Screening For The Interaction Proteins With Cap Protein Of DuCV By Yeast Two Hybrid System

Cap protein of DuCV is the virus structural protein, constructs the virus capsid which is important for immune response in host. In order to find the interacting host proteins with Cap protein, it could be better understrand the protein fuction in virus invading host. In this paper, we used Cap protein as bait and constructed yeast two hybrid library of duck spleen to screen the interaction host proteins, which made a foundation for the further study the protein fuction in invadeing host.1. Expression and purification of a codon optimized duck circovirus Cap protein and production the polyclonal antibodies The full-length CapY108 gene with optimizing codon 1-108 bp and wild 109-774 bp were cloned into pGM-T vector, and then subcloned into pET-32a(+) expression vectors. The pET32-CapY108 vector was transformed into E.coli Rosetta (DE3) pLys after PCR, restriction enzyme digesting and sequence analysis. The expressed proteins were analysised by SDS-PAGE and further purified using the Ni-chelating affinity chromatography. The concentration of the purified protein was measured by Bradford method, and which was further analyzed by western blot with DuCV-infected duck serum. The result showed that the whole Cap gene was successfully amplied by PCR, restriction enzyme digesting and sequence analysis showed that the pET32-CapY108 vector constructed successfully. The pET32-CapY108 plasmid was further transformed into and expressed in Rosetta (DE3) pLys. After SDS-PAGE, a distinct band of approximately 50 KD was observed in precipitate, corresponding to the expected size of the Cap. The concentration of the purified protein was 0.289 mg/mL. ELISA assay showed the antibody tite is above 1:80000. Western blot showed Cap protein was successfully expressed and purified, furthermore, it was used to produce the polyclonal antibodies for dectecting subsequently.2. Construction the bait vector cap gene in duck cirovirus To construct the bait vector of Cap gene in yeast two-hybrid system, the whole Cap gene was inserted into pGBKT7 vector with optimizing codon, and then were confirmed by PCR, restriction enzyme analysis. After transformating into Y2HGold yeast stain, the expression of Cap protein was analyzed by Western blotting. Toxicity and self-activation of the bait protein were detected by different dropout minimal base. The results showed that the products were inserted into pGBKT7 correctly by PCR reaction and restriction enzyme digestion. Western blotting showed that the whole Cap protein expressed about 50 KD. The recombinant plasmids had no toxicity and self-activation. The results indicated that the bait vector of Cap gene constructed successfully, which makes a foundation for the further study of screening the interaction proteins in the yeast two-hybrid system.3. Construction of yeast two hybrid library To construct the yeast two hybid library, the total mRNA of the duck spleen with DuCV-infected was extracted and then synthesized the first-strand cDNA by PCR reverse transcription. The cDNA was amplied to double-strand by LD-PCR and purified, and then co-transformed into yeast strain Y187 with pGADT7-Rec. The strains were coated on SD/-Leu plate, and then collected all clones after 5 days and detected the library quality. The results showed that titer of the library were 1.6 × 107 cfu/mL and the transformation efficiency was 1.29 × 106/4.64 μg, the titer of amplied library were 3.7 × 108 cfu/mL.22 clones vector were selected randomly, the result showed about the insert fragments were arrange from 500-3500 bp by PCR.15 clones were selected ramdonly and then sequenced analysis, the results showed that the library recombinantion rate was about 93.3%. Above all, the library constructed successfully which could be used for screening the interaction proteins with Cap protein.4. Screening the interaction proteins with Cap protein To screen the interaction protein with Cap protein, the bait and constructed library were hybrided. The products were coated on TDO plate. After 3 day, the big clones were selected and transferred on QDO/X/A plate,subculturing 3 times,the blue and big clones as positive strain to sequencing analysis. The no frameshift mRNA vectors were transformed into Y2HGold with pGBKT7-Cap, and incubated on QDO/X/A. The results showed that a positive clone was screened successfully, spermidine/spermine N1-acetyltransferase 1 (SAT1), and co-transformation experiment further showed the protein interacted with Cap protein.