盐藻LHCB3蛋白的表达纯化及抗体制备

Expression, Purification and Antibody Preparation of LHCB3 Protein in Dunaliella salina

为了解盐生杜氏藻(Dunaliella salina)主要光捕获蛋白LHCB蛋白的功能,将已获得的盐藻Lhcb3基因构建到原核表达载体pET32a-DsLhcb3,通过优化表达条件,建立了高效的重组系统.pET32a-DsLhcb3在大肠杆菌中的优化表达条件为1 mmol/L IPTG在37℃下诱导4 h.采用镍离子亲合层析纯化获得LHCB3蛋白,并以此为抗原制备了多克隆抗体,经琼脂糖扩散检测效价,在1:16处有明显沉淀.提取盐藻总蛋白,经过制备的LHCB3抗体杂交,在29 000处获得两条明显的杂交条带,为进一步研究盐藻LHCⅡ蛋白表达机理奠定了基础.

Light harvesting chlorophyll a/b-binding protein （LHCB） is the major component of antenna complex of photosystem Ⅱ. In order to investigate its functions, Lhcb3 gene in Dunaliella salina was expressed in Escherichia coli and the fusion protein was obtained. The recombinant vector pET32a-DsLhcb3 was transformed into E. coli, and the preparation for expression was optimized in order to construct a more effective recombinant system. This experiment suggested that the optimized expression procedure of pET32a-DsLhcb3 in E. coli was induced by 1 mmol/L IPTG at 37 ℃ for 4 hours. LHCB3 protein was purified by Ni2＋ NTA agarose beads, which in turn the polyclonal antibody was achieved. Significant precipitation was detected at 1 ： 16 by the agarose diffusion test. The LHCB3 antibody was then hybridized with total protein, and the test showed two hybridization bands at 29 000. This research laid a favorable foundation for further explanations of LHCⅡ mechanisms in D. salina. Fig 4, Ref 16