[Isolation and cloning of the beta subunit of human follicle stimulating hormone [hFSH beta] with its native gene's signal sequence in methylotroph yeast Pichia pastoris pPIC9 shuttle vector]

Follicle Stimulating Hormone [FSH] is one of the pituitary glycoproteines that it consists of two subunits; alpha and beta. The beta subunit is responsible for the biological activity of FSH. The aim of present study was isolation of the beta subunit coding sequence containing its signal sequence from human genome and then cloning of the isolated sequence in pPIC9 shuttle vector under the control of AOX1 promoter and ? factor signal sequence. the gene sequence of interest was isolated as a 2kb DNA fragment and cloned in pTZ57R vector resulting to pTV-2019 plasmid. The construct was used as template for modification of 5? region of gene upstream to ATG codon using PCR. Finally, amplicon was cloned in pPIC9 and the new construct named pPIC9F1. The sequence of FSH beta gene in pTV-2019 was confirmed by restriction analysis and DNA sequencing. In addition, restriction analysis and AOX1 primer-mediated PCR showed that pPIC9F1 has correct construction. The new construct, pPIC9F1, contains the coding sequence of FSH beta gene and its signal sequence [E2-IVS2-E3]. Therefore, this construct can be used for integration of FSH beta gene into yeast genome exactly downstream to AOX1 promoter. Under this condition, a fusion protein is produced that it contains two signal peptides, ? factor and FSH signal peptides. Yeast expression system is able to cleavage ? factor. It seems this is the first attempt for cloning of human FSH beta in yeast expression system

Cloning of glucose oxidase gene in PKK233-3

Glucose oxidase [GO] has found a variety of industrial applications such as food, chemical and personal care industries. However one of the most important application of GO is used as diagnostic kits. The aim of study was isolation of GO gene from a recombinant vector [PET21aGO] and sub cloning and expression in PKK233-3 vector. Recombinant PET21a GO was extracted from E.coli DH5alpha and was digested with Restriction Enzymes; BamHI, Hindlll then isolated GO gene [1.8kb] and cloned in PKK233-3. PKK233-3GO was transformed in to E.coli DH5alpha. Our data demonstrated that the GO gene has expected size in agarose gel electrophoresis and also the cloned Go has a correct size after restriction analysis. The GO gene was cloned in prokaryotic host. This is a report of cloning of GO gene in Iran that can be used for further cloning of that gene in expression vectors for production of recombinant Enzyme

Escherichia coli heat-labile toxin B subunit: Construction and evaluation of plasmids providing controlled high level production of the protein

With the plasmid DNA from a clinical isolate of enterotoxigenic Escherichia coli [ETEC] H10407 as template, PCR -mediated cloning of the sequence encoding the heat-labile toxin B subunit [L T -B] has been carried out Then this sequence was recloned into the pTrc 99A and pET23a expression vectors to give the plasmids pTRCLTB and pETLTB, respectively. After induction, the former plasmid provides for the production of rL T -Bin a yield of up to 15 mg per liter of bacterial culture. The recombinant protein was shown to be structurally and immunologically identical with the native L TB. High titer antibodies capable of neutralizing the native toxin were raised in mice by oral administration of the rL T - B. Hence the constructed plasmids provide the basis for an oral ETEC vaccine, as well as for genetic fusion of foreign antigens with the aim of developing polyvalent vaccines