念珠藻葛仙米藻蓝蛋白α与β亚基基因克隆、表达及其单体结构模拟

Cloning, Expression and Protein Monomers Structure Modeling of the Phycocyanin α and β Genes in Nostoc sphaeroides küzting

天然抗氧化剂对于清除人体内自由基、保障健康具有重要作用。藻蓝蛋白(phycocyanin,PC)是蓝藻等藻类细胞内的一类特殊色素蛋白,具捕获光能的功能。体外研究发现,藻蓝蛋白具有较显著的抗氧化、抑癌细胞增殖等生物活性。试验利用食源性的拟球状念珠藻葛仙米(Nostoc sphaeroides)的基因组DNA为材料,借助PCR扩增克隆葛仙米藻蓝蛋白两个重要亚基α与β基因,Ns-PC-α和Ns-PC-β。结果表明,葛仙米基因组中,Ns-PC-α和Ns-PC-β基因编码框长度分别为492 bp和522 bp;Ns-PC-α和Ns-PC-β基因以双顺反子形式存在,Ns-PC-α位于Ns-PC-β下游。Ns-PC-α和Ns-PC-β分别编码由163与173个氨基酸组成的蛋白。Ns-PC-α和Ns-PC-β亚基蛋白多肽链中共含有3个半胱氨酸残基(Ns-PC-α的第85位与Ns-PC-β中的第83位氨基酸残基和第154位氨基酸残基),很可能是藻蓝胆素的结合位点。原核表达显示Ns-PC-α和Ns-PC-β分子量为近18 kD,Ns-PC-α比Ns-PC-β略小,与预测结果一致。模拟其高级结构,发现Ns-PC-α和Ns-PC-β单亚基均含有7个α-螺旋,除N端α-螺旋外,其它几个α-螺旋进一步折叠形成疏水核心区;并且,Ns-PC-α和Ns-PC-β结构上能够较好吻合,形成一稳定而封闭的单体,弧度近2π/3。Ns-PC-α和Ns-PC-β组成的这种单体结构是其进一步建构圆盘状三聚体Ns-PC-(α/β)3的重要基础。本研究结果为食源性念珠藻葛仙米藻蓝蛋白抗氧化功能的进一步应用提供了重要依据。

Natural antioxidants play important roles in clearing free radicals and keeping healthy in human bodies.Phycocyanin(PC)is a special pigment protein in cyanobacteria and other algae cells,which expert the action of capturing light energy.In vitro studies have showed that PC possesses important activity of antioxidation and inhibition of tumor cell proliferation.In the assays,two important subunits of PCαandβgenes,Ns-PC-αand Ns-PC-β,were amplified and cloned using edible Nostoc sphaeroides genomic DNA by PCR.The results showed that the length of coding frames of Ns-PC-αand Ns-PC-βgenes were 492 bp and 522 bp,respectively.Ns-PC-αand Ns-PC-βgenes existed in the form of double cistron,and Ns-PC-αwas located downstream of Ns-PC-β.Ns-PC-αand Ns-PC-βencoded proteins with 163 and 173 amino acids,respectively.There are three cysteine residues in Ns-PC-αand Ns-PC-βpolypeptide chains(the 85 th position of Ns-PC-αand the 83 rd and 154 th position of amino acid residues in Ns-PC-β),which may be the binding site of phycocyanobilin.Prokaryotic expression showed that the molecular weights of Ns-PC-αand Ns-PC-βwere nearly 18 kD,and Ns-PC-αwas slightly smaller than Ns-PC-β,which was consistent with the predicted results.Stucture modeling indicated that both Ns-PC-αand Ns-PC-βsingle subunits contain sevenα-helixes.Except for the N-terminalα-helix,several otherα-helixes are further folded to form hydrophobic core regions.Moreover,Ns-PC-αand Ns-PC-βsingle subunits are well matched in structure,forming a stable and relative closed monomer with an arc of nearly 2π/3.The monomer structure composed of Ns-PC-αand Ns-PC-βis important for the trimer Ns-PC-(α/β)3 construction.The results of this study provide an important basis for the further application of antioxidant function of phycocyanin from the edible Nostoc sphaeroides küzting.