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

Introduction: Cut aneo us lesihmahiasis (L) is a world wide infect io us diseas e . Several approach toward vaccine have been taken around the world . Recombinant vaccines using gp63 in cocta il form is one of the candidates. Because, a significan t 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 th e development of Th-I subset of CD4+ T cells. Since expression of eukaryotic genes are similar to Pichia pastoris as a eukaryotic cell, refolding and glycosylation may be an alogous to native form. GP63 gene from L. major (NIH strain) cloned and expressed in Pichia pastoris . Materials and Methods: Modified gp 63 ge ne th at encoded mature protein o nly (478 aa ) cloned in shuttle vector pHIL-Sl. Under contro l of alcohol oxidase 1 gene promotor (pAOXI) with yeast acid phosphatase signal sequence (PHO I). KM71 and GSI 15 strains of Pichia pastoris transformed with it and selected on histidin e minus medium. PCR and Southerbn blotting were done on chromosomal DNA of transform ant s yeast. Expressio n of rgp63 was evaluated by using Northern blotting, SDS-PAGE, Western-blott ing and immuno electro n micro sco py. Its activity was evaluated by using SDS-PAGE gelatin gel. Results: PCR and Southern blott ing a na lys is o n chromosomal DNA o f t rnsfo rma nts have bee n demons tra ted th at gp63 gene in te grated into chro mosomal DNA of ye as t. Expressio n of rgp63 in transformants of Pichia past oris confirmed by using Nor thern blott ing and SDS-PAGE. The findings were cinfirmed by Western blotting analysis and immunoelectron microsco py too. Conclusion: Rgp63 expressed in Pichia pastori s had glcosylated form too. It was act ive on SDS-PAGE gelatin gel. Thus, it was more similar to native gp63.