Guangdong Technology Research Center for Marine Algal Bioengineering, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China.
Shenzhen Engineering Laboratory for Marine Algal Biological Development and Application, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen 518060, China.
Mar Drugs. 2023 Jun 4;21(6):346. doi: 10.3390/md21060346.
Anti-lipopolysaccharide factor 3 (ALF3) possesses a wide antimicrobial spectrum and high antibacterial and viral activities for broad application prospects in the aquaculture industry. However, the application of ALF3 is limited by its low production in nature, as well as its low activity when expressed in and yeast. Although it has been proven that its secretory expression can be used to produce antimicrobial peptides with strong antimicrobial activity, there is no study on the high-efficiency secretory expression of ALF3 in . In this study, signal peptides ARS1 and CAH1 were fused with ALF3 and inserted into the pESVH vector to construct pH-aALF and pH-cALF plasmids, respectively, that were transformed to JUV using the glass bead method. Subsequently, through antibiotic screening, DNA-PCR, and RT-PCR, transformants expressing ALF3 were confirmed and named T-JaA and T-JcA, respectively. The peptide ALF3 could be detected in algal cells and culture medium by immunoblot, meaning that ALF3 was successfully expressed in and secreted into the extracellular environment. Moreover, ALF3 extracts from the culture media of T-JaA and T-JcA showed significant inhibitory effects on the growth of , , , and within 24 h. Interestingly, the inhibitory rate of c-ALF3 from T-JcA against four Vibrio was 2.77 to 6.23 times greater than that of a-ALF3 from T-JaA, indicating that the CAH1 signal peptide was more helpful in enhancing the secreted expression of the ALF3 peptide. Our results provided a new strategy for the secretory production of ALF3 with high antibacterial activity in , which could improve the application potentiality of ALF3 in the aquaculture industry.
抗脂多糖因子 3（ALF3）具有广谱抗菌谱和高效的抗菌和抗病毒活性，在水产养殖行业具有广阔的应用前景。然而，由于其在自然界中的产量低，以及在 和酵母中表达时活性低，其应用受到限制。尽管已经证明其分泌表达可用于产生具有强抗菌活性的抗菌肽，但尚未研究 ALF3 在 中的高效分泌表达。在本研究中，ARS1 和 CAH1 信号肽与 ALF3 融合，并分别插入到 pESVH 载体中，构建了 pH-aALF 和 pH-cALF 质粒，然后使用玻璃珠法转化到 JUV 中。随后，通过抗生素筛选、DNA-PCR 和 RT-PCR，证实并分别命名为表达 ALF3 的转化子为 T-JaA 和 T-JcA。免疫印迹可在藻类细胞和培养基中检测到肽 ALF3，表明 ALF3 在 中成功表达并分泌到细胞外环境中。此外，T-JaA 和 T-JcA 培养物中的 ALF3 提取物在 24 h 内对 、 、 和 的生长表现出显著的抑制作用。有趣的是，T-JcA 中的 c-ALF3 对四种弧菌的抑制率比 T-JaA 中的 a-ALF3 高 2.77 至 6.23 倍，表明 CAH1 信号肽更有助于增强 ALF3 肽的分泌表达。我们的结果为在 中具有高效抗菌活性的 ALF3 的分泌生产提供了一种新策略，可提高 ALF3 在水产养殖行业中的应用潜力。
