神经营养素4前导序列介导Exendin-4表达质粒的构建及鉴定

Identification and Construction of Plasmid Expressing Exendin-4 Mediated NT4 Leading Sequence

目的:构建神经营养素4前导序列介导的可分泌表达Exendin-4的重组腺伴病毒载体,为探究Exendin-4用于2型糖尿病临床治疗提供实验基础。方法:使用互补引物/模板法及T载体克隆法扩增Exendin-4的cDNA克隆,连接于NT4前导肽序列的3′端,融合基因NT4-Exendin-4亚克隆于穿梭质粒pSSHG,转染HEK-293细胞,包装病毒,斑点杂交方法测定重组病毒滴度。结果:经酶切、测序证实克隆出Exendin-4 cDNA,成功构建同一开放读码框ORF的NT4前导肽Exendin-4 cDNA克隆,得到高滴度(2.5×109pfu·L-1)的重组腺相关病毒。结论:通过分子克隆技术成功制备了Exendin-4的重组腺伴病毒载体pSSHG/NT4-Exendin-4并成功包装了较高浓度的重组病毒,为进一步研究Exendin-4功能及应用于临床打下基础。

Objective:To identify and construct a recombinant adeno-associated virus(AAV) vector that can secrete Exendin-4 mediated by NT4 leading sequence. Methods:Exendin-4 cDNA was cloned by complementary primer/template PCR and T carrier cloning method. The Exendin-4 gene was linked to terminal of the signal peptide of NT4 leading sequence. The fusion gene of NT4- Exendin-4 was subcloned into the shuttle plasmid of pSSHG. The recombinant virus was transfected and packaged by human embryo kidney 293 cells and the virus titer was measured by quantitative dot-blot hybridization. Results:Exendin-4 cDNA cloning was confirmed by restriction enzyme digestion and DNA sequencing. The NT4 leading sequence and Exendin-4 cDNA clone were successful constructed in the same ORF and high titer of recombinant AAV was obtained(2.5×109pfu·L-1). Conclusion:The recombinant virus pSSHG/NT4-Exendin-4 was successfully constructed by molecular cloning. The relatively high levels of recombinant virus were obtained. These results provide the basis for further studies that examine the function of Exendin-4.