本文選題：N-型鈣通道　切入點(diǎn)：ω-芋螺多肽　出處：《安徽大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：MVIIA為ω-芋螺多肽,含25個(gè)氨基酸、3對(duì)全交叉二硫鍵,已在2004年由美國(guó)FDA批準(zhǔn)于上市,用于神經(jīng)病理疼痛、癌癥及艾滋病晚期病人鎮(zhèn)痛。SO-3為本實(shí)驗(yàn)室從中國(guó)南海線紋芋螺(Conus striatus)發(fā)現(xiàn)的新ω-芋螺多肽,也含有25個(gè)氨基酸、3對(duì)全交叉二硫鍵。SO-3與MVIIA具有71%的結(jié)構(gòu)同源性,且C端的12個(gè)氨基酸完全相同。前期研究結(jié)果表明,SO-3鎮(zhèn)痛活性與MVIIA相當(dāng),但對(duì)金魚毒性顯著低于MVIIA。MVIIA具有許多嚴(yán)重的副作用,如幻想、共濟(jì)失調(diào)及震顫等,降低了其用藥適從性。目前雖然MVIIA的結(jié)構(gòu)-活性關(guān)系已有較多研究,但其毒性來源不明確。SO-3雖已進(jìn)行藥物研發(fā),其結(jié)構(gòu)-活性關(guān)系尚不清楚。為了研究MVIIA的毒性來源及SO-3結(jié)構(gòu)-活性關(guān)系,本論文合成MVIIA與SO-3雜合體以及MVIIA突變體,利用膜片鉗、金魚毒性試驗(yàn)、小鼠震顫、自發(fā)活動(dòng)、運(yùn)動(dòng)功能測(cè)定等實(shí)驗(yàn)研究了其毒性來源氨基酸。此外,我們合成了系列SO-3突變體,利用膜片鉗手段檢測(cè)了各突變體活性。應(yīng)用分子模擬方法研究了MVIIA及SO-3與N-鈣通道的結(jié)合作用。此外在學(xué)習(xí)了華中科技大學(xué)N-鈣通道膜片鉗技術(shù)之后,利用HEK293細(xì)胞表達(dá)了N-型鈣通道,分離培養(yǎng)含天然N-鈣通道的海馬神經(jīng)元細(xì)胞,并開展了非ω-芋螺毒素類N-型鈣通道抑制劑篩選工作。本實(shí)驗(yàn)主要取得了如下結(jié)果：(1)重新合成或新設(shè)計(jì)合成了MVIIA及其5個(gè)突變體、SO-3及其12個(gè)突變體,通過多肽4℃下的氧化折疊,反相柱的富集、純化,得到各肽純品純度均在95%以上；(2)各多肽圓二色譜在210nm處均顯示較強(qiáng)負(fù)峰,表明含有p折疊二級(jí)結(jié)構(gòu)；(3)膜片鉗方法檢測(cè)MVIIA毒性突變體對(duì)N-型鈣通道體外電生理活性,結(jié)果表明將MVIIA loop2被SO-3對(duì)應(yīng)部分后取代后或MVIIA的Asp14替換SO-3相應(yīng)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突變體的金魚毒性均低于MVIIA,MVIIA對(duì)小鼠的震顫毒性、自發(fā)活動(dòng)、運(yùn)動(dòng)功能的影響均大于SO-3及MVIIA loop2被SO-3對(duì)應(yīng)部分取代后的突變體ω-2；(5) MVIIA、SO-3及MVIIA突變體與N-鈣通道后的通道結(jié)合后恢復(fù)實(shí)驗(yàn)及多肽與N-型鈣通道的結(jié)合作用計(jì)算表明,Met12可能是毒性的主要來源氨基酸。MVIIA的Asp14被SO-3的Asn氨基酸取代后,活性提高但毒性降低；(6)膜片鉗方法檢測(cè)SO-3活性突變體對(duì)N-型鈣通道體外電生理活性,結(jié)果表明：SO-3 loopl的Ala3、Ala4被ω-芋螺多肽CVID對(duì)應(yīng)的Ser及Lys取代后,活性提高1倍,SO-3的Ala4及Pro7對(duì)調(diào)互換后,活性提高。Asn14被Asp替換后活性下降了約4倍,Ile11被Ala替換后活性下降約60倍；(7)本實(shí)驗(yàn)初步建立了N-型鈣通道膜片鉗試驗(yàn)平臺(tái),并開始用于非ω-芋螺多肽類N-型鈣通道抑制劑的篩選。
[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，

本文編號(hào)：1604262