【作者】 李继东；

【导师】 林跃鑫； 孔祥平；

【作者基本信息】 福建师范大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 本论文研究了重组人可溶性肿瘤坏死因子相关的凋亡诱导配体（TNF—related apoptosis inducing ligand，TRAIL）的毕赤酵母发酵工艺优化、蛋白纯化工艺及生物活性分析，并利用生物软件和Internet对其二、三级结构预测等方面进行了研究。 摇瓶培养确定了最适培养基为BMGY/BMMY，最适pH为5.5—6.0，最佳甲醇诱导浓度1％，最适诱导时间为96小时。在此基础上，进行了5L发酵罐的高密度培养，采用补料分批培养，最终确定了高密度发酵工艺。 利用30％—60％饱和度（NH₄）2SO₄分级沉淀对发酵上清预处理，经Sephadex G25凝胶脱盐，Q-Sepharose-Fast Flow阴离子层析和SephadexG50 fine凝胶层析分离，最终得到的rhsTRAIL蛋白纯度为95％。经过电泳和ESI-MS确定蛋白的单体分子量为22,000Da，二聚体分子量为44,000Da，等电点为4.8—5.2，并且证明了其没有发生N-糖基化，与所克隆的TRAIL氨基酸序列中没有存在N糖基化位点是一致的。 形态学上观察了rhsTRAIL对肝癌细胞SMMC7721和BEL7402的诱导凋亡活性，发现rhsTRAIL在0.5μg/ml时显示了很好的诱导凋亡活性，并通过电镜观察rhsTRAIL诱导后的SMMC7721细胞，证实了凋亡；同时提取凋亡细胞DNA，进行琼脂糖电泳分析，在分子水平证明凋亡；最后利用SMMC7721细胞分组加入纯化后的rhsTRAIL蛋白，结果显示纯化蛋白含量在0.01μg/ml时细胞就呈现了诱导凋亡活性。 利用Antheprot软件和Internet对克隆的rhsTRAIL片段（114aa—281aa）进行了二级和三级结构预测，显示rhsTRAIL是一种混合型蛋白，三级结构模拟显示胞外C段（114aa—281aa）形成了8条β折叠结构，主要是平行β折叠，形成了典型的β夹心结构域。更多还原

【Abstract】 Optimization of fermentation conditions, protein purification craft and assay of biological activities of recombinant human soluble TNF梤elated apoptosis inducing ligand (TRAIL) were studied in this research. Meanwhile, the tertiary structure of TRAIL was predicted by using biological soft wares and Internet.Cultures in shaking bottle showed the optimum conditions as follows: the culture medium is BMGY/BMMY; the pH is 5.5-6.5; methanol concentration is 1%; inducement period is 96h. High-density fermentation in 5L fermentor was further studied. High-density fermentation craft of TRAIL were determined with fed-batch fermentation.The supernatants of fermentation were treated with 30% - 60% (NH4)2SO4 to gradiently precipitate the proteins followed by desalination through a Sephadex G25 gel colume. The desalted proteins were purified by Q-Sepharose-Fast Flow ion exchange chromatography and Sephadex G50 fine gel filtration. The purity of target protein-rhsTRAIL was as high as 95%. SDS-PAGE electrophoresis showed the molecular weights of monomer and dimmer based on were 22,000Da and 44,000Da, respectively. The iso-electric point of was 4.8-5.2. The amino acid analysis indicated that the recombinant protein is non- N-glycosylated.The ability of 0.5 ug/m rhsTRAIL to induce apoptosis of liver cancer cells SMMC7721 and BEL 7402 could be visioned morphologically. This was further verified by Electrical microscope observision of induced SMMC7721 cells and agarose gel electrophoresis of apoptosis DNA. MTT test results also showed that 0.01 u g/ml purified rhsTRAIL has strong effect to induce apoptosis of SMMC7721 cell.Antheprot software and Internet were used to predict secondary and 3D structure of recombinant TRAIL segment (114aa-281aa). Mixing type structure was showed. The edextracelluar C segment (114aa-281aa) formed eight paralleled 3 -folding typical 3 sandwich structure domain.更多还原