芋螺多肽MVIIA毒性以及SO-3结构—活性关系研究

发布时间：2018-03-13 01:34

  本文选题：N-型钙通道　切入点：ω-芋螺多肽　出处：《安徽大学》2014年硕士论文　论文类型：学位论文

【摘要】：MVIIA为ω-芋螺多肽,含25个氨基酸、3对全交叉二硫键,已在2004年由美国FDA批准于上市,用于神经病理疼痛、癌症及艾滋病晚期病人镇痛。SO-3为本实验室从中国南海线纹芋螺(Conus striatus)发现的新ω-芋螺多肽,也含有25个氨基酸、3对全交叉二硫键。SO-3与MVIIA具有71%的结构同源性,且C端的12个氨基酸完全相同。前期研究结果表明,SO-3镇痛活性与MVIIA相当,但对金鱼毒性显著低于MVIIA。MVIIA具有许多严重的副作用,如幻想、共济失调及震颤等,降低了其用药适从性。目前虽然MVIIA的结构-活性关系已有较多研究,但其毒性来源不明确。SO-3虽已进行药物研发,其结构-活性关系尚不清楚。为了研究MVIIA的毒性来源及SO-3结构-活性关系,本论文合成MVIIA与SO-3杂合体以及MVIIA突变体,利用膜片钳、金鱼毒性试验、小鼠震颤、自发活动、运动功能测定等实验研究了其毒性来源氨基酸。此外,我们合成了系列SO-3突变体,利用膜片钳手段检测了各突变体活性。应用分子模拟方法研究了MVIIA及SO-3与N-钙通道的结合作用。此外在学习了华中科技大学N-钙通道膜片钳技术之后,利用HEK293细胞表达了N-型钙通道,分离培养含天然N-钙通道的海马神经元细胞,并开展了非ω-芋螺毒素类N-型钙通道抑制剂筛选工作。本实验主要取得了如下结果：(1)重新合成或新设计合成了MVIIA及其5个突变体、SO-3及其12个突变体,通过多肽4℃下的氧化折叠,反相柱的富集、纯化,得到各肽纯品纯度均在95%以上；(2)各多肽圆二色谱在210nm处均显示较强负峰,表明含有p折叠二级结构；(3)膜片钳方法检测MVIIA毒性突变体对N-型钙通道体外电生理活性,结果表明将MVIIA loop2被SO-3对应部分后取代后或MVIIA的Asp14替换SO-3相应的Asn后,活性提高三倍；MVIIA loop 2氨基酸的Leu11、Met12被SO-3的Ile及Ala取代后,相比MVIIA活性略有下降；(4)各MVIIA突变体的金鱼毒性均低于MVIIA,MVIIA对小鼠的震颤毒性、自发活动、运动功能的影响均大于SO-3及MVIIA loop2被SO-3对应部分取代后的突变体ω-2；(5) MVIIA、SO-3及MVIIA突变体与N-钙通道后的通道结合后恢复实验及多肽与N-型钙通道的结合作用计算表明,Met12可能是毒性的主要来源氨基酸。MVIIA的Asp14被SO-3的Asn氨基酸取代后,活性提高但毒性降低；(6)膜片钳方法检测SO-3活性突变体对N-型钙通道体外电生理活性,结果表明：SO-3 loopl的Ala3、Ala4被ω-芋螺多肽CVID对应的Ser及Lys取代后,活性提高1倍,SO-3的Ala4及Pro7对调互换后,活性提高。Asn14被Asp替换后活性下降了约4倍,Ile11被Ala替换后活性下降约60倍；(7)本实验初步建立了N-型钙通道膜片钳试验平台,并开始用于非ω-芋螺多肽类N-型钙通道抑制剂的筛选。
[Abstract]:MVIIA, a 蠅 -Conoca polypeptide containing 25 amino acids and 3 pairs of fully crossed disulfide bonds, was approved by FDA of the United States in 2004 for neuropathic pain. Analgesia of advanced cancer and AIDS patients. SO-3 is a new 蠅 -Conus striatus polypeptide found from Conus striatus in South China Sea. It also contains 25 amino acids. SO-3 has 71% structural homology with MVIIA. The 12 amino acids at C-terminal were identical. The previous studies showed that the analgesic activity of TSO-3 was similar to that of MVIIA, but the toxicity to goldfish was significantly lower than that of MVIIA.MVIIA, such as fantasy, ataxia and tremor. Although the structure-activity relationship of MVIIA has been studied, the source of its toxicity is not clear. SO-3 has been researched and developed. In order to study the source of MVIIA toxicity and the structure-activity relationship of SO-3, we synthesized MVIIA, SO-3 heterozygote and MVIIA mutants, using patch clamp, goldfish toxicity test, mouse tremor and spontaneous activity. In addition, we synthesized a series of SO-3 mutants. The activity of the mutants was tested by patch clamp method. The binding of MVIIA and SO-3 to N- calcium channel was studied by molecular simulation. In addition, after studying the patch clamp technique of N- calcium channel in Huazhong University of Science and Technology, N- type calcium channel was expressed in HEK293 cells. Hippocampal neurons containing natural N- calcium channel were isolated and cultured. The screening of non-蠅 -conotoxin N-type calcium channel inhibitors was also carried out. The main results of this experiment were as follows: 1) MVIIA and its 5 mutants, namely, so-3 and its 12 mutants, were resynthesized or newly designed. By oxidative folding of peptides at 4 鈩，

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