中国生物工程杂志

pPIC9 was used as a template and α-factor gene of Saccharomyces cerevisiae was amplified by PCR.The gene was cloned in the intracellular expression vector pYES2/CT for Saccharomyces cerevisiae and the secreting expression vector named pYES2/CT/α-factor（pYCα）was constructed.The gene of mannase(man)from recombinant vector of pKLAC1-man（pKLman）was cut by restriction enzymes and linked with pYCα.This recombinant vector pYCα-man was used to determine the secretory ability and stability of pYCα.The excellent secretory ability of pYCα was proved by two experiments.One showed that INVSc1/pYCα-man clones formed the clear rings around the clones on the medium contained trypan blue,while INVSc1/pYCα clones had no rings.Further analysis of mannase activity of extracellular supernatant and intracellular extracts showed that both extracellular and intracellular mannase activities of INVSc1/pYCα were not detected,while INVSc1/pYCα-man had evident extracellular mannase activities and no intracellular mannase activities.The stability of pYCα was also very good proved by continuous cultivation for about 150h.