论文部分内容阅读
目的进一步了解尼罗罗非鱼免疫球蛋白的结构和功能。方法通过RACE技术得到Ig T c DNA全长,从m RNA水平检测其在不同发育时期和不同组织中的表达情况,并通过q PCR检测在嗜水气单胞菌刺激情况下Ig T和Ig M的免疫应答规律,构建重组表达载体体外表达部分恒定区序列并制备单克隆抗体。结果克隆得到全长为1 759 bp的c DNA序列,编码506个氨基酸,表达重组蛋白,纯化得到目标蛋白,并获得CH3的单克隆抗体。RT-PCR结果显示,Ig T在受精后的第8天即可检测到,Ig T在4月龄鱼头肾和脾脏中表达,在6月龄鱼中,除在以上2个组织中表达之外,在皮肤、肠、鳃、卵巢和精巢中也可检测到。嗜水气单胞菌刺激后,q PCR结果显示:Ig T和Ig M在头肾、肾、脾脏、肠、鳃和皮肤中均有不同程度的上调。结论克隆得到尼罗罗非鱼Ig T重链c DNA序列并成功制备了单克隆抗体,为免疫球蛋白及其免疫系统的研究奠定了基础。
Objective To further understand the structure and function of Nile tilapia immunoglobulins. Methods The full length of Ig T c DNA was obtained by RACE technique. The expression of Ig T c DNA was detected by m RNA levels at different developmental stages and in different tissues. Ig T and Ig M The immune response rule was constructed, and a recombinant expression vector was constructed to express part of the constant region sequence in vitro and to prepare a monoclonal antibody. Results The full-length 1 759 bp c DNA sequence was cloned, encoding a protein of 506 amino acids. The recombinant protein was expressed and the target protein was purified. A monoclonal antibody against CH3 was obtained. RT-PCR results showed that Ig T was detectable on the 8th day after fertilization, Ig T was expressed in 4-month-old head kidney and spleen, and in 6-month-old fish, except for the expression in the above two tissues In addition, it is also detectable in the skin, intestine, gills, ovaries and testes. Q-PCR results showed that Ig T and Ig M were up-regulated to varying degrees in head kidney, kidney, spleen, intestine, gill and skin after stimulated by Aeromonas hydrophila. Conclusion Cloning of the Ig T heavy chain cDNA sequence of Nile tilapia and the successful preparation of the monoclonal antibody laid the foundation for the study of immunoglobulin and its immune system.