Abstract: 【Aim】 To prepare polyclonal antibody of the fungal-resistance factor BmSPI39 in Bombyx mori and to analyze its expression in response to Beauveria bassiana invasion. 【Methods】 Prokaryotic expression and immobilized nickel ion affinity chromatography were used to obtain the highly-purified recombinant BmSPI39 protein. The inhibitory activity of the recombinant BmSPI39 protein to Bacillus subtilis protease A was detected by in-gel activity staining. Polyclonal antibodies to BmSPI39 were prepared by immunizing New Zealand white rabbit with the recombinant BmSPI39 protein. Based on the open database SilkDB 3.0 of B. mori, the spatiotemporal expression characteristics of BmSPI39 in the immune-related tissues, integument, midgut and fat body, of B. mori from the day-3 4th instar larva to 1 day-old pupa were analyzed. And the expression of BmSPI39 in response to B. bassiana invasion in the integument of B. mori larvae and pupae at different time after B. bassiana infection was assayed using the prepared BmSPI39 antibodies by Western blot. 【Results】 The results of in-gel activity staining showed that the recombinant BmSPI39 protein could strongly 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 of Western blot and in-gel activity staining showed that the purified BmSPI39 antibody could effectively bind to the BmSPI39 antigen protein in different multimerization states. The heat map analysis of the expression data of BmSPI39 showed that BmSPI39 was expressed in the integument of B. mori at the wandering stage and the pre-pupal stage, with a higher expression level in the fat body at the pre-pupal stage, but not in the midgut at all the tested developmental stages. The results of Western blot showed that the expression of BmSPI39 in the integument of B. mori larvae was up-regulated at 84 h and down-regulated at 108 and 120 h after B. bassiana infection. 【Conclusion】 Polyclonal antibody to BmSPI39 was successfully prepared in this study. The expression of BmSPI39 protein can respond to B. bassiana invasion and may be involved in the process of resisting fungal invasion in the pupal stage of B. mori.

Key words:  Bombyx mori, Beauveria bassiana, fungal-resistance factor, polyclonal antibody; wandering stage, pupal stage, integument