棉铃虫颗粒体病毒增效蛋白基因克隆及在大肠杆菌中表达

MOLECULAR CLONING OF ENHANCIN GENE FROM HELICOVERPA ARMIGERAGRANULOSIS VIRUS AND ITS EXPRESSION IN E.COLI

用PCR技术从棉铃虫颗粒体病毒基因组中扩增得到增效蛋白基因DNA序列。PCR产物酶切后克隆至 pBluescript质粒中 ,核苷酸序列测定表明 ,有 8个核苷酸、5个氨基酸与Genbank发表的序列不同。继而构建了表达质粒 pET 30a En ,诱导后经SDS PAGE和West ernblot检测 ,表明获得了增效蛋白基因在大肠杆菌BL2 1 (DE3)中包涵体形式的特异表达。生物测定结果表明 ,初步纯化的重组增效蛋白具有明显的增效活性 ,可提高棉铃虫核多角体病毒对 3龄幼虫致死率 31 7% - 34 1 %。重组增效蛋白的获得对研究其增效机理及构建新型杀虫剂提供了条件。

In order to provide recombinant enhancin for studying its mechanism of increasing the mortality of larvae infected by HaNPV and creating new insecticide, enhancin gene from \%Helicoverpa armigera\% granulosis virus was amplified by PCR technique. 2.7kb fragment of enhancin gene was cloned into \%Eco\%RI/\%Xba\%I site of plasmid pBluescript KS. Sequence analysis revealed that enhancin gene was similar with that reported in the literature except eight nucleotides and five amino acids. Thereby enhancin gene was inserted into vector pET\|30a and expressed successfully in \%Escherichia coli\% BL21(DE3). The preliminary bioassay of expressed product and HaNPV indicated that mortality of larvae increased 31.7%～34.1% in 7 post\|infection days and the LT 50 decreased at least 1.5～2.1 days.