山羊羔肠道淋巴集结提取物的分离、效应检测及特异表达基因的获得

【作者】 陈小鹏；

【导师】 张燕君；

【作者基本信息】 山东大学 ， 发育生物学， 2005， 硕士

【摘要】 水产养殖业在我国国民经济发展中占有重要地位,在病害防治方面,通常使用大量抗菌素,造成环境污染并易产生抗药性,对人类健康造成危害。用生物免疫活性因子作为饲料添加剂来提高水产养殖生物抗病害能力,不破坏养殖区水体的生态,不损害养殖生物的生理机能,材料廉价易得,生产成本低。 本研究取出生15(±2)日的山羊羔,分离其肠道淋巴集结(peyer’s patch,PP),包括回肠淋巴集结(Ileum peyer’s patch, IPP)和空肠淋巴集结(Jejunumpeyer’s patch, JPP),经反复冻融,离心收集得到PP粗提物,作为饲料添加剂,检测了其对淡水白鲳多项免疫指标的影响。通过对白鲳白细胞吞噬活性、血清抗菌活力、血清溶菌酶及血清溶血素活性的测定结果发现,添加PP粗提物的实验组白鲳,各项免疫指标较只喂基础饲料的对照组显著提高,其中实验组白细胞吞噬活性达到52.33%,而对照组仅为20.67%,实验组与对照组相比增长达153%;实验组血清抗菌活力达0.675,而对照组仅为0.331,实验组与对照组相比增长达104%;溶菌酶活性基本呈同样变化规律,实验组达到0.15,而对照组平均在0.12,实验组与对照组相比增加了25%。这些结果表明PP粗提物可提高白鲳非特异性免疫功能。在添加PP粗提物后,实验组血清溶血素达到8.67,而对照组平均在4.33,实验组相对于对照组增加了100%,表明PP粗提物可提高白鲳特异性免疫功能。PP粗提物对白鲳血清酚氧化酶(phenoloxidase, PO)、超氧化物歧化酶(superoxide dismutase, SOD)活性提高的效果不明显。从PP粗提物对鱼体生长影响的实验结果看,添加PP粗提物饲喂15天后,白鲳总体重增长率为23.69%,而对照组的总体重增长率仅为10.98%,实验组比对照组多增重11.3%;对照组饵料系数为6.43,实验组为2.99,实验组相对于对照组饵料系数降低了53.5%,表明PP粗提物呵促进白鲳生长。这可能是由于PP提取物提高了鱼体的免疫力,使健康状况改善,或粗提物有促生长因子而表现出促进生长的综合效果。 为进一步确认PP粗提物中的活性组分,本实验采用硫酸铵分级沉淀、Sephadex G-25凝胶层析的方法对其进行了初步的分离纯化,共得18种组分。以小鼠脾淋巴细胞体外细胞培养作为特异免疫反应的模型,分别将18种PP组分更多还原

【Abstract】 Fish culture is an important industry in our country. Various chemotherapeutics have been used to treat bacterial infections in cultured fish in the last 20 years. However, the incidence of drug -resistant bacteria has become a major problem in fish culture, and results in ecological and environmental disturbances. Immune factors increase resistance to infectious disease, not only by enhancing specific immune responses, but also by enhancing non-specific defense mechanisms. Use of the Immune factors is an effective means of increasing the immunocompetency and disease resistance of fish.Cepra hircus(15±2 Day-old) was used as experimental animal. In order to measure the influence of peyer’s patch (PP) crude extract in vivo, 2 % PP crude extract was added to Colossoma brachypomum’s feed for 15 days. After 15 days several immune indexes were tested on Colossoma brachypomum. The results showed that adding PP crude extract to Colossoma brachypomum feed promoted the immunoresponse level, including phagocytic activity of phagocytes, serum antibacterial activity, serum hemolysin, serum bacteriolytic activity. Phagocytic activity of phagocytes was 52.33 % in experimental group and 20.67 % in control group, increasing by 153 %. Serum antibacterial activity was 0.675 in experimental group and 0.331 in control , increasing by 104 %. Serum bacteriolytic activity was 0.15 in experimental group and 0.12 in control group, increasing by 25 %. Serum hemoiysin value was 8.67 in experimental group and 4.33 in control, increasing by 100%. However, the increase of phenoloxiddase (PO) and superoxide dismutase (SOD) activity was not remarkable. Meanwhile, the body weight of fish in experimental group increased remarkably by 23.69 % compared with 10.98 % in control, and the conversion rate of food decreased by 53.5 % in comparison with control group.In order to further investigate the active component of PP crude extract, we purified it with ammonium sulfate fractionation and Sephadex G-25 ColumnChromatography. All 18 fractions of PP extract were collected. Lymphocyte culture was used as a main model to elucidate the effect of different fractions on the proliferation of mouse spleen lymphocytes.Four groups were included in the lymphocyte culture tests.CD Medium group: RPMI1640 cell culture medium;(2) PP group: RPMI 1640 plus PP fraction at concentration of 5 ug / ml, 10 u.g / ml and 100 ug / ml respectively;(3) PP+ConA group: RPMI 1640 plus PP concentration at 5 ug / ml and 10 ug / ml respectively and Con A concentration at 5 ug / ml;? PP+LPS group (PP concentration at 5 ug / ml, 10 ug / ml, and LPS concentration at 20 fig / ml)The results showed that 7 kinds of PP fractions directly promoted lymphocytes proliferation. Low and moderate dosage (5 and 10 ug/ml) of these PP fractions promoted mouse lymphocyte proliferation in vitro and the moderate dosage of PP fractions worked the best. Nevertheless, high dosage of PP fractions (100 ug/ml) had no effect. The results showed that PP had mitogen activity. No PP fractions could promote lymphocytes proliferation induced by ConA and LPS at any tested concentrations.Because of complexity and difficulty in purifing the PP fractions, the futher research of PP physicochemical property and immune active factor was difficult to continue. Thus we constructed a subtractive cDNA library from the peyer’s patch after subtractively hybridized with excess intestinal wall tissue cDNA. Cepra hircus(l5±2 Day-old) was used as experimental animal. Lamb PP tissue was designated as the experimental group (the tester) and the adjacent intestinal wall tissue as the control group (the driver). Tester and driver double stranded cDNAs were prepared from the two mRNA samples. Tester and driver cDNA were separately digested by Rsal to obtain short, blunt-ended cDNAs. Two tester populations were created with different adaptors while driver cDNA had no adaptors. Hybridization kinetics led to equalization and enrichment of differentially expressed sequences. Templates for PCR amplification were generated from differentially expressed sequences. Usingsuppression PCR, only differentially expressed sequences were amplified exponentially. Background was reduced and differentially expressed sequences were further enriched. The PCR products were ligated to T Vector. 160 positive clones were obtained and sequenced. Homology analysis showed that a B-cell lineage specific activator protein (BSAP) was among them. The subtractive library and the obtained ESTs will be useful for identifying immune-associated genes and in vitro expression of active proteins.更多还原