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昆虫的丝氨酸蛋白酶抑制剂(serpin)可以通过抑制丝氨酸蛋白酶的活性调控机体的免疫防御反应.依据柞蚕中肠转录组数据库中一段编码serpin的CDS序列设计正、反向引物,以柞蚕幼虫总cDNA为模板克隆得到一段全长1 239 bp的序列,经序列分析后命名为Apserpin-6(GenBank登录号:MF944108).该基因编码412个氨基酸残基,其中N端第1～17位氨基酸为信号肽序列,在C端第357～391位氨基酸之间有一个反应中心环,第374～375位丝氨酸之间是预测的蛋白质裂解位点.半定量RT-PCR检测Apserpin-6在柞蚕5龄幼虫脂肪体中的表达量最高,在血淋巴和头部组织的表达量次之,在中肠、丝腺和马氏管中的表达量较低.荧光定量PCR检测在受到柞蚕肠球菌(Enterococcus pernyi)、白僵菌(Beauveria bassiana)以及柞蚕核型多角体病毒(Antheraea pernyi nucleopolyhedrovirus)侵染后的柞蚕幼虫血淋巴中,Apserpin-6的表达量均明显上升.利用在原核表达系统表达的重组Apserpin-6蛋白,对柞蚕幼虫血淋巴中的酚氧化酶原(proPO)和酚氧化酶(PO)活性进行体外抑制试验,结果显示该重组蛋白能够抑制proPO的活性,但对PO活性没有影响.试验结果提示,Apserpin-6可能作为一种负调控因子参与调控柞蚕血淋巴对于病原菌的免疫防御反应.“,”In insects,serine protease inhibitors regulate the immune defense response of the organism by inhibiting the activity of serine proteases.We here cloned the open reading frame (1 239 bp) for a serpin from Antheraea pernyi based on the transcriptome data of midgut in Antheraea pernyi,named as Apserpin-6 (GenBank accession No.MF944108).The ORF of Apserpin-6 encodes 412 amino acid residues.The predicted signal peptide sequence is located at N-terminal from the first to seventeenth amino acid residues.There is a reaction center loop from the 357th to the 391th amino acid residue at C-terminal.The predicted protein cleavage site is between 374 serine and 375 serine.Apserpin-6 is mainly expressed in fat body detected by semi-quantitative RTPCR,followed by hemolymph and head,and had the lower expression level in midgut,silk gland and malpighian tubule.The results of qRT-PCR showed that the expression level of Apserpin-6 in hemolymph of A.pernyi increased significantly after induced by Enterococcus pernyi,Beauveria bassiana and Antheraea pernyi nucleopolyhedrovirus.The recombinant Apserpin-6 protein expressed in prokaryotic expression system was used to inhibit the activity of pro-phenoloxidase (proPO) and phenoloxidase (PO) in hemolymph of A.pernyi larvae in vitro.The results showed that Apserpin-6 inhibited proPO activity but did not affect PO activity in A.pernyi hemolymph.Our results suggested that Apserpin-6 acted as a negative regulator to regulate the immune response of A.pernyi hemolymph to pathogens.