人Smad4克隆及真核表达载体的构建与鉴定

目的：克隆人抑癌基因Homo sapiens SMAD family member 4（NM_005359 1659 bp）mRNA构建人Smad4的真核表达载体。方法：从膀胱癌患者膀胱组织中提取总RNA，经RT-PCR得到Smad4基因开放阅读框架eDNA序列，并将其定向克隆到真核表达载体pcDNA3．0，构建含目的基因的pCDNA3．0-Smad4重组质粒。结果：经基因序列测定、PCR和酶切分析，证实插入载体pCDNA3．0的片段为目的基因开放阅读框的核苷酸序列。结论：重组质粒pCDNA3．0 Smad4构建成功，为该基因的蛋白表达及其相关功能研究奠定了基础。

Clone of Human Smad4 Gene and Construction of its Eukaryotic Expression Vector

Objective：To clone the suppressor gene of Smad4（NM_005359 1659 bp） and to construct its eukaryotie expression vector. Methods：Total RNA was extracted from human bladder cancer tissue. After RT-PCR, the opening reading frame of Smad4 cDNA was isolated, and then it was cloned into the eukaryotic expression vector pcDNA3.0（＋）. The expression plasmid pcDNA3.0 （＋） Smad4 was constructed. Results： The recombinant plasmid was identified by PCR, restriction enzyme and sequencing analysis, insertion vector pcDNA3.0 （＋）- Smad4 was consistant to the nucleotide sequence of target gene open reading frames. Conclusions： The successful construction of the recombinant pasmid peDNA3.0（ ＋）-Smad4 will be benefit to further study of protein expression and correlated function investigation.