Cloning expression purification and cytotoxic assay of sTRAIL in A549 cell line / 中国药理学通报

ABSTRACT

Aim To clone,express sTRAIL gene,then purify sTRAIL(114-281 amino acid) and assay its cytotoxic activity in human A549 cell line.Methods The intact full human TRAIL gene was amplified using PCR method from the human placenta and lung cDNA library.The full human TRAIL cDNA gene was inserted into pUC19 vector and sequenced.The extracellular DNA fragment was amplified using PCR,which was cloned to pET-11a vector and transformed into E.coli BL21.The denatured and refolded sTRAIL was purified and cytotoxic activity of sTRAIL was assayed using crystal violet staining and fluorescence-activated cell sort(FACS) in A549 cell line.Results The full length TRAIL cDNA gene was amplified from the human placenta and lung cDNA library,which was identical to the published TRAIL sequence.The extracellular DNA fragment was cloned to pET-11a.The expression level reached 50% of the total protein of BL21.The purity of sTRAIL was about 98%,while IC_(50) was about(24?5.2) ?g?L~(1) in TRAIL-treated A549 cells with crystal violet staining method.The time-dependent relationship of sTRAIL-induced apoptotic death in A549 cells was significant with FASC analysis.Conclusion sTRAIL gene has been cloned and successfully expressed.The process of refolding and purification of sTRAIL has been established.sTRAIL demonstrated cytotoxicity in A549 cell line.