Expression And Purification Of VP110, An Envelope Protein Of WSSV

Based on sequence analysis, we found that WSSV vp110 shared similarity with pif2 that is one of per os infectivity factors of baculoviruses and functions as transporting viruses into host epitheliums, and N terminus of both VP110 and PIF2 is high hydrophobic regions. Small VP110(s VP110), from 150 to 600 aa, was most similar to that of PIF2 homologs in insect DNA viruses of baculoviruses, nudiviruses and hytrosaviruses. After removing the hydrophobic N-terminal regions, svp110 was cloned into expression vector of p ET-16 b, and expression conditions, such as temperature, time, IPTG concentration and E. coli strains, were considered for better expression of recombinant proteins. As a result, the fusion proteins of s VP110-His were highly expressed and remaned integrated. After purified by using GE AKTA, the purity of s VP110-His was more than 80%. Refolding conditions was optimized, the optimal refolding conditions was selected: 6 mol / L urea solution of 4 ℃ dialysis 1 h, then 4 mol / L urea solution was dialyzed 4 h, and finally with 2 mol / L urea dialysis solution about2 h, At last, completion of dialysis refolding was succeed. Concentrated purified s VP110-His by ultrafiltration using ultrafiltration to get high concentration s VP110-His. s VP110-His protein’s concentration reached high concentration of 600.83 mg / m L, the total amount of preparation fully meet the requirements of immuning rabbit for polyclonal antibody ≥ 3 mg. The purified and concentrated fusion protein of s VP110-His immunized rabbits successfully and prepare high specific, good affinity of polyclonal antibodies that antibody titers reached 1:234 000. High titer antisera can verify VP110 specific during WSSV infection and can be used to study WSSV interactions mechanism and verified our speculating that VP110 was per os infectivity factor of WSSV. We constructed the functional domains Svp110 to four different expression vectors p QE80-ZZtev, p ET-16 b, p GEX-4T1 and p ET-32 a, finally we succed making fusion protein soluble when selected p ET-32 a that had a thioredoxin tag at N terminus enhancing validity of folding of protein and two his tags at both end of sequence used for purification. At last, we succed purifying soluble fusion protein s VP110-Trx.Based on the research process, try to express the full-length protein VP110, chose different expression vectors, different expressing strains, and optimized expression conditions because VP110 included high hydrophobic region of the N-terminal, envelope protein VP110 all expressed as inclusion bodies that had no activity. There wa a very small amount or no expression in supernatant. Three expression strains BL21, Rosetta and Origami B was used when VP110 fusion protein was expressed and found that the highest expression level was Rosetta strains and second was BL21 and Origami B worst; 1 mmol / IPTG was optimal VP110-His fusion protein expressed significantly better by Origami B at 30 ℃comparing with BL21 and Rosetta. Under all conditions, best expression conditions was used 1mmol/L IPTG at 30 ℃ temperature. Comparing three different labels of expression vector, we found that p GEX-4T1 had higher expression than p ET-16 b vector when comparing p ET-16 b and p ET-32 a. VP110-Trx fusion protein expressed by p ET-32 a was best. But no matter how optimize the condition, the full-length of vp110 only be expressed in inclusion bodies, inactive, that can be used as guidance for later experiments.