【作者】 曹晓华；

【导师】 吴信忠；

【作者基本信息】 浙江大学 ， 水产养殖， 2009， 硕士

【摘要】 铁蛋白是一切生物体内主要的铁贮存蛋白质,在一些生理和病理过程中具有重要的作用。本文成功地克隆了九孔鲍铁蛋白全长cDNA序列。其结果如下：(1)cDNA序列全长879bp,含有一个516bp的阅读框,编码171个氨基酸。该蛋白具有铁蛋白的特征性结构。氨基酸序列与无脊椎动物皱纹盘鲍同源性高达97.07%、与合浦母贝、加州海兔、长牡蛎分别为81.3%、80.7%和78.9%；(2)半定量和定量RT-PCR表明铁蛋白在健康鲍组织(消化腺,外套膜,鳃,足肌)均有表达,且消化腺中的表达量明显高于其它组织。LPS刺激后铁蛋白基因的表达量随着时间的延长而呈现逐渐的明显增加,当刺激24h时表达量增加达到最高峰,存在明显的时间依赖性,这些结果表明鲍铁蛋白参与了LPS刺激后的免疫反应；(3)通过构建重组表达质粒pET-FER,采用大肠杆菌BL21对铁蛋白进行了诱导表达,并进行了蛋白纯化和制备了多克隆抗体,结果表明：铁蛋白在大肠杆菌中得到了高效表达,制备的兔多克隆抗体的效价为1：14000；(4)鉴于铁蛋白的调控受铁、氧化应激、生物信使分子、细胞因子、激素等的影响,为了探讨在无脊椎动物呼吸爆发过程中铁蛋白受ROS和RNS的调节作用,本文培养了鲍的血淋巴细胞,经LPS刺激诱导呼吸爆发产生大量的ROS和RNS,结果表明：刺激后过氧化氢和一氧化氮呈现明显的增加,且H202在铁蛋白的调节中起主导作用,并能抑制铁蛋白的表达,而NO阳离子能抵抗过氧化氢介导的铁蛋白的降低。更多还原

【Abstract】 Ferritin is an iron storage protein that plays a key role in the processes of physiology and pathology. In the present study, we report the ferritin gene from abalone Haliotis diversicolor supertexta. The results indicated:(1) The full-length of ferritin cDNA consists of 879bp with an ORF encoding a 171 amino acids. Amino acid sequence analysis revealed that ferritin shares highly homology of 97.07%,81.3%,80.7%,78.9%, respectively with Haliotis discus hannai, Pinctada fucata, Aplysia californica and Crassostrea gigas. (2) Semi-quantitative PCR and real-time-PCR show that ferritin is distributed ubiquitously in abalone tissues and has the highest expression level in digestive glands. The mRNA expression in haemolymph treated with LPS was increased gradually, and has a sharp increase at 24h after challenge. The time dependency after LPS stimulation was obvious. (3) The recombinant plasmids (pET-FER) were constructed and identified by sequencing, then transformed into competent cells for expression. The recombinant fusion proteins were purified by affinity chromatography, then the antiserum against New Zealand White rabbits was prepared. The results showed that the recombinant protein was successfully expressed in Escherichia coli BL21, and the titre of anti-ferritin antibody is about 1:14000. (4) Considering that the ferritin synthesis is normally regulated not only by the intracellular iron concentration, but also by cytokines, oxidants, oncogenes, growth factor and so on, we decided to carry out the futher experiments to investigate the modulation of ferritin by ROS and RNS. Therefore, the abalone haemolymph cells were cultured and activated by LPS, and effects of ROS and RNS on ferritin were analyzed in present study. The results of modulation by reactive species show that H2O2 plays an important role in decreasing ferritin expression, however NO cation appear to have a protecting effect on H2O2-medied reduction of ferritin expression.更多还原