Oral recombinant Pichia pastoris P-I20H combined with immunopotentiator curcumin remarkably inhibits and clears LMBV in largemouth bass (Micropterus salmoides)

Abstract

Largemouth bass ranavirus (LMBV) causes massive losses in the largemouth bass industry. A promising therapeutic approach involves the oral administration of novel antimicrobial peptides (AMPs) and immunopotentiators. In this study, the Pichia pastoris expression system was employed for intracellular expression of AMP I20H. After meticulous screening, we successfully developed a strain, P -I20H-G7, demonstrating a high level of expression. Subsequently, we further optimized the induction conditions for this strain, including temperature, pH, and methanol concentration. Through these screening and optimization processes, we achieved an impressive nearly threefold increase in the production of I20H. Transmission electron microscopy , scanning electron microscopy and colony counting assays were utilized to confirm that P. pastoris effectively shielded I20H from the complex gastric and intestinal environments. Afterwards, P -I20H-G7 was lyophilized and co-fed with curcumin to largemouth bass infected by LMBV, the results indicated that oral administration of P -I20H-G7 combined with immunopotentiator curcumin effectively improve the survival rate of largemouth bass, with a 38% increase compared to the control group. qPCR demonstrated that dietary P -I20H-G7 + curcumin significantly inhibited and cleared LMBV within tissues. Additionally, oral administration of P -I20H-G7 + curcumin significantly upregulated the expression of key immune genes (IL-1β, IFN-γ, Mx, and IgM) and enhanced serum enzyme activities (TSOD, TAOC , and C3). Histopathological analysis revealed that P -I20H-G7 + curcumin treatment effectively mitigated tissue damage caused by LMBV infection . In conclusion, we successfully expressed and orally delivered I20H by the P. pastoris system and combined it with curcumin for efficiently curing diseased largemouth bass. These results underscore the potential of P -I20H-G7 + curcumin as a promising antiviral biological agent.