Identification Of A Single-chain Antibody Fragment Against White Spot Syndrome Virus With Phage Displayed Antibody Library And Expression Of Fusion Protein EGFP-VP28

White Spot disease is at present the most serious viral disease for cultured shrimp, causing very devarsting and widespread losses in the shrimp industry around the world. The viral agent is White Spot Syndrome Virus (WSSV). Since poor understanding of WSSV and no sensitive cell line to this virus, up to now there is still no effective treatment applied.The purpose of this study was to select a phage-displayed single-chain variable fragment (scFv) that can bind WSSV. Coated as antigen, intact WSSV virions were panned against the single fold scFv libraries I+J (Tomlinson I+J). 75 positive monoclonal scFvs were obtained. Five scFvs designed as IE2, IH4, JA2. JA3 and JC5 with highest OD values in ELISA were selected to be sequenced. IE2 and JA3, JC5 share an identical sequence, while 1H4 and JA2 share another identical sequence. So svFv IE2 and 1H4 were chose to do subsequent test. Both IE2 and IH4 were composed of 807 nucleotides, including V_H fragment of 351 bp and V_L fragment of 327 bp. Deduced amino acid sequences were composed of 268 amino acids and deduced molecular weights were both 28kD. IE2 have the sequence homology of 94.4% with IH4. The difference between the sequence of these two scFvs locus on the complementary-determining regions (CDR) of V_H and V_L CDR2 of V_H with 23.1%difference, CDR3 of VH with 20.0% difference and CDR3 of VL with 30.8% difference. The plasmids including positive scFv insert were isolated and transformed to expression bacteria E.Coli HB2151. ScFvs IE2 and IH4 were induced expression by IPTG and purifed by affinity chromatography with Ni resin. The concentration of purified scFvs were 0.89mg/mL and 0.90mg/mL respectively. Purified scFvs IE2 and IH4 were tested capable of binding with WSSV by immunoblot assay. Further primary cell experiments indicated that IH4 could inhibit the infection of shrimp primary lymph-like cell by WSSV to some degree. Furthermore, animal test showed WSSV infection was significantly delayed by IH4 in vivo. But IE2 can not delay or inhibit infection of WSSV both jn cell culture and in animal test. The results suggest that IH4 have potential for exploitation as diagnostic or even antiviral therapeutic reagents.The gene of envelope protein VP28 was amplified from WSSV DNA. The PCR product was added one restriction site EcoRI to one terminal and added another restriction site Sail to the other terminal by designed primers. Consequently vp28 was inserted into expression vector pET28a. Then egfp fragment was cut from pEGFP-Nl by Sail and Notl and inserted into the site between Sail site and NotI site of pET28a. Thus egfp gene insert behind vp28 gene. Because both genes were inserted into the same open reading frame promoted by the same T7 promoter, VP28 and EGFP can be fused expression. Fusion protein VP28-EGFP was purified by affinity chromatography with Ni resin. This fusion protein VP28-EGFP was expressed in order to explore the involvement of VP28 in entrance and infection of WSSV to shrimp cells indicated by green fluenscence marker.