家蚕抗真菌因子BmSPI39对球孢白僵菌入侵的表达响应

【目的】制备家蚕Bombyx mori抗真菌因子BmSPI39的多克隆抗体，分析BmSPI39对球孢白僵菌Beauveria bassiana入侵的表达响应。【方法】利用原核表达和固定化镍离子亲和层析技术获得高纯度的BmSPI39重组蛋白，利用胶内活性染色检测重组蛋白BmSPI39对枯草杆菌Bacillus subtilis蛋白酶A的抑制活性；利用重组蛋白BmSPI39，通过免疫新西兰大白兔制备BmSPI39的多克隆抗体。基于家蚕开放性数据库SilkDB 3.0，分析BmSPI39在4龄第3天至化蛹后1 d家蚕免疫相关组织体壁、中肠和脂肪体中的时空表达特征。利用制备的BmSPI39抗体通过Western blot分析感染球孢白僵菌不同时间后家蚕幼虫和蛹体壁中BmSPI39应答球孢白僵菌入侵的表达响应。【结果】胶内活性染色结果显示，重组BmSPI39蛋白能够强烈抑制枯草杆菌蛋白酶A的活性。酶联免疫吸附结果显示，BmSPI39抗血清的抗体效价达1∶128 000。Western blot和胶内活性染色结果显示，纯化后的BmSPI39抗体能够有效结合不同多聚体化状态下的BmSPI39抗原蛋白。BmSPI39表达数据的热图分析显示，BmSPI39在游走期和预蛹期的家蚕体壁中有表达，在预蛹期的脂肪体中的表达量较高，但在各发育阶段的中肠中均不表达。Western blot分析结果表明，家蚕幼虫体壁中的BmSPI39的表达在感染球孢白僵菌后84 h上调表达，108和120 h下调表达。【结论】本研究成功制备了BmSPI39的多克隆抗体。BmSPI39蛋白的表达能够响应球孢白僵菌入侵，可能参与蛹期家蚕抵御真菌入侵的过程。

Expression of the fungal-resistance factor BmSPI39 inBombyx moriin response toBeauveriabassianainvasion

【Aim】 To prepare polyclonal antibody of the fungal-resistance factor BmSPI39in Bombyx mori and to analyze its expression in response to Beauveria bassiana invasion. 【Methods】 Prokaryotic expression and immobilized nickel ion affinity chromatography wereused to obtain the highly-purified recombinant BmSPI39 protein. The inhibitory activity ofthe recombinant BmSPI39 protein to Bacillus subtilis protease A was detected by in-gelactivity staining. Polyclonal antibodies to BmSPI39 were prepared by immunizing New Zealandwhite rabbit with the recombinant BmSPI39 protein. Based on the open database SilkDB 3.0 ofB. mori, the spatiotemporal expression characteristics of BmSPI39 in the immune-relatedtissues, integument, midgut and fat body, of B. mori from the day-3 4th instar larva to 1day-old pupa were analyzed. And the expression of BmSPI39 in response to B. bassianainvasion in the integument of B. mori larvae and pupae at different time after B. bassianainfection was assayed using the prepared BmSPI39 antibodies by Western blot. 【Results】The results of in-gel activity staining showed that the recombinant BmSPI39 protein couldstrongly inhibit the activity of subtilisin A. Enzyme-linked immunosorbent assay (ELISA)showed that the antibody titer of BmSPI39 antiserum reached 1∶128 000. The results ofWestern blot and in-gel activity staining showed that the purified BmSPI39 antibody couldeffectively bind to the BmSPI39 antigen protein in different multimerization states. Theheat map analysis of the expression data of BmSPI39 showed that BmSPI39 was expressed inthe integument of B. mori at the wandering stage and the pre-pupal stage, with a higherexpression level in the fat body at the pre-pupal stage, but not in the midgut at all thetested developmental stages. The results of Western blot showed that the expression ofBmSPI39 in the integument of B. mori larvae was up-regulated at 84 h and down-regulatedat 108 and 120 h after B. bassiana infection. 【Conclusion】 Polyclonal antibody to BmSPI39was successfully prepared in this study. The expression of BmSPI39 protein can respond toB. bassiana invasion and may be involved in the process of resisting fungal invasion in thepupal stage of B. mori.