基因治疗用pUC118-ski质粒DNA纯化工艺的建立及其质量控制  

Development of purification procedure and quality control of therapeutic pUC118-ski plasmid DNA

作　　者：彭艳 李平[1] 刘苹[1] 周元国[1] 

机构地区：[1]第三军医大学附属大坪医院野战外科研究所分子生物学中心创伤烧伤与复合伤国家重点实验室,重庆400042

出　　处：《中国生物制品学杂志》2013年第10期1483-1487,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30801195);重庆市自然科学基金面上项目(CSTC2011jjA10016)

摘　　要：目的建立基因治疗用pUC118-ski质粒DNA的纯化工艺,并进行质量鉴定。方法采用碱裂解法粗提质粒,PEG/MgCl2沉淀法初步纯化后,依次通过Sepharose 6 FF分子筛层析、PlasmidSelect Xtra亲和层析和SOURCE30Q离子交换层析纯化质粒DNA。纯化的质粒DNA经琼脂糖凝胶电泳分析超螺旋质粒的比例;紫外分光光度计测定质粒的A260和A280值,以A260/A280比值评价其纯度;CCK-8法检测质粒促原代培养的大鼠皮肤成纤维细胞的增殖活性;BCA法和鲎试剂凝胶法分别检测质粒DNA中残留的蛋白质和内毒素含量;PCR法检测质粒中细菌基因组DNA的含量。结果经Sepharose 6 FF分子筛层析可有效去除质粒DNA中的RNA,经PlasmidSelect Xtra亲和层析能将超螺旋质粒DNA(scDNA)与开环质粒DNA(ocDNA)有效分离,经SOURCE30Q离子交换层析获得的质粒DNA纯度很高,但出现了少量ocDNA。获得的质粒DNA纯品中超螺旋质粒所占比例大于90.5%;纯度(A260/A280)为1.83;促原代培养的大鼠成纤维细胞的增殖活性与空载体组相比提高了22.9%(P<0.05),与纯化前的质粒促细胞增殖活性一致;内毒素含量小于50 Eu/mg;几乎检测不到蛋白质残留;细菌基因组DNA残留量小于1.2μg/mg。结论建立了基因治疗用pUC118-ski质粒DNA的纯化工艺,纯化产品的质量指标均符合国家食品药品监督管理局(SFDA)的标准,为申请该基因药物的临床试验及大规模制备质粒DNA奠定了基础。Objective To develop a purification procedure and control the quality of therapeutic pUCllS-ski plasmid DNA. Methods Plasmid DNA was crudely extracted by alkaline lysis, precipitated with PEG/MgC12, and purified by Sepharose 6 FF size exclusion, PlasmidSelect Xtra affinity and SOURCE30Q ion exchange chromatography, then analyzed for the proportion of supercoil plasmid by agarose gel eleetrophoresis, for A260 and A280 values by UV spectrometry, and evaluated for purity by the ratio of AM to A280 （A260/A280）. The activity of the plasmid in promoting the proliferation of rat skin fibroblasts in primary culture was determined by CCK-8 method. The residual protein and endotoxin contents in plasmid DNA were determined by BCA and limulus test. The bacterial genomic DNA content in plasmid was determined by PCR. Results The RNA in plasmid DNA was effectively removed by Sepharose 6 FF size exclusion chromatography. The supercoil DNA （scDNA） was effectively isolated from open circle DNA（oeDNA） by PlasmidSelect Xtra affinity chro- matography. The plasmid DNA purified by SOURCE30Q ion exchange chromatography reached a high purity, while a small quantity of ocDNA appeared. The proportion of supercoil plasmid in purified plasmid DNA, with a purity （A260/A280） of 1. 83, was more than 90. 5%. The activity of purified plasmid pUCll8-ski in promoting the proliferation of rat skin fi- broblasts in primary culture increased by 22. 9% as compared with that of empty plasmid （P 〈 0. 05）, which was consis- tent with that before purification. The endotoxin content in purified plasmid DNA was less than 50 Eu/mg. Few residual proteins were detected, and the bacterial genomic DNA content was less than 1. 2 μg/mg. Conclusion A purificationprocedure for therapeutic pUC118-ski plasmid DNA was developed, and the quality indexes of purified product met the re- quirements issued by SFDA, which laid a foundation of application for clinical trial and large-scale production of the plas- mid DNA.

关 键 词：ski基因 重组质粒 基因治疗 层析 质量控制 

分 类 号：Q819[生物学—生物工程] R392-33[医药卫生—免疫学]