球孢白僵菌HsbA蛋白的原核表达及免疫定位

【目的】明确虫生真菌球孢白僵菌（Beauveria bassiana）疏水表面结合蛋白HsbA（hydrophobic surface binding protein A）的致病过程。【方法】对HsbA进行克隆、亚克隆和原核表达，根据纯化后的蛋白制备多克隆抗体并进行抗原性分析，利用免疫电镜对HsbA蛋白进行定位观察。【结果】原核表达系统成功诱导了白僵菌的HsbA融合蛋白，制备多克隆抗体经Western杂交分析表明，该多抗能特异性识别目的蛋白。免疫电镜结果显示，HsbA蛋白在孢子和菌丝中呈随机分布。正常生长状态下，菌丝中的HsbA蛋白数量要明显多于孢子中被标记的数目，此外，孢子在侵染状态和正常生长状态下的HsbA蛋白数量也有显著差异，侵染状态下孢子的HsbA蛋白表达量更高，但菌丝在这2种状态下均呈现高表达。感染虫体中HsbA蛋白被标记的部位集中在体壁。【结论】原核表达实现了HsbA蛋白的高效表达，且制备的多克隆抗体具有较好的免疫原性。此外，免疫定位表明HsbA蛋白与菌丝的生长有关，并在白僵菌致病过程中参与了吸附作用，其作用部位位于昆虫体壁。

Prokaryocyte Expression and Immune Localization of HsbA in Beauveria bassiana

【Objective】The objective of this study is to clarify the pathogenic process of hydrophobic surface binding protein A (HsbA) in Beauveria bassiana which is an important fungus used for agricultural pests control.【Method】 Cloning, subcloning and prokaryotic expression were conducted in HsbA. Polyclonal anti-peptide antibody of HsbA was generated according to the purified protein, tested using Western blot, and then used for immune-localization of HsbA protein under transmission electron microscope (TEM). 【Result】HsbA fusion protein was successfully expressed in pET-30a, and the polyclonal antibody specifically bound purified HsbA protein in a Western blot experiment. The immune-localization demonstrated that HsbA protein was randomly distributed in both spore and hypha. The concentration of HsbA protein in hypha was significantly greater than that in spores. The concentration of HsbA protein in spores that in the pathogenic process was significantly higher than in spores that under normal condition, while the concentration of HsbA protein in hypha under either conditions was very high. In addition, at the infected host insect (Frankiniella occidentalis Pergande), HsbA protein was mainly located in the cuticle, where the spores infected the insect body. 【Conclusion】HsbA was efficiently expressed in prokartotic system and also the polyclonal anti-peptide antibody of HsbA had good immunogenicity. In addition, the results of immune localization that HsbA is related to hypha growth. Also, during the pathogenesis, it may promote the spores’ infection at the cuticle of host insects.