DiCiommo D P, Bremner R
Eye Research Institute of Canada, Toronto, Ontario M5T 2S8.
J Biol Chem. 1998 Jul 17;273(29):18060-6. doi: 10.1074/jbc.273.29.18060.
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. We 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 10-cm plate, generated enough virus (10(7) particles) to produce >500 microg of protein. Wild 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.
辛德毕斯病毒（SFV）载体的制备速度比杆状病毒载体更快，宿主范围也更广。然而，最初的SFV系统需要对RNA进行体外操作。我们构建了一个完全基于DNA的系统。编码SFV聚合酶和目的蛋白的复制子载体以及编码病毒结构蛋白的辅助载体都经过了修饰，使其表达依赖于RNA聚合酶II。单独转染修饰后的复制子质粒所产生的蛋白比简单表达载体多20至30倍。表达需要SFV复制酶，它能扩增复制子RNA。基于SFV的载体产生的蛋白比基于辛德毕斯病毒的质粒多10至20倍。共转染SFV复制子和辅助载体可产生约10⁶个感染性颗粒/毫升的病毒滴度。单次电穿孔，接种在一个10厘米的培养皿上，产生的病毒量（10⁷个颗粒）足以产生超过500微克的蛋白。在使用近10⁸个病毒颗粒的三次测试中均未检测到野生型、具有复制能力的病毒，这相对于基于DNA的辛德毕斯病毒系统具有明显优势，在该系统中，超过一半产生的病毒颗粒具有完全感染性。新的SFV载体显著提高了该表达系统的实用性。
