EXPRESSION OF ScFv-A1 OF AGAINST-WHITE SPOT SYNDROME VIRUS(WSSV)IN YEAST 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 is 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.In our research,a gene of ScFv-A1 against White spot syndrome virus(WSSV)from shrimp was amplified by PCR from phagemid vector pCANTAB5E and cloned into the E.Coli-yeast shuttle vector-pPIC9K,yielding expression vector pPIC9K-ScFv-Etag.The correct inserting was confirmed by PCR,BglⅡ enzyme digestion and DNA sequencing.Then the recombinant vector pPIC9K-ScFv-Etag was transformed into the yeast strain GS115(Pichia pastoris GS115)by sephroplasting and the ScFvA1 was expressed under the control of the AOX1 promoter.After induced by 2%methanol every 24 hours for 96 hours,the ScFvA1 could be secreted into the supernatant.From the SDS-PAGE we found that the molecular weight of the protein was about 32KD.Its WSSV binding activity could be detected by ELISA.And we found that its WSSV binding was 6—7 times higher than negative comparison.The result suggests that the genetically engineered single chain antibody can be expressed in the eucaryotic system successfully.It means that we can use yeast as a expression system to produce the antibody to detect,prevent,and inhibit the White spot syndrome virus in the near future.