Iranian Journal of Biotechnology
Volume:1 Issue: 3, Summer 2003

Hepatitis B virus (HBV) is a serious global health problem. The development of a safe and effective vaccine would help infection prevention. Previous hepatitis B vaccine production involved the isolation of the non-infectious particle from chronic HBV carriers. DNA recombinant technology has been used for vaccine production without having been contaminated with blood-born infectious agents. Vaccine production in mammalian cells has the advantage of being correctly modified and folded in comparison to other lower hosts. The surface protein coding genes, S (Major protein) and pre s2+s (Middle protein) of hepatitis B virus (HBV), were amplified from the mother plasmid containing the adr serotype virus genome. The s and pre s2+s amplicons were separately cloned in pBlueskript IIks (+) vector as pNM-sa2 and pNM-Psa2 intermediat-es respectively, then released and recloned in pcDNA3 mammalian expression vector. The correct pNM-Sb2 and pNM-Psb2 constructs containing s and pre-s2, respectively, were used to transfect the mammalian Cos-7 cell line. The major and middle proteins were secreted by this cell line and collected from the culture medium. Some features of gene cloning strategy and expression of these proteins are discussed.

The commercial availability of random peptide libraries displayed on the M13 phage is increasing their use for studies on epitope identification, enzyme inhibitors, receptor ligands, etc. In this study two experiments where planned for selection of peptides. First with sheep antibodies, the positive selector was IgG, prepared on Protein G column from a pool of 11 sheeps immunized, with a vaccine prepared from larvae of Lucilia cuprina, against blowfly strike. The negative selector was IgG from the same sheeps before vac-cination and IgG from vaccinated non-immunized sheeps. Four rounds of positive and negative selections with IgG from sheep were done respectively. In the second experiment using human IgG prepared on a fresh Protein G column, again four rounds of positive selection with either IgG from MS and schizophrenia patients were performed. This assay was alternated with two negative selections of schizophrenia IgG on MS-associated peptides and vice versa and two negative selections on each with a control IgG. After the fourth negative selection in each experiment, the phage was amplified and random clones were picked for nucleotide sequencing. A total of 44 peptides were sorted with the PILEUP program and there was a large number of 12-mer peptides containing either 4 or 5 methionine residues.

Preimplantation genetic diagnosis (PGD) is a very early form of prenatal diagnosis (PND) by which gene-tic diagnosis is performed on a single embryonic blastomer obtained by embryonic biopsy. Hence the genetic status of the embryo can be defined prior to embryo transfer; thus termination of pregnancy would not happen. The objective of the present paper was to establish preimplantation diagnosis for spinal muscular atrophy (SMA), a prevalent monogenic disorder in Iran, and to find the efficacy and sensitivity of the developed protocol. The fluorescent duplex single cell PCR technique for detection of SMN gene exon 7 deletion was developed first on single lymphocyte and then on a single blastomer. The protocol was duplexed with one of the three linked polymorphic markers namely, D5S112, D5S435 and D5S679. The PCR products of SMN exon 7 were restricted with DraI restriction endonuclease. Linkage analyses through polymorphic markers were also used for diagnosis. The amplification rates for SMN exon 7 deletion on a single lymphocytes and single blastomers were 96 and 88% respectively, using the aforementioned methods. The mean amplification rates of 3 polymorphic markers were 94% on single lymphocytes and 84% on single blastomers, respectively. Mean allele drop out (ADO) for the 3 markers on single lymphocytes was 4%. In conclusion the developed duplex single cell fluorescent PCR seems to be an accurate and feasible method at single cell level and could be easily applied in clinical preimplantation genetic diagnosis.

Ralstonia eutropha accumulates poly (?-hydroxybutyrate) up to 80% of its dry weight (as a carbon and energy source) and is the best-known PHB producer. Although PHB has many potential applications in medicine, veterinary practice, agriculture and surgery, its high cost limits the widespread use of it. The recent researches have focused on reducing these costs by optimizing fermentation process. In this study the Placket Burman experimental design was used to test the relative importance of medium components and process variables on cell growth and PHB production. The optimum values of the variables, selected as the best conditions for further studies in the development of a low cost and effective fermentation process for PHB production, were as follows: initial fructose concentration, 15 g/l; C/N ratio, 7.4; shaking rate, 200 rpm; fermentation time, 40 h; temperature, 30C; seed age, 15 h.

Pure mesophilic bioleaching bacteria were isolated to compare their potential for oxidizing ferrous and sulfur in synthetic media and copper extraction from low grade ore with mixed bacterial community. A total of 160 samples were collected from various sites of different mines. Enrichment and isolation of ferrous-/sulfur- oxidizing bacteria were done in specific media. A total of 68 isolates were screened, 63 of which oxidized Fe2+; the rest oxidized sulfur at different rates. Three important types of bacteria were identified to be Thiobacillus ferrooxidans, Thiobacillus thiooxidans and Leptospirillum ferrooxidans on the basis of morphological and physiological tests. The oxidation characteristics of both sulfur and ferrous were studied in pure isolates and mixed cultures. Oxidation behavior for all pure Fe2+-oxidizing isolates was properly modeled with Monod equation and a specific Fe2+-oxidation rate (?m) of 0.076 to 0.737 / h was reached. The rate of sulfur oxidation for pure sulfur-oxidizing isolates was 9 mg of sulfur / l/ h. Results of ferrous- or sulfur- oxidation for mixed cultures were in agreement with their bacterial community and pure isolates. The role of bacteria in releasing of copper was evaluated for pure isolates and mixed cultures. The results obtained showed that the simultaneous use of three types of isolates leads to more copper release and lower acid consumption compared to other communities. The positive effect of the initial concentration of Fe2+ showed that major portion of copper extraction is via indirect reactions.

Wheat, although moderately tolerant to salt, can not be cultivated in many areas. However, in the triticeae tribe, some of the wild wheat relatives are highly tolerant, e.g. Thinopyrum bessarabicum, which grows on the sea shore. Eight primary hexaploid tritipyrum lines, amphiploids between Triticum durum and Thinopyrum bessarabicum have been produced which can set seed in at least 250 mM NaCl. These tritipyrums (2n=6x=42, AABBEbEb) due to reasons such as brittle rachis, continuous production of tillers, late maturity, tall stature and meiotic instability will not fulfill the requirements of a successful commercial salt tolerant crop. To overcome such problems the substituted tritipyrum, in which selected Eb chromosomes are replaced by D genome chromosomes of 6x wheat, was produced from 6x tritipyrum x 6x wheat hybrids (F1: 2n=6x=42, AABBDEb) followed by selfing and backcrossing with 6x tritipyrum. The fertile plants among the above progenies were screened by the genomic fluorescent in situ hybridization technique to identify their Eb and D chromosome constitution. This study showed that producing tritiprum with variable numbers of Eb and D genome chromosomes is feasible and that FISH is a useful technique for determining the number of Eb chromosomes present.

There are evidence showing a relationship between host Src kinase activation and viral (CVB3) replication, which are based on the observation that inhibition in the enzyme activity could result in inhibition of viral replication. The present study assessed the effect of Src kinase inactivation on viral replication at different stages of infection. It was observed that the Src kinase activity is necessary for the initiation of viral replication. In this study HeLa cell lines were treated with 5 and 10? M herbimycin A (Src kinase inhibitor) with a time schedule of -90, -60, -30, 0, +15, +30, +45,. .. +210 minutes. All cultures were infected with CVB3 at zero-minute (+ve sign indicates that herbimycin A was added after infection with CVB3). The reaction was terminated after 24 h, cells were then detached from petri plates with trypsin/EDTA. Viral replication was monitored using a set of specific primers and the plaque formation unit (PFU) count. In cells pretreated with herbimycin A before infection viral replication was inhibited. However addition of herbimycin A after infection did not affect viral replication.

Soil samples were screened for alkaline protease producing bacteria on alkaline agar plates containing casein. Alkaline protease production was identified by clear zones of casein hydrolysis around colonies. Such colonies were grown in an alkaline broth for 48-72 h and the enzyme activity of the culture supernatants was determined by measuring the amount of tyrosine released from casein after 10 min at 35C, at a pH of 10.5. A spore forming Gram positive aerobic Bacillus sp. which showed the best enzyme production was chosen (strain L2). The production medium was optimized for this strain. The best developed medium contained: 1% glucose, 0.5 % peptone, 1% yeast extract, 0.1% KH2PO4 and 0.02 % MgSO4. 7H2O. Alkaline protease activity was highest at pH 11 and 45C. Biochemical tests tentatively identified the organism as Bacillus licheniformis.