Kong Wei-Wei, Yan Yu-Liang, Hou Cai-Ping, Hong Tao, Wang Yi-Sheng, Xu Xin, Liu Shi-Huo, Xu Jia-Ping
Anhui Province Key Laboratory of Resource Insect Biology and Innovative Utilization, School of Life Sciences, Anhui Agricultural University, Hefei, 230036, China; Anhui International Joint Research and Developmental Center of Sericulture Resources Utilization, Hefei, 230036, China.
Anhui Province Key Laboratory of Resource Insect Biology and Innovative Utilization, School of Life Sciences, Anhui Agricultural University, Hefei, 230036, China; Anhui International Joint Research and Developmental Center of Sericulture Resources Utilization, Hefei, 230036, China.
Dev Comp Immunol. 2025 Jan;162:105301. doi: 10.1016/j.dci.2024.105301. Epub 2024 Dec 16.
Serine proteases (SPs) are important proteases in the digestive system of lepidopteran insects. They play important roles in protein digestion, coagulation, signal transduction, hormone activation, inflammation and development. Blood-borne pyosis caused by Bombyx mori nuclear polyhedrosis virus (BmNPV) has caused serious harm to sericulture. At present, the scientific problems of BmNPV infection and silkworm resistance to BmNPV infection have been the focus of many scientists, but the molecular mechanism needs further research and exploration. Based on the results of label-free quantitative protein proteomics of the midgut digestive juice of different resistant strains in our laboratory, we successfully screened a differentially expressed candidate protein (DEP), B. mori chymotrypsin-like serine protease (BmCLSP), and comprehensively analyzed the biological characteristics and anti-BmNPV function of BmCLSP. The open reading frame (ORF) of BmCLSP is 891 bp, encoding 296 amino acid residues. The analysis of the domain structure showed that there was a signal peptide and a trypsin-like serine protease domain, Tryp_SPC, in the BmCLSP protein. Semi-quantitative and real-time fluorescence quantitative PCR analysis showed that the BmCLSP gene was highly expressed in the fifth instar larvae of silkworm, and specifically expressed in the midgut. The expression level of BmCLSP in the BmNPV resistant strain A35 was higher than that in the sensitive strain P50. Virus amplification analysis showed that the relative expression level of VP39 was significantly lower than that of the control group after infection of silkworm larvae and BmN cells with BmNPV treated with recombinant BmCLSP at an appropriate concentration. Furthermore, our overexpression of BmCLSP in BmN cells significantly inhibited the expansion of BmNPV. In summary, the results of this study indicate that BmCLSP has anti-BmNPV activity in silkworm, and can significantly inhibit the proliferation of BmNPV in silkworm. It offers a promising avenue for silkworm anti-virus breeding.
丝氨酸蛋白酶（SPs）是鳞翅目昆虫消化系统中的重要蛋白酶。它们在蛋白质消化、凝血、信号转导、激素激活、炎症和发育中发挥重要作用。家蚕核型多角体病毒（BmNPV）引起的血源性脓病给养蚕业造成了严重危害。目前，BmNPV感染及家蚕对BmNPV感染的抗性等科学问题一直是众多科学家关注的焦点，但其分子机制仍需进一步研究和探索。基于本实验室不同抗性品系中肠消化液的无标记定量蛋白质组学结果，我们成功筛选出一个差异表达候选蛋白（DEP），即家蚕类胰凝乳蛋白酶丝氨酸蛋白酶（BmCLSP），并对BmCLSP的生物学特性和抗BmNPV功能进行了综合分析。BmCLSP的开放阅读框（ORF）为891 bp，编码296个氨基酸残基。结构域分析表明，BmCLSP蛋白中有一个信号肽和一个类胰蛋白酶丝氨酸蛋白酶结构域Tryp_SPC。半定量和实时荧光定量PCR分析表明，BmCLSP基因在家蚕五龄幼虫中高表达，且在中肠中特异性表达。BmCLSP在抗BmNPV品系A35中的表达水平高于敏感品系P50。病毒增殖分析表明，用适当浓度的重组BmCLSP处理家蚕幼虫和BmN细胞后，再用BmNPV感染，VP39的相对表达水平显著低于对照组。此外，我们在BmN细胞中过表达BmCLSP显著抑制了BmNPV的扩增。综上所述，本研究结果表明BmCLSP在家蚕中具有抗BmNPV活性，能显著抑制BmNPV在家蚕中的增殖。这为家蚕抗病毒育种提供了一条有前景的途径。
