Expression Of Spring Viremia Of Carp Virus Glycoprotein In Insect Cells And Selection Single-chain Recombinant Antibodies Against SVCV And IHNV

Spring viremia of carp (SVC), a severe viral disease of cyprinid, is caused by spring viremia of carp virus (SVCV). Infectious Hematopoietic Necrosis (IHN), a severe viral disease of mainly Pacific salmon and trout in enzootic areas in western North America, is caused by infectious hematopoietic necrosis virus (IHNV). SVCV and IHNV are the members of Rhabdoviridae. The outbreaks of the diseases caused great economic losses with the mortality as high as70%-90%and became factors restricting aquaculture and the development of fisheries. Glycoprotein of SVCV is a major antigen which elicits immune responses. Most monoclonal antibodies against SVCV can react with SVCV/G and DNA vaccines containing the SVCV/G gene can protect fish against SVCV. So SVCV G expressed in insect cells by recombinant baculovirus is important for the study of SVCV glycoprotein biological activity. The antibody phage display technology for the production of recombinant antibodies has several advantages:rapid culture of phage clones; easy handling and detection of secreted antibodies; lower costs compared with monoclonal antibodies (MAbs). Therefore it is a powerful technology for selecting and engineering polypeptides with novel functions and has been used widely to produce antibodies.In this study, baculovirus expression system was used to express glycoprotein of SVCV. The glycoprotein gene was cloned into the pFastBacHTA vector, followed by transformation into competent cells DH10αBac for three antibiotics and blue-white selection. The positive recombinant bacmid-G was transfected to Sf9cell in logarithmic growth phase with Cellfectin Reagent induction to obtain recombinant baculovirus AcNPV-G. A specific57kDa protein band and hybridizing reaction between expressed protein and positive serum of SVCV were observed by SDS-PAGE and Western blot. For indirect immunofluorescence, specific fluorescence signals were observed in Sf9cells infected by AcNPV-G. The results indicated that SVCV glycoprotein had been expressed in Sf9cells with immunological activity.Antibody-displaying phage library was selected after three rounds of panning against SVCV by phage display technology. Eight positive clones which could produce soluble single-chain fragment variable (scFv) antibody induced by IPTG were obtained. Dot blot results showed that the eight scFv antibodies could recognize SVCV. The soluble scFv antibodies showed a molecular weight29kDa by Western blot. All scFv antibodies could specifically recognize SVCV proteins without cross-reaction with other virus proteins by ELISA. Indirect immunofluorescence results showed that all of these scFv antibodies reacted positively with virus in the SVCV-infected cells. These scFv antibodies will be useful tools to establish immunological detection methods for SVCV.Six single-chain fragment variable (scFv) antibodies against infectious hematopoietic necrosis virus (IHNV) were selected from an antibody phage display library by phage display technology. The soluble scFv antibodies showed a molecular weight32kDa by Western blot. Dot blot analysis revealed that the six scFv antibodies could specifically recognize IHNV. For enzyme linked immunosorbent assay (ELISA), four scFv antibodies (P1A4, P1A12, P1D5and P3E2) showed cross-reactivity with spring viremia of carp virus (SVCV). And, none of the six scFv antibodies had cross-reaction with pike fry rhabdovirus (PFRV), soft-shelled turtle iridovirus (STIV), viral haemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). Indirect immunofluorescence results showed that all of these scFv antibodies reacted positively with virus in the IHNV-infected cells. These scFv antibodies will be useful in diagnostic test development and pathogenesis studies for IHNV.