Optimization Of Bitter Melon Ribosome Inactivating Protein-map30 Gene And Transformation Into Pichia Pastoris

Map30 (Momordica anti-HIV protein of 30KD) is a single-strand, type I ribosome inactivating protein, which isolated firstly from the fruit of balsam pear by Lee in 1990, reported to possess antiviral, anti-tumor protein and antimicrobial activity. MAP30 can affect the ribosomes of the virus and interference protein synthesis. In addition, MAP30 can induce tumor or tumor cells into cell apoptosis. MAP30 significantly inhibited HIV, herpes virus, hepatitis B, epidemic cerebrospinal meningitis, breast cancer, melanoma, influenza, also have difference among the inhibition. And what's more, its cytotoxicity is specific to tumor-transformed or viral-infected cells, while showing no adverse effects on normal cells, and MAP30 play a role in conserved regions of the substrate, it can not developed immunity to drugs.We have cloned the gene encoding the mature MAP30 protein from the total DNA of bitter melon and optimized gene by PCR amplification. After sequenced, the map30 gene was cloned into the vector pMD18T-Vector to construct the expression vector map30/pPGAPHa with the N-terminal His-tag. The map30/pPGAPHa vector was transformed into GS115 by electrotransformation, and the recombinant was identified by SDS-PAGE. At last, we confirmer recombinant plasmid has transferred into the host, and obtain the expression.Through SDS-PAGE analysis on supernate of recombinant yeast expression, it shows that recombinant protein was expressed. Western blot analysis indicated that the recombinant protein expression in a specific 30KD band, and the controls did not see that expression in 30KD. It confirms that the product of expression have antigenicity of His-tag and it also illustrates the fusion protein is map30 protein. Through SDS-PAGE analysis found that the level of gene expression of optimizing gene is more than no optimizing genes.