| 薛玲1 李航2 张静1 王净2 吴雅岚3 姬秋和1 叶菁2.脂肪储存小滴蛋白5 重组腺病毒的制备[J].,2011,11(6):1083-1086 |
| 脂肪储存小滴蛋白5 重组腺病毒的制备 |
| Construction and Identification of Recombinant Adenovirus ContainingLipid Storage Droplet Protein 5 |
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| DOI： |
| 中文关键词: AdEasy 腺病毒 LSDP5 小鼠 |
| 英文关键词: Ad Easy adenovirus LSDP Mouse |
| 基金项目:国家自然科学基金（30700268,81070249） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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 vector
and sequenced after enzyme digestion. Then LSDP5 gene was cloned into adenovirus shuttle plasmid pShuttle-CMV to construct a recombinant
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 fragment,
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. |
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