Construction Of Expression System Of Pseudozyme Antarctica And P. Flocculosa And Expression Of Platelet-derived Growth Factor

Both Pseudozyme antarctica and Pseudozyme flocculosa are Basidiomycetes species belonging to filamentous fungi. There are many advantages using them in a recombinant protein expression system: 1) The fermentation of filamentous fungi is a mature industry technology, which is beneficial to produce heterologous proteins using filamentous fungi as hosts in industry; 2) They are ideally suitable to improve protein production because of their strong secretion ability; 3) The new expression system can overcome many limitations of bacterial recombinant protein production; 4) The glycosilation to the protein is more approachable to that of mammalian. In this paper, the first objective is to screen the hsp70 promoter which can transcript the hygromicine gene, and to construct the co-transformation system of Pseudozyme antarctica with the hsp70 promoter and hygromicine gene. The co-transformation system includes two plasmids: pSceI-hyg and pVectour libre. The plasmid pSceI-hyg consists of Hsp70 promoter, hygromicine gene and hsp70 terminator while the plasmid pVectour libre only contains hsp70 promoter and terminator in order to be inserted by interesting genes.The green fluorescent protein gene (GFP) was used as a report gene to test the expression system. The specific gene primers were designed based on GFP sequence, then GFP gene was obtained by PCR, and inserted into pVectour libre to achieve the recombinant vector pgGFP.hsp, which was ready for the co-transformation with pSceI-hyg.Meanwhile, the specific primers were designed to amplify the gene including hsp70 promoter, GFP gene and hsp70 terminator using pgGFP.hsp as a template. Then the gene was inserted into vector pSceI-hyg to construct the single-transformation system. Specific fluorescence can be observed from both co-transformation and single-ransformation system in P. antarctica and P. flocculosa using a fluorescent microscope.Two experiments were performed for increasing the protein expression in single transformation system: 1) The hsp70 promoter was successively deleted each 100bp from 5'end in order to weaken the promoter, which increased the copy numbers of the interesting gene. The experiment results showed, the numbers of the colonies decreased with the shortening of the promoter. The Southern-blot revealed the copy numbers of the transformation gene increased by 1/3 in -100 and -200 transformants.