State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo, 315211, China; Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo, 315211, China; Key Laboratory of Applied Marine Biotechnology of Ministry of Education, Ningbo University, Ningbo, 315211, China.
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo, 315211, China; Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo, 315211, China; Key Laboratory of Applied Marine Biotechnology of Ministry of Education, Ningbo University, Ningbo, 315211, China.
Fish Shellfish Immunol. 2024 Jun;149:109531. doi: 10.1016/j.fsi.2024.109531. Epub 2024 Apr 9.
In this study, we present the first cloning and identification of perforin (MsPRF1) in largemouth bass (Micropterus salmoides). The full-length cDNA of MsPRF1 spans 1572 base pairs, encoding a 58.88 kDa protein consisting of 523 amino acids. Notably, the protein contains MACPF and C2 structural domains. To evaluate the expression levels of MsPRF1 in various healthy largemouth bass tissues, real-time quantitative PCR was employed, revealing the highest expression in the liver and gut. After the largemouth bass were infected by Nocardia seriolae, the mRNA levels of MsPRF1 generally increased within 48 h. Remarkably, the recombinant protein MsPRF1 exhibits inhibitory effects against both Gram-negative and Gram-positive bacteria. Additionally, the largemouth bass showed a higher survival rate in the N. seriolae challenge following the intraperitoneal injection of rMsPRF1, with observed reductions in the tissue bacterial loads. Moreover, rMsPRF1 demonstrated a significant impact on the phagocytic and bactericidal activities of largemouth bass MO/MΦ cells, concurrently upregulating the expression of pro-inflammatory factors. These results demonstrate that MsPRF1 has a potential role in the immune response of largemouth bass against N. seriolae infection.
在这项研究中，我们首次克隆并鉴定了大口黑鲈（Micropterus salmoides）中的穿孔素（MsPRF1）。MsPRF1 的全长 cDNA 跨越 1572 个碱基对，编码一个 58.88 kDa 的蛋白质，由 523 个氨基酸组成。值得注意的是，该蛋白含有 MACPF 和 C2 结构域。为了评估 MsPRF1 在各种健康大口黑鲈组织中的表达水平，我们采用实时定量 PCR 技术，结果显示其在肝脏和肠道中的表达水平最高。在大口黑鲈感染链霉菌后，MsPRF1 的 mRNA 水平通常在 48 小时内升高。值得注意的是，重组蛋白 MsPRF1 对革兰氏阴性菌和革兰氏阳性菌均具有抑制作用。此外，在注射重组 MsPRF1 后，大口黑鲈在链球菌感染挑战中的存活率更高，组织细菌载量降低。此外，rMsPRF1 对大口黑鲈 MO/MΦ 细胞的吞噬和杀菌活性具有显著影响，同时上调了促炎因子的表达。这些结果表明 MsPRF1 在大口黑鲈对链球菌感染的免疫反应中具有潜在作用。
