A New Recombinant Allophycocyanin And Primary Study Of Its Bioactivities

Phycobiliproteins are important light-harvesting pigment proteins in some alga. These proteins are widely used as food additives, dye, medical diagnose, medicine, fluorescence probes and so on, because of their strong fluorescence and many bioactivities, such as anti-oxidative, anti-inflammatory, anti-tumor or cell-growth promoting. Now, phycobiliproteins are mainly prepared from alga by isolation and purification. But this traditional method has some disadvantages, so it is essential to produce these proteins by genetic engineering.In this work, allophycocyanin gene (ape) was subcloned into an expression vector pET28(a), to express a new recombinant allophycocyanin. SDS-PAGE and Western blot analyses identified that the fusion protein his-tag allophycocyanin (HAPC) was highly expressed in Escherichia coli. Then the expression optimization of recombinant allophycocyanin gene in E.coli JM109(DE3) using lactose as inducer was studied. The influences of culture conditions were analyzed in shake flask, and the recombinant E.coli was finally cultured in a 5L fermentor. The result showed lactose could be used to induce expression of HAPC in E.coli JM109(DE3). And the expression could reach the level by using with EPTG as inducer. After adding lactose, the low culture temperature could increase the expression level of soluble target protein. So the inexpensive lactose could be used as an inducer to substitute IPTG in the expression of HAPC in E.coli JM109(DE3).The expressing product was then purified by Ni2"1" affinity chromatography gel. After Ni2+ affinity chromatography, the purity of HAPC reached 90%.The bioactivities of HAPC were also studied in this paper. As to the leucocytopenia mice model with cyclophosphamide(CTX), both of two concentrations of HAPC(1.5 > 4.5mg/kg) could resist to the decrease of white blood cells remarkably. And antitumor effect and leukopenic action of different concentrations of HAPC were used Sarcomal 80 mice. It was found the activity of raising white blood cells was very strong, expecially the concentration of 4.5mg/kg. And the effect of antitumor of HAPC did not apparently enhance combined with CTX, but could resist to the decrease of white blood cells induced by CTX. In addition. HAPC also exerted antioxidative effects in vitro experimental models.