ABSTRACT
Recently, specific attention has been paid to aptamers, short DNA or RNA, as a tool for cancer diagnosis and therapy. In the present study MCS nanogels were prepared by Myristate: Chitosan at 1:9 ratio and were characterized by several techniques. A selected ssDNA aptamer [Apt] capable of detecting LNCaP cells was linked to Myristilated Chitosan nanogels [Apt-MCS] by glutaraldehyde and loaded with Doxorubicin [DOX] to be used in targeted drug delivery against the Prostate cancer cells. LNCaP and PC-3 cells were treated with Apt-MCS-DOX complex and the binding efficiency was estimated by flow cytometry. The binding affinity of the selected aptamers was above 70% compared to the initial library. The loading capacity of the nanogel was as high as 97% and up to 40% of DOX were released from MCS within 15 days. Cytotoxicity of nanodrug on LNCaP cells was determined by MTT assay. Apt-MCS-DOX was specifically binded to LNCaP cells whereas it didn't show any specificity to PC-3 cells as a negative control. Both MCS-DOX and Apt-MCS-DOX showed a lethal effect on LNCaP cells. Our results can lead to an aptamer based simple and applicable technique for early diagnosis and treatment of cancerous cells
ABSTRACT
Background: Enterohemorrhagic Escherichia coli [EHEC] O157:H7 is an infectious zoonotic pathogen causing human infections. These infections, in some cases, can lead to hemolytic uremic syndrome and its life-threatening complications and even death worldwide. The first intimate bacterial adhesion, intimin [I], with its own receptor translocated intimin receptor [Tir] and E. coli secreted protein A, acting as Tir conduit, are highly immunogenic proteins for vaccine development against E. coli O157:H7
Methods: A chimeric trivalent recombinant protein was previously found to be a suitable strategy for developing vaccines against E. coli O157:H7. In this study, the recombinant EIT [rEIT] was used to design a protective EHEC nasal nanovaccine. Chitosan and its water-soluble derivative, trimethylated chitosan [TMC], as muco-adhesive biopolymers, are good candidates for preparation of nanovaccines. Using the electrospraying technique, as a novel method, we could obtain particles of rEIT loaded with chitosan and TMC on a nanometer scale. Mice were immunized with intranasal administration or intrapretoneal injection of rEIT
Results: The rEIT-specific immune responses [IgG and IgA] were measured by indirect ELISA. Only nasal administration of chitosan electrospray and TMC formulation produced significant secretion IgA. Intranasal administration of nanovaccine reduced the duration of bacterial fecal shedding on mice challenged with E. coli O157:H7
Conclusion: Since development of mucosal vaccines for the prevention of infectious diseases requires efficient antigen delivery; therefore, this research could be a new strategy for developing vaccine against E. coli O157:H7
ABSTRACT
Background: Nowadays, highly specific aptamers generated by cell SELEX technology[systematic evolution of ligands by exponential enrichment] are being applied forearly detection of cancer cells. Prostate Specific Membrane Antigen [PSMA], over expressedin prostate cancer, is a highly specific marker and therefore can be used fordiagnosis of the prostate cancer cells. The aim of the present study was to select singlestrandedDNA aptamers against LNCap cells highly expressing PSMA, using cell-SELEX method which can be used as a diagnostic tool for the detection of prostatecancer cells
Methods: After 10 rounds of cell-SELEX, DNA aptamers were isolated against PSMAusing LNCaP cells as a target and PC-3 cell lines for counter SELEX. Five DNA aptamerswith more than 70% affinity were selected up on flow cytometry analysis of positiveclones
Results: Dissociation constants of two selected sequences [A12-B1] were estimated inthe range of 33.78-3.77 and 57.49-2.214 pmol, respectively. Conserved secondary structuresof A12 and B1 sequences suggest the necessity of these structures for binding withhigh affinity to native PSMA. Comparison of the secondary structures of our isolatedaptamers and aptamer A10 obtained by protein SELEX showed similar stem-loopstructures which could be responsible for the recognition of PSMA on LNCap cell surface
Conclusion: Our results indicated that selected aptamers may turn out to be idealcandidates for the development of a detection tool and also can be used in targeteddrug delivery for future smart drugs
ABSTRACT
Objective: Drug delivery systems related to different cancer therapies is now expanding. Chitosan [CS] is currently receiving enormous interest for medical and pharmaceutical applications due to its biocompatibility in animal tissues. In this study, two nanogels were prepared from CS. Some of the critical factors such as controlling the release, adsorption and specially targeting drug delivery are considered while preparing the nanogels
Methods: Phosphorylated CS [PCS] and Myristilated CS [MCS] nanogels were prepared by reacting CS with tripolyphosphate [TPP] and Myristate as cross-linking agents respectively and then were loaded with Doxorubicin [DOX]. The nanogels were characterized by different techniques such as scanning electron microscopy, dynamic light scattering and Fourier-transform infrared. The cytotoxicity of free DOX, MCS nanogels and DOX loaded MCS was evaluated by the MTT assay
Results: The result of DOX loading and releasing of the nanogels showed high loading capacity and drug loading efficiency of about 97%. Results indicated slow release of about 16-28% of DOX from PCS within 5 days and 18-40% from MCS within 15 days. DOX and MCS-DOX showed the same toxic effect on the prostate cancer cells [LNCaP]
Conclusion: Both PCS and MCS nanogels were qualified on the basis of size, loading and releasing capacity
ABSTRACT
OBJECTIVES: Enterotoxigenic Escherichia coli (ETEC), a major cause of diarrhea in children under 5, is an important agent for traveler's diarrhea. Heat-labile enterotoxin (LT) and colonization factors (CFs) are two main virulence mechanisms in ETEC. CS6 is one of the most prevalent CFs consisting of two structural subunits viz., CssA, CssB, necessary for attachment to the intestinal cells. METHODS: In the present research, a chimeric trivalent protein composed of CssB, CssA and LTB was constructed. The chimeric gene was synthesized with codon bias of E. coli for enhanced expression of the protein. Recombinant proteins were expressed and purified. Mice were immunized with the recombinant protein. The antibody titer and specificity of the immune sera were analyzed by ELISA and Western blotting. Efficiency of the immune sera against ETEC was evaluated. RESULTS: Antibody induction was followed by immunization of mice with the chimeric protein. Pretreatment of the ETEC cells with immunized animal antisera remarkably decreased their adhesion to Caco-2 cells. DISCUSSION: The results indicate efficacy of the recombinant chimeric protein as an effective immunogen, which induces strong humoral response as well as protection against ETEC adherence and toxicity. .
Subject(s)
Animals , Female , Mice , Antigens, Bacterial/genetics , Chimerin Proteins/immunology , Enterotoxigenic Escherichia coli/genetics , Escherichia coli Infections/microbiology , Escherichia coli Proteins/genetics , Blotting, Western , Chimerin Proteins/chemistry , Enterotoxigenic Escherichia coli/immunology , Escherichia coli Infections/immunology , Mice, Inbred BALB CABSTRACT
Enterotoxigenic Escherichia coli [ETEC] strains are the major causes of diarrheal disease in humans and animals. Colonization factors and enterotoxins are the major virulence factors in ETEC pathogenesis. For the broad-spectrum protection against ETEC, one could focus on colonization factors and non-toxic heat labile as a vaccine candidate. A fusion protein is composed of a major fimbrial subunit of coli surface antigen 3, and the heat-labile B subunit [LTB] was constructed as a chimeric immunogen. For optimum level expression of protein, the gene was synthesized with codon bias of E. coli. Also, recombinant protein was expressed in E. coli BL21DE3. ELISA and Western tests were carried out for determination of antigen and specificity of antibody raised against recombinant protein in animals. The anti-toxicity and anti-adherence properties of the immune sera against ETEC were also evaluated. Immunological analyses showed the production of high titer of specific antibody in immunized mice. The built-in LTB retains native toxin properties which were approved by GMi binding assay. Pre-treatment of the ETEC cells with anti-sera significantly decreased their adhesion to Caco-2 cells. The results indicated the efficacy of the recombinant chimeric protein as an effective immunogen inducing strong humoral response. The designated chimer would be an interesting prototype for a vaccine and worthy of further investigation
ABSTRACT
Acinetobacter baumannii [A. baumannii] has a good potential to colonize on various surfaces. As a virulence factor, adhesion to surfaces is the first step in colonization. The Two-Partner Secretion System [TPS] proteins are key factors for bacterial attachment. The purpose of this study is to identify and study the role of this family of proteins in adhesion of A. baumannii to human epithelial cells. Gene homologues that encoded the TPS were analyzed by bioinformatics tools and the primers were designed accordingly. The constructs synthesized in the pET22b vector were transferred to BL21[DE3]. The transformed cells were named FhaB1 and FhaB2. The protein expression on the cell membrane was studied in addition to bacterial adhesion and biofilm formation by recombinant strains, A. baumannii and E.coli BL21[DE3]. Bioinformatic studies showed the bacterial potential of producing two exoproteins [FhaB1 and FhaB2]. Expression of the recombinant proteins on the outer membrane was confirmed by Western Blot Analysis and whole cell ELISA. The results revealed an association between the recombinant cells and bacterial adhesion and biofilm formation. FhaB1, FhaB2 and A. baumannii exhibited enhanced adherence to human lung epithelial cells compared to E.coli BL21[DE3] TPS in A.baumannii is of adherence and colonization factors and is one of the bacterial virulence factors
ABSTRACT
Infection with Escherichia coli O157:H7 rarely leads to bloody diarrhea and causes hemolytic uremic syndrome with renal failure that can be deadly dangerous. Intimin, translocated Intimin receptor [Tir], and enterohemorrhagic E. coli [EHEC] secreted protein A [EspA] proteins are the virulence factors expressed by locus of enterocyte effacement locus of EHEC. This bacterium needs EspA as a conduit for Tir delivery into the host cell and the surface arrayed Intimin, which docks the bacterium to the translocated Tir. Here we used triplet synthetic gene [eit] which was designed from three genes: espA coding EspA 120 lacking 36 amino acids from the N-terminal of the protein, eae coding Intimin constructed of 282 amino acids from the C-terminal and tir coding Tir 103, residues 258-361 which interacts with Intimin. The multimeric gene was cloned in two eukaryotic vectors pAAV-multiple cloning site-green fluorescent protein and pCI-neo. The pAAV was used for gene expression assay in cell line 293T and pCI-neo-EIT [EspA, Intimin, Tir] was used as DNA vaccine in mice. Test groups were injected intramuscularly with pCI-neo-EIT four times and mice control group was injected under the same conditions with PBS or pCI-neo vector. The titration of serums showed that BALB/c mice were successfully immunized with DNA vaccine compared to control groups and also they were protected against challenges of live oral using E. coli O157:H7. Conclusion: The results suggest that the DNA vaccine could induce protective immunity either alone or in combination with purified antigens to reduce EHEC infection
ABSTRACT
Clostridium Botulinum Type E neurotoxin heavy chain consists of two domains: the translocation domain as the N-terminal half and the binding domain as the Cterminal half [Hc]. One effective way to neutralize botulinum neurotoxin is to inhibit binding of this toxin to neuromuscular synapses with antibodies against binding domain. Two synthetic genes, coding for Hc [the full length binding domain] and the c-terminal quarter of binding domain [HcQ], were cloned in pET-28a vector and over-expressed in E. coli BL21 [DE3] cells. These recombinant proteins were purified by affinity Ni-NTA column [under native condition]. Mice were vaccinated with 2 microg of purified proteins, respectively; at step one with complete adjuvant, steps two and three with incomplete adjuvant and step four only with phosphate buffered saline [PBS]. Enzyme-linked immunosorbent assay [ELISA] has been performed with mice serum samples 14 days following their third and final vaccination. Binding activity of the purified proteins to ganglioside and synaptotagmin II was analyzed by ELISA. The results showed that HcQ and Hc could bind with ganglioside. Based on challenge experiments it was revealed that HcQ, Hc and BoNT/E toxoid could give protections in mice challenged with 10[2], 10[4] and 10[5] minimum lethal dose [MLD] dose of BoNT/E
ABSTRACT
Helicobacter pylori multiplies and causes infection in human gastric mucosal layer. New approaches have focused on using specific treatments, such as immunotherapy, to limit this infection. Urease, as one of the most important virulent and antigenic factors of the bacterium, is a suitable target for this purpose. In order to prepare recombinant proteins, the synthetic genes for total ureC protein [UreCt] and its N [ureCn] and C [ureCc] terminal fragments were ligated into pET28a. The recombinant proteins were expressed in E. coli BL21[DE3]. White leghorn hens were injected with the purified recombinant proteins. IgY recovered from egg yolk, using PEG precipitation. Finally, urease neutralizing ability of the antibodies was evaluated by urease activity assay in presence of the purified IgY. SDS-PAGE analysis revealed a good expression and purification of the recombinant proteins. Indirect ELISA observation demonstrated high antibody titer in sera and egg yolks and high ability of IgY Anti-UreCt and IgY Anti-UreCc antibodies in recognition of urease subunit C. Anti-UreCT and Anti-UreCc IgYs were more potential H. pylori urease inhibitors than Anti-UreCn. While all three UreC fragments induce prophylactic responses. UreCt and UreCc possess almost equal responses. Anti-UreCc IgY has advantage of smaller size and is preferred for its activity and easier protein recovery and purification process. These features emphasize on importance of simpler, easier and cost effective antibody production
Subject(s)
Animals , Immunotherapy , Urease , Egg Yolk , Helicobacter pylori , Enzyme-Linked Immunosorbent Assay , ChickensABSTRACT
Profound consumption of medicinal plants products worldwide and public misconception of the products safety puts the urgent need forward as to evaluation of their safe and harmful aspects. In the present study the Lavandula angustifolia essential oil was studied with a view to the foregoing criteria. The antimicrobial, antioxidative, hematologic and cytotoxic properties of Lavandula angustifolia essential oil were studied. The bacterial strains sensitive to Lavandula angustifolia oil were in the following order: S. aureus>E. coli>K. pneumonia>Streptococcus faecalis>P. aeruginosa. Antioxidative property of the oil was carried out using beta carotene bleaching test and the results were compared with the standard synthetic antioxidants. Lipid peroxidation inhibitions were lower than the synthetic antioxidant BHT and BHA. The oil concentration required for 50% [IC50] free radical scavenging of DPPH was 56 micro g/ml with total phenol contents of 85.43 micro g GAE/mg for L. angustifolia oil. Ferricreducing antioxidant power [FRAP] in the blood sera of the rats gavaged with a daily dose of 100 micro l oil increased by 167.57%. Adverse therapeutic effects were noted as a result of feeding the rats with the essential oil. The volatile oil displayed cytotoxic effects on the human tumor cell line [HeLa cells] and peripheral blood cells with the IC50 of 26 and 21 micro g/ml respectively. The mutagenic and antimutagenic properties of various concentrations of Lavandula angustifolia oil on TA98 and TA100 strains Salmonella typhimurium in the presence and absence of S9 fraction were determined. The results show that the Lavandula oil used in the present study may not be consumed without dose determination
ABSTRACT
Escherichia coli O157:H7, Vibrio cholerae, and Salmonella typhimurium are pathogenic bacteria found inn contaminated water and food. No assay method is currently available on simultaneous detection or identification of all the three pathogens. Our aim was to develop a rapid and reliable method for this purpose. A protocol for sample collection, and a PCR procedure was designed specifically for the assay. Selected fragments of 239 bp, 432 bp, and 360 bp for E. coli O157 lipopolysaccharide [LPS] gene [rfbE], V. cholerae toxin gene [ctx], and Salmonella typhimurium putative cytoplasmic protein gene [STM4497], respectively, were amplified from the extracted bacterial DNA samples in a single tube by multiplex PCR. The multiplex PCR products were analyzed by gel electrophoresis. All unknown samples were verifiably identified. The assay was sensitive enough to detect and identify as few as 100 cells of E. coli O157:H7, V. cholerae and Salmonella typhimurium. The presence of other bacteria did not interfere with the analysis. This assay is a specific and reliable tool which allows cost-effective detection o all three bacterial pathogens in one reaction tube
Subject(s)
Vibrio cholerae/isolation & purification , Salmonella typhimurium/isolation & purification , Polymerase Chain Reaction , Electrophoresis, Agar GelABSTRACT
Escherichia coll O157:H7 is a gram-negative rod-shaped bacterium. E coll O157:H7 is an enterohemorrhagic [EHEC] strain of the bacterium Escherichia coli and a cause of foodborne illness. Infection often leads to bloody diarrhea by producing a toxin called Shiga toxin, which damages the intestines, and occasionally leads to kidney failure, especially in young children and elderly people. A 2241 bpfepA gene of E. coli O157:H7 codes for production of a ferric enterobactin binding membrane protein that is essential for iron uptake by the bacterium. Inhibition of iron uptake can protect invasion of host by the bacterium. In this study we attempted to evaluate immunogenicity of the membrane protein, FepA. In order to produce recombinant FepA protein, the genomic, fepA gene of 2241 bp long was amplified by PCR from E coli O157:H7. The PCR product was ligated to pET28a. The recombinant protein was then expressed in E. coli BL21DE3 by IPTG induction. SDS-PAGE analysis was carried out and the recombinant protein was purified by Ni-NTA affinity chromatography. The purified recombinant protein was injected to Balb/C mice in order to induce immunity. Antibody titer was determined by ELISA. The recombinant protein of 85 KD was produced and purified. Immunogenicity of the recombinant protein was determined by injecting Balb/C mice. The antibody produced therein could efficiently recognize and bind ferric enterobactin binding protein, thus heaving mice tolerance of 10[6] LD[50]. With a view to the significant recognition by the antibody of ferric enterobactin binding protein, the notion of its application in restriction of enterobacteriacea propagation could be feasible