Construction of a prokaryotic expression vector for apoptin and activity determination

doi:10.3969/j.issn.2095-4344.2015.18.025

Abstract

Abstract:

BACKGROUND: Apoptin is a protein which is synthesized in vitro or expressed by genetic engineering, without toxic and transformation activity of normal cells. Apoptin can specifically induce the apoptosis of tumor cells 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 cells was detected.
RESULTS AND CONCLUSION: Apoptin gene was successfully 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 cells H460 and H1299. The prokaryotic expression vector pET-28b-apoptin is successfully constructed. The apoptin protein with bioactivity is obtained, which allows further functional study of apoptin.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

全文链接：

Key words: Apoptosis, Lung Carcinoma

CLC Number:

R318

Zhang Yan-ling, Xu Xia, Jiang Lu-han, Du Jing-chun. Construction of a prokaryotic expression vector for apoptin and activity determination [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(18): 2928-2932.

Figures/Tables 2

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