柞蚕丝氨酸蛋白酶抑制剂6基因Apserpin-6的克隆及功能分析

发布时间：2018-07-16 13:57

【摘要】：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c DNA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(Gen Bank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(pro PO)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pro PO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。
[Abstract]:Insect serine protease inhibitor (serpin) can regulate the immune defense response by inhibiting the activity of serine protease. According to a sequence of serpin coding in Antheraea pernyi (Antheraea pernyi) midgut transcriptional database, a sequence of 1,239bp was cloned from total cDNA of tussah larvae by reverse primers. The sequence was named as Apserpin-6 (GenBank accession number: MF944108). The gene encodes 412 amino acid residues, in which N terminal 1 amino acid is a signal peptide sequence, there is a central ring between C terminal 357 and 391 amino acids, and between 374 and 375 serine is the predicted protein cleavage site. Semi-quantitative RT-PCR was used to detect the expression of Apserpin-6 in the fat body of 5th instar larvae of Antheraea pernyi, followed by hemolymph and head tissue, and lower in midgut, silk gland and Markov tube. The expression of Apserpin-6 in the haemolymph of Antheraea pernyi larvae infected by Enterococcus pernyi), Beauveria bassiana and Antheraea pernyi nuclear polyhedrosis virus (Antheraea pernyi nucleopolyhedrovirus) was detected by fluorescence quantitative PCR. Using recombinant Apserpin-6 protein expressed in prokaryotic expression system, the activity of phenol oxidase (pro) and phenol oxidase (PO) in hemolymph of Antheraea pernyi larvae was tested in vitro. The results showed that the recombinant protein could inhibit the activity of pro PO. But there was no effect on PO activity. The results suggest that Apserpin-6 may be a negative regulatory factor in regulating the immune defense response of tussah hemolymph to pathogenic bacteria.
【作者单位】：沈阳农业大学生物科学技术学院沈阳农业大学柞蚕研究所辽宁省昆虫资源工程技术研究中心;
【基金】：现代农业产业技术体系建设专项(No.CARS-18) 辽宁省教育厅科学研究项目(No.LSNYB201613)
【分类号】：S885.1

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