جعفر امانی

APC gene in ctDNA has been proposed as a potential biomarker for cancer diagnosis. A biosensor based on a multi-walled carbon nanotube (MWCNT) and DNA probe with fluorophore FAM (6-carboxyfluorescein) for detection of APC gene in ctDNA was developed to identify patients with colorectal cancer (CRC).This method was designed based on the adsorption and immobilization of FAM-labeled single-stranded DNA (ssDNA) on MWCNT, which leads to the quenching of FAM fluorescence emission. By adding its cDNA could release single-stranded DNA probe (ssDNA) from the MWCNT surface and a double-stranded DNA (dsDNA) was formed. It led to the return of FAM fluorescence emission. While in the case of non-complementary DNA the corresponding dsDNA was not formed and therefore we did not have the return of FAM fluorescence emission. The results of this study showed that the biosensor based on carbon nanotubes can be used as a high-sensitivity method for the early detection of CRC.

Necrotic enteritis (NE) is an economically important disease, caused by Clostridium perfringens type G strains, and is one of the major targets of antibiotics used in poultry feed.

This study aimed to genotypically characterize virulent strains of C. perfringens isolated from healthy and diseased birds in Iran.

Eleven isolates were derived from necrotic enteritis cases, and 27 were from healthy chickens. Isolations were performed using blood agar. To assess whether zmp is generally associated with avian NE, 38 C. perfringens isolates were screened using PCR and western blotting. The involvement of these toxins as virulence factors was investigated using cytotoxicity assays.

All isolates carried the phospholipase c (plc) gene regardless of their origin and virulence. The zinc metallopeptidase (zmp) gene was found in the isolates collected from birds affected by necrotic enteritis. Furthermore, Necrotic enteritis like B (NetB) was only found in 36.36% of the isolates derived from necrotic enteritis-infected birds. Western blot analysis further confirmed the expression of Alpha toxin, NetB, and Zmp in different isolates. Incubation of Leghorn Male Hepatoma (LMH) cells with crude C. perfringens toxins indicated that the supernatants of all bacterial strains were toxic toward LMH cells at different dilutions. In addition, crude toxins of the Cp28 strain expressing Alpha toxin, Zmp, and NetB showed an approximately 50% cytotoxic dose (CD50) at a 1:34 dilution. Strain Cp119.2, which produces both ZMP and the Alpha toxin, and strain Cp48, which only produces the Alpha toxin, showed CD50 at 1:23 and 1:4 dilutions, respectively.

It seems that both NetB and Zmp play major roles in the cytotoxicity and pathogenicity of this organism.

The chimer structure consisting of the extracellular domain of CTLA4 and the FC fragment of human IgG1 antibody binds to CD80 (B7-1) and CD86 (B7-2) ligands on APCs as a result inhibits CD28 binding and T lymphocyte activation. This structure, called Abatasept, was approved by the US FDA in 2005 to treat rheumatoid arthritis.

In this study, the CTLA4-FC chimer structure was designed and then some of its features were investigated using bioinformatics tools. The gene was then cloned into the pET-28a plasmid and transformed into E.coli BL21DE3. Recombinant protein was expressed and purified, and flow cytometry was used to evaluate the binding affinity. Mixed lymphocyte culture (MLR) method was also used to evaluate the biological activity of recombinant protein.

Bioinformatics results showed that the designed structure of CTLA4-FC is suitable in terms of physicochemical properties, second and third structure, how it binds to the receptor, antigenic properties and mRNA properties. Recombinant protein was well expressed in bacteria. Purification was performed as required. Western blotting and ELISA results were positive and flow cytometry and MLR tests were acceptable.

Recombinant CTLA4-FC protein was produced in bacterial cells and bound well to CD80 and CD86 receptors and was able to inhibit T cells. So it can be a good candidate for drug production.

COVID-19, an infectious viral disease caused by severe acute respiratory syndrome (SARS-CoV-2) with more than 260 million infections as of December 2019, is a serious threat to the health and economy of human societies. Designing and producing a suitable vaccine can Reduce the incidence of this disease. Therefore, it is very important to find effective and safe neutralizing antibodies and vaccines for COVID-19. The RDB section of the spike protein is a suitable option for the production of subunit vaccines and neutralizing antibodies. This research aims to introduce the RBD section as a vaccine candidate.

The RBD was recombinantly expressed in two hosts, Escherichia coli (E.coli) and insect cells, and its production rate was compared. Using the western blotting method, protein production was confirmed. To evaluate the function of the protein expressed in two prokaryotic and eukaryotic hosts, using the serum of patients recovered from COVID-19 (wild type and delta), the ELISA method was used.

Despite the higher production of recombinant protein by E.coli, the affinity of the antibodies in the serum of the patients to the protein expressed in the insect cell was higher.

In this study, the RBD was expressed in two different hosts. The results show that RBD is mentioned as a vaccine candidate. Homozygous in the insect cell preserves the biological activity of the protein by carrying out the process of post-translational changes such as glycosylation.

Given the global epidemic of COVID-19, it is important to design a vaccine for prevention. The virus belongs to the beta-coronavirus family and forms appendages on the surface of the glycoprotein spike virus membrane. Studies on SARS-CoV-1 and related MERS-CoV vaccines have shown that the spike protein on the virus surface is a suitable target for the vaccine. In this experimental study, we compare the recombinant fragment of spike protein (rfsp) expressed in the eukaryotic host CHO-K1 cell and prokaryotic E. coli in terms of immunogenicity, neutralizing activity, and epitopes recognition Similar to the virus strain and the ability to bind to the serum of improved patients, in two types of alpha and delta variants. The results showed that both rfSP proteins are a potential new antigen candidate for the development of the Covid 19 vaccine, but the CHO cell maintains the biological activity of the protein by performing post-translational modification processes such as glycolysis. This increases the present likelihood of developing virus-like epitopes and increases the titer of rfsp-specific antibodies in the serum of immunized mice. Therefore, priority is given to rfsp expressed in CHO cells to evaluate vaccine efficacy.

Shigella and Enterohemorrhagic Escherichia coli are among the most common causes of bacterial diarrhea, and no effective vaccine candidate for these bacteria have approved yet. Due to the role of IpaD protein and Shigella enterotoxin B subunit (StxB) in Shigella and E. coli O157: H7 pathogenicity, STX1B-IpaD chimeric protein can be used as a suitable molecule to produce a recombinant vaccine candidate. This study aimed to clone, express, and purify STX1B-IpaD chimeric protein to develop an effective vaccine candidate against Shigella and E. coli O157: H7 species.

IpaD gene with NdeI and BamHI restriction enzyme sites was isolated from a recombinant vector and subcloned into the pET28a -STX1B expression vector. Vector was transferred to E.coli strain Rosetta (DE3) and confirmed by PCR and restriction enzyme digestion. SDS-PAGE and western blotting were used to confirm the recombinant protein. The recombinant STX1B-IpaD protein was purified by affinity chromatography, and its concentration was measured by the Bradford method.

The PCR and restriction enzyme digestion showed the accuracy of the gene cloning. The protein electrophoresis showed the proper expression and correct molecular weight (27 kDa) of STX1B-IpaD. The western blot analysis confirmed the recombinant protein. The recombinant protein concentration was estimated at more than 0.3 gr/L.

An effective method for the production of recombinant proteins is codon optimization and effective expression in heterologous hosts. After the immunogenicity in the animal model, this recombinant protein can be used as a chimeric vaccine candidate against EHEC and Shigella bacteria.

APC gene in ctDNA and miR-21-5p have been proposed as potential biomarkers for cancer diagnosis. In this above study, a biosensor based on multi-walled carbon nanotube (MWCNT) and DNA probe with fluorophore FAM (6-carboxyfluorescein) to detect APC gene in ctDNA, and DNA probe with fluorophore ROX (5-carboxy-x-rhodamine ) to detect miR-21-5p, was designed to identify patients with colorectal cancer (CRC).This method is based on the adsorption and stabilization of single-stranded DNA (ssDNA) labeled with FAM and ROX on MWCNT, which leads to the quenching of the fluorescence emission of FAM and ROX. By adding their complementary DNA (cDNA), which led to the release of single-stranded DNA (ssDNA) probes from the MWCNT surface and a double-stranded DNA (dsDNA) was formed, which led to the return of FAM and ROX fluorescence emission. In this study, the sensitivity of FAM fluorophore was 50 nM (50 nM) and the sensitivity of ROX fluorophore was 25 nM (25 nM). The response time in the presence of the target strand is 12 minutes. The LOD and LOQ of DNA biosensor individually were 1.12 nM and 3.2 μM, respectively. The results of this study showed that the biosensor based on carbon nanotubes and DNA probe, in addition to existing methods, can be used as a high-sensitivity method for early detection of CRC.

Botulism, a syndrome caused by food poisoning, results from use of food contaminated with the botulinum toxin, which is very dangerous and deadly. Botulism is caused by the effects of bacterial toxins on the terminals of the motor nerves. Botulinum neurotoxins are among the most potent toxins. The aim of this study was to investigate expression, purification and, evaluation of the immunogenicity of the recombinant BoNT/B-HcC protein as an immunogen candidate in mice.

The C-terminus of the receptor-binding domain of  botulinum neurotoxin type B(BoNT/B-HcC) was selected as the antigen for bioinformatics evaluations. The pET17b-BoNT/B-HcC plasmid was transferred to E. coli BL21(DE3) by heat shock. The recombinant protein was purified and analyzed by SDS-PAGE. After verification of the recombinant protein by western blot, immunization of mice was performed. Antibody titers of recombinant proteins were evaluated by indirect ELISA and the results were compared using t-test.

The codon adaptation index (CAI) of the optimized gene was 0.99. The percentage of codons having high-frequency distribution was improved to 78%. Restriction analysis confirmed the 1119 bp construct gene and the recombinant protein with a molecular weight of 43.8 kDa was expressed in the prokaryotic host. The total yield of purified protein was 23 mg of protein per liter of culture. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer was significantly different compared to that of the control sample.

The designed recombinant antigen showed high antigenicity that could be considered as an immunogen against botulinum type B neurotoxin in future studies.

Toxins produced by Clostridium botulinum are among the deadliest compounds known that cause botulism. Currently, the detection of BoNTs in food using bioassays on laboratory mice is a very sensitive method with a detection range of 7 to 20 pg.mL-1. However, bioassay for mice is time consuming. This method is fast, highly specific, and sensitive to experiments on mice. The aim of this study was to use the modified Sandwich ELISA method to detect BoNT/B toxin.

Recombinant 370 amino acid protein was expressed from the carboxyl terminus of the binding moiety of BoNT / B toxin with a molecular weight of 45 kDa as antigen and purified by Ni-NTA affinity chromatography. IgG antibodies were isolated from mouse and rabbit sera byG protein affinity chromatography. The sensitivity and specificity of the method designed to detect recombinant BoNT/B-HcC antigen and botulinum toxin type B were evaluated.

Purified rat and rabbit antibody concentrations were 3 and 4.5 mg / ml serum, respectively. The minimum concentrations of detectable protein were determined by indirect ELISA with purified mouse and rabbit antibodies at 475 and 118 pg. By optimizing the sandwich ELISA method, at least 30 ng of recombinant BoNT/B-HcC antigen and146 pg of highly specific BoNT/B were detected.

sandwich ELISA method can be used for accurate and sensitive identification of Clostridium botulinum toxin type B. It is necessary to evaluate the effectiveness of this method in the future to detect botulinum toxin in environmental and food samples.

 Cancer is mainly caused by defect in natural regulation of programmed cell death (PCD). On the other hand, it is proved that, the EGFR is overexpressed on the surface of many malignant cell. Therefore, immunotoxin against EGFR, which consisting an antibody or fragment of an antibody linked to the toxic moiety is one of the most reliable strategies for cancer treatment. Here we evaluate the effect of a novel immunotoxin molecule harboring ricin, as a toxic moiety on the mRNA level of two main apoptotic molecules on cancerous cell lines.

 The Effects of immunotoxin molecule and it’s components including ricin toxin and antibody fragment on the mRNA level of pro- apoptotic BAX and anti-apoptotic BCL-2 and EGF receptor genes on the breast MDA-MB-468 and colorectal HCT-116 cancer cell lines were assayed by qRT-PCR. We used glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference gene.

 Treatment with immunotoxin significantly increased the expression of BAX gene and decreased the expression of EGFR and BCL-2 genes (P<0.05) and induced apoptosis. Also, ricin toxin caused a significant decrease in BCL-2 and no significant increase in BAX in cancer cells and decreased EGFR only in breast cancer cells. The scFv part had no significant changes on genes expression.

 The present findings showed that the recombinant Panitumumab-Ricin protein, may be an appropriate candidate for the treatment of EGFR overexpressed cancers by induction in apoptosis pathway.

Covid-19 epidemic results from an infection caused by SARS-CoV2. Evolution-based analyses on the nucleotide sequences show that SARS-CoV2 is a member of the genus Beta-coronaviruses and its genome consists of a single-stranded RNA, encoding 16 proteins. Among the structural proteins, the nucleocapsid is the most abundant protein in virus structure, highly immunogenic, with sequence conservatory. Due to a large number of mutations in the spike protein, the aim of this study was to investigate bioinformatics, expression of nucleocapsid protein and evaluate its immunogenicity as an immunogenic candidate

B and T cell epitopes of nucleocapsid protein were examined in the IEDB database. The PET28a-N plasmid was transferred to E. coli BL21(DE3) expression host, and IPTG induced recombinant protein expression. The protein was purified using Ni-NTA column affinity chromatography, and the Western blotting method was utilized to confirm it. Finally, mice were immunized with three routes of purified protein. Statistical analysis of the control group injection and test results was carried out by t-test from SPSS software.

The optimized gene had a Codon adaptation index (CAI) of 0/97 Percentage of codons having high- frequency distribution was improved to 85%. Expression of recombinant protein in E.coli led to the production of BoNT/B-HCC with a molecular weight of 45 kDa. The total yield of purified protein was 43 mg/L. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer ratio was significantly different compared to the control sample and the antibody titer was acceptable up to a dilution of 1.256000

According to the present study results, the protein can be used as an immunogenic candidate for developing vaccines against SARS-CoV2 in future research.

Cyanobacteria are ubiquitous photosynthetic microorganisms that cause blooms in surface waters; some species endanger the health of humans and other animals. To date, more than 65 cyanobacterial neurotoxins have been discovered, of which Saxitoxins (STXs) is the most well-known and have many variants. Saxotoxins target voltage-gated ion channels that cause skeletal or respiratory muscle block and cause death due to the inability to breathe. Saxotoxins, by accumulating in marine food sources such as snails, shellfish, and finfish, cause poisoning of aquatic mammals and humans. The incidence of saxitoxin has been reported in several different regions, including the Persian Gulf, Australia, and North and South America, which should be given special attention due to the high lethality of this type of poisoning. In addition to the risk to human health, it is also very dangerous for wildlife, especially aquatic animals. Due to the high toxicity of saxitoxin and some reports in Iran, more precise and extensive preventive and control measures should be taken for this dangerous toxin, which has been somewhat facilitated due to sensitive diagnostic methods.

Aim and Background

Enterohemorrhagic E. coli O157: H7 is an important human and animal pathogen that causes hemolytic uremic hemorrhagic diarrhea in humans. The binding to host cells is the first step in stabilizing this bacterium through the secretion system of the type (T3SS) III and through the interactions between secretory proteins. Tir is a protein that is transferred to the host cells after expression in the bacterium and via T3SS and is located in the membrane and plays an important role in the bacterial binding to the host. The purpose of this research is to clone a gene construct that has Tir virulence factors.

Specific primer design for tir gene was performed using oligo softwares and gene amplification was performed by PCR reaction on a DNA template. After the enzyme digestion, the gene was inserted into expression vector pET28a; the construct was transformed to the expression host. After confirmation of gene cloning, the Tir recombinant protein was expressed by induced IPTG induction. The western blot analysis was carried out to confirm Tir protein. The gene of tir was cloned inside pET28a vector.

Cloning of tir gene in pET28a vector was performed appropriately between desired sites and after induction protein, the Tir recombinant protein showed appropriate and significant expression. The antibody used in Western blot was also able to properly detect Tir.

After the induction, the protein of Tir showed a remarkable expression of the recombinant protein. As a result, this structure can be used to produce Tir protein for immunological studies against E.coli O157: H7.

Diarrhea is the second most common cause of under-5 mortality. The most important strains of Entrotoxigenic Escherichia coli causing Lt and St toxin cause diarrhea and Entrohemoragic E.coli causing Shiga-like toxin secretion. Chlorine enterotoxin B subunit (Ctx) plays a key role in the development of diarrhea in Vibrio cholerae. More specific antibodies could be developed to counter these toxins by combining the CtxB, LtB and StB (LSC) epitopes and the production of trivalent vaccine. The aim of this study was to cloning lsc gene into pcDNA3.1 to design a vaccine DNA.

The lsc gene sequence was transferred to pcDNA3.1(+) vector after primer design and amplification by PCR. The pcDNA3.1(+) vector and the PCR product were digested using HindIII and EcoRI enzymes. Cloning of lsc gene was performed in pcDNA3.1(+) vector and PCR. The clones were digested enzymatically. To ensure expression of lsc gene, it was transferred to HEK-293T cell and confirmed by Western blotting.

The lsc gene was confirmed by PCR and cloning in pcDNA3.1(+) vector using enzymatic digestion and a fragment length of 933 bp was detected and confirmed. Transfection kit was then transferred to HEK-293T cell and expression of the recombinant protein was confirmed by Western blotting and the protein was 39 kDa.

The results of the chimeric gene are well expressed in the cell line and confirmed by Western blotting that can be a good candidate for the fight against bacterial infection.

Enterotoxigenic Escherichia coli (ETEC) and Enterohemorrhagic Escherichia coli O157:H7 , the most common strains,  are causes diarrhea that can kill  hundreds of thousands of children annually. The development of an effective combination vaccine for enterohemorrhagic Escherichia coli and Enterotoxigenic Escherichia coli is very important.

In this study, the sicl gene was amplified and Subcloned as a DNA vaccine. The sequence of antigen encoding genes was evaluated from the genebank and their epitopes were evaluated to design of primer for the synthetic chimeric gene and was amplified by PCR. Subcloning of  a multipartal chimeric gene in eukaryotic expression vector was performed to make a DNA vaccine and finally the protein was purified by nickel chromatography and evaluated by Western blotting.

The immunoblotting results of the expression of SICL chimeric protein indicated the presence of a 76 kDa band in the form of insoluble particles after 12 hours induction. Purification of the recombinant protein using His tag sequencee and confirmation of the purified protein with the recombinant protein specific antibody demonstrated the accuracy of the protein expression.

Protein expression, purification and verify by western blotting showed that this recombinant chimeric protein (SICL) can be expressed in eukaryotic host.

Data suggests that elderly people are more easily infected by COVID-19 and develop a more serious prognosis than other age groups. The current study compared the clinical, laboratory, and radiological findings between groups of elderly and non-elderly Iranian patients with COVID-19.

Data was collected on 598 patients that had been diagnosed and confirmed with COVID-19 in Baqiyatallah hospital, Tehran, Iran from February 26 to March 26, 2020. Patients were assigned to the elderly (≥65 years old) and the non-elderly (<65 years old) groups and clinical characteristics, laboratory findings, imaging manifestations, and outcomes were compared between two age groups.

Data from a total of 139 elderly patients (23.2%) with a mean age of 73.1±6.5 years (range: 65-94 years), and 459 non-elderly patients (76.8%) with a mean age of 49.1±10.2 years (range: 22-64 years) were evaluated. Comorbidities including; hypertension, liver disease, rheumatism, asthma, and allergy were more common in the elderly than in the non-elderly. Early symptoms in the elderly and non-elderly patients included cough and dyspnea, as well as fever, muscle weakness, and chills. The RBC and hemoglobin in the elderly were significantly lower than in the non-elderly patients. While other laboratory findings were not significant differences between the two groups. The number of neutrophils and the ESR, AST, LDH, and CRP levels were higher in all elderly and non-elderly patients. The incidence of multilobe lesions in CT scan and mechanical ventilation in elderly patients was significantly greater than in non-elderly patients with COVID-19. Elderly and non-elderly patients with COVID-19 received the same symptomatic comprehensive supportive therapies. Eleven (7.9%) deaths occurred in the elderly group and 30 (6.5%) occurred in the non-elderly group.

Elderly patients with COVID-19 are more likely to have a complicated clinical condition and worse in-hospital outcomes than the non-elderly. It’s suggesting that elderly patients should seek early screening and early diagnosis to reduce the mortality rates and worse prognosis associated with COVID-19.

Cancer is one of the most deadly diseases in the present age and its conventional therapies have had low success. Toxin therapy of cancer is a new therapeutic approach, which has attracted the attention of pharmaceutical specialists. Diphtheria toxin consists of three functional, transducing, and binding domains, that the functional part inhibits protein synthesis and causes cell death. The purpose of this study was to produce the functional domain of the diphtheria toxin fused with a TAT penetrating peptide and to investigate its degree of lethality on cell line.

In this study, primer was designed for tat-diph gene and the gene construct containing the sequence of the functional chain of diphtheria toxin and the TAT peptide sequence, were amplified and cloned into prokaryotic expression vector pET-28a containing a sequence of histidine. After transferring into bacterial host (E. coli BL21 DE3), induction of expression was performed using IPTG, then, the resulting protein was purified and the protein expression was confirmed by anti-histidine antibody attached to horseradish peroxidase (HRP) using western blotting technique. In the next step, the lethality of the produced chimeric protein, was evaluated on MCF7 cell line by MTT assay. Data were analyzed by uni pirate and oligo 6 software.

The results of MTT assay indicated that the TAT-Diph recombinant protein adjacent to the MCF7 cell line increased the rate of mortality of the cells.

The results of this study revealed that the recombinant protein with functional domain of diphtheria toxin and AT peptide could have lethal effect on MCF7 cell line.

Escherichia coli is the first bacteria on which cloning and protein expression are carried out. The purpose of this study was to summarize the numerous articles published in the field of protein expression in Escherichia coli. In studies, researchers in a new project require the production of pure protein, which at the theory level is easy to obtain a recombinant protein, but   there are virtually many problems, including low growth of host, formation of inclusion, protein not being active, and there is even no protein production in the exprimental scale. To enhance the expression of the protein, factors such as suitable host, vector design, transcriptional settings, selective markers, and reluctant labels are effective. There are a range of fusion proteins that can effect on accurate folding, solubility, and proteolytic resistance. Also the ability to target produced proteins to cytoplasm, periplasm, membranes and the culture medium, and some techniques grant the ability of post translational modifications in prokaryotic cells. Therefore, the key to the successful expression of recombinant proteins in E. coli is a combination of expert manipulation of the components of a broad genetic apparatus.

Breast cancer is one of the most common types of cancer among women. HER-2 molecule as the receptor of tyrosine kinase from the family of epithermal growth factor is a major cause of cancer. The Herceptin protein molecule, which is an anti-HER-2 antibody, can play an important role in the diagnosis and treatment of breast cancer. This study was done to subcloning of Herceptin gene, expression in the prokaryotic system (E.coli) and produce Herceptin recombinant protein for use in the treatment of breast cancer. In this descriptive – laboratory study, Herceptin gene from synthesized construct was isolated by enzyme digestion, and then subcloned to the expression vector pET28a. Subcloning of the gene was confirmed using PCR and enzyme digestion. After transferring the vector into E.coli BL21 DE3, expression of the recombinant Herceptin gene was induced by IPTG. The recombinant protein was evaluated by SDS-PAGE and purified with Ni-NTA column chromatography and finally verified by western blotting using anti-histidine antibody. The survival of cells adjacent to recombinant Hercaptin by MTT was investigated. Following the subcloning of the Herceptin gene, PCR and enzyme digestion, the 741 fragment of the Herceptin gene was confirmed. Confirmation of Herceptin's recombinant protein and its evaluation on SDS-PAGE gel about 27 kDa was done. The recombinant protein was also confirmed with anti-histidine tag. The purified protein adjacent to the SKBR3 cell line was able to block the growth of cancer cells. Regarding the expersion of HER2 antigen on surface of breast cancer cells, Herceptin can act as antibody blocker and it arrests the growth of breast cancer cells.

 Enterotoxigenic Escherichia coli (ETEC) is the most important bacteria causing traveler’s diarrhea. The bacterium has several virulence factors, including colonization factors (CFs) or Escherichia coli adhesins, heat-labile (LT), and heat-stable (ST) toxins. The design and production of vaccine against this disease is one of the goals of the World Health Organization due to increased antibiotic resistance and a reduction of healthy water sources. An effective subunit vaccine against ETEC could include a toxoid from both toxins and colonization factors. The aim of the current study was to express, purify, and encapsulate the recombinant protein in chitosan nanoparticles.

 In the present experimental study, the E. coli BL21DE3 harbring pET- 28a-cscl vector was used. The chimeric cscl gene is composed of cfab along with st toxin, cfae, and ltb. After the expression and purification of recombinant protein, using Ni-NTA column, Western blotting was performed with anti-His antibody. Then, the CSCl protein was encapsulated in chitosan nanoparticles and the particle size was measured.

 The recombinant CSCL protein was purified by Ni-NTA column and urea denaturation method. Then, this purified protein (~57kDa) was confirmed by Western blotting and the size of the nanoparticles was estimated as 112.0 nm with 98.8% of encapsulation efficiency.

 With some advantages, including the presence of surface and important antigens of ETEC and encapsulating in chitosan nanoparticles, the CSCL recombinant protein can be considered as a candidate for producing oral nanovaccine and stimulating of mucosal and systemic immune response.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common bacterial causes of diarrhea deaths among children and travelers in developing countries. The ETEC colonization factors, such as CFA/I and CS2 play an important role in the development of the disease. In this study, to produce the CFaE fusion recombinant protein, the tip subunits CFA/I(CfaE) and sub structural unit of CS2 (CotD) from ETEC, were used. Since mucosal immune responses to CFs can prevent disease, the aim of this study was to develop a chimeric antigen for developing the effective vaccine. In order to amplify the cfae-cotd gene, a dual gene construct consisting of cfae and cotd, the PCR reaction was performed by designed primers. The propagated gene was cloned in the expression vector pET28a. Following the induction of a recombinant gene construct with IPTG, the recombinant protein was expressed and purified by Ni-NTA chromatography column and confirmed by western blotting by Anti-Histag. Ethical Considerations: This study with research ethics code IR.IAU.SRB.REC.1397.066 has been approved by research ethics committee at Islamic Azad University, Science and Research Branch of Tehran, Iran. Cloning accuracy was confirmed by PCR and enzyme digestion reaction. The presence of the band in the SDS-PAGE 10% gel in the 68 kDa region, the expression of the recombinant protein, and the presence of the band on the nitrocellulose paper in the Western blotting test confirmed the production of recombinant protein. Optimization of codon and expression in heterologous hosts is a useful method for the production of recombinant proteins. The production of ETEC antigens as a candidate for vaccination against this bacterium is also prominent.

Enterotoxigenic Escherichia coli (ETEC) is a major causative agent of diarrhea. Enterotoxins and the colonization factors (CFs) are major virulence factors in ETEC infections. The bacterium binds to the intestinal epithelial cell surface through colonization factors and produces enterotoxins that cause excessive fluid and electrolyte secretion in the lumen of the intestine, which ultimately leads to diarrhea. Due to the difficult of treatment and the high prevalence of this disease, the design of the vaccine against this organism is one of the goals of the World Health Organization. The CooD-CotD protein, as adhesion tip subunits of CS1 and CS2, plays an important role in bacterial attachment to the intestinal epithelial cells. In this study, the expression and purification of chimeric protein CooD-CotD was carried out with the aim of investigating as candidate vaccine. In this study, codon optimization of cooD-cotD chimeric gene was performed by Gene Designer software. The gene was amplified by PCR and cloned into pJET1.2 / blunt then it was subcloned into pET28a to express chimeric protein. Recombinant protein was purified following expression, using Ni-NTA affinity chromatography and confirmed by western blotting analysis. The presence of 82kDa in the SDS-PAGE gel showed that expression of CooD-CotD chimeric protein and confirmed by western blotting analysis. Yield of purified protein was 121 mg/ml. Expression and protein purification studies showed that this protein has expression in the homologous host.

Enteropathogenic Escherichia coli from Enterobacteriaceae family is one of the most common causes of chronic diarrhea in children and infants. Polymerase Chain Reaction (PCR) method is commonly used for detection of enteropathogenic Escherichia coli species, but there are some disadvantages with this method due to the use of gel electrophoresis and staining with ethidium bromide, including being time consuming, limits on the number of samples, and toxicity of ethidium bromide. The aim of this study was to evaluate the PCR-ELISA technique for detection of enteropathogenic E.coli. In this study, Streptavidin was loaded in ELISA plate, and a specific Biotinylated probe was used to connect the PCR product. Biotinylated probe was connected to Streptavidin and the amplified gene was attached to the probe. Finally, the digoxigenin antibodies were used to identify the PCR product. The reaction was measured with an ELISA reader. eae of enteropathogenic Escherichia coli was amplified using the gene specific primers which resulted in a fragment of a 999 bp. The results of PCR-ELISA showed that this technique does not cross-react with the bacteria in their families and its sensitivity was 11 pg. PCR-ELISA technique is an accurate and rapid test for detection of infectious agents by the specific gene. PCR-ELISA could be used as an alternative method instead of time-consuming, less sensitive, and expensive techniques.

Newcastle disease virus (NDV) is an infectious agent of a large variety of birds, including chickens, which poses a real threat to the poultry industry. This virus is a member of the avian Paramyxoviridae. NDV is enveloped with membrane-embedded spikes consisting of glycosylated hemagglutinin (HN) and fusion (F) proteins. The mean death time after vNDV infection is 2-6 days, hence, the presence of preexisting antibodies prior to infection appears to be the most critical protection from this disease. Antibodies produced against the HN and F trans-membrane surface glycoproteins are able to neutralize NDV upon subsequent infection and inhibition of viral fusion with the host cell membrane, respectively. In this experimental study, the immunogenic epitopes of the F protein of NDV were designed artificially and were expressed in the heterologous system (Escherichia coli), using the appropriate vector (pET32a). In order to evaluate the immunogenicity of the recombinant f fragment, the protein was injected into the animal model. Immune response and the rise of specific antibodies titers were determined in immune sera. The results showed that immunization of mice with this recombinant protein could elicit significant serum IgG antibody up to 1/204800 titer. We show that the recombinant F protein was recognized by the mice sera immunized with the commercial vaccine. Moreover, the reactivity of vaccine strain virus with sera from F protein immunized mice suggested that the F protein is able to present similar epitopes with viral vaccine strain and hopefully could stimulate the immune system of the animal against the infectious viruses.

High prevalence of Methicillin Resistant Staphylococcus Aureus isolates (MRSA) as well as the multi-drug resistance in this bacterium causes difficulties in the treatment of infections due to these bacteria. Hence, detection of MRSA isolates by rapid and accurate methods is necessary. PCR-ELISA is an accurate and molecular technique that is used for the detection of several pathogens. The aim of this study is the detection of MRSA using PCR-ELISA. Specific primers for mecA gene were designed. Then, dNTP labeled with Digoxigenin was applied for amplifying mecA gene. DIG-labeled PCR products were seeded on the well coated streptoavidin and identified by anti-DIG–peroxidase conjugate. Furthermore, Biotin-labeled DNA probe specific for mecA gene was used. Sensitivity and specificity of the method was determined. Resistance to methicillin among 70 clinical isolates was determined by the disk diffusion, agar dilution and PCR-ELISA methods. MecA gene of S. aureus was amplified using gene specific primers resulted in a fragment with 310 bp length. Findings from the PCR-ELISA technic showed no cross-reactivity with Klebsiella Pneumoniae, Bacillus subtilis and Esheriashia coli as control bacteria and its sensitivity was 0.5 ng. The prevalence of MRSA clinical isolates by the disk diffusion, agar dilution and PCR-ELISA methods was 60%, 58.5% and 60%, respectively. The PCR-ELISA technique was known as an accurate and rapid test for the detection of infection agents using their specific gene. This technic can applied as an appropriate alternative method for time-consuming, less sensitive and expensive techniques such as Real-time PCR and differential biochemical tests which are currently used in laboratory.