Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene
Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene
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DOI：
10.1099/0022-1317-78-12-3073
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发表时间：
1997-12-01
影响因子：
3.8
通讯作者：
Oda, K
中科院分区：
文献类型：
--
作者：
Suzuki, T;Kanaya, T;Oda, K
家蚕（家蚕）幼虫感染杆状病毒（家蚕核多角体病毒，BmNPV）作为一种生产有用蛋白质的高效表达系统，然而，在感染后期，感兴趣的蛋白质的表达量趋于减少，部分原因可能是幼虫血淋巴中产生的蛋白酶。从BmNPV感染的幼虫血淋巴中纯化的蛋白酶的n端氨基酸序列与病毒半胱氨酸蛋白酶基因的内部氨基酸序列相同，表明血淋巴中的半胱氨酸蛋白酶起源于BmNPV基因，构建了缺失半胱氨酸蛋白酶基因的突变病毒（CPd）。在多面蛋白启动子的控制下，分别将萤火虫荧光素酶和人类生长激素基因引入CPd，这些构建体可以非常有效地产生这些蛋白，因为这些蛋白的降解程度大大降低，利用CPd构建的BmNPV载体系统提高了外源表达蛋白的稳定性。尤其是那些对半胱氨酸蛋白酶敏感的人。
Infection by a baculovirus (Bombyx mori nuclear polyhedrosis virus, BmNPV) in silkworm (Bombyx mori) larvae is highly efficient as an expression system for the production of useful proteins, However, the amount of the protein of interest expressed tends to decrease in the later stages of infection presumably due, in part, to a proteinase produced in the larval haemolymph. The N-terminal amino acid sequence of a proteinase purified from the haemolymph of BmNPV-infected larvae was identical to the internal amino acid sequence of the viral cysteine proteinase gene of BmNPV, suggesting that the cysteine proteinase in the haemolymph originated from the BmNPV gene, We constructed a mutant virus (CPd) which had a deletion in the cysteine proteinase gene, No proteinase activity corresponding to this proteinase was detected in the haemolymph of silkworm larvae infected with CPd, The firefly luciferase and the human growth hormone genes were separately introduced into CPd under control of the polyhedrin promoter, These constructs produced these proteins very efficiently, because of a greatly reduced degree of degradation of these proteins, A BmNPV vector system using CPd enhances the stability of foreign expressed proteins, especially for those that are cysteine proteinase-sensitive.