[A study on polymerase chain reaction site-directed mutagenesis of prepro-parathyroid hormone in vitro].
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
A correctly mutated prepro-parathyroid hormone cDNA was obtained successfully, transfected, and expressed efficiently in human cells.
Zhu
Yi-fan
YF
Department of Endocrinological Surgery, First Affiliated Hospital of Sun-Yet-Sen University, Guangzhou 510080, China. zyf0924@126.com
Wang
Shen-ming
SM
D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D010281:Parathyroid Hormone / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
D018076:DNA, Complementary; C470125:PTH protein, human; D010281:Parathyroid Hormone