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利用苜蓿银纹夜蛾核型多角体病毒表达载体实现非融合外源基因的高水平表达。

High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors.

作者信息

Luckow V A, Summers M D

机构信息

Department of Entomology, Texas A&M University, College Station.

出版信息

Virology. 1989 May;170(1):31-9. doi: 10.1016/0042-6822(89)90348-6.

Abstract

High levels of nonfused chloramphenicol acetyltransferase, beta-galactosidase, and beta-glucuronidase expressed under the control of new vector constructs of the polyhedrin promoter in Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus were investigated by SDS-PAGE and RNA dot blot analysis of total cytoplasmic RNA. When the polyhedrin ATG start codon was converted to ATT by site-directed mutagenesis, translation initiated at downstream ATG codons resulting in high yields of nonfused foreign proteins. When a stop codon was inserted downstream from and in phase with the polyhedrin ATG codon but upstream from the ATG of a foreign gene, nonfused proteins were also produced, but at lower levels. The level of steady-state polyhedrin gene-promoted mRNA was not affected by the mutation from ATG to ATT or the insertion of in phase stop codons downstream from the polyhedrin ATG.

摘要

通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和对总细胞质RNA的RNA斑点印迹分析,研究了在感染苜蓿银纹夜蛾核型多角体病毒的草地贪夜蛾细胞中,由多角体蛋白启动子的新载体构建体控制下表达的高水平非融合氯霉素乙酰转移酶、β-半乳糖苷酶和β-葡萄糖醛酸酶。当通过定点诱变将多角体蛋白的ATG起始密码子转换为ATT时,翻译在下游的ATG密码子处起始,从而产生高产率的非融合外源蛋白。当在多角体蛋白ATG密码子下游且与之同相位但在外源基因的ATG上游插入一个终止密码子时,也会产生非融合蛋白,但产量较低。多角体蛋白基因启动的mRNA的稳态水平不受从ATG到ATT的突变或在多角体蛋白ATG下游插入同相位终止密码子的影响。

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