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首页> 外文期刊>BMC Biotechnology >Highly efficient concentration of lenti- and retroviral vector preparations by membrane adsorbers and ultrafiltration
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Highly efficient concentration of lenti- and retroviral vector preparations by membrane adsorbers and ultrafiltration

机译：通过膜吸附器和超滤高效浓缩慢病毒和逆转录病毒载体制剂

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Background Lentiviral vectors (LVs) can efficiently transduce a broad spectrum of cells and tissues, including dividing and non-dividing cells. So far the most widely used method for concentration of lentiviral particles is ultracentrifugation (UC). An important feature of vectors derived from lentiviruses and prototypic gamma-retroviruses is that the host range can be altered by pseudotypisation. The most commonly used envelope protein for pseudotyping is the glycoprotein of the Vesicular Stomatitis Virus (VSV.G), which is also essential for successful concentration using UC. Results Here, we describe a purification method that is based on membrane adsorbers (MAs). Viral particles are efficiently retained by the anionic exchange MAs and can be eluted with a high-salt buffer. Buffer exchange and concentration is then performed by utilizing ultrafiltration (UF) units of distinct molecular weight cut off (MWCO). With this combined approach similar biological titers as UC can be achieved (2 to 5 × 109 infectious particles (IP)/ml). Lentiviral particles from small starting volumes (e.g. 40 ml) as well as large volumes (up to 1,000 ml) cell culture supernatant (SN) can be purified. Apart from LVs, vectors derived from oncoretroviruses can be efficiently concentrated as well. Importantly, the use of the system is not confined to VSV.G pseudotyped lenti- and retroviral particles and other pseudotypes can also be purified. Conclusions Taken together the method presented here offers an efficient alternative for the concentration of lenti- as well as retroviral vectors with different pseudotypes that needs no expensive equipment, is easy to handle and can be used to purify large quantities of viral vectors within a short time.
机译：背景慢病毒载体（LV）可以有效地转导广泛的细胞和组织，包括分裂和非分裂细胞。迄今为止，最广泛使用的浓缩慢病毒颗粒的方法是超速离心（UC）。源自慢病毒和原型伽玛逆转录病毒的载体的重要特征是宿主范围可通过假型化改变。最常用于假型化的包膜蛋白是水泡性口腔炎病毒（VSV.G）的糖蛋白，这对于使用UC成功浓缩也是必不可少的。结果在这里，我们描述了一种基于膜吸附器（MAs）的纯化方法。病毒颗粒被阴离子交换MA有效地保留，并可以用高盐缓冲液洗脱。然后通过利用截留分子量不同的超滤（UF）单元（MWCO）进行缓冲液交换和浓缩。通过这种组合方法，可以实现与UC类似的生物滴度（2至5×10 9 感染性颗粒（IP）/ ml）。可以纯化小起始体积（例如40 ml）以及大体积（最大1,000 ml）细胞培养上清液（SN）的慢病毒颗粒。除左心室外，衍生自癌病毒的载体也可被有效地浓缩。重要的是，该系统的使用不限于VSV.G假型慢病毒和逆转录病毒颗粒以及其他假型也可以纯化。结论综上所述，该方法为浓缩具有不同假型的慢病毒和逆转录病毒载体提供了一种有效的替代方法，不需要昂贵的设备，易于操作，可用于在短时间内纯化大量病毒载体。

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