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Genetic modification of a baculovirus vector for increased expression in insect cells 收藏

杆状病毒载体的昆虫细胞中表达增加的遗传修饰

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作者

作者单位

1. Oxford Expression Technologies Ltd, Oxford Brookes University, Oxford, OX3 0BP, UK 2. Centre for Ecology & Hydrology, Mansfield Road, Oxford, Oxfordshire, OX1 3SR, UK 3. Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK 6. School of Life Sciences, Oxford Brookes University, Oxford, OX3 0BP, UK 4. EMD Biosciences, Inc., 441 Charmany Dr., Madison, WI, 53719, USA 5. Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, Roosevelt Road, Oxford, OX3 7BN, UK

关键词

Baculovirus ;Gene deletion ;Insect cells ;Recombinant protein expression ;Chitinase ;Cathepsin

关键词译文

杆状病毒;基因缺失;昆虫细胞;重组蛋白表达;几丁质酶;组织蛋白酶

页码

57-68

DOI

10.1007/s10565-009-9133-y

来源信息

Cell Biology and Toxicology ISSN：0742-2091, 2010年, 26卷, 1期, 57-68页

摘要

Generating large amounts of recombinant protein in transgenic animals is often challenging and has a number of drawbacks compared to cell culture systems. The baculovirus expression vector system (BEVS) uses virus-infected insect cells to produce recombinant proteins to high levels, and these are usually processed in a similar way to the native protein. Interestingly, since the development of the BEVS, the virus most often used (Autographa californica multi-nucleopolyhedovirus; AcMNPV) has been little altered genetically from its wild-type parental virus. In this study, we modified the AcMNPV genome in an attempt to improve recombinant protein yield, by deleting genes that are non-essential in cell culture. We deleted the p26, p10 and p74 genes from the virus genome, replacing them with an antibiotic selection cassette, allowing us to isolate recombinants. We screened and identified recombinant viruses by restriction enzyme analysis, PCR and Western blot. Cell viability analysis showed that the deletions did not improve the viability of infected cells, compared to non-deletion viruses. However, expression studies showed that recombinant protein levels for the deletion viruses were significantly higher than the expression levels of non-deletion viruses. These results confirm that there is still great potential for improving the BEVS, further increasing recombinant protein expression yields and stability in insect cells.

摘要译文

产生大量的重组蛋白在转基因动物是往往具有挑战性，并且具有许多缺点相比细胞培养系统。杆状病毒表达载体系统（BEVS）使用受病毒感染的昆虫细胞中生产重组蛋白以高水平，而这些通常是处理以类似的方式与天然蛋白质。有趣的是，由于BEVS，最常用的（苜蓿银纹夜蛾多nucleopolyhedovirus病毒的发展;AcMNPV的）已经被小基因从它的野生型亲代病毒的改变。在这项研究中，我们修改了AcMNPV的基因组中，以试图改善重组蛋白的产量，删去基因是非必需的细胞培养物。我们从病毒基因组中被删除的P26，P10和P74的基因，与抗生素选择盒替换它们，让我们以分离重组体。我们筛选并确定了重组病毒的限制性内切酶分析，PCR和Western blot。细胞生存力分析表明，缺失没有改善感染的细胞的生存力，相对于非缺失病毒。然而，表达的研究表明，重组蛋白的水平对删除的病毒比非缺失病毒的表达水平显著高。这些结果证实，仍有改善的BEVS，进一步增加在昆虫细胞中重组蛋白的表达产率和稳定性的巨大潜力。

Richard B. Hitchman [1] Robert D. Possee [2] Andrew T. Crombie [1] [3] Adam Chambers [1] [6] Kim Ho [1] [6] Evangelia Siaterli [1] Olga Lissina [1] Heather Sternard [4] Robert Novy [4] Kathryn Loomis [4] Louise E. Bird [5] Raymond J. Owens [5] Linda A. King [6]. Genetic modification of a baculovirus vector for increased expression in insect cells[J]. Cell Biology and Toxicology, 2010,26(1): 57-68