论文部分内容阅读
增效蛋白(ENHANCIN)是一种主要在昆虫杆状病毒中存在的杀虫增效因子,黄地老虎颗粒体病毒(Agrotis segetum granulovirus,AgseGV)基因组中有一个ORF的编码产物为杆状病毒ENHANCIN同源物,命名为en-hancin-like。生物信息学分析结果表明,AgseGV的enhancin-like基因编码产物含有ENHANCIN的保守域,无信号肽序列,不存在a-螺旋跨膜区,在其C-末端拥有类似哺乳动物的脯氨酸富集区。为了确定AgseGV的EN-HANCIN-like蛋白是否具有增效功能,构建了AgseGV的enhancin-like全基因和5′端片段(1 017bp)的原核表达载体。利用层析技术纯化截短片段的原核表达产物,以其为抗原制备了抗体。利用自制抗体和His-tag抗体,Western blot检测了enhancin-like全基因的表达产物。结果表明,在融合蛋白预期大小处显示出阳性条带,证明目的基因得到了良好的表达。以棉铃虫为供试虫进行生物测定,证明在棉铃虫核多角体病毒(HaNPV)浓度为1.17×102 PIBS/mL时,AgseGV enhancin-like的全基因原核表达产物能使其感染3龄初幼虫的死亡率提高16.25%,增效作用显著。但是,5’端截短片段的原核表达产物对HaNPV没有增效作用。
ENHANCIN, a pesticidal synergistic factor mainly present in insect baculoviruses, encodes an ORF in the Agrotis segetum granulovirus (AgseGV) genome as the baculovirus ENHANCIN Homologues, named en-hancin-like. The results of bioinformatics analysis showed that the enhancin-like gene of AgseGV contained conserved domain of ENHANCIN with no signal peptide sequence, no a-helical transmembrane region and similar Mammalian proline enrichment at its C-terminal Area. In order to determine whether the EN-HANCIN-like protein of AgseGV has synergistic effect, a prokaryotic expression vector of enhancin-like gene and 5 ’end fragment of AgseGV (1017bp) was constructed. The prokaryotic expression product of the truncated fragment was purified by chromatography, and the antibody was prepared as antigen. Using self-made antibody and His-tag antibody, Western blot was used to detect enhancin-like whole gene expression product. The results showed that a positive band was shown at the expected size of the fusion protein, demonstrating that the gene of interest was well expressed. The bioassay of Helicoverpa armigera showed that the prokaryotic expression product of AgseGV enhancin-like gene could induce the infection of early instar larvae of 3rd instar larvae at the concentration of 1.17 × 102 PIBS / mL The mortality rate increased by 16.25%, synergy effect was significant. However, the prokaryotic expression product of the 5 ’truncated fragment had no synergistic effect on HaNPV.