ABSTRACT
Background: Archaea are Extrtermophile microorganisms and for several decades it has been believed that they are only found in harsh environments, such as volcanoes, deep oceans and salt lakes. However, at present time, their existence in human and mammal's intestine has been proved. The most important Archaea in human intestine is Methanobrevibacter smithii, which has a major role is some gastrointestinal disorders, as well as obesity. Therefore, Methanogens isolation and detection has such a crucial clinical importance. In this study, we isolated this microorganism for the first time using local technique
Materials and methods: In this study, Archaea DNA was extracted from healthy subject's stool samples, considering the specific criteria for choosing the healthy group. PCR reaction was performed to amplify the rpoB. Enzyme digestion was operated using restriction enzyme to confirm the rpoB gene. The PCR product was then cloned in E.coli [DH5alpha] host and sequencing process was performed
Results: Of 20 stool samples, the rpoB gene was confirmed in 18 samples [90%] and also the AVAII enzyme digestion results proved the gene identity. Sequencing results in NCBI site proved that isolated microorganisms were Methanobrevibacter smithii
Conclusion: This study revealed that by considering the microorganisms' variety in intestine, the precise gene detection methods for selecting the specific microbiota, in order to prevent existing similarities between homolog microbiota is vital in microbiota isolation
ABSTRACT
Helicobacter pylori is a spiral, microaerophilic gram negative bacterium, that multiplies and causes infection in human gastric mucosal layer. H.pylori infection, followed by destruction of gastric epithelial tissue, leads to gastric chronic inflammation, which can cause gastric and peptic ulcers. New approaches have focused on using specific treatments, such as immunotherapy, to eradicate this infection. Urease, as one of the most important virulent and antigenic factors of the bacterium, is a suitable target for this purpose. This study is aimed at production of specific IgY against urease UreC subunit. In this study, initially for preparing recombinant UreC, after purification of the genomic DNA, ureC gene was amplified by polymerase chain reaction [PCR]. The PCR product was ligated to pET28a. The recombinant protein was expressed followed by transformation of recombinant construct into E.coli BL21DE3. SDS-PAGE analysis was carried out and the recombinant protein was purified by Ni-NTA affinity chromatography. The purified recombinant protein was injected to hens. IgY recovered from egg yolk, was purified by PEG precipitation at >70% purity. The purified IgY was analyzed by ELISA and SDS-PAGE. SDS-PAGE analysis revealed a good expression and >70% purification of the recombinant protein. ELISA observation demonstrated high immunogenicity of the recombinant protein. With a view to higher potential of IgY-HpUc in recognition of UreC subunit, the results are in favour of the oral administration of the IgY obtained from hens immunized by H.pylori may provide a novel approach to the management of H.pylori infections