Fusion Of Single Chain Fragment Variable Against White Spot Syndrome Virus Of Shrimp With Glucose Oxidase And Expression In Pichia Pastoris

White spot syndrome virus (WSSV) from shrimp was first found in Taiwan in 1992. Since 1993, white spot syndrome disease of shrimp caused by this virus was widely spread throughout China, Asia and Pan-Pacific Ocean. It's a great harm to the aquaculture of shrimp. Unfortunately, we still haven't found a powerful method to protect or treat shrimp from this disease. So it is important to detect this virus in short time. Our research purpose is to fusion scFv A1 against WSSV with glucose oxidase, and make this expressing product to detect the viruse directly by color. It may reduse a step of second antibody as normal immunological methods and makes the testing method more convenient. The cDNA fragment of Single chain antibody was fused to the 3,end of Aspergillus niger glucose oxidase gene with the insertion of a flexible linker peptide [(Ser-Gly)5] coding sequence. The insertion of a [(Ser-Gly)5] linker peptide was expected to minimize the steric hindrance between ScFv and GOD. At the same time, we amplified the ScFv-Etag gene from the phagemid vector pCANTAB5E- ScFvA1-Etag. The ScFvA1-Etag and the glucose-linker-ScFvA1 (GLS) fusion gene were cloned into the vector pPIC9K and expressed in methylotrophic yeast Pichia pastoris GS115, respectively, under the control of the AOX1 promoter. We found that the GLS had been degraded completely in the expressing system. So we turn to use methylotrophic yeast Pichia pastoris SMD1168, which is proteinase A deficient, as host cell. We found that not all of the GLS fusion protein had been degraded in thistime. The GLS fusion protein were purified using Q SepharoseTM Fast Flow ion exchange chromatography. The resulting GLS fusion protein with a molecular 130kDa maintained GOD activity and WSSV binding activity by scFv A1. The resulting ScFvA1-Etag protein has a molecular 32kDa weight. Through ELISA analysis, it was found that WSSV could be detected by both GLS fusion protein and ScFv-Etag.Our findings suggest that ScFv and GOD can be effectively used as fusion partners to construct a genetically artificial fusion protein and made the detecting method for WSSV more convenient.