| 徐倩,王宁宁,白莉,史鲲鹏,沙珍霞.俄罗斯鲟肝脏表达抗菌肽2基因的分子特征及抗菌活性分析.渔业科学进展,2023,44(3):37-51 |
| 俄罗斯鲟肝脏表达抗菌肽2基因的分子特征及抗菌活性分析 |
| Molecular characterization and antimicrobial activity analysis of liver- expressed antimicrobial peptide 2 (LEAP-2) gene in Russian sturgeon (Acipenser gueldenstaedti) |
| 投稿时间：2022-02-28 修订日期：2022-03-23 |
| DOI：10.19663/j.issn2095-9869.20220228001 |
| 中文关键词: 肝脏表达抗菌肽2 (LEAP-2) 基因表达 抗菌活性 细菌感染 俄罗斯鲟 |
| 英文关键词: Liver-expressed antimicrobial peptide 2 (LEAP-2) Gene expression Antibacterial activity Bacterial challenge Acipenser gueldenstaedti |
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| 肝脏表达抗菌肽2 (liver-expressed antimicrobial peptide 2, LEAP-2)在鱼类先天免疫系统中发挥重要作用。本研究通过RACE技术获得了俄罗斯鲟LEAP-2 (Acipenser gueldenstaedti LEAP-2, AgLEAP-2)的全长cDNA序列，对其序列特征和表达水平进行了分析；构建AgLEAP-2原核表达载体并对重组蛋白进行纯化；进一步采用琼脂稀释法初步检测AgLEAP-2蛋白的抑菌活性。结果显示，AgLEAP-2基因cDNA全长为622 bp，开放阅读框(ORF)为246 bp，预测编码81个氨基酸，分子量约为11.2 kDa，5′端非编码区为184 bp，3′端非编码区为192 bp。AgLEAP-2包含一个信号肽(1~25 aa)和一个成熟肽(26~81 aa)，成熟肽含有4个保守的半胱氨酸残基，在Cys58-Cys69和Cys64-Cys74的相对位置之间各形成一个二硫键的核心结构。实时荧光定量PCR (qRT-PCR)结果显示，AgLEAP-2在所有健康组织中广泛表达，在肝脏中表达水平最高，在肠道和肌肉中表达水平次之，在鳃中表达量最低。与0 h相比，AgLEAP-2在感染嗜水气单胞菌(Aeromonas hydrophila)后的不同时间点的肝脏、脾脏、肠道、头肾、鳃和血液组织中均显著上调。此外，重组AgLEAP-2蛋白(rAgLEAP-2)在体外对革兰氏阳性菌和革兰氏阴性菌都具有抗菌活性并存在剂量效应。本研究表明，AgLEAP-2可能在俄罗斯鲟的先天免疫系统中发挥重要作用且对多种病原菌有一定的抗菌效果，本研究为开发新的抗菌制剂提供了依据。 |
| 英文摘要: |
| Antimicrobial peptides (AMPs) are an important part of the innate immune defense system; they can effectively kill viruses, fungi, and bacteria, preventing infection (and even sepsis). AMPs also have other functional roles in immune regulation, anti-tumor activity, angiogenesis, and wound healing. Liver-expressed antimicrobial peptide 2 (LEAP-2) is an AMP that has been extensively studied in mammals, birds, and fishes. The earliest fish LEAP-2 study reported the cloning of LEAP-2A and LEAP-2B of rainbow trout. So far, LEAP-2 has been studied in various fishes, including blunt snout bream, grass carp, golden pompano, ayu, and large yellow croaker. These studies found that LEAP-2 generally contains four highly conserved cysteine residues and two disulfide bonds; it can disrupt the structural integrity of bacterial cell membrane, revealing its antibacterial activity with an important role in the innate immune system of fishes. Russian sturgeon (Acipenser gueldenstaedtii) is cultivated on a certain scale in China, and Aeromonas hydrophila is its main pathogen. Hemorrhagic ascites and tissue hemorrhage appear after infection, causing huge economic losses to Russian sturgeon aquaculture. As an important innate immunity component, LEAP-2 research is of great significance. At present, studies on AgLEAP-2 have not been reported. To study the molecular characteristics and the transcriptional expression patterns of AgLEAP-2, as well as its antibacterial activity in vitro, the full-length AgLEAP-2 cDNA sequence was obtained by RACE. AgLEAP-2 was cloned and its sequence characteristics were analyzed by bioinformatics. The qRT-PCR method was used to detect AgLEAP-2 in 13 different tissues (liver, intestine, spleen, head kidney, blood, gill, skin, ovary, brain, heart, stomach, metanephros, and muscle) of healthy Russian sturgeon, and the AgLEAP-2 transcriptional expression pattern in immune tissues after infection with A. hydrophila. The AgLEAP-2 prokaryotic expression vector was constructed and the recombinant AgLEAP-2 protein (designated rAgLEAP-2) was purified. The antibacterial activity of rAgLEAP-2 was preliminarily detected by the agar dilution method. The results showed that the full-length cDNA of the AgLEAP-2 gene was 622 bp in length, of which the 5'-UTR was 184 bp, the 3'-UTR was 192 bp, and the ORF was 246 bp in length, encoding an 81-amino acid peptide. The molecular mass of the rAgLEAP-2 protein was predicted to be 11.2 kDa, and the theoretical isoelectric point was 9.15. AgLEAP-2 contained a signal peptide (1~25 aa) and a mature peptide (26~81 aa). The mature peptide contained four conserved cysteine residues and formed a core structure is consistent with two disulfide bonds between the Cys58-Cys69 and Cys64-Cys74. The AgLEAP-2 structure agreed with the LEAP-2 family characteristics. Moreover, the results showed that the AgLEAP-2 sequence was highly evolutionary conserved. Amino acid sequence alignment and phylogenetic analysis showed that AgLEAP-2 was clustered into a clade with the LEAP-2C of fishes, with the highest similarity with the LEAP-2C of Yangtze sturgeon and Chinese sturgeon. The qRT-PCR analysis showed that AgLEAP-2 was widely expressed in all healthy tissues, with the highest expression level in the liver, followed by the intestine and muscle, with the lowest expression in the gill. The expression patterns of LEAP-2 in different fish species were slightly different, with the highest expression level in the liver of Russian sturgeon and the intestine of large yellow croaker. The similar LEAP-2 expression patterns in different species suggested that its functions may be consistent, although it also had tissue and species specificity. The AgLEAP-2 expression reached its maximum within 72 h in the liver, spleen, head kidney, gill, and blood immune tissues. Among them, the expression AgLEAP-2 changed most significantly on the gill, which increased 5 000-fold compared with 0 h. The highest relative expression level was observed in the spleen at 72 h, corresponding to a 1500-fold induction relative to 0 h, and in the intestine at 48 h, with a 900-fold induction compared to 0 h kept constant until 72 h. In addition, the rAgLEAP-2 protein exhibited good antibacterial effects against both Gram-positive bacteria (Streptococcus sp. and S. aureus) and Gram-negative bacteria (E. coli, Vibrio anguillarum, and Shewanella spp.) in a dose-dependent manner in vitro. The rAgLEAP-2 protein at 500 μg/mL could inhibit the growth of most bacteria, significantly reducing the number of colonies on the plate. In conclusion, cloning and homology analysis revealed that AgLEAP-2 was closely related to LEAP-2C. AgLEAP-2 plays an important role in the immune response of Russian sturgeon against bacterial infection, and the rAgLEAP-2 protein could inhibit the growth of Gram-positive and Gram-negative bacteria in vitro. This study enhanced our current understanding of the nonspecific immune response in fish and provided a research basis for further studies on the antibacterial mechanism of LEAP-2 found in Russian sturgeon. |
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