The Cloning Of Fasl Gene And The Observation Of Its Expression In Human Lumbar Disc Tissue

Objective: A 352 bp of FasL fragment was isolated by reverse transcription-PCR(RT-PCR) from health human peripheral blood mononuclear cell (PBMC). After recombinant plasmid had been confirmed, digoxigenin labeled FasL cRNA probe was in vitro transcripted by means of T7 RNA polymerase.Materials and Methods: PBMC was collected from peripheral blood by density-gradient centrifuge. Treated by 50mM PHA-P, total RNA was extracted from actived PBMC. As a template, RNA was reverse transcripted and PCR amplificated by one-step RT-PCR using specific primers. PCR product was inserted into the MCS of pSPT19 with T4 ligase to construct recombinant plasmid. The recombinant pSPT19-FasL was transformed into E coli JM109, then. DNA sequencing showed an unique sequence data with GenBank (NM₀00639.1) The recombinant plasmid pSPT19 was linearized by EcoR/ to synthesize the digoxigenin -cRNA probe in vitro with T7 RNA polymerase.Results: The insert FasL cDNA fragment was identified by DNA sequencing. The labeling efficiency of Dig was detected by dot assay.Conclusions: 1, The cloning of FasL gene plays an important role in the study of mechanic of apoptosis in human lumbar intervertebral disc cells; 2, Dig-FasL cRNA probe can be used for further research on the expression and localization of FasL mRNA in all kinds of cells and tissues.Postgraduate: Si Chunxiang (Orthopaedics) Directed by: Chen Bohua (Professor)Lu Zhenhua (Associate Professor)...