关键词: 丝状真菌, 粗糙脉孢菌, 蛋白表达系统, 纤维二糖, 强启动子

Abstract: To demonstrate the potential of the model filamentous fungus Neurospora crassa as a recombinant protein expression system,we used fungal hybrid technology to develop a sextuple gene disruptant LQ-1（Δ3βG∷Δ2cbh∷Δhis3）of it as the host strain of expressing the protein. The cellobiose was used to induce the expression of cellulase promoter in host strain,which removed the background band of glucosaccharase and was conducive to the expression and purification of target protein. We constructed 3 new efficiently-expressed vectors respectively consisting of its own strong inducible promoters（Pcbh-1,Pcbh-2 and Ptef-1）from N. crassa. Those vectors had fusion tag protein tev-6×his-gfp and efficiently and conveniently screened positive transformants,which was favourable for the purification of later target protein. Two endogenous cellulase（GH3-4 and CBH-1）were chosen as test proteins for recombinant expression. Then enzymatic activity measured by cellobiose-induced fermentation broth of recombinant strain,SDS-PAGE analysis,and Western blotting indicated that the recombinant proteins GH3-4-GFP and CBH-1-GFP were successfully expressed with secreted levels reaching as high as approximate 2.77 and 2.83 mg/L. The expression of recombinant protein with Pcbh-1 promoter was the highest,suggesting that promoting efficiency of Pcbh-1 in the cellobiose-induced system was the highest,and a protein expression system in cellobiose-induced N. crassa was preliminarily constructed.

Key words: filamentous fungi, Neurospora crassa, recombinant protein expression system, cellobiose, strong inducible promoters