| 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. |
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