Biotechnology & nanotechnology
Amirhossein Barghi; Hossein Honari; Mohamadali Ebrahimi; Gholamreza Bakhshi Khaniki; Seyed Mojtaba aghaee
Volume 27, Issue 1 , May and June 2020, , Pages 103-112
Abstract
Introduction: Saponaria officinalis have various saponin isoforms. Saponin is a ribosome-inactivating protein (RIP). The SO9 isoform of saponins depurinates the adenine 4324 in the preserved GAGA sequence resulting in impairment of protein production. In this study, the S09 isoform was expressed in E. ...
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Introduction: Saponaria officinalis have various saponin isoforms. Saponin is a ribosome-inactivating protein (RIP). The SO9 isoform of saponins depurinates the adenine 4324 in the preserved GAGA sequence resulting in impairment of protein production. In this study, the S09 isoform was expressed in E. coli and its antibody titers were evaluated in Mouse. Methods: The S09 gene was synthesized and isolated from the pUC57-S09 recombinant plasmid using the restriction enzymes BamH1 and Sal1, and then cloned in the expression vector pET28a(+). Expression of the new recombinant protein was induced by IPTG. The recombinant S09 protein was purified by Ni affinity chromatography. The recombinant protein was confirmed through western blotting. The Mouse were vaccinated through intraperitoneal injection of the purified protein and serum IgG titer was measured through ELISA.Results: Subcloning of S09 gene in the pET28a(+) expression vector was confirmed by PCR and enzymatic digestion. The presence of 29 kDa protein band in SDS-PAGE showed the high expression of recombinant protein. The recombinant S09 protein was detected by polyclonal antibody. After injection of the protein to the test groups, the antibody titer was measured by ELISA. Conclusion: The adjuvant property and immunogenicity of the purified recombinant S09 antigen showed that this antibody can be used to detect the presence of S09 in Saponaria officinal is, as a candidate for vaccine, for production of diagnostic kits, and in human cells anticancer studies.
Seyed Masih E'temad Aubi; Hossein Honari
Volume 23, Issue 1 , May and June 2016, , Pages 95-102
Abstract
Background: Anthrax is a common illness between human and animal which the agent of that is Bacillus anthracis. The modified protective antigen cab be used in treatment and vaccination. The aim of this study is the recombinant expression of modified protective antigen in E. coli. Material and Methods: ...
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Background: Anthrax is a common illness between human and animal which the agent of that is Bacillus anthracis. The modified protective antigen cab be used in treatment and vaccination. The aim of this study is the recombinant expression of modified protective antigen in E. coli. Material and Methods: Gene fragments were amplified with PCR from pXOI and fusion gene from SOEing PCR was cloned in a cloning vector and finally sub cloned in pET28a(+) vector and transferred to E. coli BL21(DE3) competent cells. Recombinant expression of modified PA was induced by IPTG and after protein purification with affinity chromatography, resulted antigen was injected to mice in 4 repeats. Polyclonal antibodies produced in mice serum was accessed. Results: The modified protective antigen cloned in expression vector pET28a(+) was confirmed by PCR, enzymatic digestion and sequencing. Recombinant protein was confirmed by SDS-PAGE and western blot. Serum was separated from mice blood and titre of antibody against modified PA was assessed by indirect ELYSA. Conclusion: resulted protein has high inhibitory property for LF and EF virulence factors. By consideration in PA detection by polyclonal antibody against modified PA from Bacillus anthracis, it can be used in for treatment and prophylaxis against anthrax in individual or combination forms with other PA domains.
MohammadEbrahim Minaee; Mojtaba Saadati; Mostafa Najafi; Hossein Hinari
Volume 22, Issue 5 , November and December 2015, , Pages 797-804
Abstract
Background & Objectives: Nanobiosensors could be used instead of traditional detection methods of Escherichia coli O157:H7. In this paper, a nanobiosensor for detection of rfbE gene of the Escherichia coli O157:H7 has been studied by immobilization and hybridization single strand DNA (ssDNA) sequences ...
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Background & Objectives: Nanobiosensors could be used instead of traditional detection methods of Escherichia coli O157:H7. In this paper, a nanobiosensor for detection of rfbE gene of the Escherichia coli O157:H7 has been studied by immobilization and hybridization single strand DNA (ssDNA) sequences on the electrode surface modified with gold nanoparticles.
Materials & Methods: Electrochemical impedance spectroscopy technique was used to study the properties of the sensing modified electrode. The working electrode surface was modified by electrochemical method with gold nanoparticles. The single-stranded DNA sequence using self-assembled monolayer was immobilized on the gold electrode for two hours. A mixed monolayer comprising both mercaptohexanol (MCH) and mercaptopropionic acid (MPA) were used as blocking layer. The hybridization DNA/DNA was performed by immersion of the modified gold electrode into ssDNA at a concentration of 1 μM target DNA.
Results: The results showed that the electrode modified with gold nanoparticles after immobilizing ssDNA effectively detected rfbE gene of Escherichia coli O157:H7 by DNA hybridization . The nanobiosiosensor showed suitable selectivity for the detection of target DNA complementary sequence compared with the mismatched oligonucleotides sequence and the noncomplementary oligonucleotides sequence.
Conclusion: According to the results obtained and similar studies, the electrochemical nanobiosensor based on DNA hybridization has advantages such as low cost, simplicity, and miniaturization and can provide a basis for the development of genomic detection tools.
Hossein Honari; Mehdi Baranvand; Mohammad Ebrahim Minaee
Volume 21, Issue 6 , January and February 2015, , Pages 1103-1112
Abstract
Background: Shigellosis is an acute intestinal infection from Shiga toxin and Shiga-like toxin, which is caused by Shigella and enterohemorrhagic Escherichia coli (EHEC) and entrotoxinogenic Escherichia coli (ETEC). The disease has a high prevalence rates in the world and is known as a bioterrorist agent. ...
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Background: Shigellosis is an acute intestinal infection from Shiga toxin and Shiga-like toxin, which is caused by Shigella and enterohemorrhagic Escherichia coli (EHEC) and entrotoxinogenic Escherichia coli (ETEC). The disease has a high prevalence rates in the world and is known as a bioterrorist agent. STxB is a part of Shiga toxin and have the property of immunogenicity.
Materials and Methods: In this experimental study, the vector pET28a (+) containing the stxB gene was used and was transformed into E. coli BL21 DE3. The bacteria were grown on antibiotic medium and were confirmed with direct PCR, protein expression and SDS-PAGE gel. The recombinant protein purified by nickel column and was confirmed with SDS-PAGE gel and immunoblotting. The chitosan nanofibers containing STxB protein were synthesized by the electrospinning device. The Intranasal and injectable prescription of STxB protein and nanofibers containing STxB protein were performed in mice for four consecutive times and their Antibody titer were assessed. By ELISA, increased IgG antibody titers were observed in injectable and nasal view mode, which may not capture antigen by nasal epithelial cells in mice.
Results: By ELISA, The increase in IgG antibody titers was observed in injectable and intranasal states but not in naonnasal one, which may be due to the lack of antigen captured by nasal epithelial cells in mice. Immunized mice were able to tolerate five times of the Shiga toxin LD50 of E. coli O157: H7.
Conclusion: The results of this study indicate that with nasal and injection prescribed of STxB protein, the immunized mice can tolerate E. coli O157: H7 Shiga toxin.