Ou Yaohui, Zhuang Huilin, Chen Ruoyu, Huang Danqiong, Wang Chaogang
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.
Bioengineering (Basel). 2023 May 8;10(5):564. doi: 10.3390/bioengineering10050564.
Anti-lipopolysaccharide factor is a class of antimicrobial peptides with lipopolysaccharide-binding structural domains, which has a broad antimicrobial spectrum, high antimicrobial activities, and broad application prospects in terms of the aquaculture industry. However, the low yield of natural antimicrobial peptides and their poor expression activity in bacteria and yeast have hindered their exploration and utilization. Therefore, in this study, the extracellular expression system of , by fusing the target gene with the signal peptide, was used to express anti-lipopolysaccharide factor 3 (ALFPm3) from in order to obtain highly active ALFPm3. Transgenic T-JiA2, T-JiA3, T-JiA5, and T-JiA6, were verified using DNA-PCR, RT-PCR, and immunoblot. Additionally, the IBP1-ALFPm3 fusion protein could be detected not only within the cells but also in the culture supernatant. Moreover, the extracellular secretion containing ALFPm3 was collected from algal cultures, and then its bacterial inhibitory activity was analyzed. The results showed that the extracts from T-JiA3 had an inhibition rate of 97% against four common aquaculture pathogenic bacteria, including and . The highest inhibition rate of 116.18% was observed in the test against . Finally, the minimum inhibition concentration (MIC) of the extracts from T-JiA3 to and were 0.11 μg/μL, 0.088 μg/μL, 0.11 μg/μL, and 0.011 μg/μL, respectively. This study supports the foundation of the expression of highly active anti-lipopolysaccharide factors using the extracellular expression system in , providing new ideas for the expression of highly active antimicrobial peptides.
抗脂多糖因子是一类具有脂多糖结合结构域的抗菌肽，具有广谱抗菌性、高抗菌活性，在水产养殖业方面具有广阔的应用前景。然而，天然抗菌肽产量低及其在细菌和酵母中的表达活性差阻碍了它们的开发利用。因此，在本研究中，通过将目标基因与信号肽融合，利用[具体生物名称]的细胞外表达系统来表达[具体生物名称]的抗脂多糖因子3（ALFPm3），以获得高活性的ALFPm3。利用DNA-PCR、RT-PCR和免疫印迹对转基因[具体生物名称]T-JiA2、T-JiA3、T-JiA5和T-JiA6进行了验证。此外，不仅在细胞内而且在培养上清液中都能检测到IBP1-ALFPm3融合蛋白。而且，从藻类培养物中收集了含有ALFPm3的细胞外分泌物，然后分析其抑菌活性。结果表明，T-JiA3的提取物对四种常见水产养殖病原菌，包括[病原菌名称1]和[病原菌名称2]，具有97%的抑制率。在对[病原菌名称3]的测试中观察到最高抑制率为116.18%。最后，T-JiA3的提取物对[病原菌名称1]、[病原菌名称2]、[病原菌名称3]和[病原菌名称4]的最低抑菌浓度（MIC）分别为0.11μg/μL、0.088μg/μL、0.11μg/μL和0.011μg/μL。本研究为利用[具体生物名称]的细胞外表达系统表达高活性抗脂多糖因子奠定了基础，为高活性抗菌肽的表达提供了新思路。
