受磷蛋白重组腺病毒的制备与鉴定

Generation of Recombinant Adenovirus Expressing Phospholamban

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,PmeⅠ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,PacⅠ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6.25×10^(10)/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。

Objective To construct the recombinant adenovirus overexpressing phospbolamban 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 Pme I and subsequently electrotransfected into E. coll. 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 Pac I - 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 × 10^10/ml. The adenoviruses can effectively mediate PLB expression in rat myocardial ceils. Conclusion The obtained recombinant adenoviruses overexpressing PLB gene can be used as a useful tool for investigating the biological function of PLB.