ST8SiaV基因启动子的克隆及功能鉴定

Molecular cloning and functional identification of ST8SiaV promoter

目的:克隆ST8SiaV基因启动子及构建ST8SiaV基因启动子-pGL3-Basic-GFP表达载体,并对该载体进行功能鉴定。方法:①基于NCBI公布的大鼠基因组ST8SiaV基因序列,以SD大鼠肝基因组DNA为模板,采用PCR方法,扩增ATG起始密码子上游1.7kb片段。②分离、纯化PCR产物,与pMD18-T载体相连,构建克隆载体,转化大肠杆菌(DH5α),筛选阳性克隆并测序。③启动子片段进行SacⅠ和BglⅡ双酶切,克隆进pGL3-Basic质粒载体。为探索该基因在神经干细胞分化过程的活体表达,将pGL3-Basic的luciferase报告基因替换为GFP,构建成promoterpGL3-Basic-GFP表达载体。④将构建的表达载体电转染SD大鼠神经干细胞,对该表达载体进行生物学功能鉴定。结果与结论:成功地克隆到1.7kb的具有生物学活性的ST8SiaV基因启动子,并构建了适于活体研究的promoterpGL3-Basic-GFP表达载体,为进一步研究该基因的启动子区转录及调控作用奠定了基础。

Objective: To clone ST8SiaV promoter,construct ST8SiaV gene promoter-pGL3-Basic-GFP expressing vector and identify the constructed vector. Methods:① Based on the ST8SiaV rat genomic DNA sequence obtained from NCBI by BLAST software, a 1.7 kb targeted sequence from ATG original code was amplified by PCR method.② The product of PCR was inserted into pMD 18-T vector then transferred into E.coli DH5α. The positive clone was picked out and identified by DNA sequencing.③ The identified target promoter was digested with SacⅠ and Bgl Ⅱ, and then inserted into pGL3-Basic plasmid vector. In order to study this gene expressed in vivo during the differentiation of neural stem cells, the luciferase report gene in pGL3-Basic was replaced by GFP.④The recombinant plasmid was transfected into SD neural stem cell to identify the biologic activity.Results and Conclusion: The 1.7 kb 5′flanking region of ST8SiaV gene with activity was successfully cloned and the promoter-pGL3-Basic-GFP vector for studying in vivo was well constructed, laying a foundation for further research into the function of the ST8SiaV gene promoter.