Construction of a bait plasmid containing human telomerase RNA mutant in yeast three-hybrid system / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To construct a bait plasmid containing human telomerase RNA with multiple point mutations in a yeast three-hybrid system and evaluate the toxicity of the recombinant bait plasmid.</p><p><b>METHODS</b>The primers were designed according to the hTR sequence and the target mutation sites for inducing T-->A mutations at the 41st, the 80th and 102nd nucleotides of the hTR gene using the overlapping extension PCR (OE-PCR) method. The mutant was cloned into PMD18T vector, confirmed by sequencing, sub-cloned into the bait plasmid PRH3' and identified with PCR and restriction enzyme digestion. The recombinant bait plasmid was then transformed into yeast L40 ura3/pHyblex/ZeoMS2 for toxicity test.</p><p><b>RESULTS</b>Sequence analysis demonstrated successful introduction of point mutations at the target sites without causing random mutation. The recombined bait plasmid constructed showed no obvious toxicity against the host yeast cells.</p><p><b>CONCLUSIONS</b>The recombinant plasmid containing the human telomerase RNA mutant (PRH3'-hTRm) has been successfully constructed and can be used as the bait plasmid in yeast three-hybrid system.</p>