The Construction of the Pichia Pastoris Expression Vector with the Enhanced Green Fluo-rescent Protein Gene as the Report Gene
Objective:To construct Pichia pastoris expression vector with eGFP as report gene . Methods:A fusion expression vector pPICZαB-eGFP was constructed to express enhanced Green Fluorescent Protein eGFP and the right Pichia pastoris transformants were selected on YPD plates containing Zeocin(100ug/ml) after lithium chloride transformation of strain X33.Then X33-eGFP was checked by the polymerase chain reaction using 3′-AOX/5′-AOX and eGFP-F/eGFP-R as the primer, and by agarose gel electrophoresis and sequencing .Finally, a new strain expressing eGFP named X33-eGFP was induced by formaldehyde .Results: Results from PCR and electrophoresis showed that the eGFP fragment had been successfully inserted into the X 33.Green fluorescence could be successfully observed when the mutant strain exposed in the blue fluorescence .Conclu-sion:Pichia pastoris expression vector with eGFP as report gene was constructed successfully , which can express GFP by its AOX promoter .
国家科技期刊平台
NSTL
万方数据
