| 邹海1 韩骅1△ 高方2 曹秀丽2 徐骁2.小鼠EVL 基因的克隆和真核表达载体的构建[J].,2012,12(24):4606-4610 |
| 小鼠EVL 基因的克隆和真核表达载体的构建 |
| Clone of EVL and Construction of EVL Eukaryotic Expression Plasmid |
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| DOI： |
| 中文关键词: EVL 蛋白 基因克隆 真核表达 |
| 英文关键词: EVL protein Gene cloning Eukaryotic expression |
| 基金项目:国家重点基础研究发展规划（973 项目）资助（001CB509906）；国家自然科学基金资助项目（30200148） |
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| 中文摘要: |
| 目的：构建小鼠EVL(Ena/VASP like)基因的真核表达载体，为深入研究EVL 的功能奠定基础。方法：采用PCR 方法，从小鼠
cDNA 文库中，扩增出1245bp 的EVL 编码区片段，经电泳、胶回收后连接入pMD-18T 载体中，测序鉴定正确。用BamH I 和Hinc
II 双酶切，定向克隆EVL 编码区片段到真核表达载体pcDNA3.1 中，用限制性内切酶酶切鉴定重组质粒正确后。将重组质粒转染
入HELA 细胞中，以RT-PCR 检测EVL 的mRNA 的表达，以Western Blot 检测EVL 蛋白的表达。结果：酶切鉴定结果显示小鼠
EVL 编码区基因被成功克隆入真核表达载体pcDNA3.1 中；RT-PCR 和Western Blot 结果以及免疫荧光染色显示Hela 细胞中有
EVL 的mRNA 和蛋白的表达。结论：成功获得pcDNA3.1-EVL 的真核表达载体，为进一步深入研究EVL 蛋白的功能奠定了基
础。 |
| 英文摘要: |
| Objective: To generate eukaryotic expression plasmid of EVL (Ena/VASP like) gene, to lay foundation for further investigation
of the function of EVL gene. Methods: EVL coding region of 1272/1245bp was amplified from mouse cDNA library by PCR,
and was then cloned into pMD-18T-vector after being electrophoresed and gel-extracted. After identifying the T-EVL plasmid by sequencing,
EVL CDS fragment was cut down by double digestion with BamH I and Hinc II, and then was subcloned into eukaryotic expression
plamid pcDNA3.1. The recombinant plasmid was identified with restriction enzymes and was transfected into HELA cells via liposome
mediation. The expression of EVL was detected by RT-PCR for the mRNA transcription and byWestern Blot for the protein expression.
Results: Identification by restriction enzymes showed that EVL coding region fragment had been successfully cloned into eukaryotic
expression plasmid pcDNA3.1. After transfected into HELA cells, the recombinant plasmid could produce EVL mRNA and protein indicated
by RT-PCR results and Western Blot results. Conclusion: Eukaryotic expression plasmid pcDNA3.1-EVL was successfully cloned,
which is helpful for a further investigation of the function of EVL protein. |
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