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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: Ad-GFP-SHP-2 and Ad-GFP-SHP-2-E76A were packaged in HEK293 cells and the Ad-GFP recombinant adenovirus was amplified and infected into neonatal rat cardiomyocytes. The recombinant plasmids were purified by restriction endonuclease digestion and linearized with PacⅠ. The recombinant plasmids were transfected into HEK293 cells for packaging. The recombinant adenovirus was isolated from diseased HEK293 cells. The neonatal rat cardiomyocytes were infected with the recombinant adenovirus and the expression of green fluorescent protein (GFP) was observed under a fluorescence microscope. Results After digestion with Pac Ⅰ, a 4.5 kb band was displayed on the agarose gel. The recombinant adenovirus vector was transfected into HEK293 cells. HEK293 cells were repeatedly frozen and thawed. The supernatant contained the recombinant adenovirus. When the number of murine cardiomyocytes is 100, the infection efficiency is more than 90%. Conclusion The recombinant adenovirus was successfully packaged and the packaged adenovirus could efficiently infect neonatal rat cardiomyocytes.