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 .