The Study Of Obtaining Human Transforming Growth Factor-beta 1 Mature Peptide Gene From Different Materials

Objective: To seek methods of obtaining human transforming growth factor-beta 1( TGF- β1) mature peptide gene. To amplify human TGF- β1 mature peptide gene from human skin fibroblasts, peripheral blood mononuclear cells, human platelets, peripheral blood cells and GLC-82 cells. And to do the groundwork of constructing prokaryotic and eukaryotic expression vector of human TGF- β1.Methods: We cultured human foreskin fibroblasts and GLC-82 cells and collected peripheral blood mononuclear cells, human platelets and peripheral blood cells. Then we extract total RNA from different materials. Design a pair of primers based on the sequence of complementary deoxyribonucleic acid (cDNA) of TGF- β1.The upstream primer is P1 5'-CGGAATTCGCCCTGGACACCAACTATTGCTT-3', which contain EcoR I restrictionsite .The downstream primer is P2 5'-TAAGCTTTCAGCTGCAC TTGCAGGAGCG-3', which contain Hind III restrictionsite .Oligonucleotide primers were obtained from TaKaRa Biotechnology Company. TGF β1 mature peptide gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) using RNA LA PCR^TM Kit(TaKaRa Co.).Reverse transcription conditions: 30℃, 10minuts; 42 ℃, 50minutes; 70℃, 15 minutes. Polymerase chain reaction conditions: 94 ℃ degeneration reaction 2 minutes, 1 cycle; 94℃ degeneration reaction 30 seconds, 55℃ renaturation reaction 30 seconds, 72℃ extension reaction 90 seconds, totally 30 cycles, 72 ℃ extension reaction 10 minutes. The PCR products were subjected to agarose gel electrophoresis, and amplified products with conformity to expecting DNA fragment that has 336 base pairs were, retrieved by using UNIQ-10 column DNA Gel Extraction Kit (Shanghai Sangon). The DNA fragments were ligated with pMD18-T vectors (TaKaRa Co.), and the ligated products were sent for gene sequencing in TaKaRa Biotechnology Company.