乳源免疫调节肽分离、纯化与鉴定及其功能的初步研究

发布时间：2018-09-19 06:58

【摘要】：1．乳源免疫调节肽的制备、纯化及鉴定 目的 探讨酪蛋白酶解产生免疫调节肽的最适条件，建立酶解产物的分离及其鉴定免疫调节肽的方法。方法 将酪蛋白酶解，其酶解产物经超滤膜过滤，用十二烷基磺酸钠——聚丙烯酰铵凝胶电泳(SDS-PAGE)方法分析其酶解情况。用反相高效液相色谱层析(RP-HPLC)，C_(18)柱，以免疫调节肽为标准已知样，利用保留时间外标法与内标法，与已知对照物进行定性，鉴定酪蛋白酶解产物中的免疫调节肽。酶解上清液经Sephadex G-15凝胶层析分离，得到不同级分，然后用RP-HPLC鉴定各级分中的免疫调节肽。结果 酪蛋白在胰蛋白酶与糜蛋白酶组合下水解90分钟，能产生PGPIPN量最多，大约5％。酶解产物经Sephadex G-15凝胶层析后，酶解产物中小分子物质被分开，经RP-HPLC检测，Sephadex G-15的第5级分含有免疫调节肽。结论 酪蛋白经过胰蛋白酶和糜蛋白酶酶解后可以生成免疫调节肽，并可以通过Sephadex G-15粗分免疫调节肽。 2．乳源免疫调节肽对大鼠生长和机体免疫的影响 目的 探讨乳源免疫调节肽对大鼠机体免疫功能，增重和采食量的影响。方法 42只40日龄的SD大鼠(雌雄各一半)随机分为两个试验组和一个对照组，每组14只雌雄各一半，，分开饲养。预饲一周后，实验组分别被灌喂2.5×10~(-3)g·L~(-1)免疫调节肽(试验组Ⅰ)、2.5×10~(-2)g·L~(-1)免疫调节肽(试验组Ⅱ)：对照组灌喂生理盐水，并记录大鼠日采食量和体重。饲养一个月后分别检测对照组和试验组的淋巴细胞转化、红细胞免疫、巨噬细胞吞噬、日采食量和体重等的差别。结果 乳源免疫调节肽对大鼠腹腔巨噬细胞吞噬和红细胞免疫有显著促进功能(p＜0.05)；而对淋巴细胞转化作用有增长的趋势，但未达到显著水平(p＞0.05)。与对照组相比，实验组Ⅱ的大鼠日采食量显著地增加(P＜0.05)，雌雄大鼠分别提高了
[Abstract]:1. Preparation, purification and Identification of Milk-derived Immunoregulatory peptides objective to study the optimal conditions for casein enzymatic hydrolysis to produce immunomodulatory peptides. To establish a method for the isolation and identification of immunomodulatory peptides. Methods casein was hydrolyzed by ultrafiltration membrane, and the enzymatic hydrolysis of casein was analyzed by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE). The immunoregulatory peptides in casein enzymatic hydrolysis products were identified by reversed-phase high performance liquid chromatography (RP-HPLC) with C _ (18) column, using immunoregulatory peptides as standard known samples, using retention time external standard method and internal standard method, and qualitative analysis with known controls. The supernatant of enzymatic hydrolysis was separated by Sephadex G-15 gel chromatography, and the different fractions were obtained, and then the immunomodulatory peptides were identified by RP-HPLC. Results casein hydrolyzed in combination of trypsin and chymotrypsin for 90 minutes resulted in the highest amount of PGPIPN, about 5%. After Sephadex G-15 gel chromatography, the small molecules in the enzymatic hydrolysis products were separated. The fifth fraction of Sephadex G-15 contained immunomodulatory peptides by RP-HPLC. Conclusion casein can be hydrolyzed by trypsin and chymotrypsin to produce immunomodulatory peptide. The effect of milk source immunomodulatory peptide on the growth and body immunity of rats was studied. Objective to study the immune function of milk source immunomodulatory peptide in rats. The effects of weight gain and feed intake. Methods Forty-two 40-day-old SD rats (half of male and female) were randomly divided into two experimental groups and one control group. After one week of prefeeding, the experimental group was fed with 2.5 脳 10 ~ (-3) g L ~ (-1) immunomodulatory peptide (test group 鈪

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