SHP-2重组腺病毒的包装及对心肌细胞的感染

Package of recombinant adenovirus of SHP-2 and transfection to the cardiomyocytes

目的采用HEK293细胞包装Ad-GFP-SHP-2和Ad-GFP-SHP-2-E76A并扩增Ad-GFP重组腺病毒并感染乳鼠心肌细胞。方法纯化两种载体;PacⅠ酶切线性化;线性化的载体转染入HEK293细胞进行包装;从病变的HEK293细胞分离重组腺病毒。利用重组的腺病毒感染乳鼠心肌细胞,并在荧光显微镜下观察绿色荧光蛋白的表达。结果 PacⅠ酶切后,琼脂糖凝胶上显示有4.5 kb的条带;转染线性化的重组腺病毒载体入HEK293细胞中,反复冻融HEK293,其上清中含有重组腺病毒,当感染乳鼠心肌细胞感染复数为100时,感染效率大于90%。结论成功包装重组腺病毒,包装后的腺病毒可高效感染乳鼠心肌细胞。

Objective To package Ad-GFP-SHP-2 and Ad-GFP-SHP-2-E76A and amplify Ad-GFP and transfect viruses to neonatal rat cardiomyocytes.Methods Two vectors were purified and digested by Pac Ⅰenzyme.The linearized vector was transfected into HEK293 cells.Adenovirus particles were isolated from HEK293 cells and transfected into neonatal rat myocytes.Results A 4.5 kb band was shown on agarose gel after PacⅠdigestion.The supernatant of HEK293 cells contained adenovirus particles.When MOI was 100,transfection efficiency was more than 90%.Conclusion Recombinant SHP-2 adenoviruses were packaged and neonatal rat cardiomyocytes can be efficiently tranfected by the viruses.