重组腺病毒Ad-GFP的纯化

目的：用色谱法纯化制备携带绿色荧光蛋白基因的重组腺病毒。方法：采用5L生物反应器悬浮培养HEK-293N3S细胞进行病毒的扩增,冻融法裂解细胞,通过超滤、Source15Q离子交换层析和Sepharose4Fast Flow凝胶过滤层析进行分离纯化,采用分光光度法和高效液相色谱法分析重组腺病毒产品的纯度,采用组织培养半数感染剂量方法测定病毒的感染滴度。结果：通过两步色谱层析得到产品,经分光光度法分析,其D260nm/D280nm比值为1.21,高效液相色谱法分析其纯度达96.5%,产品滴度为1.0×1011U/mL,病毒颗粒数为1.76×1012/mL,比活性为5.68%。结论：确定了稳定可行的重组腺病毒色谱分离工艺,得到的产品在纯度、滴度和比活性等方面均符合国家食品药品监督管理局的标准。

Purification of the Recombinant Adenovirus Ad-GFP

Objective： To purify the recombinant adenovirus by using chromatography. Methods： Source 15Q ion ex- change chromatography and Sepharose 4 Fast Flow size exclusion chromatography were used to purify Ad-GFP from the lysate of engineering cells HEK-293N3S infected by recombinant adenovirus and cultured in suspension. The purity of the product was determined by D~,aJD~or= and high performance liquid chromatography（HPLC）. The infective titer was deter- mined by TCIDs0 assay. Reults： The D260nm/D280nm of the product was 1.21, and the purity determined by HPLC was 96.5%. The infective titer of the product was 1.0×10^11 U/mL, the viral particle concentration was 1.76×10^112/mL. The specific in- fectivity was 5.68%, which was higher than that required（3.3% ） by the FDA, USA. Conclusion： We established the chromatography methods to purify recombinant adenovirus, and the purity and specific infectivity of the product meeting the standards established by State Food and Drug Administration（SFDA）.