基于纽虫的自割和吐捕食器解析凝溶胶蛋白的新功能

Gelsolin is an important and effective actin binding protein which regulates the activity of actins by polymerization, depolymerization and severing them thus participates in physiological processes such as cytoplasmic movements, apoptosis, immune and tumorgenesis. As the essential component of cytoskeleton, actins function through interacting with the actin binding proteins. When the tissue are injured, cell death or apoptosis happens, G-actin were released out of the cells and forms polymers which make damage to the organs. Then gelsolin will express in abundance to disintegrate the polymer actins in the damaged tissues to buffer the damage done by actins polymerization to the body and provide with protections. In this study, the full length cDNA of gasoline genes was cloned from the cDNA library of nemertean by PCR. The gelsolin was expressed in E.coli and the purified proteins were used for polyclonal antibody making. The recombinant of gelsolin was verified by western blot analysis using antiserum from immunized mice. Adopting the FQ-PCR method we researched on the expression level of gelsolin after rupture stimulation in different peroid. We also observed the surface morphology of r-gelsolin and r-actin,the interaction between the two proteins and the in vitro self-assmbly of r-actin through the atomic force microscope (AFM) which are of great importance for analysing the interaction mechanism between the two. Besides, we also did some research on the antitumor properties of r-gelsolin by fluorescence microscope and FCM.GSN was used to genetic markers of quality of Pseudosciaena croceafarmed cultured.

凝溶胶蛋白（Gelsolin，GSN）是一种重要的肌动蛋白结合蛋白，可通过对肌动蛋白产生聚合、解聚和剪切等作用，进一步参与细胞运动、凋亡、免疫、肿瘤形成等生理过程中。当组织受损时，肌动蛋白单体大量释放到细胞外并形成多聚体，导致器官功能损伤，此时GSN则启动大量表达，通过解离损伤组织中肌动蛋白多聚体的方式起到保护作用。为了充分发挥该蛋白在水产养殖动物中的标记作用，本研究选择养殖虾塘中的纽虫为研究对象，构建纽虫cDNA文库、酵母双杂交文库；通过PCR技术克隆GSN基因的全长序列，通过原核和真核表达，制备大量GSN表达产物；利用原子力显微镜研究GSN与actin的表面形貌和相互作用间的形貌变化，直观展示其功能。利用荧光定量PCR技术研究GSN与自割和再生的关系，利用酵母双杂交系统筛选功能蛋白和新功能，利用GSN作为人工养殖大黄鱼品质的遗传标记。

本研究首先从纽虫cDNA文库从中克隆出gelsolin全长1947 bp，开放阅读框为1107 bp，编码369个氨基酸，蛋白质分子量为42.714 kDa，理论等电点为5.72；Actin全长1830 bp，开放阅读框为1131 bp，编码377个氨基酸，蛋白分子量为41.816 kDa，理论等电点为5.30。在1mmol/L IPTG和37℃条件下诱导E.coli表达gelsolin的可溶性蛋白，而actin则以包涵体为主；SDS-PAGE电泳分析表明，gelsolin和actin基因的重组蛋白的分子质量均接近45 KDa。FQ-PCR结果显示断裂后5 d，其mRNA表达量约为对照组的19.88倍，推测断裂后凝溶胶蛋白起到缓解肌动蛋白多聚体对组织造成的毒性效应的作用。AFM结果显示，纯化后的r-gelsolin蛋白直径约为50~70nm。复性后的r-actin呈球形颗粒，蛋白直径约为100~200nm；并通过AFM研究了两者之间的相互作用，r-gelsolin可以通过使r-actin聚集成环或切断F-actin等方式来控制其结构。MTT法检测30μg/mL r-gelsolin作用于人胃癌细胞MGC803后的细胞增殖抑制率可达35%，r-gelsolin能有效减缓MGC803细胞生长增殖速率，G1期细胞减少，S/G2期细胞增加并诱导其早期凋亡。双向电泳表明了70蛋白质参与自割和再生过程，有33种蛋白上调和下调。其中，约79%细胞骨架蛋白和运动蛋白。其中肌动蛋白与凝溶胶蛋白样蛋白（GSN），两者均参与了此过程。利用酵母双杂交技术筛选出一种与凝溶胶蛋白相互作用的铁蛋白Ferritin。RT-qPCR结果证明了大黄鱼随着死亡的时间，actin和gelsolin 与死亡时间呈负相关。而和炎症以及一些环境变化成正相关。