通过设计一种新型抗凋亡杆状病毒载体来抑制 Sf-caspase-1 和 Tn-caspase-1 的表达来改善蛋白质生产,Biotechnology and Bioengineering

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通过设计一种新型抗凋亡杆状病毒载体来抑制 Sf-caspase-1 和 Tn-caspase-1 的表达来改善蛋白质生产
Biotechnology and Bioengineering ( IF 3.5 ) Pub Date : 2021-05-15 , DOI: 10.1002/bit.27807
Xiaoyue Zhang _{1,

2} , Kaixia Zhao ₁ , Lan Lan ₁ , Na Shi ₁ , Hao Nan ₁ , Yanan Shi ₁ , Xiaodong Xu ₁ , Hongying Chen ₁

Affiliation

杆状病毒表达载体系统 (BEVS) 是用于在昆虫细胞中生产重组蛋白的有吸引力的制造平台。然而，杆状病毒感染通常会在 3-4 天内诱导宿主细胞凋亡，这随后会终止蛋白质表达。先前的研究已经证明，BEVS 产生的蛋白质可以在凋亡抑制的昆虫细胞中升高。我们还在之前的报告中开发了一种杆状病毒载体，以抑制Sf 9 细胞的细胞凋亡并改善蛋白质的产生。在这项研究中，我们设计了五个短发夹 RNA (shRNA) 表达盒，针对草地贪夜蛾 caspase-1 ( Sf-caspase-1 ) 和Trichoplusia ni caspase-1 ( Tn-caspase-1)，并发现在表达盒的茎区内引入 C 到 T 突变有利于异源蛋白的表达。将改进的 shRNA 表达盒之一敲入杆粒中，删除了几个非必需基因。新型杆状病毒载体在Sf 9 和 High Five 细胞中均表现出抑制细胞凋亡的能力，并表现出细胞内表达的 GFP 和萤火虫荧光素酶以及分泌的糖蛋白 OD-Fc 的卓越重组蛋白生产力。本研究开发的抗凋亡杆状病毒载体可作为科学研究和制药行业蛋白质生产的有用工具。

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Improvement of protein production by engineering a novel antiapoptotic baculovirus vector to suppress the expression of Sf-caspase-1 and Tn-caspase-1

The baculovirus expression vector system (BEVS) is an attractive manufacturing platform for recombinant protein production in insect cells. However, baculovirus infection commonly induces host apoptosis in 3–4 days which would subsequently terminate the protein expression. Previous studies have proved that protein production by BEVS can be elevated in apoptosis-suppressed insect cells. We also developed a baculovirus vector in our previous report to inhibit the apoptosis and improve protein production in Sf9 cells. In this study, we designed five short hairpin RNA (shRNA) expression cassettes targeting a conserved region in Spodoptera frugiperda caspase-1 (Sf-caspase-1) and Trichoplusia ni caspase-1 (Tn-caspase-1), and found that introduction of C to T mutations within the stem region of the expression cassette was beneficial for the heterologous protein expression. One of the improved shRNA expression cassettes was knocked into a bacmid with the deletion of several nonessential genes. The novel baculovirus vector demonstrated the ability to suppress cell apoptosis in both Sf9 and High Five cells, and exhibited superior recombinant protein productivity of intracellularly expressed GFP and firefly luciferase and secreted glycoprotein OD-Fc. The antiapoptotic baculovirus vector developed in this study could serve as a useful tool for the protein production in scientific research and pharmaceutical industries.

更新日期：2021-07-16

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