A study on polymerase chain reaction site-directed mutagenesis of prepro-parathyroid hormone in vitro / 中华外科杂志

ABSTRACT

<p><b>OBJECTIVE</b>To study PCR site-directed mutagenesis of prepro-parathyroid hormone gene in vitro and let furin convert it into mature parathyroid hormone in human cells.</p><p><b>METHOD</b>Prepro-parathyroid hormone cDNA of SD rat was cloned from its genomic gene and mutated by overlap mutant PCR, introducing furin consensus sequences (Arg-Lys-Lys-Arg). An expression pcDNA3.1/mPTH vector encoding a genetically modified prepro-parathyroid hormone cDNA was generated, and transduced to 293 cells by lipofectin-mediated DNA transfection. Forty-eight and 72 h after the transfection, the culture media were collected for further assay with radioimmunoassay.</p><p><b>RESULTS</b>A fragment of prepro-parathyroid hormone gene was cloned and one site were mutated simultaneouly. After screening and sequencing of pcDNA3.1/mPTH vectors, a correctly mutated vectors was obtained. While measuring parathyroid hormone in the medium of the expressing 293 cells by RIA method, the results of transient expression were 28.34 - 52.64 pg/2.0 x 10(6)/cells/Day, which were much higher than that in control cells.</p><p><b>CONCLUSIONS</b>A correctly mutated prepro-parathyroid hormone cDNA was obtained successfully, transfected, and expressed efficiently in human cells.</p>