Black Fungus Ribosome Inactivating Protein Gene

Ribosome-inactivating protein (RIP) is a group of toxic protein widely distributed in the nature. Because of its biological activity to human and animal cell particularly and its available utility to treat acquired immune deficiency syndrome and cancer, it causes lots of interests. Although recently RIP reported mostly come from higher plants, it was also found in edible fungi. This study is mainly revolves around Auricuralia auricular.This study has successfully separated and obtained RIP from mycelium of Auricularia auricular and its molecular weight is 26KDa by determination, and then clones the gene of RIP based on its amino acid sequence. At first, sequencing of random amino acid sequence of RIP which is obtained by purification has been done. Design degenerate primer according to the results of sequencing, preference of codon of Auricularia auricular, and principles of design of primer. Applying cDNA 3′-end full-length amplification kit, 3′-RACE reaction is performed. After procedures of electrophoresis, cDNA fragments recovering, linking, converting, blue-white screening, plasmid extraction and PCR identification, cDNA 3′-end sequencing of RIP is completed. Design the nested primers according to the results of 3′-end sequencing and analysis of sequencing. Then, applying cDNA 5′-end full-length amplification kit, 5′-RACE reaction is performed. After similar procedures of electrophoresis, cDNA fragments recovering, linking, converting, blue-white screening, plasmid extraction and PCR identification, cDNA 5′-end sequencing of RIP is completed. At last, obtain the full-length cDNA sequence of RIP of Auricularia auricular by splicing the sequences of 3′-end and 5′-end. The entire cDNA is 760 nucleotides in length and encodes a protein of 234 amino acids with less sequence identity with other RIP.This project has not only developed an available method for gene cloning, but also done necessarily previous research for additional study of expression in vitro of RIP gene of Auricularia auricular.