محمود تولایی

Nuchal translucency (NT), the ultrasound transparency detected behind the fetal neck, is considered a marker in the first trimester. Excessive increase in this marker is associated with chromosomal and structural abnormalities, as well as congenital birth defects, especially cardiovascular abnormalities. Noonan Syndrome and Spinal muscular atrophy (SMA) are two common associated syndromes with autosomal dominant and recessive inheritance respectively. PTPN11 is the most common gene whose mutation leads to Noonan syndrome. SMN2 gene is a modulating gene and in case of mutation, it leads to a benign increase in NT. The purpose of this article was to determine the causes for increased nuchal translucency in fetuses with normal karyotype by studying mutations in SMN2 and the two common exon 3 and exon 13 of PTPN11.

In this study, forty cases from Noor Genetics Clinics were entered. The cases were those who have both increased NT and normal karyotype. Amniotic fluid samples were acquired and DNA was extracted at the molecular laboratory. Then, genetic investigations were conducted using Real Time PCR and Sanger sequencing.

The investigation discovered four cases with deletion in SMN2; which none presented SMA pathogenesis. In one sample, Noonan syndrome pathogenesis through mutation in exon 13 was observed,  while no mutation in exon 3 was observed in any of the samples.

According to this study, it is suggested that molecular investigations, especially Noonan syndrome in fetuses with high NT level, should be included in the pregnancy screening program and diagnostic protocols in the society.

Warfarin is the most commonly prescribed anticoagulant drug for treating thromboembolic events. There is a variation in dose requirements for each individual due to nongenetic factors and gene polymorphisms. In this study, we performed Tetra-ARMS PCR molecular technique in order to investigate the association between 5 gene polymorphisms and patient characteristics with warfarin dose requirements in all Iranian ethnic groups.  

This study was done on 68 Iranian patients on warfarin treatment who attended the cardiovascular centers in Tehran Province for regular INR monitoring. Genotyping of VKORC1 (rs9923231 and rs2884737), CYP4F2, and CYP2C9*3 were performed using Tetra-Arms-PCR and conventional PCR was done for the detection of CYP2C9*2 (rs1799853) SNP. Multiple regression model was performed for statistical analyses and P<0.05 was considered as the significance level.

Of 5 SNPs, VKORC1 rs9923231, CYP2C9*2, and CYP2C9*3 variants alleles had a significantly lower mean warfarin daily dose compared with the wild-type genotypes, whereas no significant association was seen between VKORC1 rs288473710, CYP4F2, and warfarin maintenance doses. Furthermore, despite a significant association between patients’ age and warfarin dose (p=0.021), it was not statistically significant for other demographic characteristics.

It can be concluded that VKORC1 rs9923231, CYP2C9*2, and CYP2C9*3 gene polymorphisms and patients’ age had a significant effect on warfarin dose. The new warfarin-dosing algorithm was developed based on VKORC1 rs9923231, CYP2C9*2, and CYP2C9*3 genotypes for predicting the required dose of warfarin.

Sulfur mustard or mustard gas is a blistering agent that has been used as a war weapon. Chemical veterans are people who have multiple injuries in different organs, especially in the skin. Studies have shown that the side effects of mustard gas are the result of alkylation with DNA, RNA, protein, and lipids, which leads to metabolic and genetic changes. The respiratory system and the skin are the main targets of mustard gas alkylation. microRNAs are small non-coding and single-stranded RNAs that play a role in the spatial and spatial regulation of protein synthesis and the stability of messenger RNA. The disruption of the expression of any miRNA gene will be equal to the disruption of the expression of several protein-coding genes, each of which can play an essential role in cell biology, so to investigate the changes in the expression of microRNA, miR-20a, in the skin of chemically injured people. In this study, 30 skin biopsy samples were collected, including 10 samples from patients with moderate SM, 10 samples from patients with severe SM, and 10 control samples. RNA extraction and cDNA synthesis were performed. The expression of the miR-20a gene was investigated using the Real-time PCR method. 5s rRNA gene was used as the internal control. GraphPad Prism 6.07 software was used for the statistical analysis of data. Roc curve was used to check the biomarker value of the miR-20a gene. In this study, according to the results of the Roc curve, the miR-20a gene in the skin has a biomarker value, but it needs more study.

Mustard sulfur (SM) is a blister and has destructive effects on the lungs, eyes, and skin. MicroRNAs are non-coding RNA molecules that regulate many important cellular pathways. The clear association of microRNA expression changes in a wide range of diseases and their interception in various organs of the body, including the skin, has made them a suitable biomarker. This study aims at evaluating the changes in miR-21 gene expression in chemical warfare victims infected with SM. In this study, skin biopsy specimens including 10 veterans exposed to SM with moderate complications, 10 veterans exposed to SM with severe complications, and 10 control samples were collected, and then total RNA was extracted. cDNA synthesis was performed. MiR-21 gene expression was assessed using real-time PCR. 5 s rRNA gene was used as the internal control. Graph Pad Prism software version 6.07 was used for the statistical analysis of data. Roc curve was used to evaluate the biomarker value of the miR-21 gene. Increased expression of the miR-21 gene was observed in samples of SM veterans compared with normal samples. There was a statistically significant difference in the expression of the miR-21 gene in the skin samples of veterans exposed to SM with moderate complications and veterans exposed to SM with severe complications compared with the samples of control skin (P-value 0.0001). The expression of the miR-21 gene in the skin samples of veterans exposed to SM with severe complications and veterans exposed to SM with moderate complications in old age was not significantly related to the control sample (P-value = 0.8049, P-value = 0.3802). This study showed that the relative expression of miR-21 could be a potential biomarker in distinguishing SM veterans from healthy individuals but needs further researches..

Dental and skeletal features, unlike soft tissues, remain unchanged in events where most body tissues are destroyed such as car accidents, plane crash, crimes, etc. Panoramic and lateral cephalometric radiographs can provide useful information about dental and cephalometric indicators in human identification.

A cohort retrospective study was carried out in five stages, including collecting the samples, using a specific tooth counting system, identifying the landmarks in panoramic radiographs and designing a dental formula, designing anatomical formula with cephalometric indicators and identifying operator errors, and matching and data analysis.

We studied 180 people, including 97 (54%) women and 83 men aged 15-59 (mean age= 25.5) years in Tehran. Average indexes of missing (M), filled (F), Root Canal Therapy (RCT) teeth, crown (C), (Body/Go-Go), (Mf-Mf/Mf-Go(R+L)), De, P, ER, Go in lateral Cephalometry, SNA, SNB, Basal, N-Ans-Me, Ans-Pns/Go-Me, and S-Go/N-Me were not significantly different between cases by comparing the graphs before and after treatments (P>0.05). Examination of average indices of implant and dilacerated teeth in general and without considering the number of teeth, did not show a significant difference when the pre/post-treatment graphs were compared (P>0.05). In examining the Co-Ans/Co-Gn, Go in panoramic graph, (S-N/S-Ba) and S-N-Ba, no significant difference was observed between the cases (P>0.05).

Panoramic, lateral cephalometric graphs and designing creative formula can be used in human identification.

The role of the lung microbiome in respiratory complications associated with chemicals such as sulfur mustard or chlorine gas has yet to be determined. The aim of this study was to compare the structure and composition of the lung microbiome in chemically injured and healthy individuals in order to understand the relation between the population of the lung microbiota and respiratory complications caused by exposure to these chemicals.

To study lung microbiota, the bronchial alveolar lavage (BAL) fluids were collected from 17 chemically injured and 15 healthy cases during the bronchoscopy procedure. The diversity of lung bacteria present in BAL samples was explored using 16S rRNA gene sequencing.

The lung microbiome dominated by members of phyla Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Fusobacteria and Synergistetes which collectively accounted for > 95% of sequences. At the genus level, members of the genera Prevotella, Leptotrichia, Atopobium, Aggregatibacter, Catonella, and Oribacterium showed more than 2-fold increase in abundance in the lung microbiome of chemically injured patients. Comparing lung bacterial community at the species level, however, revealed an increased prevalence of members of Rothia mucilaginosa (3-fold), Prevotella melaninogenica (2.7-fold), Prevotella pallens (3.5-fold), Actinobacillus parahaemolyticus (2.5-fold), Veillonella parvula (2.5-fold), and Neisseria subflava (1.5-fold) in these patients.

An increased abundance of bacterial species known to associate with airway inflammation suggested their implications in respiratory failure in chemically injured patients. Monitoring and maintaining the homeostasis of the microbial population colonizing lung of chemically injured patients will pave the way to develop a more targeted treatment for these patients.

Oxygen deficiency, referred to as hypoxia, can be divided into two categories of environmental (exogenous) which is sensitized by respiratory cells, and microenvironmental (endogenous) responses by other cells in the body, like cancer cells. Cell responses to oxygen fluctuations are mediated by hypoxia-inducible factors (HIFs). Lung is known as the first organ that encounters the exogenous and environmental oxygen after breathing. However, data are limited for HIF respiratory pathways of environmental oxygen sensing inside the lung cells. Thus, we evaluated the role of both HIF-1 and HIF-2 isoforms in environmental hypoxia sensation by pulmonary vascular and bronchial cells. Then, we introduced the role of HIFs in microenvironmental and endogenous oxygen sensation in tissues and cells other than respiratory cells as well as all of involved molecular mechanisms in those non-lung cells and tissues. Next, a detailed emphasize was put on the strict role of HIF molecule in tumor cells and cancer stem cells.

 Arsenic trioxide has anti-cancer effects on a wide range of cancers, mainly by inducing apoptosis in cancerous cells. Thalidomide is an inhibitor of angiogenesis. The main purpose of this study was to investigate the combined effect of arsenic trioxide and thalidomide on the expression level of VEGF A gene.   In this experimental study, MTT test was used to investigate the effect of arsenic trioxide and thalidomide both in isolation as combined on cell viability of KG-1 and U937 cells at different times and different doses. Cell apoptosis was assessed by flow cytometry. The level of mRNA expression of VEGF A gene was assessed by Real Time PCR. Student-t, ANOVA and SPSS-17 software were used for data analysis.   The selective doses of arsenic trioxide in the KG-1 and U937 cell lines were 1.618 μM and 1 μM, respectively. The selective doses of thalidomide in the KG-1 and U937 cell lines were 80 μM and 60 μM, respectively. Combination of arsenic trioxide and thalidomide had a significant effect on both cell lines. On the other hand, the expression of the VEGF A gene significantly decreased.     The present study showed that the expression of VEGF A gene in 1-KG and U937 cell lines decreased and the combination effect of arsenic trioxide and thalidomide on the cells induced apoptosis. To confirm these results further investigation is required on the protein level.

Single nucleotide polymorphisms (SNPs) are being increasingly used by forensic laboratories. In the Iranian population, few studies have been reported that aim to investigate allelic frequencies and determine genetic variation of SNPs based on SNPforID database. The goal of the current study was to evaluate the population genetic diversity by genotyping of 25 informative autosomal single nucleotide polymorphisms residing in Gilan province in Northern Iran, using the SNaPshot assay.
In this cross-sectional study, the results of the bioinformatics analysis indicated that at least 25 autosomal loci SNP could help discriminate between Iranian ethnic groups. A total of 53 unrelated individuals from the Gilakis ethnicity group provided blood samples for extraction of genomic DNA by the RGDE method. The SNapShot method was used to genotyping 25 selected SNPs on the 3110xl ABI Genetic Analyzer. Statistical analysis was conducted using the Arlequin 3.5, GeneAlex 6.5 and GeneMapper 5.0 software.
The mean heterozygosity was 0.428. The minimum heterozygosity observed was 0.190 that related to rs938283. Minor allele frequency (MAF) for rs938283 was 0.105. There was significant deviation in allelic frequencies from Hardy-Weinberg equilibrium for all the studied SNPs except rs1382387. The calculated FST values presented among Gilak and three Iranian ethnic groups including Kurd, Persian ad Lors were not significantly genetic different.
Evaluation of the 25 autosomal SNP simultaneously enabled discrimination among ethnic groups within the population. The results presented Gilakis ethnicity genetically when compared to the other ethnicity, Lor, Persian and Kurdish, are similar. The SnapShot method has appropriate sensitivity and specificity to simultaneously genotype SNPs.

Both Chronic Obstructive Pulmonary Disease (COPD) and mustard lung victims of chemical injury (unlike asthma and allergy) are known respiratory diseases with a substantial genetic background and there are no highly effective medications for either disease. Considering the many similarities between the two diseases, both can be considered simultaneously under one category. Therefore, we assessed the expression of miR-20a and miR-92a as representatives of the miR-17/92 cluster due to the effective interference of these two miRNAs in the signaling pathways of inflammation and hypoxia in the pulmonary serum (consisting of Mustard Lung and COPD, simultaneously).
Stem-Loop Real-Time Quantitative Polymerase Chain Reaction was used to examine the expression of miR-20a and miR-92a simultaneously in patients with COPD (n=25) and mustard lung (n=50).
The results showed that the expression of both miR-20a and miR-92a was significantly downregulated in patients with COPD and chemical injuries when compared to the control group. The expression of each of two miRNAs in the moderate and severe phases of the disease was more downregulated in the mild stage, and miR-92a showed a further downregulation compared to miR-20a (p The downregulation of miR-20a and miR-92a might be due to alterations in 1- genomic and/or 2- environmental and/or 3- ecogenomic (affected by epigenomic) factors which requires intensive research in this field. Our results may shed light on new effective strategies for the treatment of respiratory diseases by regulating the mir17/92 cluster by regulating related factors. The members of the oncogenic miR17/92 cluster are significantly upregulated in lung cancer and have the potential to be used as biomarkers in the non-invasive screening of respiratory diseases. Furthermore, these miRNAs can be used for distinguishing respiratory diseases from lung cancer and cardiovascular diseases (also known as COPD comorbities).

Single nucleotide polymorphisms (SNPs) are being increasingly used by forensic laboratories. In Iranian population, few studies have been reported with aim to investigation allelic frequencies and determination of genetic variation of SNPs based on SNP for ID database. The goal of this study was to evaluate of population genetic diversity by genotyping of 25 informative autosomal single nucleotide polymorphisms residing in Khuzestan province by using the SNaPshot assay. The cross-sectional study, the results of bioinformatics analysis indicated that at least 25 autosomal loci SNPs had the power to discriminate between the Iranian Ethnicity. A total of 53 unrelated individuals from Arab ethnicity group were extracted genomic DNA from blood samples. Genotyping 25 selected SNPs by using SNapShot method on the 3110xl Genetic Analyzer were performed. Statistical analysis was calculated using the Arlequin 3.5 & Gene Alex 6.5 soft wares. The mean heterozygosity was calculated 0.428, also the minimum heterozygosity was observed 0.190 that relate to rs938283.  Minor allele frequency (MAF) for rs938283 was 0.105. There was significant deviation in allelic frequencies from Hardy-Weinberg equilibrium for all the studied SNPs except rs1382387. The calculated FST values and Evaluation of the 25 autosomal SNPs simultaneously, presented that among Arabs and three Iranian ethnic groups including Kurd, Persian and Lurs were not significantly genetic different and thay are similar to each other. Finally, it is obvious that this 25 autosomal SNPs enable to discriminate between ethnic groups within the population. The SnapShot method has appropriate sensitivity and specificity in order to simultaneously SNP genotyping.

Colorectal cancer (CRC) is one of the most common malignancies in the world with a high mortality rate. Among all of the genes that are involved in colorectal cancer, KRAS gene has a high diagnostic importance. This gene is mutated in early stages of colorectal cancer. Most of KRAS effective mutations are observed in codons 12 and 13. Therefore, detection of KRAS gene mutations (codon 12 or 13 or both codons) in patients with colorectal cancer is important for the treatment protocol of choice.
Material & DNA was extracted from the tissue and serum samples of 35 patients with colorectal cancer who had inclusion criteria. The primer of KRAS codons 12 and 13 were designed and synthesized. qPCR technique was used to determine the location and type of KRAS gene mutations. Statistical analysis was performed using the SPSS 20 software and P-value less than 0.05 was considered as significant.
Statistical analysis have shown that the incidence of mutations in genes KRAS was 37.1%. The mutations in codons 12 and 13 were 69.2% and 30.8%, respectively. The possibility of KRAS mutations in colorectal cancer patients with high grade was 1.14 times greater than the cases with low grade.
KRAS mutation analysis is a diagnostic tool for cancer with a remarkable prognostic result. Regarding the age of outbreak in Iran, screening and prevention programs and providing effective treatment protocols are very important.

In order to estimate genetic diversity of native Iranian Red deer, the complete sequence of cytochrome b gene (cytb) from three naturally reserved populations of north and northwest of Iran was analyzed. These populations are concerned due to their small population size. 25 fresh fecal samples were collected from these populations during 2015. Results indicated that in spite of geographical distances there was no diversity between cytb sequences of these three Iranian maral populations. All three populations were one haplotype. A comparison of the cytb between native Iranian maral and Turkish maral deer showed 7 polymorphic sites with one site of transversion. The nucleotide diversity between these two groups was, 0.003 with 0.02 mean genetic distance that was low and indicated low genetic differences between Iranian maral and Turkish maral deer. High Tajima's D (1.55) indicated natural selection between Iranian and Turkish maral deer populations. The cytb from Iranian maral deer and other red deer subspecies was compared. Genetic distance between these populations was 0.037±0.004. The nucleotide diversity was 0.03517 within all 46 sequences and there were 135 polymorphic sites. Tajima's D was 0.322 and non-significant, that indicated there was a genetic mutation in these populations. Results of haplotype network showed that Iranian native maral deer is classified in European red deer and was in the same cluster with Turkish maral deer. According to the low genetic diversity of reserved maral populations, it is necessary to make suitable conversational management decisions to conserve this species and for second, to increase the effective size of these populations.

The cause of epilepsy in most of cases is unknown but inherited causes can play a role in its incidence. Recently, many researches have been conducted in the field of epilepsy. The aim of this study was to identify gene(s) responsible in an Iranian family with autosomal recessive non-syndromic epilepsy using whole exome sequencing (WES) method.
In this experimental study, genomic DNA was extracted from whole blood belonging to three affected persons and a healthy individual and whole exome sequencing was performed using Illumina Hiseq2000 platform. At first, variants classification were carried out based on mutation types, position of the mutation and their allele frequencies and then, prioritization was performed via two approaches. Sanger sequencing was carried out for RNF216 confirming the variant [NM_207111.3 (RNF216): (g.5780794G > A), (c.854C > T)].
Findings: From 130855 variants, 85% were single nucleotide variants and 15% were indels. One third of them located in intergenic and intronic regions, one third located in 3’and 5’ UTRs and one third located in exonic and splice site regions. 3.8% and 3.4% of variants had allele frequencies below 0.01 in ExAC and 1000 genome project databases, respectively. By using the two prioritization approaches, a variant in RNF216 gene [NM_207111.3 (RNF216): (g.5780794G > A), (c.854C > T)] was selected as a prior candidate. However, this variant did not co-segregate with the disease in all of the members of this family.
Exome sequencing has been considered as a tool for studying genetic causes of Mendelian disorders; but it has some limitations. Mutation in the non-coding regions of the genome, clinical heterogeneity of disease, wrong clinical diagnosis, phenocopy, and technical and analytical limitations can be considered as a reason that we could not find gene(s) responsible for the disease in this family.

Due to large sized genetic database of population in a given society, researchers face serious problem to analyze genetic data and provide accurate genetic identification. Variation in molecular markers such as SNPs, mtDNAs, STRs and Y-chromosome are utilized for different purposes, including genetic identification. In our country due to natural disasters and imposed war, the use of this technology was considered and according to the requirements, an optimal database was designed that has the capability of analyzing genetic data. After obtaining individual genetic information, a software was designed for the analysis of genetic information as well as to serve as a common genetic database which contained application-specific local data search capabilities, characterization of individual genetic information, and its application. The software was named as NoorGIS. By use of NoorGIS software, it was possible to study and compare genetic relationship between individuals and a given population, investigate personal genetic information, process obtained genetic information by employing genetic similarities and information and also enhancing the capabilities of the software to achieve favorable outcomes in genetic identification. This software was assessed at the Human Genetics Research Center and successfully used to genetically identify unknown martyrs of the imposed Iraq-Iran war and military accidents. With this comprehensive software, personal and genetic data can be stored and utilized for the identification purposes. The results are also approved from the scientific and legal aspects.

Background and The 50 KDa protein (50 µg) in carboxylic domain of the neurotoxin heavy chain (BoNT/A-Hc) recognizes surface receptors on target neurons and this fragment contains the principle protective antigenic determinants. Recently, this fragment has been used as a recombinant vaccine candidate for botulism. The study aimed to compare the evaluation of BoNT/A-Hc production in fed-batch (high cell density cultivation) and batch cultivation of recombinant Escherichia coli (E. coli.). In this research, growth of recombinant E. coli in batch culture was studied. In order to maximize protein expression, induction time and Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducer concentration were optimized by the Taguchi statistical method. Then, fed-batch culture was applied to achieve high cell density cultivation. Finally, the recombinant protein expression level was determined. In optimized conditions, 62 and 486 mg/l of soluble recombinant BoNT/A-Hc were produced in batch and fed-batch cultivation. According to the results, high cell density in fed-batch cultivation is a very effective for improve of recombinant proteins productivity.

Bone marrow stromal cells (BMSC) have been successfully employed for movement deficit recovery in spinal cord injury (SCI) rat models. One of the unsettled problems in cell transplantation is the relative high proportion of cell death، specifically after neural differentiation. According to our previous studies، p75 receptor، known as the death receptor، is only expressed in BMSC in a time window of 6-12 hours following neural induction. Moreover، we have recently reported a decreased level of apoptosis in p75-suppressed BMSC in vitro. Therefore، our objective in this research was to explore the functional effects of transplanting p75: siRNA expressing BMSC in SCI rats.

Laminectomy was performed at L1 vertebra level to expose spinal cord for contusion using weight-drop method. PBS-treated SCI rats (group one) were used as negative controls، in which cavitations were observed 10 weeks after SCI. pRNA-U6. 1/Hygro- (group two، as a mock) and pRNA-U6. 1/Hygro-p75 shRNA- (group three) transfected BMSC were labeled with a fluorescent dye، CM-DiI، and grafted into the lesion site 7 days after surgery. The Basso-Beattie-Bresnehan locomotor rating scale was performed weekly for 10 weeks.

There was a significant difference (P≤0. 05) between all groups of treated rats regarding functional recovery. Specifically، the discrepancy among p75 siRNA and mock-transfected BMSC was statistically significant. P75 siRNA BMSC also revealed a higher level of in vivo survival compared to the mock BMSC.

Our data suggest that genetically modified BMSC that express p75: siRNA could be a more suitable source of cells for treatment of SCI.

Shigella dysentery producing shigatoxin (subunits A, B) is one of the most important human pathogenic intestinal bacteria. Entering to epithelial cells, the toxin inhibits protein synthesis leading to cell death. In spite of great investigation on vaccine production against S. dysentery studying to achieve significant stxA recombinant protein still remains important. The objective of this study was designing mutant stxA gene and expressing protein production as vaccine candidate against stx for further immunization studies. Three stxA mutant gene including (E167Q-A231D-G234E) were designed and the synthetic gene in pET28a plasmid was obtained and confirmed by PCR. Thereafter the plasmid was transformed into the host cell E. coli BL21 after which gene expression was optimized and protein purity assay was then performed. Preliminary studies led to mutant stop gene design after which it was confirmed by synthetic plasmid and PCR. The expression of this gene in E. coli BL21 host cell was then optimized and resulted in forming large amount of protein inclusion bodies. Purification of inclusion bodies and protein solubilization was performed with a combinatorial method. Regarding the mechanism of shigatoxin effect and simultaneous use of two different mutations in this gene, less toxicity is expected in comparison with previous mutants as vaccine candidate, posing a better vaccine candidate.

Shigella dysentery is one of the most important human pathogenic intestinal bacteria. Entrance of the shigella toxin into the epithelial cells inhibits protein synthesis leading to cell death. In spite of great investigations on vaccine production against S. dysentery, studying to achieve significant stxA recombinant protein still remains important. The objective of this study was to determine the appropriate mutation loci and designing stxA subunit synthetic gene, its further expression and optimization and ultimately assaying purification method for further immunization studies. Three mutant stxA gene including (R170L-A231D-G234E) were designed and the synthetic gene in pET28a plasmid was obtained and confirmed by PCR. Thereafter the plasmid was transformed into the host cell E.coli BL21 DE3 after which gene expression was optimized and protein purity assay was then performed. Preliminary studies led to mutant stop gene design, after which it was confirmed by synthetic plasmid and PCR. Expression and optimization were then performed which resulted in large amount of protein inclusion bodies. Purification of inclusion bodies and protein which resulted solubilization was done with a combinatorial method. With regard to the mechanism of shiga toxin effect and favorable mutation design with new arrangement, less toxicity of expressing protein is predicted than previous other mutants, posing a better vaccine candidate.

Botulism is a lethal disease which is caused by the one of the neurotoxins of the 7 types of Clostridium botulinum. The carboxylic domain of the heavy chain of Clostridium botulinum type A neurotoxin (BoNT/A-Hc), is highly capable of activating immune system and is used for production of a vaccine against botulism. The aim of this study was to express and produce soluble form of BoNT/A-Hc in recombinant E. coli.

Hc part of BoNT/A was subcloned in pET28a vector and clone was expressed in E. coli BL21 (DE3). Then the best recombinant clones were selected based on three main factors: expression, growth and plasmid stability. Then protein expression was evaluated in LB and M9 media and the protein was purified by resin column chromatography.

In flask culture, under optimal conditions of bacterial culture yielded 52mg of BoNT/A-Hc soluble protein from each liter of culture medium.

According to the results of this study, selection of suitable strains on the basis of important growth indices of the bacteria, expression of recombinant protein and plasmid stability can lead to in increased efficiency of the recombinant protein production.