关键词: 家蚕, 病原体相关分子模式, Kazal型丝氨酸蛋白酶抑制剂7, 细胞免疫, 吞噬作用

Abstract: 【Aim】 To elucidate the biological function of serine protease inhibitor Kazal-type 7 (SPINK7) and its immunorecognition mechanism of Bombyx mori against bacterial infestation. 【Methods】 Bioinformatics analysis of SPINK7 of B. mori was made followed by prokaryotic expression, and the secondary structure and heat resistance of SPINK7 were analyzed by circular dichroism. The tendency of SPINK7 to form aggregates was detected by non-reducing electrophoresis and Western blot. The 5th instar larvae of B. mori were infected by Escherichia coli and Staphylococcus aureus, respectively, and the expression levels of SPINK7 gene in hemocytes and fat bodies were detected by qPCR, and the SPINK7 content in serum was detected by Western blot. The inhibitory activities of SPINK7 against E. coli, S. aureus and Micrococcus luteus were analyzed by bacterial inhibition curves. The binding of SPINK7 to pathogen-associated molecular patterns (PAMPs) such as peptidoglycan and lipopolysaccharide, and the binding of SPINK7 to E. coli, S. aureus and M. luteus were detected by immunofluorescence localization and enzyme-linked immunosorbent assay (ELISA).【Results】 SPINK7 consists of three conserved Kazal structural domains, each of which contains six cysteines, forming three pairs of disulfide bonds. The secondary structure of SPINK7 is mainly α-helix, which is consistent with the typical secondary structure of Kazal structural domains, it is heat-resisting and remains structurally stable at 80 ℃. After induction by E. coli and S. aureus, SPINK7 formed a multimeric structure, and the expression levels of SPINK7 gene in hemocytes and fat bodies and the SPINK7 content in the serum of the 5th instar larvae of B. mori were significantly up-regulated, as compared with those of the phosphate buffer solution (PBS) control group. SPINK7 did not directly inhibit bacterial activity, and promoted bacterial aggregation by binding to bacterial PAMPs. 【Conclusion】SPINK7 is a Kazal-type immune-associated protein, whose expression is up-regulated and multimerized after bacterial infestation in B. mori, and helps hemocytes to aggregate bacteria and exert immune effect by recognizing and binding to bacterial PAMPs.

Key words: Bombyx mori, pathogen-associated molecular patterns, serine protease inhibitor Kazal-type 7, cellular immunity, phagocytosis