Studies On Neutralizing Antibody And Proteins Involved In Infection Of White Spot Syndrome Virus (WSSV) Utilizing Phage Display Technology

White Spot Syndrome, causing high mortality and economic damage, has become a major threat to shrimp culture all over the world since its outbreak in 1992. White Spot Syndrome virus (WSSV), the viral pathogens of the syndrome, has been erected as a new spice belongs to a new family, and its structure is very complex. Therefore, it is difficult to infer molecular mechanisms of infection from comparisons to known viruses. Since a shrimp continuous cell line has not been established, it is difficult to study on the neutralizing antibody and viral proteins involved in infection. Thus, we did some researches about neutralizing antibody against WSSV and viral protein involved in infection using phage display technology.An in vitro neutralizing screening method with high throughput was constructed based on shrimp lymphoid primary cell culture. From the scFv antibodies against WSSV that were obtained in previous work, one neutralizing scFv antibody, named as P1D3, was identified by using the in vitro screening method. The neutralizing ability of scFv P1D3 has been testified by in vivo neutralization test. Since scFv antibody can be expressed in large amounts in bacterial hosts, and it can be produced quickly and cheaply, such antibodies have the potential to provide unlimited supplies of specific reagents.To perfect the in vitro neutralizing screening method described above, a novel fluorescent quantitative PCR (FQ-PCR) assay was constructed for the quantification of WSSV particles and was utilized in the in vitro test. Since WSSV particles could be added directly to the assay without a separate DNA extraction step and could be quantified exactly, the in vitro neutralization test could be performed more conveniently and accurately.A phage display random decapeptide library using pCANTAB 5 E as vector with high titer was successfully constructed to select and identify mimic antigen epitope of scFv P1D3. ScFv A1, which can bind WSSV specifically, was used to pan against the random peptide library. A series of positive clonies, which can bind scFv A1 specifically, were obtained. The mimic antigen epitope of the scFv A1 was deduced...