高滴度腺病毒介导的绿色荧光蛋白和反义血管内皮生长因子165cDNA的制备

Preparation of high-titer Ad-GFP and Ad-aVEGF_(165)cDNA

目的:制备高滴度腺病毒介导的绿色荧光蛋白(Ad-GFP)和腺病毒介导的反义血管内皮生长因子165(Ad-aVEGF_(165))cDNA。方法:人胚肾293腺病毒包装细胞转染第3代Ad-GFP和Ad-aVEGF_(165)cDNA后,反复冻融293细胞,提取腺病毒悬液,用氯化铯梯度离心法纯化腺病毒,在荧光显微镜下测定Ad-GFP的滴度,用空斑法测定Ad-aVEGF_(165)cDNA的滴度。结果:获取了实验所需的第4代腺病毒液,Ad-GFP的滴度为9.5×109efu/ml,Ad-aVEGF_(165)cDNA的滴度为1.2×1010pfu/ml。结论:成功制备了高滴度的Ad-GFP和Ad-aVEGF_(165)cDNA。

Aim： To obtain high titer of adenovirus mediated GFP（Ad-GFP） and antisense-VEGF165 （Ad-aVEGF165 ）cDNA. Methods：Ad-GFP and Ad-aVEGF165 cDNA were transfected into 293 cells, then adenovirus suspension was extracted by freezing and thawing 293 cells repeatedly and purified by CsCl2 gradient centrifugation and titrated with fluorescence microscopy or cytopathetic effect （CPE）. Results- Enough fourth adenovirus suspension was harvested. The titers of Ad- GFP and Ad-aVEGF cDNA were 9.5 × 10^9 efu/ml and 1.2 × 10^10 pfu/ml, respectively. Conclusion： Ad-GFP and Ad- aVEGF165 cDNA of high titers were obtained successfully.