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 subti...

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.