【作者】 陈一；

【导师】 曹小红；

【作者基本信息】 天津科技大学 ， 食品科学， 2003， 硕士

【摘要】 开发功能性食品或新药用以增强机体的免疫功能是近年来食品生化领域的研究重点，当前的免疫调节制剂多从植物和哺乳动物的血液中提取，成本高且安全性难以保障，工程蝇蝇作为具有强大免疫功能的资源昆虫，具有养殖简单，繁殖迅速等特点，有望成为低廉且安全的免疫制剂来源。 本论文采用家蝇作为工程蝇种，对于家蝇免疫活性物质—凝集素的纯化和生理活性进行了系统的研究，首次采用戊二醛固定化红细胞吸附法粗提凝集素，以及研究了家蝇凝集素的抗菌和免疫调节功能，为开发家蝇凝集素这一新型免疫制剂提供了理论依据。同时，为了保证资源的综合利用，研究了家蝇中所含的另一类免疫活性物质—甲壳素与壳聚糖的提取工艺。 采用戊二醛固定化红细胞吸附法结合Sepharose-4B亲和层析纯化凝集素。戊二醛固定化红细胞吸附法为第一步粗提，红细胞浓度为15％，将红细胞与血淋巴混合吸附，离心后得到红细胞—凝集素混合物，再用半乳糖解吸附，所得粗提物血凝比活力提高了533倍，可用于其性质的初步研究。进一步用Sepharose-4B亲和层析柱（1.1cm×30cm）纯化，加样量2ml，样品浓度1g/L，所得纯化产物比活力提高到血淋巴的1066倍。 亲和层析后的家蝇凝集素电泳表现为单一糖蛋白，其分子量为84KD，SDS聚丙烯酰胺凝胶电泳（考马斯亮蓝G-250染色）表明家蝇蛹凝集素含有三个亚基，分子量分别为16KD，18KD，和50KD。对凝集素用shiff’s试剂进行糖蛋白染色，表明分子量为16KD和18KD的两个亚基含有糖结构。同时用SephadexG-200凝胶过滤柱（1.1cm×30cm）测定纯化产物的纯度和分子量，结果凝胶过滤图谱上表现为单一峰，通过标准曲线测得家蝇凝集素分子量为86KD。 本纯化方法得率为1.5mg凝集素/30g蛹，用苯酚—硫酸法所得凝集素含有12.3％中性糖。 血凝活力和糖结合实验表明家蝇凝集素专一性识别D-半乳糖，最低结合浓度为100mmol/L，不识别D-葡萄糖、D-甘露糖、果糖、N-乙酰-D-葡萄糖胺、乳糖、麦芽糖、蔗糖和D-核糖，其血凝活力对热不稳定，不依赖Ca²⁺，在pH6～9之间稳定。 家蝇凝集素对Escherichia coli，Bacillus subtilis和Salmonella typhi有不同程度的抑制作用。利用平板计数测得家蝇蛹凝集素对三种菌的抑制比率为39.8％，45.3％和66.2％。 对小白鼠的T淋巴细胞转化实验表明家蝇凝集素具有促进哺乳动物细胞免疫功能的作用在浓度大于 15石 n g／ml时，作用显著，溶血空斑实验表明家蝇凝集素具有同时促进体液免疫功能的作用。 通过对不同盐酸浓度，不同反应时问的除灰分的测定，确定以 2 molth为盐酸浓度，在室温下反应 lh即可。以 1：10的比例门 ig蛆粉刊 mol／L盐酸)进行反应。 通过对不同碱浓度，不同反应时间，不同温度及不同实验方法，确定除蛋白的反应条件为，在沸水浴中，先用 2．smol／L NaOH与样品反应 4h后，过滤，再用 1．5 mol／L NaOH与样品反应 4h，则蛋白能够基本脱除。 通过对不同碱浓度，不同反应时问，不同温度及不同实验方法，确定脱乙酚的反应条件为，加以浓度为40％的NaOH，在电炉上直接加热（40％NaOH沸腾温度为 118℃），反应 4h后，过滤，再加相同浓度的碱液，同样反应 4h，可得脱乙酚率较高的壳聚糖。更多还原

【Abstract】 Developing functional foods and new drugs to enhance the immunity is a main project in the field of food science and biotechnology. Nowadays immunity enhancers are mainly extracted from plant and blood of mammal. Their cost is high and their safety always calls problems. Musca domestic is an insect with high immune system which is easily to get. It is prosperous to be a new source of immune reagent.This paper systematically studied the purification and bioactivity of from Musca domestic pupae lectin(MPL). Isolation by glutaraldehydeized red blood cells (GRBC) adsorption and study of antibacterial activity and immunoactivity were first reported in this paper. To maximumly utilized the resource, another immunoactive compounds (chitin and chitosan) were isolated from Musca domestic.The purification procedure included glutaraldehydeized red blood cells (GRBC) adsorption and then affinity chromatography on Sepharose-4B. Diluted hemolymph was mixed with GRBC and incubated for 2.5h at RT under gentle agitation. Then, GRBC was precipitated to and the mixture of lectin-GRBC was obtained. Desorption of the proteins on GRBC was carried out using D-galactose solutions. The specific activity of crude extract was 533 times larger than the hemolymph. The specific activity of affinity-purified MPL was 1066 times larger than the hemolymph.The purified MPL was thought to be pure as it appeared as a single band in native PAGE. By SDS-PAGE with 2-mercaptoethanol, MPL yielded 3 bands corresponding to 16 KDa, 18 KDa and 50 KDa, respectively. By gel filtration, the affinity-purified MPL eluted as a single symmetrical peak. Compared with standard proteins its molecular mass was estimated to be 86 KDa .About 1.5 mg of MPL was obtained from 30 g of pupae. Carbohydrate estimation indicated that MPL contained about 12.3% neutral sugar.Hemagglutination and hemagglutination inhibition assay showed that MPL recognized D-galactose and did not recognize D-glucose, D-mannose, fructose, N-acetyl-D-glucosamin, lactose, maltose, sugar and D-ribose.Antibacterial activity of MPL has been observed against Escherichia coli, Bacillus subtilis and Salmonella typhi. The results of lymphocyte transformation test showed that MPL can increase cellular immunity and the result of plaque-forming cell assay showed that MPL can increase humoral immunity.Different conditions were studied to remove ash and protein from Musca domestic and to remove acetyl from chitin. The best condition is: 1g Musca Domestic powder was mixed with 20 ml 2mol/L HCL and incubated for 1h at RT under agitation. Then it was mixed with 2.5 mol/L NaOH and incubated for 8h in two sessions surrounded by boiling water. Musca domestic chitin was mixed with 40%NaOH and heated directly on the electric cooker for 8h in two sessions.更多还原