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【目的】研究转宿主粘虫颗粒体病毒(Pseudaletia unipuncta granulovirus,Pu GV-Ps)增效蛋白基因截短片段优化及其增效作用,探索增效蛋白基因的合理利用途径。【方法】生物信息学分析增效蛋白结构域,构建增效蛋白基因截短片段原核表达载体,分析目的基因片段表达产物的表达水平、围食膜蛋白降解效能和增强活性,进一步明确Pu GV-Ps增效蛋白基因的功能区域。【结果】Pu GV-Ps增效蛋白含有M60-like结构域、锌离子催化域和糖蛋白结合域,并包含13个潜在的糖基化位点。以此为依据设计P69(短截M60-like结构域)和P77(短截糖蛋白结合域)2个截短片段,构建了表达载体p ET15b-P69和p ET15bP77,原核表达量明显高于全长基因P104。表达产物纯化蛋白围食膜降解活性表明,P69对斜纹夜蛾围食膜大分子蛋白降解程度高于P77,但两者均低于P104。病毒增强苏云金杆菌(Bt)实验表明,截短片段的表达产物提高了Bt对小菜蛾的毒力,但增强活性显著低于P104。【结论】研究结果表明,Pu GV-Ps增效蛋白基因N端M60-like结构域和C端糖蛋白结合域对其增效作用的发挥都具有一定功能,这些结构对维持增效蛋白的构象也发挥了一定的作用,截短片段P69有利于保持Pu GV-Ps增效蛋白的活性、提高表达水平。该研究结果对增效蛋白的工业化生产具有一定的指导意义。
【Objective】 The purpose of this study was to investigate the optimization and synergistic effects of the truncated fragment of Phagocytophilous unipuncta granulovirus (Pu GV-Ps) and to explore the rational utilization of the enhancer protein gene. 【Method】 Bioinformatics analysis of the putative protein domain and construction of a prokaryotic expression vector of the truncated fragment of the putative protein gene revealed that the expression level of the expressed product of the target gene fragment, the degradation efficiency and the enhanced activity of the peritrophic membrane protein, further clarified that Pu GV- Functional regions of Ps augmenting protein genes. 【Result】 The Pu GV-Ps potentiating protein contains M60-like domain, zinc ion catalytic domain and glycoprotein binding domain and contains 13 potential glycosylation sites. Based on this, two truncated fragments of P69 (short M60-like domain) and P77 (short glycoprotein binding domain) were designed and the expression vectors p ET15b-P69 and p ET15bP77 were constructed. Long gene P104. The results of peritoneal degradation showed that P69 degraded peritoneal macrophages proteins more than P77, but both of them were lower than P104. Virus-enhanced Bacillus thuringiensis (Bt) experiments showed that the expression products of truncated fragments increased the toxicity of Bt against Plutella xylostella, but the enhancing activity was significantly lower than that of P104. 【Conclusion】 The results showed that both the N-terminal M60-like domain and the C-terminal glycoprotein binding domain of Pu GV-Ps potentiating protein gene have some functions in enhancing their synergistic effects. These structures play an important role in maintaining the conformation of synergistic protein Also played a certain role, truncated fragment P69 is conducive to maintaining the activity of Pu GV-Ps booster protein, improve the expression level. The results of this study on the efficiency of the industrial production of protein has a certain significance.