cDNA CLONING AND IDENTIFICATION OF HUMAN G-CSF WITH HIGH ACTIVITY

mRNA was isolated and purified from total RNA of human peripherial blood mononuclear cells stimulated by LPS in vitro, and was used as templates in reverse transcription reaction to synthesize single strand cDNA. A fragment (544bp) with deleted signal sequence and 3'-untranslated region of G-CSF cDNA was then produced through PCR amplification with the help of a pair of specific primers of interest. The result of Southern blot, in situ hybridization and direct sequence determination after recombination of cDNA into pBV 220 showed that the amplified DNA encoded G-CSF with 174aa, which was reported to be 20 times more active than that of 177aa. Key words: G-CSF; cDNA, cloning; polymerase chain reaction