Research on Secretion Expression in Pichia pastoris and Function of the HC-Pro Gene of Watermelon Mosaic Virus

Abstract:

HC-pro gene of Watermelon Mosaic virus was obtained by RT-PCR was 1371bp in length. It was cloned into pPIC9K, then the eucaryotic recombinant expression plasmid pPIC9K-WHC was constructed. After being linearized with restriction endonuclease SalⅠ, the recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation. The high copy transformants with Mut⁺/His⁺ phenotype were selected by RT-PCR and screening on G418, MD and MM medium. Induced by methanol for 5 days, the culture supernatant was analyzed by SDS-PAGE，the results showed that a specific protein with a molecular weight of about 66kD was expressed. Western blot analysis proved that the expression protein could specifically bind to HC-Pro polyclonal antibody. Far western blot analysis proved that the expression protein could bind to coat protein, given support to “bridge" hypothesis that HC-Pro help aphid transmission of non-persistent viruses.