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

Sequence analysis of ORF94 in different white spot syndrome virus (WSSV) isolates of Iran

White spot syndrome virus (WSSV) is a pathogen that causes high mortality in shrimp culture in the whole world. Sequence analysis of WSSV has shown similarity of WSSV isolates in different countries with exception of a few variable genomic loci. This study investigated the sequence variation of some Iranian WSSV isolates and previously identified isolates. Samples were collected during target surveillance and were feed, broodstock, post-larvae, artemia, crabs, and wild and cultured shrimp of northern Persian Gulf (Boushehr and Khuzestan provinces). The open reading frame (ORF) 94 sequence of different Iranian WSSV isolates were amplified using specific primers from positive samples. The ORFs 94 sequence of positive samples were sequenced and registered in the Gene Bank and then compared to other WSSV isolates. The number of repeat units in ORF94 showed that WSSV isolates were varied in number. There are SNPs (G and T) in position 48 of RUs that varies in different Boushehr and Khuzestan isolates. Also these sequences were compared to Gene Bank WSSV isolates and showed a high similarity (>90%) to Southeast Asian countries. To our knowledge this is the first report of sequence analysis in Iranian WSSV isolates applications

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

Phylogenetic relationships of Iranian Infectious Pancreatic Necrosis Virus (IPNV) based on deduced amino acid sequences of genome segment A and B cDNA

Infectious Pancreatic Necrosis Virus (IPNV) is the causal agent of a highly contagious disease that affects many species of fish and shellfish. This virus causes economically important diseases of farmed rainbow trout, Oncorhynchus mykiss, in Iran which is often associated with the transmission of pathogens from European resources. In this study, moribund rainbow trout fry were collected during an outbreak of IPNV in three different fish farms in one northern province (Mazandaran), and two west provinces (Chaharmahal and Bakhtiari, and Kohgiluyeh and Boyer Ahmad) of Iran. We investigated full genome sequence of Iranian IPNV and compared it with previously identified IPNV sequences. The sequences of different structural and non-structural protein genes were compared with other aquatic birnaviruses sequenced to date. Our results showed that the Iranian isolate fall within genogroup 5, serotype A2 strain SP, having 99 % identity with the strain 1146 from Spain. These results suggest that the Iranian isolate may have originated from Europe

Isolation and expression of recombinant viral protein (VP2) from Iranian isolates of Infectious Pancreatic Necrosis Virus (IPNV) in Escherichia coli

Infectious Pancreatic Necrosis Virus (IPNV) is a member of the family Birnaviridae that has been linked to high mortalities in salmonids. Bacterial based systems as live vectors for the delivery of heterologous antigens offer a number of advantages as vaccination strategies. VP2 is a structural viral protein of IPNV with immunogenicity effects. In this study IPNV was isolated from diseased fry of rainbow trout Oncorhynchus mykiss (Walbaum) using CHSE-214. Then an expression vector was constructed for expression of viral protein VP2. The designed vector was constructed based upon pET-26b (+) with T7 promoter. A fragment containing the full length of the VP2 gene of Iranian Sp strain was amplified by PCR using genomic RNA of IPNV as template and cloned inpET-26b(+) plasmid. Recombinant structural viral protein VP2 was expressed as a soluble, N-terminal PelB fusion protein and secreted into the periplasmic space of Escherichia coli BL21(DE3) and Rosetta (DE3). The glucose, Isopropyl-β-D-thiogalactopyranoside (IPTG) was used as a chemical inducer for rVP2 production in 37º C. The rVP2 was extracted from the periplasm by osmotic shock treatment. The presence of gene in bacterial system of E. coli was confirmed by gel electrophoresis technique. The constructed vector could efficiently express the rVP2 into the periplasmic space of E. coli. The successful cloning and expression of the structural viral protein gene into E. coli can be used for developing a useful and safe vaccine to control IPNV infection in Iranian fish industry

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