卵黄高磷蛋白磷酸肽的制备及其对小鼠免疫功能的影响

发布时间：2018-06-26 20:41

本文选题：卵黄高磷蛋白磷酸肽 + 生长性能　；参考：《四川农业大学》2009年硕士论文

【摘要】： 为了初步探索卵黄高磷蛋白磷酸肽(PPP)是否具有免疫作用,本研究在制备了PPP的基础上,考察了PPP对小鼠体内外免疫功能的影响,并初步分析PPP影响机体免疫功能的机理。本研究包括以下三个试验。试验一,PPP的制备。本试验首先从鸡蛋黄中提取卵黄高磷蛋白(PV),经胰蛋白酶水解,水解液经截留分子量3 kDa和1 kDa的超滤膜超滤,取分子量在1 kDa-3 kDa之间的截留液冷冻干燥得到PPP。经SDS-PAGE电泳测PV的纯度,用N/P(摩尔比)检测PPP的磷酸丝氨酸残基密度。结果表明:所制备的PV样品与纯品电泳模式相近,在1 kDa-3 kDa范围内存在PPP电泳条带,且N/P比例为5.02。说明所提取PV样品相对较纯,PPP样品含有磷酸丝氨酸残基。试验二,PPP对小鼠体外免疫功能的影响。为了评价PPP对细胞增殖、吞噬功能、细胞因子以及免疫球蛋白生成的影响,分离小鼠的脾淋巴细胞和腹腔巨噬细胞并进行培养。结果显示:添加LPS或ConA后,显著地促进了细胞的增殖、细胞吞噬功能、细胞因子的分泌以及免疫球蛋白的生成(p＜0.05)。终浓度为0.02 mg/mL的PV组不能显著地影响细胞功能(p＞0.05)。然而,终浓度为0.02 mg/mL的PPP组和0.1 mg/mL PPP组显著地提高了刺激指数(SI)和吞噬指数(PI),以及IgA、IL-1β、IL-2和IL-6的生成。LPS或ConA诱导组比无丝裂原组刺激作用显著增强。结果表明,PPP显著提高了SI、PI、细胞因子以及免疫球蛋白生成的影响,无丝裂原组相比,LPS或ConA诱导下,以上各指标均显著提高。试验三,PPP对小鼠体内免疫功能的影响。为了确定胃饲PPP对小鼠免疫功能的影响,将48只小鼠随机分到3个处理中,每个处理设4个重复,每个处理的小鼠在基础日粮的基础上分别隔天灌胃0.2mL生理盐水(对照组)、PPP和PV溶液,按体重喂给0.03mg/(g·d)。14天后取样确定小鼠的生长性能和免疫功能。结果显示:与对照组和PV组相比,PPP组小鼠的体增重极显著提高(p＜0.01),血清总蛋白、白蛋白、球蛋白和葡萄糖均升高,但差异不显著(p＞0.05),而血清尿素氮、总胆固醇和甘油三酯呈下降趋势,脾脏指数和胸腺指数呈显著提高(p＜0.05)。PPP组的SI和PI显著提高(p＜0.05),血清和脾淋巴细胞培养液中的IgA、IL-2和IL-6浓度,以及血清和腹腔巨噬细胞培养液中的IL-1β均显著地上升(p＜0.05)。PV组与对照组相比,这些指标均无显著变化(p＞0.05)。在LPS或ConA诱导下,培养液上清中的SI、PI、IgA、IL-1β、IL-2和IL-6均有显著的提高(p＜0.05)。结果表明,胃饲PPP不仅提高了体增重,脾脏指数和胸腺指数,还显著增强了小鼠SI和PI,血清和脾淋巴细胞培养液中的IgA、IL-2和IL-6浓度,以及血清和腹腔巨噬细胞培养液中的IL-1β浓度。与无丝裂原组相比,LPS或ConA诱导组以上各指标均显著提高。综合以上各试验可看出,制备所得的PPP不仅提高了小鼠的生长性能,还增强了小鼠体内外免疫功能。
[Abstract]:In order to explore the immune effect of phosphopeptide (PPP) on yolk high phosphorous protein (PPP), the effect of PPP on immune function of mice in vivo and in vitro was investigated on the basis of preparation of PPP, and the mechanism of PPP affecting immune function in vivo and in vitro was preliminarily analyzed. This study includes the following three experiments. The preparation of PPP. In this experiment, egg yolk high phosphorous protein (PV) was extracted from egg yolk. The protein was hydrolyzed by trypsin, and then purified by ultrafiltration membrane with molecular weight of 3 kDa and 1 kDa. PPP was obtained by freeze-drying of the retention solution with molecular weight between 1 kDa and 3 kDa. The purity of PV was measured by SDS-PAGE, and the PPP's serine residue density was measured by N / P (molar ratio). The results showed that the electrophoretic patterns of the prepared PV samples were similar to those of the pure samples. PPP bands were found in the range of 1 kDa-3 kDa, and the ratio of N / P was 5.02. The results showed that the extracted PV samples contained relatively pure PPP containing phosphate serine residues. The effect of PPP on the immune function of mice in vitro. To evaluate the effects of PPP on cell proliferation, phagocytosis, cytokine and immunoglobulin production, spleen lymphocytes and peritoneal macrophages of mice were isolated and cultured. The results showed that LPS or Cona significantly promoted cell proliferation, phagocytosis, cytokine secretion and immunoglobulin production (p < 0.05). PV with 0.02 mg / mL final concentration did not significantly affect cell function (p > 0.05). However, the stimulation index (SI) and phagocytic index (Pi) were significantly increased in the PPP group with 0.02 mg / mL final concentration and 0.1 mg / mL PPP group, as well as the production of IL-2 and IL-6 in IgA IL-1 尾, IL 2 and IL 6 production. LPS or Cona induced stimulation was significantly higher than that in the mitogen free group. The results showed that PPP significantly increased the production of SIPI, cytokines and immunoglobulin, and the above indexes were significantly higher in mitogen free group than that in LPS or Cona group. The effect of PPP on immune function in mice. In order to determine the effect of gastric feeding PPP on immune function of mice, 48 mice were randomly divided into 3 treatments, each of which was divided into 4 replicates. Each treated mice were fed with 0.2 mL normal saline solution (control group) every other day on the basis of basal diet. The growth performance and immune function of the mice were determined by 0.03mg/ (g d). 14 days later, the growth performance and immune function of the mice were determined. The results showed that compared with the control group and PV group, the body weight gain of PPP group was significantly increased (p < 0.01), the serum total protein, albumin, globulin and glucose were all increased, but the difference was not significant (p > 0.05), but the serum urea nitrogen. Total cholesterol and triglyceride decreased, spleen index and thymus index increased significantly (p < 0.05). The SI and Pi of PPP group increased significantly (p < 0.05). IL-1 尾 in serum and peritoneal macrophage culture medium were significantly increased (p < 0.05). Compared with control group, these indexes had no significant change in PV group (p > 0.05). Under the induction of LPS or Cona, the levels of IL 2 and IL 6 in the supernatant of Sipi Pia IgA 尾, IL 2 and IL 6 were significantly increased (p < 0 05). The results showed that PPP not only increased the body weight gain, spleen index and thymus index, but also significantly increased the levels of IgA, IL-2 and IL-6 in serum and splenic lymphocyte culture medium, and IL-1 尾 in serum and peritoneal macrophage culture medium. The above indexes were significantly higher in LPS or Cona-induced group than in non-mitogen group. From the above experiments, we can see that the prepared PPP not only improves the growth performance of mice, but also enhances the immune function of mice in vivo and in vitro.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1

【引证文献】