| In recent years,Takifugu rubripes as important economic mariculture fish be known as ’the king of fish’.bacause Takifugu rubripes meat tender,tastes delicious, high protein content and nutrient-rich.The most of Takifugu rubripes throughout China’s yellow sea, bohai sea and the sea of Japan is one of superior specimen can be breeding in the family of Tetraodontidae.however,a variety of diseases caused by the damage is becoming more and more big With the expansion of the scale of farming, leading to a large number of chemical drugs of abuse, the consequences are drug residues,disease and insect resistance, and the spread of drugs in vast areas directly affect the food safety and environmental public health security.Urgent need to develop pollution-free to the environment and no resistance drug,at the same time to reseatch Takifugu rubripes immune mechanism,expect to disease outbreaks can be defense by immune mechanisms.Cryptocaryon irritans disease as the main breeding Takifugu rubripes parasitic disease, caused great economic losses to aquaculture.But what about prevention and control of Cryptocaryon irritans method mainly by chemical drugs for prevention and control, but give the fish itself and the environment have caused great negative consequence.This experiment mainly through observation of Cryptocaryon irritans and filtered from a large number of Chinese herbal medicines, to get the most effective to Cryptocaryon irritans Chinese herbal medicines.Also learned that when Takifugu rubripes infections Cryptocaryon irritans, the immune system plays an important role.So purification and analysis serum IgM which play most directly role in immune response and preparation polyclonal antibody lay a good foundation to research Takifugu rubripes immunology further.This paper is completed with the support of National Science and technology support program.The killing effects of 15 Chinese herbal medicines including of Betel nut (Areca catechu),Bitter ginseng (Sophora flavescens),Rhubarb (Rheum officinale),Cyrtomium rhizome (Cyrtomium Rhizoma), Radix scutellariae(Scutellaria baicalensis) and Wild chrysanthemum (Dendranthema indicum) on trophonts and theronts in vitro of the ciliate Cryptocaryon irritans were studied, and the optimum temperature and salinity for the cysts rupture were also explored as well. At the same time separate and purify Takifugu rubripes serum immunoglobulin IgM and the preliminary research and analysis on their characteristics, by the the purified IgM of Takifugu rubripes immune BALB/c mice and prepare antiserum.An immunoglobulin M(IgM)was purified from serum of Takifugu rubripes with ammonium sulfate precipitation,followed with DEAE-Sepharose Fast Flow column chromatogsaphy, HiTrap IgM Purification Column chromatogsaphy and HiTrap Protein A Sepharose Column chromatogsaphy. Protein concentration determination,Indirect ELISA titer,SDS-PAGE and Western blotting analysis.The specific research results are as follows:(1) The collection of the Cryptocaryon irritans trophonts,the cultivation of the theronts and cysts, and morphological observation.The trophonts are rounded or pear-shaped, parasitic trophonts in tissue slow rotation, the u-shaped arrangement big nucleus can be observed from Cryptocaryon irritans,The trophonts get round after in vitro turn into cyst. The 20 trophonts,theronts and cysts body size were observed and measured, found that trophont body similar to the cyst size, about 200 to 500 μm, the theront size is 30～50μm.(2) The most effective to Cryptocaryon irritans Chinese herbal medicines were filtered and the optimum temperature and salinity for the cysts rupture were studied.The results showed that Betel nut could kill trophonts and theronts,Rhubarb and Wild chrysanthemum could only kill theronts of the ciliate at the herbal concentration of 4.55g/L; Both Bitter ginseng and Cyrtomium rhizome could kill trophonts and theronts at the herb concentration of 9.09g/L.At the concentration of 18.18g/L, Radix scutellariae, Toosendan fruit(Melia toosendan) and Fructus aurantii(Citrus aurantium) could kill trophonts and theronts and Wild chrysanthemum was able to kill trophonts. The other herbs such as Astragalus root(Astragalus membranaceus),Heartleaf houttuynia herb (Houttuynia cordata),Isatis. root(Isatis tinctoria),Chinese bulbul (Anemone chinensis),Honeysuckle(Lonicera japonica),Rehmannia glutinosa(Rehmannia glutinosa)and Rhizoma coptidis (Coptis chinensis) had killing effects on trophonts and theronts when the herb concentration reached at a higher level of 45.45g/L to 90.91g/L. It was showed that the killing effects of the herbs of Betel nut,Bitter ginseng, Rhubarb, Cyrtomium rhizome,Radix scutellariae, Toosendan fruit,Fructus aurantii and Wild chrysanthemum were better than the other selected herbs. They could be selected as herbal medicines to control the ciliate. The experiments also revealed that the optimum temperature and salinity for the cysts rupture were 26℃ and 20～30‰, separately.(3) Purify and analysis Takifugu rubripes serum immunoglobulin IgM.IgM was purified from serum of Takifugu rubripes with ammonium sulfate precipitation was found that the best ammonium sulfate saturation was 50% and the best purifying method was HiTrap IgM Purification Column chromatogsaphy;SDS-PAGE electrophoresis showd that the purified IgM was composed of 68.4 kD heavy chains and 27.9 kD light chains.(4) Preparetion Takifugu rubripes serum immunoglobulin IgM of polyclonal antibodies of mice and the cross-reaction with Sebastods schlegelii, Lateolabrax japonicus,Pagrosomus major and Scophthalmus maximu. Antiserum against the purified IgM was prepared by repeatedly immunized BALB/c mice with the purified protein.An ELISA method was established to detect the antiserum title show that the titer of the antibody reached to 1:128000. Western blot analysis showed that the heavy chain of the purified IgM reacted well with the rabbit antiserum while the light chain hardly reacted with it.ELISA cross reaction analysis also showed that the polyclonal antibody anti-IgM of Takifugu rubripes by immuning mice in addition to react with Takifugu rubripes, with Sebastods schlegelii, Lateolabrax japonicus,Pagrosomus major,Scophthalmus maximu serum IgM leads to positive reaction. |
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