ADAM9基因真核表达质粒的构建及表达

Construction and eukaryotic expression of ADAM9 gene

目的:构建人源性去整合素金属蛋白酶9(ADAM9)基因真核表达载体并表达其编码蛋白(转染293T细胞)。方法:利用包含ADAM9全长编码核酸序列的质粒PCR4-TOPO作为模板,用PCR扩增其核心编码区,将其克隆至PMD18-TVector中构建PMD18-T-ADAM9质粒,同时双酶切PMD18-T-ADAM9及PXJ40-HA后构建PXJ40-HA-ADAM9真核表达载体,经酶切鉴定及测序后将载体转染至293T细胞中,通过免疫印记法检测ADAM9蛋白的表达。结果:经过双酶切和测序结果鉴定PXJ40-HA-ADAM9载体构建成功,经过West-ern blot法证实ADAM9基因表达。结论:成功克隆ADAM9基因并构建其真核表达载体,该载体的构建为后期建立稳定表达ADAM9细胞模型,并稳定表达ADAM9蛋白,为进行ADAM9功能研究提供可能。

Objective： To construct human a disintegrin and metalloprotease 9（ADAM9） gene into a eukaryotic expression vector PXJ40-HA and study the expression of ADAM9 gene in transfected 293T cells.Methods： ADAM9 gene was amplified from a vector PCR4-TOPO ordered from Open Biosystems and cloned into PMD18-T vector to construct a vector named PMD18-T-ADAM9.The PMD18-T-ADAM9 and the PXJ40-HA double digested by the same restrict enzymes,the recombinant eukaryotic expression vector PXJ40-HA-ADAM9 subsequently yielded.The expression of ADAM9 gene in tansfected 293T cells were identified by Western blot.Results： The vector PXJ40-HA-ADAM9 was constructed successfully and the expression of ADAM9 gene was detected by Western blot finally.Conclusion： ADAM9 gene is obtained as well as its recombinant eukaryotic expression plasmid is successfully constructed and expressed in 293T cells.The expression vector would be a useful material for the ADAM9 stable expression cell model and further funciton investigation.