鱼类白介素(IL-1β)及干扰素(IFN)重组质粒构建及联合应用研究

Construction of interleukin(IL-1 beta)and interferon(IFN) recombinant plasmid and joint application in fish

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。

To enhance prevention disease resistance of the fish, we synthetic cytokine interleukin（IL-1beta）and interferon（IFN）gene sequence, respectively build fluorescence plasmids connected with PIRES 2 EGFP- eukaryotic expression vector. the plasmids transfection Hela, fall under the fluorescent microscope fluorescence reaction, then intramuscular immunization fish. Within 2 weeks after the immune, collecting each group ascites samples, Muscle tissue and serum, Through the fluorescence, RT-PCR and SDS-PAGE testing purpose protein expression. then the plasmids intramuscular immunization trout broodstock obtained expectantstill in the process of artificial insemination for secondary immune. Trout fry sample collection, applying fluorescence microscope testing purpose protein expression. The results showed that both plasmids Transfection Hela visible fluorescence reaction, ck without fluorescence; Laboratory animal test results show that there are only two fluorescent signal detected in the experimental group ascites, RT-PCR result shows two patients in each group of 10 tail pieces can be detected purpose, blank group without a purpose. Sds-page shows that two group at 40 kd visible banding. After the second immunization larvae can be detected by fluorescent protein expression. Experimental group survival rate 97%. Blank control group reproductive survival rate 56%. These results prove that synthetic IL-1β、IFN fluorescent plasmids immune fish, Significantly improves the fish itself immunity, Can effectively enhance prevention disease resistance of the fish.