Ekspresija i pročišćavanje površinskog lipoproteinskog antigena Lipl41 iz patogenih bakterija roda Leptospira

Pathogenic species of Leptospira lead to a zoonotic disease called leptospirosis, which is spread worldwide. A major topic of investigation is to detect the antigens that induce an immune response and to utilize them in diagnostic kits or vaccine development. The outer membrane proteins (OMPs) of Leptospira are potential candidates for this purpose. Lipl41 is an OMP that is conserved among pathogenic Leptospira. The aim of this study was to express and purify the Lipl41 recombinant protein in Iranian isolates. All collected Lipl41 protein sequences were compared and analyzed using bioinformatics tools from NCBI databases. Complete codon sequences of the Iranian pattern of Lipl41 recombinant protein were codon optimized and sub-cloned into a pET32a+ expression vector, and transformed into Escherichia coli BL21 (DE3). Optimal expression of recombinant Lipl41 (47kDa) was achieved post-induction with IPTG within the inclusion body. It was then purified by denaturation using serial concentrations of urea, and the recombinant protein was confirmed by western blot. In this study, sufficient amounts of Lipl41 were expressed and purified to be used for the development of a diagnostic kit and subunit vaccine.Patogene vrste Leptospira uzrokuju zoonotsku bolest leptospirozu koja je raširena u cijelom svijetu. Cilj istraživanja bio je dokazati antigene koji potiču imunosni odgovor i mogu se primijeniti u dijagnostičkim kompletima ili za razvoj cjepiva. Vanjska proteinska membrana (OMPs) leptospira potencijalni je kandidat za tu svrhu, a gen Lipl41 je dobro očuvan među patogenim leptospirama. Ekspresija i pročišćavanje rekombinantnog proteina Lipl41 provedeno je u izolatima iz Irana. Uspoređene su sve prikupljene sekvencije proteina Lipl41 i analizirane bioinformatičkim alatom iz baze podataka NCBI. Kompletne sekvencije kodona rekombinantnog proteina Lipl41 u izolatima su optimizirane, zatim subklonirane u pET32a+ vektor i pretvorene u bakteriju Escherichia coli BL21 (DE3). Optimalna ekspresija rekombinantnog Lipl41 (47kDa) postignuta je post-indukcijski pomoću isopropyl β-d-1-thiogalactopyranoside (IPTG). Zatim je pročišćena denaturacijom što je uključilo primjenu serijskih koncentracija ureje. Rekombinantni protein je potvrđen metodom western blot. Istraživanje potvrđuje mogućnost ostvarenja dostatne količine i ekspresije pročišćenog Lip141 da se može upotrijebiti za razvoj dijagnostičkih kompleta i subjediničnih cjepiva

The Neuroprotective Effects of Curcumin Nanoparticles on The Cerebral Ischemia-Reperfusion Injury in The Rats-The Roles of The Protein Kinase RNA-Like ER Kinase/Extracellular Signal-Regulated Kinase and Transcription Factor EB proteins

Objective: Reduction of cerebral ischemia-reperfusion injury (IRI)/re-oxygenation injury, is defined as the paradoxicalexacerbation 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 withnovel therapies, which have fewer side effects, are of great importance. Therefore, this study aimed to investigate theeffects of curcumin nanoparticle (CN) pre-treatment on the cerebral I/R rat model.Materials and Methods: In this experimental study, CN was administered to rats orally five days before the bilateralcommon carotid artery occlusion (BCCAO) and continued for three days. The intensity of oxidative stress, the activities ofantioxidant enzymes, glutathione (GSH) content, the activity of mitochondrial enzymes, including succinate dehydrogenase(SDH), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), curcumin bioavailability, pERK/ERK expressionratio and TFEB protein were studied. Data analysis was performed using Graphpad Prism V.8 software, one-way analysisof variance (ANOVA) with the statistical package for the social sciences (SPSS V.26 software).Results: Cerebral IRI-damage significantly increased the oxidative stress (P=0.0008) and decreased the activityof the antioxidant enzymes including catalase (CAT) (P<0.001), super oxide dismutase (SOD) (P<0.001), reducedGSH (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/ERKexpression ratio and TEFB expression.Conclusion: CN has neuroprotective effects on the cerebral IRI condition due to its antioxidant properties and is ableto overexpress the pERK and TFEB proteins; thus, it can be considered as a suitable treatment option during and afterthe incidence of stroke

Analyzing Bacterial Agents of Keratoconjunctivitis in Patients Referred to Ophthalmology Ward of Feiz Hospital in Isfahan

Background & Objectives: Keratoconjunctivitis is considered as the most prevalent ocular disease which is caused by multiple microorganisms. Considering the fact that identifying etiologic agents of Keratoconjunctivitis in a specific geographical area, and determining their antibiotic resistance pattern could be very important in specifying treatment strategy of such patients, the present research has been conducted to discriminate and evaluate etiologic microbial agents of this disease in patients referred to ophthalmology ward of Feiz health center in Isfahan. Materials & Methods: This descriptive study has been conducted in an 18-month period on 196 patients, referred to ophthalmology ward of Feiz hospital with positive symptoms of Keratoconjunctivitis. Ocular secretions were sampled with sterile swap and from conjuctival sac. Then the related samples were transferred and cultured in the medium for bacteria. Antibiogram of isolated strains was performed by method of disc diffusion. One of the samples related to patients was transferred to virus transport media and was used for direct immunofluorescence test. The possible content of IgM anti-adenovirus was investigated by ELISA method and on serum samples of patients. Resulst: From among the total 196 evaluated samples, 75 cases were infected with bacterial agents, 37 cases with adenovirus, and 58 cases with both bacterial and adenovirus agents. The isolated bacterial agents were as follows with respect to their prevalence, Staphylococcus aureus (28.1%), Coagulase negative Staphylococci (16.8%), Bacillus spp (6.6%), Pseudomonas aeruginosa (5.1 %), Enterobacter (4.2%), Klebsiella (3.6%) and Streptococcus group D (3.6%). From among antibiotic drugs, the highest rate of sensitivity for S. aureus and P. aeruginosa was observed in Ciprofloxacin and Tobramycin respectively. Conclusion: The results of the study could be applied in specifying treatment strategies for patients suffering from Keratoconjunctivitis in Isfahan city

Evaluation of three different administration routes (IM, SC and IN) on humoral immune responses against Mycobacterium Tuberculosis ESAT-6/CFP-10 fusion protein

Introduction: 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. Methods: 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. Results: 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. Conclusion: The results could be used for formulation of mucosal vaccines against tuberculosis in future studies

Higher Expression Level and Lower Toxicity of Genetically Spliced Rotavirus NSP4 in Comparison to the Full-Length Protein in E. coli

Background: 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. Objectives: The aim of this study is the evaluation of rotavirus ANSP4 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. Materials and Methods: 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. Results: 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. Conclusions: This is the first report of production of rotavirus NSP4 lacking the diarrhea-inducing domain (S-NSP4). SNSP4 shows less toxicity in this expression system and potentially could be a promising goal for rotavirus immunological and vaccine studies in the future. Keywords: Diarrhea; Enterotoxin; Expression; NSP4; Rotavirus; Splicing by overlap extension PC

Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-&#947; was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj) ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model

Cloning, expression, and purification of recombinant protein MPT-64 from a virulent strain of Mycobacterium bovis in a prokaryotic system

Objective: Tuberculosis (TB) is a zoonotic infectious disease common to humans and animals that is caused by the rod-shaped acid-fast bacterium Mycobacterium bovis. Rapid and sensitive detection of TB is promoted by specific antigens. Virulent strains of the TB complex from M. bovis contain 16 regions of difference (RD) in their genome that encode important proteins, including major protein of Mycobacterium tuberculosis 64 (MBT-64, which is a primary immune-stimulating antigen encoded by RD-2. In this study, we cloned, expressed, and purified MPT-64 as a potent M. bovis antigen in a prokaryotic system for use in future diagnostic studies. Methods: The antigenic region of the Mpt64 gene was investigated by bioinformatics methods, cloned into the PQE-30 plasmid, and expressed in Escherichia coli M15 cells, followed by isopropyl β-d-1-thiogalactopyranoside induction. The expressed protein was analyzed sodium dodecyl sulfate polyacrylamide gel electrophoresis and purified using a nickel-affinity column. Biological activity was confirmed by western blot using specific antibodies. Results: Our data verified the successful cloning of the Mpt64 gene (687-bp segment) via the expression vector and purification of recombinant MPT-64 as a 24-kDa protein. Conclusion: These results indicated successful expression and purification of recombinant MPT-64 protein in a prokaryotic system. This protein can be used for serological diagnosis, improved detection of pathogenicity and non-pathogenicity between infected cattle, and for verification of suspected cases of bovine TB

Evaluation of specific antibodies against Mycobacterium tuberculosis recombinant antigens for detection of recent infection

Introduction: Since an accurate test for detection of Mycobacterium tuberculosis early infection is urgently needed, this study was designed for development of an efficient screening test in diagnosis of tuberculosis infection. Materials and methods: In the present study, two recombinant proteins CFP-10, ESAT-6 were tested as antigens for the diagnosis of recent tuberculosis. The proteins were produced in Escherichia coli, purified and tested in indirect ELISAs with sera from 63 subjects with positive clinical results. Also, 56 sera from healthy persons were tested as controls. The results were compared with molecular and culture. Results: The levels of antibodies against M. tuberculosis antigens in patients with tuberculosis were significantly higher than those in healthy subjects. Among 63 patients, 58 were positive for ESAT-6, 54 for CFP-10. Conclusion: Altogether, the role of M. tuberculosis recombinant proteins, as a suitable candidate for early diagnosis of tuberculosis infection was supported in this study. However, these strongly offer the potential of mixture or fusion of these recombinant proteins for better sensitivity and specificity

Evaluation of Serum Specific Antibody against Recombinant ESAT-6 Antigen in Patients with Tuberculosis and Comparing to Normal Controls

Background & Objective: 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). Materials & Methods: 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. Results: 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. Conclusion: 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

Evaluation of Immunological Parameters in Purified Protein Derivative Positive Tuberculin Workers

Objective(s): According to the occupationally risk of infection in staff workers who have direct contact with mycobacterium species, we investigated their immunological parameters and compared with healthy purified protein derivative (PPD) negative volunteers.   Materials and Methods : We investigated 20 PPD positive volunteers working at Tuberculin Unit of Razi Vaccine and Serum Research Institute and PPD negative healthy controls with no exposure or history of active tuberculosis. The percentages of circulating lymphocyte subpopulations were detected by flowcytometry. IL-4 and IFN-γ production levels were measured by ELISA in supernatants of PPD-stimulated peripheral blood mononuclear cells (PBMCs) culture. Results : Tuberculin workers showed an increase in IFN-γ level and significant decrease of CD4+ T cells percentage and CD4/CD8 ratio compared to PPD negative normal individuals. However the IL-4 production and percentage of other lymphocyte population has been unchanged. Discussion: These observations suggest that the immunological parameters of tuberculin workers with PPD positive reaction, who are occupationally exposed to mycobacterium antigens, could be changed. Future studies will be directed towards cytokine networking and regulatory lymphocytes, which will help us validate the significant data presented in this study