Genetic modification of a baculovirus vector for increased expression in insect cells
Genetic modification of a baculovirus vector for increased expression in insect cells
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DOI：
10.1007/s10565-009-9133-y
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发表时间：
2010-02-01
影响因子：
6.1
通讯作者：
King, Linda A.
中科院分区：
文献类型：
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作者：
Hitchman, Richard B.;Possee, Robert D.;King, Linda A.
在转基因动物中产生大量重组蛋白通常具有挑战性，并且与细胞培养系统相比具有许多缺点。杆状病毒表达载体系统（BEVS）使用病毒感染的昆虫细胞来产生高水平的重组蛋白，并且这些重组蛋白通常以与天然蛋白类似的方式加工。有趣的是，自从BEVS的发展以来，最常用的病毒（苜蓿银纹夜蛾多核多角体病毒; AcMNPV）与其野生型亲本病毒相比几乎没有遗传改变。在这项研究中，我们修改了AcMNPV基因组，试图提高重组蛋白的产量，删除基因，是非必需的细胞培养。我们从病毒基因组中删除了p26，p10和p74基因，用抗生素选择盒取代它们，使我们能够分离重组体。通过限制性内切酶分析、PCR和Western blot等方法对重组病毒进行了筛选和鉴定。细胞活力分析显示，与非缺失病毒相比，缺失病毒没有改善感染细胞的活力。然而，表达研究表明，缺失病毒的重组蛋白水平显著高于非缺失病毒的表达水平。这些结果证实，仍然存在改善BEVS的巨大潜力，进一步提高重组蛋白在昆虫细胞中的表达产量和稳定性。
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.