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目的 应用分步克隆法构建人拓扑异构酶Ⅲalpha (hTOP 3alpha)真核反义表达载体 ,为进一步阐明其与控制细胞周期的相关基因ATM之间的相互作用 ,研究ATM的生物学功能奠定基础。方法 采用分步克隆法首先用HindⅢ和EcoRI双酶切构建插入 2 .6kb的片段 ,再用EcoRI酶切插入另一 1kb的片段 ,最终获得人拓扑异构酶Ⅲ真核反义表达的重组载体。结果 经酶切、测序鉴定 ,人拓扑异构酶Ⅲ真核反义表达载体构建成功。结论 为研究ATM基因与人拓扑异构酶Ⅲ的相互作用 ,阐明其生物学功能奠定了基础 ,同时为大片段的基因的表达载体构建探索了新的方法和途径
Objective To construct the eukaryotic antisense expression vector of human topoisomerase Ⅲalpha (hTOP 3alpha) by step-by-step cloning. To further clarify the interaction between hTOP 3alpha and its ATM related genes and to study the biological function of ATM . Methods Using step-by-step cloning method, a fragment of 2.6 kb was inserted into Hind Ⅲ and EcoRI double digestion to construct another eukaryotic antisense expression vector of human topoisomerase Ⅲ . Results After digestion and sequencing, the human topoisomerase Ⅲ eukaryotic antisense expression vector was successfully constructed. Conclusions In order to study the interaction between ATM gene and human topoisomerase Ⅲ and elucidate the biological functions of ATM gene, we explored new methods and approaches for the construction of expression vector of large fragments