| Shrimp cultivation has been suffering serious problems due to the outbreak of diseases in the whole world. It is an emergent and necessary work for us to explore the immune system of shrimps and to find effective methods to control the shrimp diseases. Fortilin, a multifunction protein, is proved to perform several functions including involving in cell cycle, calcium-binding and anti-apoptotic activities. Furthermore, shrimp fortilin is reported to be involved in the antiviral response. The recent research showed that injection of shrimp with recombinant fortilin protein, after infection with WSSV, resulted in 80-100% survival. However, the recombinant fortilin mixed with commercial pellets was fed to shrimp, which subsequently were challenged with WSSV. The dietary fortilin supplements only had low survival (10%).The intramuscular injection is costly and laborious. More effective and practical methods to deliver protein such as oral administration would be highly desirable in shrimp culture.The present study focused on improving transportation efficiency of fortilin protein using cell-penetrating peptides, and attempted to seek for a feasible method for its application in feeds. TAT was fused to fortilin by PCR procedure. Then, fusion proteins were expressed in yeast Pichia pastoris. And the culture supernatants of recombinant yeasts were lyophilized for analysis of immune responses of white shrimp Litopenaeus vannamei in vitro and in vivo. The study are summarized as follows:1. Firstly, the fortilin coding sequence (GenBank ID: DQ231062) was amplified by PCR from shrimp (Litopenaeus vannamei). Meantime, the TAT sequence was fused in the terminus of fortilin directly by PCR. The GFP coding sequence was created by PCR using the vector pTracer-CMV2 as a template. Then, the different combinations of GFP, fortilin and TAT genes were amplified by overlap PCR approach. Fortilin-GFP, GFP-Fortilin, TAT-Fortilin-GFP and GFP-Fortilin-TAT were used for the cell-penetrating study. Meanwhile, Fortilin, TAT-Fortilin and Fortilin-TAT were used to study the immune effects. The purified PCR fragments were cloned into the EcoR I/Xba I sites of the pGAPZαA vector. Sequencing analysis verified whether the target genes were correctly inserted in right reading frame. The construct was linearized and was integrated into the yeast Pichia pastoris X-33 by electroporation under the selection of Zeocin. The expressed proteins were identified by SDS-PAGE and characterized by matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS).2. For localization observation, green fluorescence protein (GFP) was selected as a fluorescence mark reporter, which can retain its intrinsic visible fluorescence when fused to the protein of interest. In the present study, hemocytes were incubated with fusion proteins. The intracellular green fluorescence was detected starting at 30 min, increasing progressively till to 24 h. The results showed that the TAT can deliver fortilin into primary shrimp hemocytes. Moreover, TAT-Fortilin-GFP and GFP-Fortilin-TAT showed successful TAT-mediated penetration through the intestinal membrane. TAT peptide fused to fortilin either at the N-terminus or at the C-terminus, can mediate transcellular protein transduction of fortilin, and the transduction efficiency was similar.TAT can deliver fortilin into primary shrimp hemocytes. Furthermore, how about the immune responses of hemocyte? Four immune enzyme were selected to analyse immune responses of cultured hemocytes of white shrimp Litopenaeus vannamei. Results showed that Fortilin , TAT-Fortilin and Fortilin-TAT significantly improved the activities of phenoloxidase and superoxide dismutase of cultured hemocytes (P<0.05). The respiratory burst activity of hemocytes treated with 200, 500μg/ml Fortilin, TAT-Fortilin and Fortilin-TAT was significantly higher than that in control group (P<0.05). TAT-Fortilin and Fortilin-TAT significantly improved the NOS activities, only the NOS activities of 500μg/ml Fortilin group significantly higher than that in control group (P<0.05). The respiratory burst activity and SOD activity of hemocytes incubated with 200μg/ml Fortilin-TAT was significantly higher than that in same concentration Fortilin group (P<0.05). Hemocytes treated with 100,200μg/ml TAT-Fortilin and Fortilin-TAT showed significantly higher PO and NOS activity than those in same concentration Fortilin treatment (P<0.05). After infection with WSSV, the SOD activity of hemocytes incubated with 200μg/ml TAT-Fortilin and Fortilin-TAT, the PO activity of hemocytes incubated with 500μg/ml TAT-Fortilin and the NOS activity of hemocytes incubated with 500μg/ml Fortilin-TAT were significantly higher than that in same concentration Fortilin group (P<0.05). These results showed that the presence of TAT fusion peptide improved the immune effect of fortilin.3. The effects of recombinant proteins on immune response and disease resistance in white shrimp (L. vannamei) were studied. The respiratory burst activity, PO and SOD activity of the recombinant fortilin group were significantly higher than that in control group (P<0.05). Meanwhile, four enzyme activities of Fortilin-TAT group were significantly higher than that in control and fortilin group (P<0.05). Based on these observations, it was speculated that fortilin enhanced the immune ability of shrimp, and TAT fusion peptide improved the immune effect. Except the respiratory burst activity, other enzyme activities of TAT-Fortilin group were significantly higher than those in Fortilin group (P<0.05). However, the SOD activities of TAT-Fortilin group were significantly lower than that in Fortilin-TAT group (P<0.05). So, maybe TAT peptide fused to fortilin at the C-terminus can work better. Meanwhile, there was no significant difference between enzyme activities of the culture supernatants of recombinant yeasts and the recombinant yeast cultures. The results of WSSV infection experiment showed that oral administration of recombinant proteins enhanced the immune ability of shrimp and improved the shrimp survival challenged with WSSV. The survival of Fortilin-TAT group was highest, and the TAT-Fortilin supplementation followed it. The survival of Fortilin group was lower than the previous two group, but there were no significant difference between three groups (P>0.05).Based on the above observations, it was speculated that TAT could promote transportation and absorption of fortilin to TAT in shrimp, and improved the immune effect of fortilin. This result has opened the way to a number of possible applications of TAT-mediated transcellular protein transduction using yeast as a vehicle in shrimp.
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