Research on secretion expression in Pichia pastoris and function of the HC-pro gene of watermelon mosaic virus.
- Author:
Jian-Xin ZHANG
1
;
Yun-Feng WU
;
Xiu-Min WANG
Author Information
1. Colllege of Plant Protection and Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, Yangling 712100, China.
- Publication Type:Journal Article
- MeSH:
Citrullus;
virology;
Cysteine Endopeptidases;
biosynthesis;
genetics;
Mosaic Viruses;
genetics;
Pichia;
genetics;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
metabolism;
Viral Proteins;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2007;23(6):1102-1106
- CountryChina
- Language:Chinese
-
Abstract:
HC-pro gene of Watermelon Mosaic virus was obtained by RT-PCR was 1371bp in length. It was cloned into pPI(9K, then the eucaryotic recombinant expression plasmid pPIC9K-WHC was constructed. After being linearized with restriction endonuclease Sal I , 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 66 kD 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.
