Wu Chunxiao, Soh Ker Yin, Wang Shu
Institute of Bioengineering and Nanotechnology, Singapore.
Hum Gene Ther. 2007 Jul;18(7):665-72. doi: 10.1089/hum.2007.020.
Significant progress in the application of baculoviral vectors for gene delivery into mammalian cells calls for the development of powerful methods for virus purification and concentration. We report here a novel and efficient method based on membrane chromatography to prepare baculoviral stocks. On a strong cation-exchange membrane unit, baculovirus in insect cell culture supernatants was captured at a flow rate of 3 ml/min and efficiently eluted at the same flow rate with a physiological buffer containing 150 mM NaCl as a desorption reagent. The procedure allowed for a final recovery of 78% of infective viral particles from the original supernatant and 30-fold enrichment. The high purity of the viral preparation was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Baculovirus gp64 proteins could be purified by the same method, indicating the importance of the protein in mediating the binding of baculovirus to the cation-exchange membrane. The method developed should be suitable for preparing baculoviral stocks, and probably other gp64-pseudotyped viral vectors, for gene therapy applications.
杆状病毒载体在将基因导入哺乳动物细胞中的应用取得了重大进展，这就需要开发强大的病毒纯化和浓缩方法。我们在此报告一种基于膜色谱法制备杆状病毒储备液的新颖且高效的方法。在强阳离子交换膜装置上，昆虫细胞培养上清液中的杆状病毒以3毫升/分钟的流速被捕获，并以相同流速用含有150 mM氯化钠的生理缓冲液作为解吸剂进行高效洗脱。该方法最终能从原始上清液中回收78%的感染性病毒颗粒，并实现30倍的富集。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析证明了病毒制剂的高纯度。杆状病毒gp64蛋白也可通过相同方法纯化，这表明该蛋白在介导杆状病毒与阳离子交换膜结合中具有重要作用。所开发的方法应适用于制备用于基因治疗应用的杆状病毒储备液，可能也适用于其他gp64假型化病毒载体。
