鳕鱼皮免疫活性肽的制备

Preparation of Immunoregulatory Peptides from the Skinof Gadus macrocephalus

本研究旨在探究鳕鱼皮免疫活性肽的最优制备条件以及对RAW 264.7巨噬细胞免疫功能的影响。实验以鳕鱼皮为原料,选用胃蛋白酶、碱性蛋白酶、胰蛋白酶以及木瓜蛋白酶对其进行酶解,通过MTT法细胞增殖实验,以巨噬细胞RAW 264.7的相对增殖率为筛选指标,从4种实验酶种里选出最佳酶种;通过单因素和L9(34)正交试验在最佳酶种的基础上确定最佳酶解条件。将酶解液超滤分成4组分子段,检测不同分子段对RAW 264.7细胞的增殖率,并在倒置显微镜下观察细胞形态的变化。结果表明提取鳕鱼皮免疫活性肽的最优蛋白酶为碱性蛋白酶,其最优酶解条件:酶解时间7 h、加酶量2500 U·g^(-1)、pH为8、酶解温度50℃,经超滤得到的<1 kD的肽段对RAW 264.7细胞有明显的促增殖作用。随着酶解液浓度的升高,细胞出现分化和伪足的数量也明显增多。因此碱性蛋白酶酶解所得的鳕鱼皮活性肽能够增强RAW 264.7细胞的免疫活性。

The purpose of this study is to explore the optimal preparation conditions of immunoregulatory peptides from the skin of Gadus macrocephalus and its effect on the immune function of RAW 264.7 cells.The skin of G.macrocephalus were used as raw materials and pepsin,alkaline protease,trypsin and papain were used for enzymatic hydrolysis.The proliferation rates of RAW 264.7 cells were used as the index for enzyme screening.Single-factor and L9(34)orthogonal tests were used to optimize the extraction conditions of immunoregulatory peptides from the skin of G.macrocephalus.The hydrolysates were isolated by ultrafiltration membranes and then divided into four parts according to Mw distributions.The proliferation rate of different molecular segments on RAW 264.7 cells was detected,and the changes in cell morphology were observed under inverted microscope.The results showed that alkaline protease was the best protease to extract immunoregulatory peptides from the skin of G.macrocephalus,and the optimized conditions were as follows:hydrolysis time of 7 h,volume of enzyme of 2500 U·g^(-1),pH of 8,and hydrolysis temperature of 50℃.Furthermore,it was found that peptides with a Mw of less than 1 kD had a significant proliferation effect on RAW 264.7 cells, and the number of cell differentiation and pseudopod also increased significantly. Therefore, the immunoregulatory peptides from the skin of G. macrocephalus hydrolyzed by alkaline protease can enhance the immune function of RAW 264.7 cells.