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为增强鱼类自身防病抗病能力,本研究人工合成了细胞因子白介素(IL-1β)及干扰素(IFN)基因序列,分别与p IRES2-EGFP真核表达载体连接,构建荧光表达质粒。两种质粒分别接种Hela细胞,倒置于荧光显微镜下观察,确定质粒在细胞内的表达情况。之后将两种质粒注射免疫鱼体,于免疫后第2周抽样采集腹水、肌肉组织及血清,通过荧光、RT-PCR及SDS-PAGE方法检测目的蛋白表达情况。最后将两种质粒联合免疫待产虹鳟亲鱼,同时在人工授精过程中进行二次免疫,抽样采集孵化出的虹鳟鱼苗,检测目的蛋白表达情况。结果表明,两种质粒转染Hela细胞均可见荧光反应,空白对照无荧光,表明两种质粒在细胞内成功表达;室内动物试验结果表明只有两试验组腹水中检测到荧光信号,RT-PCR结果可见两试验组每组10尾均可检测到目的片段,空白组未检测到目的片段。SDS-PAGE显示,两试验组在40KD处可见目的条带;中试试验幼鱼可检测到荧光蛋白表达;试验组繁殖成活率97%,空白对照组繁殖成活率56%。以上结果证明人工合成IL-1β、IFN荧光质粒免疫鱼体,显著提高了鱼体自身免疫力,可有效的增强鱼体自身防病抗病能力。
In order to enhance their anti-disease and anti-disease ability of fish, the IL-1β and IFN gene sequences were artificially synthesized and linked with pIRES2-EGFP eukaryotic expression vector respectively to construct a fluorescent expression plasmid. The two plasmids were inoculated Hela cells, inverted fluorescence microscope to determine the expression of the plasmid in the cell. After the two plasmids were injected into the immunized fish, the ascites, muscle tissue and serum were sampled and sampled at the second week after immunization. The expression of the target protein was detected by fluorescence, RT-PCR and SDS-PAGE. Finally, the two plasmids were co-immunized to produce rainbow trout broodstock, and the second immunization during artificial insemination was carried out. The hatched rainbow trout seedlings were sampled for detecting the expression of the target protein. The results showed that both plasmids transfected Hela cells were fluorescent reaction, blank control without fluorescence, indicating that the two plasmids were successfully expressed in the cells; indoor animal test results show that only two test groups ascites fluorescence signal detected, RT-PCR results Visible two test groups each group of 10 can detect the purpose of the fragment, the blank group did not detect the target fragment. The results of SDS-PAGE showed that the bands were visible at 40KD in both groups. The fluorescent protein expression was detected in the pilot test juveniles. The survival rate of the experimental group was 97%, while that of the blank control group was 56%. The above results demonstrate that artificial synthesis of IL-1β, IFN fluorescent plasmid immune fish significantly increased the body’s own immunity, which can effectively enhance the ability of fish to prevent disease.