家蚕丝氨酸蛋白酶抑制剂Bmserpin2对酚氧化酶原激活和抗菌肽基因表达的抑制作用

Inhibition of the activation of prophenol oxidase and the expression of antimicrobial peptide genes by the serine protease inhibitor Bmserpin2 in Bombyx mori

【目的】丝氨酸蛋白酶抑制剂家族蛋白是昆虫中调控自身免疫反应的重要蛋白酶抑制剂,本研究旨在研究家蚕Bombyx mori丝氨酸蛋白酶抑制剂2(Bmserpin2)在家蚕2个重要的自身免疫通路即酚氧化酶原(prophenol oxidase,PPO)激活通路和革兰氏阳性菌诱导抗菌肽的TOLL通路中的调控作用。【方法】PCR扩增家蚕Bmserpin2基因片段后原核表达并通过镍柱纯化。利用纯化后的重组Bmserpin2蛋白分别与胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶和蛋白酶K反应,检测Bmserpin2对上述蛋白酶活性的影响。通过RT-qPCR检测Bmserpin2在家蚕5龄第3天幼虫头、中肠、脂肪体、血淋巴、丝腺和表皮组织中表达的模式。往家蚕5龄第3天幼虫注射Bmserpin2重组蛋白,检测Bmserpin2对其血淋巴中PPO活性的影响。通过滕黄微球菌Micrococcus luteus诱导家蚕5龄第3天幼虫产生抗菌肽并注射Bmserpin2重组蛋白后,RT-qPCR检测其血淋巴中抗菌肽基因gloverin2和moricin表达量。【结果】成功构建重组质粒并表达纯化目的蛋白Bmserpin2。通过与不同蛋白酶反应得出Bmserpin2可极显著抑制消化酶胰蛋白酶和弹性蛋白酶活性,对胰凝乳蛋白酶和蛋白酶K活性影响不显著,提示Bmserpin2对不同蛋白酶具有生物学活性和催化特异性。基因表达模式显示Bmserpin2在家蚕5龄幼虫血淋巴和脂肪体中表达量最高。家蚕5龄幼虫注射重组Bmserpin2蛋白后发现目的蛋白能有效抑制血淋巴中PPO活性。利用滕黄微球菌诱导家蚕5龄幼虫产生抗菌肽后,滕黄微球菌和Bmserpin2混合注射组中血淋巴中抗菌肽基因gloverin2和moricin的转录表达与只注射滕黄微球菌的比较被显著下调。【结论】Bmserpin2可能参与家蚕酚氧化酶原激活和TOLL途径的胞外级联反应的免疫通路。

【Aim】Serine protease inhibitor family proteins are important protease inhibitors that regulate the autoimmune response in insects.This study aims to investigate the regulatory role of Bmserpin2 in two important autoimmune pathways,i.e.,the prophenol oxidase(PPO)activation pathway and the TOLL pathway of antimicrobial peptides(AMPs)induced by gram-positive bacteria,in Bombyx mori.【Methods】Bmserpin2 gene fragment of B.mori was amplified by PCR,and the target protein expression was induced by prokaryotic expression system and purified by nickel column.The effect of Bmserpin2 on the activities of trypsin,chymotrypsin,elastase and protease K was determined after the reaction between the purified recombinant Bmserpin2 and the above proteases,respectively.The expression patterns of Bmserpin2 in the head,midgut,fat body,hemolymph,silk gland,and integument of the day-35th instar larvae of B.mori were detected by RT-qPCR.The recombinant Bmserpin2 was injected into the day-35th instar larvae of B.mori,and the effect of Bmserpin2 on the PPO activity in their hemolymphs was determined.After the day-35th instar larvae of B.mori was induced by Micrococcus luteus to produce antimicrobial peptides(AMP)and injected with Bmserpin2,the expression levels of AMP genes gloverin2 and moricin in their hemolymphs were detected by RT-qPCR.【Results】The recombinant plasmid was successfully constructed,and the target protein Bmserpin2 was expressed and purified.Bmserpin2 significantly inhibited the activities of trypsin and elastase,but had no significant effect on the activities of chymotrypsin and proteinase K,suggesting that Bmserpin2 has biological activity and catalytic specificity to different proteases.The expression levels of Bmserpin2 were the highest in the hemolymph and fat body of the 5th instar larvae of B.mori.Most importantly,Bmserpin2 was found to inhibit the PPO activity in the hemolymph of the 5th instar larvae of B.mori after injection of the recombinant Bmserpin2.AMP was produced in the 5th instar larvae of B.mori induced by M.luteus.The transcription levels of AMP genes gloverin2 and moricin in the hemolymph in the Bmserpin2 and M.luteus mixedly injected group was significantly down-regulated as compared with that in the M.luteus injected group.【Conclusion】Bmserpin2 may be involved in the immune pathway of the activation of PPO and the extracellular cascade reaction of TOLL pathway in B.mori.