The Pharmaceutical Research And Mechanism Exploration Of Recombinant Allophycocyanin (rAPC)

The recombinant allophycocyanin (rAPC) was expressed in our lab, and developed as a novel anti-tumor drug. The anti-tumor mechanism of rAPC was studied, and different allophycocyanin subunits were expressed in Escherichia coli to evaluate their anti-tumor activities in vivo. The amp gene was replaced with kan coding sequence in expression vector pMAL-p2X/APC of rAPC according to Chinese Pharmacopoeia. The constructed strain TB1 containing pMAL-p2X/APC had been sub-cultured serially to 100th generation in Kan+ plate to study the heredity stability of the recombinant plasmid. The result showed that there were no significant differences in growth rate, restriction enzyme digesting chromatogram and expression level of rAPC in different generations. The bench scale fermentation and purification process of rAPC was scaled up into pilot scale process, and further optimized. The pilot scale process gave the same result with the bench scale. The expression rate of rAPC reached 41.3% of the total soluble proteins and the final optical cell density of the broth was 78, yielding up to gram level of rAPC with the purity over than 98% per batch operation. The quality control system was built up using different analytical methods, such as HPLC and ARB assay. Peptide mapping was used to assure the identity of rAPC in different batch. The impurities, such as endotoxin and residual host proteins, were also assayed according to Chinese Pharmacopoeia. The rAPC showed significant inhibition effect on human hepatoma cell line SMMC-7721 in nude mice. It could also increase the proliferation of T cells, enhancing the production of both IL-2 and IFN-γ, and the cytotoxicity of NK cells in vivo. The deoxyribose assay showed that rAPC could scavenge the hydroxyl radical remarkably. It was suggested that antioxidant and immunomodulatory mechanism would be partially responsible for the anti-tumor efficacy. Different subunits of the hybrid protein were cloned and expressed in E. coli, and their anti-tumor activities were assayed with S180 sarcoma model in vivo. The efficacy results showed that, within all of the expressed proteins, His-βAPC could inhibit the growth of S180 sarcoma and H22 hepatoma remarkably, which also showed significant inhibition on human hepatoma cell line SMMC-7721 in nude mice.