Construction of a prokaryotic expression vector for apoptin and activity determination / 中国组织工程研究

ABSTRACT

BACKGROUND:Apoptin is a protein which is synthesized in vitro or expressed by genetic engineering, without toxic and transformation activity of normal cel s. Apoptin can specifical y induce the apoptosis of tumor cel s and provide the opportunity of inhibiting the growth of cancer. OBJECTIVE:To construct a prokaryotic expression vector for apoptin, optimize the expression conditions, and detect the activity of the purified protein. METHODS:The apoptin gene that had been constructed was cloned into prokaryotic expression vector pET-28b (+), which was transformed into E.coli host bacteria. Apoptin was induced by isopropyl-beta-D-thiogalactoside, and analyzed by polyacrylamide gel electrophoresis. The inhibition activity of apoptin on tumor cel s was detected. RESULTS AND CONCLUSION:Apoptin gene was successful y cloned into pET-28b (+). Apoptin protein was induced to express in form of inclusion body by isopropyl-beta-D-thiogalactoside (0.5 mmol/L) at 26 ℃. And the expression of apoptin with relative molecular mass of about 15 000 was identified by polyacrylamide gel electrophoresis. The target protein was purified by denaturation-renaturation and affinity chromatography, which has pro-apoptotic effect on lung cancer cel s H460 and H1299. The prokaryotic expression vector pET-28b-apoptin is successful y constructed. The apoptin protein with bioactivity is obtained, which al ows further functional study of apoptin.