Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in .

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.