|Other Abstract||Since 1992, shrimp White Spot Syndrome Virus (WSSV) has caused great losses to the shrimp farming industry all over the world. WSSV can not only cause up to 100% mortality of shrimp within seven days , but also infect many other aquatic organisms, such as copepod, crabs. To date, there is no good method to cure this disease, therefore, the most important strategy is to prevent in advance.
Single chain fragment variable (scFv) consists of the variable fragment of the heavy chain and the light chain of antibodies, with a soft linker between them. It remains the ability to bind to the antigen as the native antibody, and is small, stable, easy to penetrate but hardly arises immune response. Further more, it is easy for gene manipulation and can be expressed in many hosts. All these virtues make scFv have a great potential in diagnosis and therapy of diseases.
In order to study the mechanism of WSSV infection, and a new way to prevent the disease, we constructed a phage-displayed scFv cDNA library against WSSV , and have got some scFvs specifically binding to WSSV. Among these scFvs, one scFv, designated as P1D3, could neutralize WSSV in the shrimp lymphoid primary cell culture. So, we think about the feasibility to enhance the ability of shrimp to resist WSSV by passive immunization with P1D3.
When E. coli is used for the scFv expression, the scFv product may be contaminated by something of E.coli. E. coli is harmful to health of shrimp and environment. However, yeast is a kind of shrimp’s food and environmental friendly. In order to introduce scFv into shrimp body by feeding way, we tried to express P1D3 in Pichia pastoris. At last, we got a yeast colony that expressed P1D3 with high production. Simultaneously, we tried a neutralization test against WSSV with the yeast-expressed P1D3.
The results showed that we succeeded in expressing P1D3 in Pichia pastoris, intracellularly and secretively. The P1D3 expressed secretively showed more activity binding to WSSV than the P1D3 expressed intracellularly in yeast or in E. coli. After optimizing inducing conditions, the production of P1D3 by yeast secretion could reach 302 mg/L.
Neutralization test was carried out by intramuscular injection and oral way with P1D3 expressed by Pichia pastoris. The result showed that WSSV could not be neutralized evidently by scFv P1D3. The low density and instability of P1D3 may be related to this result. Otherwise, coaction of several antibodies may finally create an excellent neutralization of WSSV. Although we could not get a successeful result in this research, it still provided some basic methods and reference date for future research of virus neutralization in shrimp body.|