Pichia pastoris ليشماماژورد در مخمر متيلوتروف )gp36( توليد متالوپروتييناز 36 كيلودالتوني

The production of recombinant 63kDa glycoprotein of Leishmania major by methylotropic yeast Pichia Pastoris

Introduction: Cutaneous lesihmahiasis (L) is a world wide infectious disease. Several approach toward vaccine have been taken around the world. Recombinant vaccines using gp63 in coctail form is one of the candidates. Because, a significant protection against a challeng with L. major was elicited in senssitive BALB/C mice after vaccination with recombinant BCG producing gp63, as well as, gp63 delivered orally by salmonella typhimurium C an preferably induce the development of Th-1 subset of CD4+ T cells. Since expression of eukaryotic genes are similar to Pichia pastoris as a eukaryotic cell, refolding and glycosylation may be analogous to native form. GP63 gene from L. major (NIH strain) cloned and expressed in Pichia pastoris. Materials and Methods: Modified gp63 gene that encoded mature protein only (478 aa) cloned in shuttle vector pHIL-Sl. Under control of alcohol oxidase 1 gene promotor (pAOXl) with yeast acid phosphatase signal sequence (PHOl). KM71 and GS115 strains of Pichia pastoris transformed with it and selected on histidine minus medium. PCR and Southerbn blotting were done on chromosomal DNA of transformants yeast. Expression of rgp63 was evaluated by using Northern blotting, SDS-PAGE, Western-blotting and immunoelectron microscopy. Its activity was evaluated by using SDS-PAGE gelatin gel. Results: PCR and Southern blotting analysis on chromosomal DNA of trnsformants have been demonstrated that gp63 gene integrated into chromosomal DNA of yeast. Expression of rgp63 in transformants of Pichia pastoris confirmed by using Northern blotting and SDS-PAGE. The findings were cinfirmed by Western blotting analysis and immunoelectron microscopy too. Conclusion: Rgp63 expressed in Pichia pastoris had glcosylated form too. It was active on SDS-PAGE gelatin gel. Thus, it was more similar to native gp63.