粘虫颗粒体病毒增效蛋白基因片段优化及功能

Optimized expression and functional analysis of enhancin gene from Pseudaletia unipuncta granulovirus (PuGV-Ps)

摘要:

[目的]研究转宿主粘虫颗粒体病毒（Pseudaletia unipuncta granulovirus, PuGV-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PuGV-Ps增效蛋白基因的功能区域。[结果] PuGV-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pET15b-P69和pET15bP77，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，PuGV-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PuGV-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。

Abstract:

[Objective] To explore the feasibility of using enhancin as synergist to Bacillus thuringiensis (Bt), the truncated fragments of enhancin gene from Pseudaletia unipuncta granulovirus (PuGV-Ps) were optimized and the enhancing effects were studied.[Methods] Based on bioinformational analysis of the function domain of PuGV-Ps enhancin, the prokaryotic expression vectors were constructed, and the protein expression levels as well as their enhancing effects on the degradation of peritrophic membrane (PM) proteins were analyzed, and the function domains of PuGV-Ps enhancin were confirmed.[Results] Three domains were found in the enhancin of PuGV-Ps, including M60-like domain, Zincins catalytic domain and putative mucin or carbohydrate-binding domain. Thirteen predicted N-glycosylation sites were also identified. Based on the sequences of truncated M60-like domain (P69) and carbohydrate-binding domain (P77), two expression vectors, pET15b-P69 and pET15b-P77, were constructed. The expressed P69 and P77 abundance were higher than that of full length enhancing (P104). The degradation activity of purified P69 on the PM proteins of Spodoptera litura was higher than that of purified P77, but both showed lower degradation activities than P104. Both P69 and P77 improved the toxicity of Bt against larvae of Plutella xylostella. However, their synergistic effects were significantly lower than that of P104.[Conclusion] The results revealed that the M60-like domain in N-terminus and carbohydrate-binding domain in C-terminus of PuGV-Ps enhancin all contributed to the enhancing effects of enhancin as well as the maintenance of its native conformation. The truncated P69 fragment may function in keeping the activity of enhacin and improving prokaryotic expression levels. These results provide some useful guidance for the industrialized production of enhancin.