Rapid, high level protein production using DNA-based semliki forest virus vectors
Rapid, high level protein production using DNA-based semliki forest virus vectors
复制标题
DOI：
10.1074/jbc.273.29.18060
复制
发表时间：
1998-07-17
影响因子：
4.8
通讯作者：
Bremner, R
中科院分区：
文献类型：
--
作者：
DiCiommo, DP;Bremner, R
与杆状病毒载体相比，Semliki森林病毒(SFV)载体的生产速度更快，宿主范围更广，但原始SFV系统需要在体外操作RNA。Me已经建立了一个完全基于DNA的系统，编码SFV聚合酶和目的蛋白的复制子载体和编码病毒结构蛋白的辅助载体都被修改，使其表达依赖于RNA聚合酶II。单独转染修饰的复制子载体产生的蛋白质是简单表达载体获得的蛋白质的20-30倍，表达需要SFV复制酶，它扩增复制子RNA。基于SFV的载体产生的蛋白质是基于辛德比斯病毒的质粒的10-20倍。SFV复制子和辅助载体的共转染产生了约10(6)个感染性颗粒/毫升的病毒滴度。一个IO-cm平板上的单次电穿孔产生的病毒(10(7)个颗粒)足以产生500毫克蛋白质、轻型性、复制熟练的病毒，在使用近10(8)个病毒颗粒的三次测试中未检测到病毒，这与基于DNA Sindbis的系统相比是一个明显的优势，在该系统中产生的病毒颗粒的一半以上是完全具有传染性的。新的SFV载体显著增强了该表达系统的实用性。
Semliki Forest virus (SFV) vectors can be produced faster, and have a wider host range, than baculovirus vectors, However, the original SFV system requires in vitro manipulation of RNA. me have generated a system that is wholly DNA-based, Both the replicon vector, encoding SFV polymerase and the protein of interest, and the helper vector, encoding viral structural proteins, were modified so that expression was RNA polymerase II-dependent. Transfection of the modified replicon plasmid alone generated 20-30-fold more protein than obtained from a simple expression vector, Expression required the SFV replicase, which amplifies replicon RNA. The SFV-based vector generated 10-20-fold more protein than a plasmid based on Sindbis virus. Cotransfection of SFV replicon and helper vectors generated viral titers of around 10(6) infectious particles/ml. A single electroporation, plated on one IO-cm plate, generated enough virus (10(7) particles) to produce >500 mu g of protein, mild type, replication proficient virus was not detected in three tests utilizing almost 10(8) viral particles, a distinct advantage over a DNA Sindbis-based system in which over half the virus particles generated are fully infectious. The new SFV vectors significantly enhance the utility of this expression system.