Lu M, Farrell P J, Johnson R, Iatrou K
Department of Medical Biochemistry, The University of Calgary, Calgary, Alberta T2N 4N1, Canada.
J Biol Chem. 1997 Dec 5;272(49):30724-8. doi: 10.1074/jbc.272.49.30724.
It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based expression cassette results in an augmentation of transgene expression in transfected cells by two orders of magnitude relative to the control recombinant expression cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the expression cassette and occurs only when the HR3 element is linked to the expression cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell expression system for continuous high-level expression of recombinant proteins. Such a system should provide levels of expression of recombinant proteins comparable to those obtained from baculovirus expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant proteins and the capability of expression of proteins from genomic as well as cDNA sequences.
先前已有报道称，杆状病毒同源区域（即杆状病毒基因组中包含DNA复制起点的区域）可在体外增强少数杆状病毒基因的表达。我们现在报道，家蚕核型多角体病毒（BmNPV）基因组中包含同源区域3（HR3）的一个区域可作为非病毒基因（家蚕细胞质肌动蛋白基因）启动子的增强子。将BmNPV的HR3序列整合到基于肌动蛋白启动子的表达盒中，相对于对照重组表达盒，可使转染细胞中的转基因表达增强两个数量级。这种增加是由于肌动蛋白启动子转录速率相应增加，而非表达盒的复制，并且仅当HR3元件与表达盒顺式连接时才会发生。在家蚕肌动蛋白启动子活性方面，类似程度的增强也发生在异源鳞翅目细胞中。同时用BmIE1反式激活因子补充转染细胞，先前已证明该因子在体外可作为细胞质肌动蛋白基因启动子的转录共激活因子，这会使置于肌动蛋白基因启动子控制下的重组蛋白表达水平增加1000倍以上。这些发现为开发用于重组蛋白连续高水平表达的非裂解昆虫细胞表达系统奠定了基础。这样的系统应能提供与杆状病毒表达系统相当的重组蛋白表达水平，并且还应具有在细胞环境中连续生产的额外优势，与杆状病毒感染产生的环境不同，该细胞环境支持重组蛋白持续进行正确的翻译后修饰，以及从基因组序列和cDNA序列表达蛋白质的能力。
