow indicates the recombinant epsilon toxin band .
lane1 : protein molecular weight marker ; lanes 24 : bacterial lysate 3h after induction with 0.5 , 0.1 and 1.5 mm iptg at 37 c ; lane 5 : bacterial lysate before induction ; lane 6 : negative control lysate ( e. coli rosetta containing empty pet22b+ ) ; arrow shows recombinant epsilon toxin band .
lane 1 : protein molecular weight marker ; lanes 24 : bacterial lysate at 25 , 31 and 37 c , 3 h after induction with 0.5 mm iptg ; lane 5 : bacterial lysate before induction ; lane 6 : negative control ( e. coli rosetta strain containing empty pet22b+ ) ; arrow shows recombinant epsilon toxin band . effect of time on expression of recombinant epsilon toxin .
lane1 ; protein molecular weight marker ; lanes 24 : bacterial lysate after 3 , 6 and 18h after induction with 0.5 mm iptg ; lane 5 : bacterial lysate before induction ; lane6 : negative control ( e. coli rosetta containing empty pet22b+ ) .
purified protein appeared as an approximately 35 kda protein on sds - page and western blot using sheep primary and anti - sheep hrp conjugated secondary antibody ( fig .
( a ) sds - page analysis of purified protein ; lane 1 : protein molecular weight marker ; lane 2 : bacterial lysate 3hours after induction ; lane 3 : purified epsilon toxin ; ( b ) western blot analysis of purified protein ; lane 4 : protein molecular weight marker ; lane 5 : western blot of the purified recombinant epsilon toxin .
genomic dna of c. perfringens type d was extracted and used for pcr amplification of etx gene ( approximately 927 bp ) ( fig .
1 ) . colony pcr on top10 colonies containing the recombinant pjet cloning vector using etx forward and reverse primers confirmed the presence of a 927 bp dna fragment via 1% agarose gel electrophoresis ( fig .
2 ) . identity confirmation was done through restriction digestion with ndei and xhoi ( fig .
pcr on recombinant e. coli top10fcolonies containing pjet. lanes 111 : pcr products ( 927 bp ) ; lane 6:100 bpdna ladder .
lane1 : 100 bpdna ladder ; lane 2 : undigested vector ; lane 3 : digested pjet.
double digested etx gene was purified using agarose gel extraction kit according to the manufacturer and its sequence was confirmed through sequencing .
subcloning of the gene into pet22b ( + ) expression vector was done and the recombinant vector was transformed into e. coli rosetta ( de3 ) strain .
iptg induction of protein expression was tested through sds - page analysis showing the expression of recombinant protein 3 h after induction ( fig .
different concentrations of iptg had no significant effect on protein expression ( fig . 5 ) .
the effect of different temperatures and incubation times , after iptg induction , on protein expression was significant ( figs . 6 and 7 ) .
lane 1 : protein molecular weight marker ; lane 2 : negative control ( e. coli rosettade3 strain containing empty pet22b+ ) ; lane 3 & 4 : recombinant e. coli strain before and after 3 h induction ; arrow indicates the recombinant epsilon toxin band .
lane1 : protein molecular weight marker ; lanes 24 : bacterial lysate 3h after induction with 0.5 , 0.1 and 1.5 mm iptg at 37 c ; lane 5 : bacterial lysate before induction ; lane 6 : negative control lysate ( e. coli rosetta containing empty pet22b+ ) ; arrow shows recombinant epsilon toxin band .
lane 1 : protein molecular weight marker ; lanes 24 : bacterial lysate at 25 , 31 and 37 c , 3 h after induction with 0.5 mm iptg ; lane 5 : bacterial lysate before induction ; lane 6 : negative control ( e. coli rosetta strain containing empty pet22b+ ) ; arrow shows recombinant epsilon toxin band .
lane1 ; protein molecular weight marker ; lanes 24 : bacterial lysate after 3 , 6 and 18h after induction with 0.5 mm iptg ; lane 5 : bacterial lysate before induction ; lane6 : negative control ( e. coli rosetta containing empty pet22b+ ) .
purified protein appeared as an approximately 35 kda protein on sds - page and western blot using sheep primary and anti - sheep hrp conjugated secondary antibody ( fig .
( a ) sds - page analysis of purified protein ; lane 1 : protein molecular weight marker ; lane 2 : bacterial lysate 3hours after induction ; lane 3 : purified epsilon toxin ; ( b ) western blot analysis of purified protein ; lane 4 : protein molecular weight marker ; lane 5 : western blot of the purified recombinant epsilon toxin .
in spite of using traditional methods in production of effective vaccines , large scale production of such vaccines from different clostridium species needs high safety conditions and precise quality control steps .
therefore , production of these candidate vaccines in a safe host is beneficial ( 19 ) .
recombinant dna technology has enabled researchers to produce vaccines for diseases which could not be treated using the traditional methods such as infections caused by c. perfringens species ( 19 ) . c. perfringens is a potent pathogen because of its ability to generate several toxins . in this study pjet1.2 blunt vector was used due to its advantages over the other available ta cloning vectors .
blunt ends of this vector avoided self - ligated vector products ( 16 , 17 ) . in this study ,
the recombinant vector was used in dna sequencing , protein expression or gene expression functional analysis . in a previous study , souza and
the colleagues cloned etx gene in pet11a expression vector and expressed the recombinant epsilon toxin as inclusion bodies which were successfully used in animal immunization ( 12 ) .
toxicity of the fusion epsilon - beta gene of c. perfringens types d and b revealed that toxigenesis of vaccine strains is one of the factors playing an important role in the effectiveness of the designed vaccines ( 17 ) .
this finding made us to select c. perfringens vaccine strain ( cwd cn409 ) for the present study . in 2010 , a vaccine candidate containing detoxified recombinant epsilon toxin and aluminum hydroxide as the adjuvant was developed and co - administered with sheep pox vaccine to sheep and it was shown that sheep were being protected against both sheep pox and enterotoxemia ( 20 ) . in the present study , experimental optimizations revealed that different concentrations of iptg have no obvious effect on the level of protein expression .
our findings revealed that different temperatures and incubation times had significant effects on the expression of epsilon toxin protein as optimal thermal condition was 37 c and the highest yield of the recombinant protein was achieved 6h after iptg induction .
this study enabled us to express recombinant vaccine candidates indicating the possibility of prevention of clostridial diseases through manufacturing vaccine candidates under safer conditions and lesser costs .
 background and objectives : clostridium perfringens , a gram - positive obligate anaerobic bacterium , is able to form resistant spores which are widely distributed in the environment . 
 c. perfringens is subdivided into five types a to e based on its four major alpha , beta , epsilon and iota toxins . 
 the aim of the present study was cloning and expression of c. perfringens type d vaccine strain epsilon toxin gene.materials and methods : genomic dna was extracted and the epsilon toxin gene was amplified using pfu dna polymerase . the pcr product was cloned into pjet1.2/blunt cloning vector . the recombinant vector ( pjet ) was sequenced using universal primers . at the next step epsilon toxin gene was subcloned into pet22b(+ ) expression vector and transformed into e. coli rosetta ( de3 ) host strain.results:the recombinant protein has been expressed in e. coli rosetta ( de3 ) cells after subcloning of c. perfringens etx gene ( 1008 bp ) into the expression vector.conclusion:we concluded that e. coli rosetta strain was suitable for the expression of recombinant c. perfringens epsilon toxin protein from pet22 expression vector . this recombinant cell can be used for further research on recombinant vaccine development . 
INTRODUCTION
MATERIALS AND METHODS
Bacterial strains and cultivation.
DNA extraction and gene amplification.
Cloning of 
Subcloning of 
Expression of recombinant epsilon toxin.
Purification of epsilon protein.
RESULTS
Cloning of 
Subcloning of 
Protein expression.
Purification of r-protein.
DISCUSSION
CONCLUSION