脂肪储存小滴蛋白5重组腺病毒的制备

目的：利用AdEasy腺病毒表达系统构建含有小鼠脂肪储存小滴蛋白5（LSDP5）基因的重组腺病毒。方法：从小鼠肝脏cDNA克隆出LSDP5基因全长,克隆至pMD18-T载体中,酶切测序。回收酶切产物,连接到腺病毒穿梭载体pShuttle-CMV,构建pShuttle-CMV-LSDP5重组质粒,经PmeI酶切线性化后转化至含有腺病毒骨架质粒pAdEasy-1的BJ5183中。筛选阳性克隆,提取重组质粒,PacI酶切线性化并转染AD293细胞进行包装,提取病毒DNA,鉴定重组病毒并检测病毒滴度。结果：LSDP5基因克隆经测序证实与Genebank公布一致,双酶切重组pMD18-T载体得到1400 bp左右的片段。重组穿梭载体经Kpn I和Sal I双酶切后得到预期片段。PacI酶切得到30 Kb大片段和4.5 Kb小片段。转染AD293细胞后收集病毒,经PCR鉴定,获得理想的目的片段。取病毒上清反复感染AD293细胞以扩增病毒,最后所得病毒滴度为2.5×109pfu/ml。结论：成功构建了携带脂肪储存小滴蛋白5基因的重组腺病毒载体,为进一步研究LSDP5基因功能奠定基础。

Construction and Identification of Recombinant Adenovirus Containing Lipid Storage Droplet Protein 5

Objective：To construct mouse lipid storage droplet protein 5（LSDP5） gene recombinant adenovirus using AdEasy Adenoviral Vector System.Methods：The full-length LSDP5 gene cloned from mouse liver cDNA library was inserted to pMD18-T vec-tor and sequenced after enzyme digestion.Then LSDP5 gene was cloned into adenovirus shuttle plasmid pShuttle-CMV to construct a re-combinant plasmid pShuttle-CMV-LSDP5.The linearized plasmid by PmeI was transformed into E.coli strain BJ5183 with adenovirus backbone plasmid AdEasy-1.The recombinant plasmid extracting from the positive clones was linearized by PacI and transfected into adenovirus package cells AD293.The recombinant adenovirus was harvested by several freeze-thaw cycles.The recombinant adenovirus DNA was identified by PCR,and the titer of adenovirus was detected.Results：LSDP5 gene was cloned successfully.The specific frag-ment,about 1400bp,was obtained from recombinant pMD18-T vector cleavage.The recombinant plasmid pShuttle-CMV-LSDP5 was digested by KpnI and SalI to produce anticipated fragments.A bigger fragment of 30Kb and a smaller fragment of 4.5Kb were generated when the recombinant adenovirus vector was digested by PacI.The reombinat adenovirus was constructed after packaged in AD293 cells.The target DNA fragment was gained by PCR.The extracted virus was used to infect AD293 cells repeatedly for amplification.At last,the titer was about 2.5×109pfu /ml.Conclusion：The recombinant adnenovirus containing LSDP5 was successfully eastablished,and it may lay a foundation for the further functional study of LSDP5.