majid tebianian

Two structural antigens, haemagglutinin (HA) and neuraminidase (NA), are attractive candidates for the development of a genetically engineered vaccine against influenza. Recombinant vaccines are produced by a simple and effective method, although expected to induce an immune response to a specific antigen, remain to be further improved for their high effectiveness. On the other hand, a potent and effective vaccine against influenza should be able to induce both humoral and cellular immune responses. In the present study, the NA gene, which is more stable than the HA one, was amplified by polymerase chain reaction (PCR), and the band appeared in the range of 1400 bp. Then cloned into a eukaryotic expression vector pFastBac HTA. The purity of the expressed NA protein was analyzed on SDS-PAGE electrophoresis. In the western blot, a band with a weight of about 41 kDa produced by the recombinant baculovirus containing NA is observed compared to the uninfected cells used as a negative control, and a recombinant neuraminidase is confirmed. Finally, neuraminidase (NA) gene clone can be used to make recombinant human influenza vaccine.

Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira spp., often occurring in tropical and subtropical regions. Focusing on development of rapid diagnostic methods to facilitate early diagnosis and a universal vaccine are the main critical issues to overcome the burden of leptospirosis. Here, we have studied the immunogenic potential of prepared recombinant Loa22 protein (rLoa22) of local pathogenic Leptospira species in mice and its ability to induce humoral and cellular immunity, being further evaluated by analyzing the immunoglobulin G (IgG) subclasses and cytokines produced through immunization. Based on the results, mice immunized with rLoa22/adjuvant and a trivalent vaccine, induced high titers of total IgG. All immunized groups increased IgG1 almost on the same level; but, IgG2a level was significantly higher in the vaccine and rLoa22/adjuvant groups than rLoa22 alone group. Animals immunized with the vaccine produced more interleukin 4 than rLoa22/ adjuvant group. The results of evaluating interferon gamma level showed that the rLoa22/adjuvant and vaccine groups had a significant increase compared to the rLoa22 alone group. The results also demonstrated that the rLoa22 protein, in indirect enzyme-linked immunosorbent assay, was able to detect the anti-Leptospira antibodies in mice serum that can be used as a marker in assessing the seroprevalence of leptospirosis and/or in combination with other leptospiral antigens in development of an effective vaccine against leptospirosis.

Leptospirosis is a disease that threatens the health of humans and animals, which is caused by pathogenic Leptospira. Early detection of pathogenic Leptospires prevents many problems. LipL41-OmpL1, a protected outer membrane protein (OMP) of pathogenic Leptospira, was inserted into E. coli bacteria using different software for the amino acid sequence of OmpL1 and LipL41 to design a recombinant fusion protein and then expressed to investigate immunogenicity. The selected genes were propagated and cloned as a fusion in pET32a+ plasmid vector and expressed by Escherichia coli plays S (DE3) by heat shock method. It was evaluated in the BALB/c mouse laboratory animal model. Recombinant LipL41-OmpL1 protein was confirmed using urea purification method and western blot and its immunogenicity was evaluated by measuring high humoral immune stimulation and antibody secretion in BALB/c mice by ELISA method. The results showed that animals that received both OmpL1 and LipL41 proteins had 85% immunogenicity, while the control group that did not receive the fusion protein had only 25% immunogenicity (P < 0.001). Furthermore, no evidence of infection was found in recipients of the OmpL1-LipL41 fusion protein, suggesting that this protein is safe to use. The protective effects of immunization with OmpL1 and LipL41 were synergistic, as significant levels of protection were not observed in animals immunized with OmpL1 or LipL41 alone. Overall, this study highlights the potential of recombinant OmpL1 and LipL41 fusion protein as a promising research strategy on the development of vaccines and ELISA diagnostic kit for prevention and rapid diagnosis of leptospirosis.

Leptospirosis is an infectious zoonotic disease that can result in severe complications. It is widespread, especially in hot and humid climates such as the northern region of Iran. The immune responses to leptospirosis are multifaceted. Lipl41 is an outer membrane protein that is expressed during infection and is highly conserved among pathogenic species. This makes it a good candidate for diagnosis and induction of specific immune responses. The aim of the present study was to evaluate immune responses against recombinant Lipl41 in mice.

After immunizing of different groups of mice with recombinant Lipl41 (rLipl41), the levels of specific antibodies and cytokine profiles interferon-gamma/ interleukin-4 (IFN-γ/IL-4) were measured.

The results revealed that rLipl41 showed a significant increase in antibody levels compared with the control groups (P< 0.05). Although the level of IL-4 in the groups that received Lipl41 was similar to that in the other control groups, the IFN-γ levels showed a significant increase (P<0.05).

It has been concluded that recombinant Lipl41 protein could strongly stimulate specific immune responses and be considered a potential candidate for vaccine development and diagnostic research.

One of the important issues for quality control of biological products is purity. In most cases, electrophoresis is used to determine the purity of the proteins and the quantity of related band is determined by densitometry and respective software. In order to check the purity of hyperimmune plasma and antivenom bulks, we have evaluated the albumin content of these products. Sixty samples were divided into two groups: the hyperimmune plasma group that containing thirty bulks of monovalent and polyvalent anti-snake, polyvalent anti-scorpion and anti-diphtheria toxin plasma, and plasma-derived antivenoms group, comprising thirty bulks sera obtained from the purification of relevant plasma bulks. The albumin purification from horse normal serum was performed through heat shock strategy and used as reference for the quantification of albumin content. After electrophoresis of samples, the gel images were analyzed using Image J software and the amount of protein in relevant band was determined by calculating the curve area and comparison with reference albumin. The results revealed significant changes on albumin level in hyperimmune plasma samples. Two out of 30 plasma samples had lower and sixteen samples had a higher albumin than normal levels. Using the Image J, we could not detect any residue of albumin in antivenom sera. The results demonstrated effectiveness of purification protocol for removal of albumin during antivenom production process. However, development of a quantitative evaluation method along with the semi-quantitative method used in this study can help the accuracy of these results.

In recent years, a strategy based on nanoparticles has shown that non-denatured protein toxins can be used to enhance the appropriate immune response. The toxin does not bind to its ligand on the cell surface. The results of the nanoparticle and toxin complex show that the nanoparticles facilitate the internal release of the toxin. Clostridium perfringens beta toxin is a toxin produced by Clostridium perfringens type B and C and the most important disease is bloody diarrhea of newborn lambs. The toxin loses its lethality due to its involvement, so it becomes a toxoid. The nanoparticle used in this research is PLGA, which is one of the most developed biodegradable polymers. The purpose of this study is separate, Production and purification of Clostridium perfringens beta toxin and production of its complex with PLGA nanoparticles in order to form a non-toxic structure.In this study, beta-Clostridium perfringens type B and C toxins were isolated by ammonium sulfate precipitation and gel filtration chromatography. Purified was calculated to be 10 mg / ml. In this study, beta toxin antigen was used as a basis for the preparation of nanotoxoid candidate with nanoparticle formulation. When nanoparticles are injected into mice with beta toxin, they turn the toxin into a toxoid that has no toxicity effects, and in fact the toxin cannot bind to its receptors and reveal its effects, so the mice showed no signs of disease.

Reduction of cerebral ischemia-reperfusion injury (IRI)/re-oxygenation injury, is defined as the paradoxical exacerbation of the cellular dysfunction and death, following restoration of the blood flow to previously ischemic tissues. The re-establishment of blood flow is essential to salvage the ischemic tissues. As a result, the treatment of IRI with novel therapies, which have fewer side effects, are of great importance. Therefore, this study aimed to investigate the effects of curcumin nanoparticle (CN) pre-treatment on the cerebral I/R rat model.

In this experimental study, CN was administered to rats orally five days before the bilateral common carotid artery occlusion (BCCAO) and continued for three days. The intensity of oxidative stress, the activities of antioxidant enzymes, glutathione (GSH) content, the activity of mitochondrial enzymes, including succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), curcumin bioavailability, pERK/ERK expression ratio and TFEB protein were studied. Data analysis was performed using Graphpad Prism V.8 software, one-way analysis of variance (ANOVA) with the statistical package for the social sciences (SPSS V.26 software).

Cerebral IRI-damage significantly increased the oxidative stress (P=0.0008) and decreased the activity of the antioxidant enzymes including catalase (CAT) (P<0.001), super oxide dismutase (SOD) (P<0.001), reduced GSH (P<0.001), mitochondrial enzymes, pERK/ERK expression ratio (P=0.002) and TEFB protein (P=0.005) in rats’ brains. In addition, the pre-treatment of the rats with CN resulted in a decrease in the reactive oxygen species (ROS), and an increase in the activities of antioxidants and mitochondrial enzymes. This in turn up-regulated the pERK/ERK expression ratio and TEFB expression.

CN has neuroprotective effects on the cerebral IRI condition due to its antioxidant properties and is able to overexpress the pERK and TFEB proteins; thus, it can be considered as a suitable treatment option during and after the incidence of stroke.

Mycoplasma gallisepticum, the pathogen responsible for chronic respiratory disease in chickens, is the most economically important species of Mycoplasma that causing tremendous economic losses worldwide. The most abundant membrane proteins in M. gallisepticum are pMGA, lipoproteins of about 67 kDa. The pMGA family genes have an extraordinary potential for diversifying antigenic structure on the surface of Mycoplasma gallisepticum cells. The aim of this study was to compare the pMGA protein patterns between different strains and hosts of Mycoplasma gallisepticum.

All Mycoplasma gallisepticum full genomes available in GenBank till January 2020 were considered and pMGA1.2 sequences were identified, grouped and coded. pMGA1.2 protein with a chain of 650 amino acids between two different hosts (poultry and house finch) was studied by bioinformatics software in all Mycoplasma gallisepticum full genomes.

pMGA1.2 gene among different strains of Mycoplasma gallisepticum showed five major groups with more than 10 percent divergence. Based on multiple sequence alignment, a specific pattern was identified in house finch isolates. Interestingly, two specific motifs 480DNQNVSNQ487 and 639SSNVSSPSY647 were found in the pMGA1.2 of TS-11 strain, which can probably be used as markers to identify and differentiate this vaccine strain from pathogenic Mycoplasma gallisepticum.

This study showed that pMGA1.2 protein have some B-cell epitope antigenic regions that are conserved among all isolates and might be applicable to design serological test for detection antibody against Mycoplasma gallisepticum.

Vaccination against bird flu H9N2/H5N8 is considered as an effective strategy to deal with and control these deadly pathogens. For this purpose, in this study, an effective dual chimeric vaccine against H9/H5 from the bidirectional HA2 region of H9N2 and H5N8 was designed and analyzed using bioinformatics models, physicochemical characteristics and antigenic structure analysis of selected epitopes.To carry out this study, (A/chicken/Iran/B308B/2004) H9N2 and (A/Poultry/Iran/clade 2344/2018) H5N8 strains were selected and the epitopes stimulating B and T lymphocytes were investigated and the most suitable ones were selected. Then a chimeric vaccine was designed by combining highly immunogenic epitopes. In the following, using related software, the physicochemical characteristics and secondary structure of the designed vaccine were evaluated in terms of thermal stability, hydrophilicity, isoelectric pH, antigenicity and allergenicity. Finally, the proposed vaccine was reverse transcribed and adapted to be placed in the prokaryotic expression vector pET-41 a(+). According to the results, it is predicted that the antigenic structure designed in this study can be expressed successfully in prokaryotic systems and use for immunogenic studies against H5N8 and H9N2 strains of influenza virus.

One of the highly conserved outer membrane proteins expressed only by pathogenic Lepto- spires is Loa22. The study aims is to achieve the optimum conditions for high expression of recombinant Loa22 (rLoa22) protein.

Complete coding sequence of loa22 gene sub-cloned into a pET32a (+) expression vector. BL21 competent E. coli (pLysS) used as expression host for transformation. The recombinant clones selected on ampicillin plates and subjected to PCR by using pET T7 primers. Then expression conditions optimized by adjusting parameters such as cul- ture media, induction time, temperature, and IPTG concentration.

SDS-PAGE analysis showed that high production of rLoa22 protein obtained when post induction incubation, IPTG concentration, and duration of induction were 37ºC, 0.1 M and 5 h in 2×TY medium respectively. The purification of rLoa22 protein under native conditions using Ni-NTA pull-down was optimum in one hour binding at 37°C, five times washing process and elution buffer with a pH 7.4 and a 0.3 M imidazole concentration.

The findings of the study led to high production of pure Loa22 protein, which can form the basis for future investigation on the design of rapid diagnostic tests and more effective vaccine candidates for leptospirosis.

Leptospirosis is a common zoonotic infectious disease, that cause by pathogenic Leptospires. FlaB2 is a periplasmic flagellum protein that is expressed during infection in the host. This protein is conserved among all pathogenic Leptospiral serovars. The aims of this study was molecular evalution of the gene encoding FlaB2 protein by PCR method in pathogenic Leptospira serovars in Iran. In this study, 20 pathogenic Leptospira serovars and two nonpathogenic Leptospira serovars were used. All 22 Leptospira serovars were cultured into the selective medium EMJH and genomic DNA was purified using standard phenol-chloroform method. Specific primers were designed for amplification of flaB2 gene and its sensitivity and specificity were evaluated by PCR. The results of PCR product of flaB2 gene was 1050 bp and it was observed that all 20 pathogenic leptospira serovars contained the flaB2 gene. This gene was not amplified from two non-pathogenic L. biflexa. The results of this study showed that the gene flaB2 gene was present only in pathogenic serovars. Therefore, molecular characterization of flaB2 gene is important for detection of pathogenic serovars from nonpathogenic Leptospira serovars

In recent years, due to advances in vaccine design, bioinformatic studies on Leptospira have undergone significant changes and advances. The aim of this study was to design a fusion protein based on OmpL1 and LipL41 surface proteins that have a conserved structure over time. Based on the previous field studies, the target proteins were examined and the OmpL1-LipL41 fusion protein was selected. After examining the chemical properties of the target proteins using appropriate servers, the initial structure of the fusion protein was determined and finally this protein was placed in the PET32a+ vector between the two enzymatic regions of HindIII and BamHI. The results revealed that the protein weighs 73 kDa and has 2097 bp and 610 amino acids. The isoelectric pH of the above protein is 7 and its solubility degree in the prokaryotic system is 100%. Finally, according to the above data, it is predicted that the designed construct structure has acceptable immunogenic properties and can be expressed to an acceptable and successful extent in prokaryotic systems and used for immunogenicity studies of Leptospira as a new candidate in the production of leptospirosis vaccine and in the design and manufacture of diagnostic kits.

Natural products can help in the treatment of many diseases due to their low side effects. In the present study the effect of pretreatment of nanobetanin and nanocurcumin in cerebral ischemia rats was investigated.

The present study was experimental. 40 male Wistar rats were divided into eight groups of healthy control, healthy animals receiving nanobetanin, healthy animals receiving nanocurcumin, healthy animals receiving nanobetanin+nanocurcumin, cerebral I/R, cerebral I/R receiving nanobetanin, cerebral I/R receiving nanocurcumin and cerebral I/R receiving nanobetanin + nanocurcumin. Oral administration of nanobetanin and nanocurcumin started five days before Bilateral Common Carotid Artery Occlusion induction in rats and continued three days later. Neurological disorders scores were assessed. After preparing the brain homogenate, the intensity of oxidative stress, the activity of antioxidant enzymes and the expressions of interleukin-1β (IL-1β), interleukin 6 (IL-6) and alpha tumor necrosis factor (TNF-α) cytokines were measured by relevant kits. Data analysis was done in GraphPad prism V.8 software.

Nanobetanin and nanocurcumin showed strong anti-inflammatory and antioxidant effects in cerebral ischemia/reperfusion (I/R) rats. The content of reactive oxygen species (ROS) in the brain of I/R rats receiving nanobetanin and nanocurcumin decreased (P=0.00001) and the activity of antioxidant enzyme such as superoxide dismutase improved (P=0.00002), and the content of glutathione regeneration showed an increasing trend (P=0.0004). A decrease in neurological disorders scores (P=0.009) was observed in cerebral I/R rats receiving nanobetanin and nanocurcumin. Furthermore, nanobetanin and nanocurcumin administration reduced the expression levels of inflammatory cytokines IL-1β (P=0.00003), IL-6 (P=0.00006), TNF-α (P=0.008) in the rats with cerebral I/R injury.

Nanobetanin and nanocurcumin has neuroprotective effects in cerebral I/R condition due to its antioxidant and anti-inflammation properties, which can be considered as a suitable treatment option in stroke conditions

Leptospirosis is one of the most common zoonotic diseases, which is caused by a Spiral shaped bacterium called Leptospira. The recommended diagnosis method is to perform microscopic agglutination test (MAT) which is both hazardous, due to using live bacteria, and time-consuming. As a result, many attempts have been recently made to develop other serological methods, such as ELISA.

This study aimed to develop an indirect ELISA method using leptospiral whole antigens for diagnosing pathogenic Leptospira.

In this study, four pathogenic serovars of Leptospira were used and cultured in selective culture medium. The cultured bacteria were sonicated and the extracted antigens were used as captured antigen in ELISA method. A total of 74 samples from bovine suspected to leptospirosis and 43 samples from healthy animals were examined by MAT method.

According to the study results, 42 samples (56.7%) out of 74 suspected ones were found positive while 32 ones (43.2%) were determined negative by MAT analysis. All of the 43 negative control samples were found negative after performing MAT. The sensitivity and specificity of ELISA, compared to those of MAT, were measured as 87.5% and 84.2%, respectively.

Taking into account the high sensitivity and appropriate specificity of the developed indirect ELISA method, it was recommended that ELISA be employed as an accurate method for early and rapid diagnosis of bovine leptospirosis

Utilizing subunit vaccines is one of the strategies to address influenza infection. Recent innovations have focused on high doses of vaccine antigens and immune enhancers or adjuvant to induce more robust and long-lasting immune responses. Here, an effect of the B cell-activating factor receptor (BAFF-R) to increase the magnitude and durability of immune responses of the recombinant HA1 (rHA1) protein against the H1N1 influenza virus was studied.
The HA1 protein and the effector domain of BAFF-R were expressed in the pET-28a (+) vector. Eight-week-old BALB/c mice were equally grouped into five groups (n=20). The 15 and 25 μg/μL of rHA1 were mixed with 2 μg/μL of rBAFF-R and injected three times for vaccinated groups. Three control groups were received normal saline and two concentrations of rHA1. The ability of rBAFF-R in eliciting HA-specific antibody response and stimulating T lymphocyte proliferation to induce the cell-mediated immunity was assayed. Induction of protection was evaluated following the challenge with PR8 strain.
Analysis of immune responses showed that the co-administration of rBAFF-R with rHA1 boosted HI responses to the antigen in mice, whilst it was not able to promote the T cell proliferation responses against influenza. Compared to rHA1alone, the rBAFF-R/rHA1 generated efficient protection for the animals. There were no significant differences in eliciting the immune responses in mice immunized with the lower dose of rHA1 than that with the higher dose.
The data indicate the rBAFF-R can enhance the primary and memory immune responses to protect against influenza infection.

Leptospirosis is a zoonotic disease caused by pathogenic spirochete called Leptospira that has worldwide distribution. Unfortunately, due to the variety of clinical symptoms, its diagnosis has many limitations. LipL41 is among the most abundant outer membrane proteins in Leptospira and is exclusively found in pathogenic species. A small gene called lep is located very close to lipl41 gene on the bacterial chromosome, which facilitates its expression. In this study, the expression and purification of LipL41-Lep fusion protein among prevalent pathogenic isolates in Iran was performed in a prokaryotic system for the first time.

All collected Lipl41 and Lep protein sequences were analyzed from the NCBI database. Complete codon sequences of the pattern of the Iranian serovars were designed and synthesized then sub-cloned into a pET32a+ and transformed into Escherichia coli BL21(DE3) to expression by using IPTG inducer. Thefusion recombinant protein was purified by denaturation and confirmed by western blot.

The results of PCR analysis showed a clear band of ~ 2000 bp in Agarose gel. Optimal expression of fusion recombinant protein (60kDa) was achieved post-induction at 4 h at 37 °C in the presence of 0.1 mM IPTG. Then it was purified in the insoluble form.

The results demonstrated that adequate amounts of this fusion recombinant protein were expressed andpurified to be used as a fusion antigen in serological testing, such as ELISA, or for the development of subunit vaccines against Leptospirosis in the future.

An indirect ELISA for potency assessment of Naja naja oxiana horse antivenom was developed, with the aim of monitoring of antivenom immunoglobulin levels to replace the well-established conventional in vivo neutralization assay. According to this purpose, blood sera were taken during immunization schedule of the nineteen horses used in snake antivenom manufacture at the Razi Vaccine and Serum Research Institute. In addition, a group of fifteen batches of monovalent and polyvalent anti-snake venom plasma was examined by both in vitro and in vivo assays. ELISA is based on the horse anti-Naja antibodies recognizing toxic fractions mixture, the main antigen that induces neutralizing antibody against Naja naja oxiana venom. Consistent results were found between the ELISA optical densities and corresponding neutralization potency values, demonstrating that the in vitro assay as well as in vivo assay can be used to estimate neutralizing antibody activity of the sera. It was found that venom antibodies reached a maximum level about 3rd week after immunization. The major advantage of designed ELISA is its ability to correctly separate poor or non-responder horses during initial steps of immunization which venom antibodies are very low in sera and antivenom estimation is difficult by in vivo neutralization

Multiple Sclerosis is a central nervous system disease which belongs to the category of autoimmune diseases. The prevalence of this disease in Iran is approaching the European level. Astrocyte cells are nerve tissues that regulate the immune system activity by secreting various cytokines such as IL- 17.  The aim of this study was partial purification of toxin from M.eupeus scorpion venom that has immunomodulatory effect on astrocyte cell line (1321N1) In the present study, purified crude venom of M.eupeus scorpion. Size exclusion and reverse-phase high-performance liquid chromatography was used for fractionation. The fractional molecular weight was determined by Using SDS and Tricine electrophoresis, Astrocyte cells (1321N1) were selected as functional cells in testing the immunomodulatory effect of venom. The viability of cells were determined by MTT and LDH assays. Astrocyte cells were activated by lipopolysaccharide and the release of interleukin-17 in activated cells was estimated using ELISA kit.   fraction 331 (F331) from RP-HPLC contain the purified peptide with molecular weight of about 4500 Dalton. When activated cells exposed to purified peptide the rate of interleukin-17 release was found to be 85 pg/ml which is almost similar to un-activated cells (78 pg/ml). However in activated cells by LPS without treatment with purified peptide the rate of IL-17 release was found to be 147 pg/ml which was significantly (p <0.05) higher than control group.   The purified peptide (F331) from   venom of Mesobouthus eupeus can inactivate the astrocyte 1321N1 cells activated by LPS as indicated by decreased secretion of IL-17 from the cells.

The bovine tuberculosis is one of the most zoonotic diseases which could be infected domestic and wild animals. The causative agent of this infection is Mycobacterium bovis. The correct diagnosis is very critical for effective control of this infection. The usual diagnosis is made by skin test and gamma interferon assay. But other serological techniques based on ELISA could be noted for development of a rapid and correct method. Here, we use MPT-64 recombinant protein as capture antigens for development of an indirect ELISA. The study population consists of thirty two infected and healthy (control) cattle. The infected animals were confirmed by resident veterinary surgeon according to skin test and clinical examination. The levels of specific antibody against MPT-64 were evaluated by indirect ELISA method. According to our results, the levels of specific antibodies against MPT-64 have great consistency with skin test. So that, all of 9 positive samples have high titer for MPT-64 specific antibody and no false negative results have been reported. This study could proposed that development of ELISA method with recombinant MPT-64 antigen could be considered as a rapid, simple and exact method for detection of BTB infected cattle.

Antimicrobial peptides produced by lactic acid bacteria have gained enormous attention owing to their health benefits. This study aimed to isolate, purify and characterize the antibacterial protein produced by autochthonous Lactobacillus casei TA0021 strain.

The antagonistic activity of L. casei TA0021 against a number of pathogenic bacteria was tested by agar well diffusion assay. The antimicrobial agent in the neutralized supernatant fluids was subjected to the action of proteolytic enzymes, catalase, lipase and lysozyme, and their tolerance to variable pH and temperature was estimated. The proteinaceous antagonistic compound was precipitated by 60% w/v ammonium sulphate, desalted and subjected to cation exchange and gel filtration chromatography. Approximate molecular weight of Lactocin was determined by SDS-PAGE and non-denaturing gel electrophoresis. Hemoglobin release assay and cytotoxicity effect of Lactocin TA0021 was determined. The results were statistically analyzed.

The antagonistic agent active against Salmonella Typhimurium and Shigella flexneri appeared resistant to catalase and lipase treatments, while sensitive to the tested proteolytic enzymes. Lactocin TA0021 resisted acidic pH values of 3.0, while alkaline pH values of ˃9 completely destroyed the activity. The antibacterial peptide was approximately 68 KDa and heat labile as lost its activity at 100°C after 5 minutes. The bacteriocin was non-toxic to MRC-5 cell lines and non-hemolytic. Purification method lead to increase in antibacterial activity while, subsequent decrease in recovery and yield was observed with increasing purification fold.

The purified antimicrobial protein from L. casei TA0021 might be used for application in medicinal and food products.

Tuberculosis (TB) has been considered as a main health problems of the present century. Unfortunately, it has been reported different protective responses against BCG vaccine, as only available vaccine against TB .The search for a new and improved vaccine against tuberculosis is currently a very active field of research. In this study, we have evaluated the relative effects of intranasal (i.n), subcutaneous (s.c) and intramuscular (i.m) routes of immunization on the induction of humoral immune responses against TB recombinant protein.
The recombinant ESAT-6/CFP-10 antigens has been assessed and confirmed preliminary, for this study. The Balb/C mice were immunized in 3 different routes, three times at 2-week intervals with recombinant protein, formulated with or without adjuvants (MF-59 for the i.m and s.c routes and CTB for i.n). Blood was collected from retro-orbital sinus 10 days after each immunization and after separation of sera, they were evaluated for specific antibodies against antigen by an indirect ELISA method, which had set up during the research.
Mice vaccinated against recombinant proteins had high level of specific antibodies compared to the controls. Our results showed that although the i.m and s.c injections also induce immune responses, but the antibody titer in i.n rout had graduately elevated to reach in acceptable levels.
The results could be used for formulation of mucosal vaccines against tuberculosis in future studies.

Tuberculosis (TB) is a zoonotic disease which is caused by Mycobacterium tuberculosis. Because of common structural and secretory antigens between pathogen and nonpathogenic mycobacterium, the specific diagnosis of TB is difficult. Therefore, it is very important to find a new method with high specificity and sensitivity for accurate and rapid diagnosis of tuberculosis. In this study, the serodiagnostic potential of Mycobacterium tuberculosis recombinant ESAT-6 in TB infected patients was evaluated by Enzyme Linked Immunosorbent Assay (ELISA).
55 TB patients with active disease and 28 healthy controls have been collected and evaluated in different dilutions in ELISA methods for the presence of specific anti-ESAT-6 antibody. The specificity and the sensitivity of this method was compared with the culture test.
TB patients have high levels of specific antibody against ESAT-6 antigens. The specificity and the sensitivity of this method was calculated as 80.90% and 85.45%, respectively.
These findings provide useful information on the importance of ESAT-6 protein and suggested this serologic test as a good alternative method for rapid and prefect diagnosis of tuberculosis.

Rotavirus group A (RVA) is recognized as a major cause of severe gastroenteritis in children and new-born animals. Nonstructural protein 4 (NSP4) is responsible for the enterotoxic activity of these viruses in the villus epithelial cells. Amino acids 114-135 of NSP4 are known to form the diarrhea-inducing region of this viral enterotoxin. Therefore, developing an NSP4 lacking the enterotoxin domain could result in the introduction of a new subunit vaccine against rotaviruses in both humans and animals.
The aim of this study is the evaluation of rotavirus A NSP4 expression in E. coli expression system before and after removal of the diarrhea-inducing domain, which is the first step towards further immunological studies of the resulting protein.
Splicing by overlap extension (SOEing) PCR was used to remove the diarrhea-inducing sequence from the NSP4 cDNA. Both the full-length (FL-NSP4) and the spliced (S-NSP4) cDNA amplicons were cloned into pET-32c and pGEX-6P-2. Expression levels of the recombinant proteins were evaluated in E. coli BL21 (DE3) by Western blot analysis. In addition, the toxicity of pET plasmids bearing the S-NSP4 and FL-NSP4 fragments was investigated by plasmid stability test.
For FL-NSP4, protein expression was detected for the strain containing the pGEX:FL-NSP4 plasmid, but not for the strain carrying pET:FL-NSP4. Hourly sampling up to 3 h showed that the protein production decreased by time. In contrast, expression of S-NSP4 was detected for pET:S-NSP4 strain, but not for pGEX:S-NSP4. Plasmid stability test showed that pET:S-NSP4 recombinant plasmid was almost stable, while pET:FL-NSP4 was unstable.
This is the first report of production of rotavirus NSP4 lacking the diarrhea-inducing domain (S-NSP4). S-NSP4 shows less toxicity in this expression system and potentially could be a promising goal for rotavirus immunological and vaccine studies in the future.

Bovine tuberculosis (BTB) is a zoonotic disease that caused by Mycobacterium bovis (M. bovis). The most widely used methods to detect BTB are the skin test and in vitro gamma interferon assay which do not detect anergic animals, but serological tests such as ELISA have been found promising in ancillary tuberculosis diagnosis. The overall aim was to study M. tuberculosis ESAT-6 recombinant protein as a target for serological assays in the detection of antibodies to BTB. Serum samples were collected from suspected animals where M. tuberculosis was confirmed at postmortem examination by clinicians and positive skin test. Also, control samples were collected from BTB free bovines. Circulating specific antibody levels were measured by an enzyme-linked immunosorbent assay using purified recombinant proteins of ESAT-6. The results show a mild satisfactory agreement with the skin test reflecting in 95.23 sensitivity and 81.81 Specificityin serum diluted to 1/100. This study demonstrated that ELISA with the use of the esat-6 antigens is simple and sensitive and can be used to analyze large numbers of samples for the serodiagnosis of BTB.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remain as a leading causes of mortality among infectious diseases. The only current vaccine, bacillus Calmette-Gue´rin (BCG), displays highly variable efficiency for preventing tuberculosis. Thus, identification of protective antigens could be mentioned for designing new vaccines. Among different Mtb antigens, TB10.4 and Ag85B have been identified as immune stimulator antigens which induce strong cellular responses and increase production of IFN-γ. In this study, we aimed to clone the fusion form of these antigens into a eukaryotic vector (pcDNA3). After extraction of Mycobacterium tuberculosis H37Rv genome, the selected genes were amplified by specific primers with PCR method. The gene segments were fused and cloned into eukaryotic pcDNA3 vector. Recombinant plasmid was transformed into E.coli DH5α and after purification was confirmed with restriction enzyme analysis and sequencing. The results of sequencing and digestion demonstrated that TB10.4 & Ag85B genes were successfully fused and cloned into pcDNA3 vector The recombinant plasmid was successfully constructed. In the future studies, the immunogenicity of this cassette could be assessed as a DNA vaccine.