Prokaryotic Expression Of Flammulin CDNA Gene And Purification Of Recombinant Protein And Activity Research

This thesis is focused on a part of the National Nature Science Fund Project on Flammulin, which is one anti-tumor protein from a fungus, Flammulina Velutipes. Its English name is winter mushroom, an edible fungus. Flammulin is a simple protein without saccharide, its molecular mass is 23,000 Dalton, its pI value is 8.9, and it has no enzyme activity. In 1965, flammulin was firstly isolated from the Flammulina Velutipes by an Japanese, and was considered an anti-tumor protein. In vivo studies, Flammulin showed marked anti-tumor effects on Ehrlich ascites tumor and Sarcoma 180. Cytopathogenic changes in the tumor cell happened: Inhibition of mitosis, swelling of tumor cells, formation of vacuoles in both nuclear and cytoplasm, bubbling of the cells and finally cytolysis were observed. But the flammulin hardly had side effects on the animal bodies.My main work was on prokaryotic expression of Flammulin cDNA gene, purification of the recombinant protein and investigation on the characters of the recombinant protein. Because the protein is very low content in the fungus, I chose the method that building the Flammulin prokaryotic expression vector and making the protein expressed in E.coli to get a large number of the anti-tumor protein for the further investigation of character and clinical pharmacology.I amplified Flammulin cDNA gene by PCR, digested it with restriction enzyme and ligated the insert to prokaryotic expression vector pET32a(+), Novagen, Merk company. The recombinant plasmid was transformed into the E.coli. BL21(DE3), identified position clones, sequencing analysis and stored in 10% glycerol at -20℃.