Kausar Saima, Abbas Muhammad Nadeem, Qian Cen, Zhu Baojian, Sun Yu, Sun Yuxuan, Wang Lei, Wei Guoqing, Maqsood Iram, Liu Chao-Liang
College of Life Sciences, Anhui Agricultural University, Hefei, 230036, China.
College of Wildlife Resources, Department of Wildlife Conservation and Utilization, Northeast Forestry University Harbin, China.
Dev Comp Immunol. 2017 Nov;76:45-55. doi: 10.1016/j.dci.2017.05.017. Epub 2017 May 22.
Genes encoding proteins of serpins superfamily are widely distributed in invertebrates. In insects, serpins play important roles in regulating immune responses and other physiological processes. Here, we report the cloning and characterization of cDNA of Apserpin-14 from Chinese oak silkworm (Antheraea pernyi). The Apserpin-14 gene contains 1206 bp open reading frame, encoding a predicted 401 amino acid residue protein. We expressed the recombinant Apserpin-14 protein in Escherichia coli and then purified protein was used to prepare rabbit anti-Apserpin-14 polyclonal antibodies. Quantitative real-time PCR analysis revealed that mRNA level of Apserpin-14 was highest in the fat body, whereas, among developmental stages the 5th instar and pupal stage showed greatest expression. Furthermore, Escherichia coli, Beauveria bassiana, Micrococcus luteus and nuclear polyhedrosis virus challenge enhanced Apserpin-14 transcript in both the fat body and hemocyte. Recombinant Apserpin-14 added to hemolymph inhibited spontaneous melanization and suppressed prophenoloxidase activation stimulated by M. luteus, but did not affect phenoloxidase (PO) activity. Injection of recombinant Apserpin-14 protein into A. pernyi larvae significantly reduced the transcript levels of antimicrobial peptides in the fat body, while its depletion by double stranded RNA enhanced their expression. We concluded that Apserpin-14 likely involved in regulation of proPO activation and production of antimicrobial peptides, implying its important role in the innate immune system of A. pernyi.
丝氨酸蛋白酶抑制剂超家族蛋白质的编码基因广泛分布于无脊椎动物中。在昆虫中，丝氨酸蛋白酶抑制剂在调节免疫反应和其他生理过程中发挥着重要作用。在此，我们报道了中国柞蚕（Antheraea pernyi）Apserpin - 14 cDNA的克隆与特征分析。Apserpin - 14基因包含1206 bp的开放阅读框，编码一个预测的401个氨基酸残基的蛋白质。我们在大肠杆菌中表达了重组Apserpin - 14蛋白，然后用纯化的蛋白制备兔抗Apserpin - 14多克隆抗体。定量实时PCR分析表明，Apserpin - 14的mRNA水平在脂肪体中最高，而在发育阶段中，5龄幼虫和蛹期的表达量最大。此外，大肠杆菌、球孢白僵菌、藤黄微球菌和核型多角体病毒的刺激均增强了脂肪体和血细胞中Apserpin - 14的转录本。添加到血淋巴中的重组Apserpin - 14抑制了自发黑化，并抑制了藤黄微球菌刺激的前酚氧化酶激活，但不影响酚氧化酶（PO）活性。将重组Apserpin - 14蛋白注射到柞蚕幼虫中显著降低了脂肪体中抗菌肽的转录水平，而通过双链RNA使其缺失则增强了它们的表达。我们得出结论，Apserpin - 14可能参与前酚氧化酶激活和抗菌肽产生的调节，这意味着它在柞蚕先天免疫系统中发挥重要作用。
