Recombinant Expression Of Fusion Proteins Between White Spot Syndrome Virus Envelope Proteins VP28, VP26 And Cell Penetrating Peptides In Pichia Pastoris By Constitutive Promoter

WSSV has been quickly spread across the globe. It has became one of the most severe pathogens, and been causing tremendous economic losses to shrimp culture industry worldwide. Although no effective WSSV-controlling approach is available at present, researchers are more familiar with the composition of structural genes and their functions of WSSV with the completion of WSSV genome project. This is very important for the research on the mechanism how WSSV infects shrimp. Researchers found that virus envelope proteins play important roles in interacting with host cells, initiating virus infection or mediating virus intrusion. These envelope porteins include VP26 and VP28. More and more studies proved that some WSSV envelope proteins are effective in protecting hosts against WSSV when delivered into shrimp. It has promoted the born of WSSV protein subunit vaccine. However, the protein subunit vaccine is very easy to be digested by organism. So it is difficult to provide immunity for shrimp by oral administration. For this reason, cell penetrating peptides which can deliver many types of cargos, such as peptides and proteins, into cells are used to solve the problems of oral administration. In this study, the expression system of P. pastoris was used to express fusion proteins (CPPs-GFP). It was confirmed that CPPs-GFP was able to pass through the intestinal wall tissue and go into the heart and muscle tissue of crayfish. On this basis, the VP28-TAT/R9, VP26-TAT/R9 and GFP-TAT/R9 fusion proteins were successfully expressed by P. pastoris expression system. The main process and conclusions are as follows:1. Secretory expression of TAT-GFP, R9-GFP and Penetratin-GFP fusion proteins in P. pastoris. Firstly, the GFP coding sequence was created by PCR using the vector pTracer-CMV2 as a template. Meantime, the nucleotide sequences of TAT, R9 and Penetratin were designed to be more suitable for being expressed in P. pastoris by Codon Adaptation Tool. Then the designed nucleotide sequences of TAT, R9 and Penetratin were added to the 5’end of GFP gene by general PCR. In the meantime, the sequences of EcoRⅠ(GAATTC) and Xba Ⅰ (TCTAGA) were added to the 5’and 3’end of the target genes, respectively. TAT-GFP, R9-GFP and Penetratin-GFP fusion genes were acquired by PCR amplification. 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 by BinⅠ (Avr Ⅱ) and then was integrated into P. pastoris X-33 by electroporation under the selection of Zeocin. The expressed proteins were identified by SDS-PAGE. The results showed that GFP, TAT-GFP, R9-GFP and Penetratin-GFP fusion proteins were expressed successfully, and the size of the proteins are about 29kDa.2. Detect the transmembrane effects of CPPs-GFP. Fusion proteins (GFP, TAT-GFP. R9-GFP, Penetratin-GFP) were acquired by small-scale fermentation in the laboratory. Then the desired protein were freeze-dried into a powder-like material. The powder-like fusion proteins were dissolved to 10mg/ml by PBS. Crayfish in experimental groups were individually fed the same amount of fusion proteins (TAT-GFP, R9-GFP, Penetratin-GFP, GFP) via a feeding needle in size five, and the negative control was fed the same volume of PBS. After two hours, the midgut, heart and muscle of the experimental crayfish were taken to produce the cryostat section. The results showed that strong green-fluorescent signals of GFP were detected in the mid gut, muscle and heart sections from crayfish fed with TAT-GFP, R9-GFP and Penetratin-GFP.3. Secretory expression of VP28-TAT/R9, VP26-TAT/R9 and GFP-TAT/R9 fusion proteins in P.pastoris. The VP28 and VP26 coding sequences (GenBank:AF332093.3) were amplified by PCR from WSSV. The designed nucleotide sequences of TAT and R9 were added to the 3’end of VP28, VP26 and GFP genes by general PCR amplification respectively. The sequences of EcoR I (GAATTC) and Xba I (TCTAGA) were added to the 5’and 3’end of the desired genes as well. Recombinant yeasts were acquired by a series of technology of molecular cloning just like above. The expressed proteins were identified by SDS-PAGE. The results showed that GFP, GFP-TAT and GFP-R9 recombinant proteins were detected using coomassie brilliant blue R250 stain, but not to detect the bands of VP28-TAT/R9, VP26-TAT/R9, VP28 and VP26 fusion proteins. But the bands of those fusion proteins were detected by silver staining, and the size of VP28-TAT/R9, VP26-TAT/R9, VP28 and VP26 fusion proteins are about 32kDa.