Showing papers on "XhoI published in 2011"

Molecular cloning of gluconobacter oxydans DSM 2003 xylitol dehydrogenase gene.

Abstract: Due to the widespread applications of xylitol dehydrogenase, an enzyme used for the production of xylitol, the present study was designed for the cloning of xylitol dehydrogenase gene from Glcunobacter oxydans DSM 2003. After extraction of genomic DNA from this bacterium, xylitol dehydrogenase gene was replicated using polymerase chain reaction (PCR). The amplified product was entered into pTZ57R cloning vector by T/A cloning method and transformation was performed by heat shocking of the E. coli XL1-blue competent cells. Following plasmid preparation, the cloned gene was digested out and ligated into the expression vector pET-22b(+). Electrophoresis of PCR product showed a 789 bp band. Recombinant plasmid (rpTZ57R) was then constructed. This plasmid was double digested with XhoI and EcoRI resulting in 800 bp and 2900 bp bands. The obtained insert was ligated into pET-22b(+) vector and its orientation was confirmed with XhoI and BamHI restriction enzymes. In conclusion, in the present study the recombinant expression vector containing xylitol dehydrogenase gene has been constructed and can be used for the production of this enzyme in high quantities.

TRANSGENIC MOUSE EXPRESSING REPORTER PROTEIN UNDER REGULATION OF α-FETOPROTEIN ENHANCER AND PROMOTER, METHOD FOR PREPARATION THEREOF AND METHOD FOR SCREENING COMPOUNDS INDUCING INCREASE OR DECREASE OF Α-FETOPROTEIN EXPRESSION USING THE SAME

Abstract: PURPOSE: A transgenic mouse, a method for preparation thereof, and a method for screening compounds inducing increase or decrease of alpha-fetoprotein expression are provided to express reporter protein. CONSTITUTION: A polynucleotide(sequence number two) includes an Alpha-feto protein enhancer and a promoter of the human. A primer(sequence number four) includes a recognition base sequence of the limitation enzyme MluI. A primer(sequence number four) includes a recognition base sequence of the restriction enzyme XhoI. The size of an amplified polynucleotide is 2,143 bp. The amplified polynucleotide has a sticky end by treating MluI and XhoI limitation enzyme. A recombinant vector is prepared by connecting the amplified polynucleotide and a pGL3-Basic vector using ligase.

Determination and cloning of the gene encoding EG95 protein in Iranian isolate of Echinococcus granulosus

Abstract: Background: Echinococcus granulosus is a cestode parasite that causes cystic hydatid disease in humans worldwide. The gene encoding EG95 protein may be a good candidate to design a DNA vaccine to prevent the disease. Considering the importance of EG95 gene and the scarceness of research on it in Iran, this study was carried out to determine and clone the gene encoding EG95 from Iranian isolate of E. granulosus. Materials and Methods: At the first stage, protoscoleces was isolated from hydatid cyst fluid and then RNA was extracted from protoscoleces and after performing RT-PCR, the amplified cDNA samples were detected by gel electrophoresis. In next stage, the obtained gene was cloned in pTZ57R/T vector. Two methods were used for conformation of cloning: colony PCR amplification and digestion with the EcoRI and XhoI restriction enzymes. Finally, the cloned EG95 gene in pTZ57R/T vector was sequenced. Results: Homological comparison of sequences showed that cDNA of EG95 in Iranian isolate of E. granulosus had 492 bp and was different from the standard strain of EG95 reported from New Zealand and Australia (X90928.1). Moreover, cloning of EG95 gene in pTZ57R/T plasmid was confirmed by digestion of this plasmid with the restriction enzymes. Conclusion: The EG95 gene was cloned in pTZ57R/T plasmid successfully and this plasmid can be used to design a DNA vaccine in further studies. Keywords: Echinococcus granulosus, EG95 gene, Protoscoleces, pTZ57R/T plasmid,

Development of a high-expression system for staphylococcal exfoliative toxin genes.

Abstract: We constructed a new expression system for staphylococcal exfoliative toxin (ET). The expression vector, pETA-exp2, was constructed based on Bacillus-Escherichia shuttle vector pHY300PLK. The pETA-exp2 vector includes the regulator of the ETA gene (eta), the promoter and Shine-Dalgarno (SD) sequences of eta, a SalI sequence at the end of the signal sequence of eta, a nucleotide sequence encoding mature ETA, an XhoI site, a 6x His sequence just before the stop codon and the end of the transcription sequence of eta. The nucleotide sequences coding for the mature proteins of ETB, ExhA, ExhB, ExhC, ExhD and SHETB were amplified by polymerase chain reaction (PCR) and inserted into pETA-exp2. These recombinant plasmids were transformed into Bacillus megaterium. The major protein in the culture supernatant of the transformant was recombinant ET (rET). The yields of all rETs were high, and all of them showed exfoliative activity in susceptible animals. The antigenicities of rETs and ETs were not distinguishable from each other.