Comparison of expression systems for the production of human interferon-a2b.

The production of human interferon alpha2b (IFN-α2b) in two expression systems, tobacco (Nicotiana tabaccum) and Escherichia coli, was compared in various aspects such as safety, yield, quality of product and productivity. In the E. coli system, IFN-α2b was expressed under a pelB signal sequence and a T7lac promoter in a pET 26b(+) vector. The same gene was also cloned in expression plant vector (pCAMBIA1304) between cauliflower mosaic virus promoter (CaMV35S) and poly A termination region (Nos) and expressed in transgenic tobacco plants. The expression of protein in both systems was confirmed by western immunoblotting and the quantity of the protein was determined by immunoassay. The amount of periplasmic expression in E. coli was 60 µg/L of culture, while the amount of nuclear expression in the plant was 4.46 µg/kg of fresh leaves. The result of this study demonstrated that IFN-α2b was successfully expressed in periplasm of bacterial and plant systems. The limitations on the production of IFN-α2b by both systems are addressed and discussed to form the basis for the selection of the appropriate expression platform

Cloning and periplasmic expression of peptidoglycan-associated lipoprotein (PAL) protein of Legionella pneumophila in Escherichia coli

Abstract Introduction and objective: Legionella pneumophila, the etiological agent of Legionnaires’ disease, is an important cause of both community-acquired and nosocomial pneumonia; therefore, rapid diagnosis and early antibiotic treatment of pneumonia are required. Urinary antigen testing to detect Legionella antigen has proven to be the most powerful diagnostic method. Peptidoglycan-associated lipoprotein (PAL) protein of L. pneumophila, as a component of Legionella antigens, will be detected efficiently by the PAL antigen capture assay and is considered as useful diagnostic antigen to diagnose Legionella infection. Because of the transfer of protein to the periplasmic region of Escherichia coli has numerous advantages including separation from cytoplasmic proteins and the concentration of recombinant proteins in periplasm, the aim of this study was to produce periplasmic PAL protein of L. pneumophila in E. coli. Materials and methods: The pal gene of L. pneumophila serogroup 1 was amplified with specific primers, cloned and expressed under pelB signal sequence and T7 lac promoter in pET26b+ plasmid. Results: The cloning was confirmed with digestion and sequencing of recombinant pET- 26b-pal plasmid. The expression of r-PAL protein in cytoplasm and periplasmic space of E. coli was approved by SDS-PAGE and western blotting. Conclusion: The results of this study demonstrated that the r-PAL protein successfully expressed in E. coli

MORPHOLOGICAL, MORPHOMETRIC AND MOLECULAR CHARACTERIZATION OF MERLINIUS MICRODORUS (GERAERT, 1966) SIDDIQI, 1970, SCUTYLENCHUS RUGOSUS (SIDDIQI, 1963) SIDDIQI, 1979 (MERLINIIDAE), AND PSILENCHUS CURCUMERUS RAHAMAN, AHMAD AND JAIRAJPURI, 1994 (PSILENCHIDAE) AND APPROACHES TO PHYLOGENETIC RELATIONSHIPS

Merlinius microdorus and Scutylenchus rugosus (Merliniidae), and Psilenchus curcumerus (Psilenchidae) were collected from the rhizosphere of faba bean (Vicia faba L.) fields in Khuzestan province, south-western Iran. Morphological and morphometric data are provided for these species. Additionally, sequences of the D2-D3 expansion segments of 28S rRNA gene for all species were also used for molecular phylogenetic analysis. The phylogenetic relationships of Psilenchidae and Merliniidae in relation to representatives of the superfamily Tylenchoidea, obtained from Bayesian inference (BI) and maximum likelihood (ML) analyses of the D2-D3 sequences, are presented and discussed. The results of phylogenetic analysis strongly supported (BPP = 100) Merliniidae and Psilenchidae as monophyletic. The family Tylenchidae formed a sister clade to Merliniidae/Psilenchidae with high branch support (BPP = 100). Monophyly of representatives of Merliniidae (including Pratylenchoides) was supported with maximum BPP

DESCRIPTION OF TYLENCHORHYNCHUS IRANENSIS SP. N. (NEMATODA TELOTYLENCHIDAE) FROM IRAN

A new species of stunt nematodes, Tylenchorhynchus iranensis sp. n. is described from the rhizosphere of faba bean (Vicia faba L.), from material collected in the Khuzestan province, southwestern Iran. The new species is characterized by the following combination of features: lateral fields with four non areolated incisures, cephalic region continuous to slightly offset and conformed by 6-7 fine annuli, stylet 15-18 µm long, post-anal intestinal sac extends into the entire tail cavity, epiptygma present, tail sub-cylindrical with a hemispherical to sub-hemispherical smooth terminus, 46-65 μm long and composed by 46-49 annuli. Molecular analysis based on 28S rRNA gene sequences placed T. iranensis sp. n. within a clade that contained representatives of the genus Tylenchorhynchus with high support

Cytokine Profiles and Cell Proliferation Responses to Truncated ORF2 Protein in Iranian Patients Recovered from Hepatitis E Infection

Background.The aim of this study was to evaluate hepatitis E virus (HEV) specific cellular immune responses to truncated ORF2 protein in Iranian patients recovered from HEV infection. Information about HEV-specific immune responses could be useful in finding an effective way for development of HEV vaccine. Methods. A truncated formof HEVORF2 protein containing amino acids 112-608 was used to stimulate peripheral blood mononuclear cells (PBMCs) separated from HEV-recovered and control groups. Finally, the levels of four cytokines, IFN

Effect of promoter strength and signal sequence on the periplasmic expression of human interferon-α2b in Escherichia coli

Two plasmids, pFLAG-ATS and pET 26b(+), were studied for the periplasmic expression of recombinant human interferon-α2b (IFN-α2b) in Escherichia coli. The pFLAG-ATS contains ompA signal sequence and tac promoter while pET 26b(+) contains pelB signal sequence and T7lac promoter. It was observed that periplasmic expression of IFN-α2b from pET 26b(+) was around 3000 times higher than pFLAG-ATS. Difference in the expression level was attributed to the difference in the promoters and the signal sequences. In silico analysis of mRNA secondary structures were analyzed using Vienna RNA package and MFOLD. The resultssuggested that the increase of expression would mainly due to the difference in the translation initiation associated with secondary structure of mRNA transcribed by both plasmids

Multiple overlap extension PCR (MOE-PCR): an effective technical shortcut to high throughput synthetic biology

The current study describes multiple-overlap-extension PCR (MOE-PCR) as a simple and effective approach to assembling multiple DNA fragments with various sizes and features in a single in vitro reaction. In this research, 50 bp of homology in overlapping DNA fragments and a specific touchdown PCR program resulted in successful assembly of eight different DNA fragments using a single PCR protocol. The simplicity, speed, reliability and cost-effectiveness of MOE-PCR offers it as an attractive method for assembling and/or cloning single and multiple DNA fragments. Indeed, the method is a one-step approach that eliminates some of the routine steps such as ligation and complex enzymatic reactions as well as sequence limitations of the other methods. The applications of this relatively high fidelity method could be extended to the construction of chimeric recombinant sequences that can be widely used in metabolic engineering, functional analysis of genes and genetic elements, expression studies of multi-domain proteins, protein engineering and the most recent genome editing strategies which together with synthetic biology are revolutionizing the life sciences. We expect the technique to be used as a robust, reliable and fast method in synthetic biology

Cytokine Profiles and Cell Proliferation Responses to Truncated ORF2 Protein in Iranian Patients Recovered from Hepatitis E Infection

Background. The aim of this study was to evaluate hepatitis E virus (HEV) specific cellular immune responses to truncated ORF2 protein in Iranian patients recovered from HEV infection. Information about HEV-specific immune responses could be useful in finding an effective way for development of HEV vaccine. Methods. A truncated form of HEV ORF2 protein containing amino acids 112-608 was used to stimulate peripheral blood mononuclear cells (PBMCs) separated from HEV-recovered and control groups. Finally, the levels of four cytokines, IFN-ELISPOT, and cell proliferative responses following stimulation with the truncated ORF2 protein were assessed in the both groups. Results. The truncated ORF2 protein was able to induce IFN-ELISPOT and cell proliferation responses and to produce significant amounts of IFN-and IL-12 cytokines, but low amounts of IL-10 and IL-4 cytokines in vitro. These responses were significantly higher in the recovered group compared to the control group. These results indicate the antigenic nature of the truncated ORF2 protein and production of T helper type 1 cytokines. Conclusion. The truncated ORF2 protein can effectively induce significant cellular immune responsesand can be introduced as a potential vaccine candidate. However, further studies are required to evaluate this protein in vivo

New expression vector for microalgae

The present invention relates to expression vectors for the improved production of recombinant proteins in microalgae through synthetic biology. The present invention also relates to the construction of a vector system which can be used for promoter analysis in gene expression