Cloning and expression of recombinant shrimp PmRab7 (a virus-binding protein) in Pichia pastoris.

White spot syndrome virus (WSSV) is one of the most serious pathogens in shrimp aquaculture. A shrimp WSSV-binding protein called PmRab7 has been isolated and characterized. Since injection of bacterial expressed-rPmRab7 could reduce shrimp mortality caused by WSSV from approximately 95% to 15% mortality, there was potential for its use in protection against WSSV in shrimp aquaculture. To test the feasibility of this, the Pichia pastoris yeast expression system was used for production of rPmRab7 since its expression system has eukaryote post-translational modification capability and since P. pastoris is widely accepted for use in human food or animal feed. Moreover, ß-1,3-glucan, a major cell wall component of yeast, has been reported to act as an immunostimulant in shrimp. The recombinant protein was produced intracellularly and the resulting whole yeast cells were lyophilized and stored for supplementation in shrimp feed. The yield of rPmRab7 was 20-30 mg/l of culture medium or 2.67 mg/g yeast dry weight, which was 2-3 times higher than the yield obtained from an Escherichia coli expression system. A two-copy gene expression system was developed to enhance rPmRab7 expression using expression vector pAO815 containing a two-copy PmRab7 expression cassette constructed by site-directed mutagenesis of the PmRab7 gene and two-step overlap, extension PCR. This improved the yield of rPmRab7 2-3 times (40-60 mg/l of culture medium). ELISA was developed to show that the expressed rPmRab7 had WSSV-binding activity. Although some loss of rPmRab7 was found after lyophilization of the yeast cells, projected cost calculations indicated that this production level would make it feasible to use rPmRab7 in shrimp feed for protection against WSSV.

PmRab7 is a VP28-binding protein involved in white spot syndrome virus infection in shrimp.

Our aim was to isolate and characterize white spot syndrome virus (WSSV)-binding proteins from shrimp. After a blot of shrimp hemocyte membrane proteins was overlaid with a recombinant WSSV envelope protein (rVP28), the reactive bands on the blot were detected using anti-VP28 antibody. Among three membrane-associated molecules identified by liquid chromatography-tandem mass spectrometry, there was a 25-kDa protein that bound to both rVP28 and WSSV. Since it had a primary structure with high homology to the small GTP-binding protein Rab7, we named it Penaeus monodon Rab7 (PmRab7). The full-length PmRab7 cDNA was obtained, and results from a glutathione S-transferase pull-down assay confirmed specific binding to rVP28. Reverse transcriptase PCR analysis revealed PmRab7 expression in many tissues, and real-time PCR analysis revealed that expression was constitutive. Binding of PmRab7 to rVP28 or WSSV occurred in a dose-dependent manner and was inhibited by anti-Rab7 antibody. In an in vivo neutralization assay, the number of dead shrimp after challenge with WSSV plus PmRab7 (15%) or WSSV plus anti-Rab7 antibody (5%) was significantly lower than after challenge with WSSV alone (95%). In contrast to the WSSV-injected group, shrimp injected with WSSV plus PmRab7 or WSSV plus anti-Rab7 showed no WSSV-type histopathology. We conclude that PmRab7 is involved in WSSV infection in shrimp. This is the first study to identify a shrimp protein that binds directly to a major viral envelope protein of WSSV.