| 受磷蛋白重组腺病毒的制备与鉴定 |
| 投稿时间：2016-03-14 修订日期：2016-03-21 点此下载全文 |
| 引用本文：刘梦娜,汪凯,任安经,章卫平,吕建新.受磷蛋白重组腺病毒的制备与鉴定[J].医学研究杂志,2016,45(10):48-52 |
| DOI：
10.11969/j.issn.1673-548X.2016.10.012 |
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| 基金项目:国家自然科学基金资助项目（面上项目）（31271221） |
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| 中文摘要:目的 构建含受磷蛋白PLB基因的重组腺病毒载体，为研究受磷蛋白PLB的生物学功能提供工具。方法 将RT-PCR获得的受磷蛋白PLB cDNA克隆至pShuttle-CMV载体，Pme Ⅰ酶切线性化后，电转化至含有腺病毒骨架质粒的大肠杆菌BJ5183中进行同源重组，从而获得重组腺病毒载体，Pac Ⅰ酶线性化该重组载体，转染293细胞进行病毒的包装和扩增，经超离心对重组病毒Ad-PLB进行纯化；测定病毒效价后，体外感染大鼠心肌细胞H9C2，Western blot法验证腺病毒病毒介导的重组PLB蛋白表达。结果 成功构建了重组腺病毒载体，体外包装制备了PLB的重组腺病毒，扩增纯化后的病毒效价达6.25×1010/ml，能有效介导PLB在心肌细胞中的重组表达。结论 过表达受磷蛋白PLB的重组腺病毒载体构建成功，为之后受磷蛋白PLB的相关生物学功能研究奠定了基础。 |
| 中文关键词:受磷蛋白 腺病毒 心肌 肌质网 |
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| Generation of Recombinant Adenovirus Expressing Phospholamban |
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| Abstract:Objective To construct the recombinant adenovirus overexpressing phospholamban gene for studying the biological functions of PLB. Methods Mouse PLB gene was amplified by RT-PCR, and cloned into pShuttle-CMV vector. The resultant plasmid was linearized by restriction endonuclease PmeⅠ and subsequently electrotransfected into E.coli. BJ5183 with adenovirus framework plasmid to obtain a recombinant adenovirus expression vector. Recombinants were selected by kanamycin resistance and confirmed by restriction endonuclease analysis. Then, the PacⅠ-linearized the recombinant adenovirus vector was transfected into human embryonic kidney cells 293T for packaging adenoviruses. The resultant positive adenoviruses were amplified, purified by ultracentrifugation, and tittered. Western blot was performed further to verify the protein expression of recombinant PLB. Results We have successfully generated recombinant adenoviruses expressing mouse PLB with the titers of 6.25×1010/ml.The adenoviruses can effectively mediate PLB expression in rat myocardial cells. Conclusion The obtained recombinant adenoviruses overexpressing PLB gene can be used as a useful tool for investigating the biological function of PLB. |
| keywords:Phospholamban Adenovirus Myocardium Sarcoplasmic recticulum |
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