[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 protective antigen gene from Bacillus anthracis into Bacillus subtilis

Protective antigen [PA] of Bacillus anthracis is used as anthrax vaccine. Cloning and expression of the PA gene in various strain such as E.coli and Bacillus subtilis was reported that most expression was in B. subtilis up to 160 microg/ml. The objectives of this study were: to clone the gene of PA in an expression vector [pWB980] and then transformation into the B. subtilis WB600 strain. The pXOl plasmid was separated from the strain stern of B. anthracis with alcalin method and the PA gene with 2.4kb sequence amplified by PCR. Then the amplified fragment was directly cloned into pTZ57R plasmid as T-vector and transferred into E.coli DH5 alpha using CaC12 method. After that the gene was separated from the T-vector by enzymatic digestion [Sa1I and KpnI]. Ligation between the purified gene fragment of the PA and the vector was carried out. Then it was transferred into B. subtilis WB600 by electroporation method in 1000 V. In this study we isolated PA gene from B. anthracis strain stern with PCR and was cloned into pTZ57R plasmid. The Presence of the gene was confirmed by restriction analysis, PCR and sequencing. Then the PA gene was cloned into pWB980 and B. subtilis and the presence of the gene in two kanamycin resistant colonies [AMN1 and AMN3] was confirmed by restriction analysis and PCR. We may conclude that by making modification in the methods used and using pWB980 expression vector, we were able to clone the PA gene into B. subtilis. This is the first research project in Iran that the PA gene is isolated and cloned and B. subtilis is used as host