miRNA介導(dǎo)GSPT與Survivin蛋白互作及功能調(diào)控
定 價(jià)：68 元
- 作者：肖瑞琳
- 出版時(shí)間：2025/7/31
- ISBN：9787524500858
- 出 版 社：知識(shí)產(chǎn)權(quán)出版社
- 中圖法分類(lèi)：Q786
- 頁(yè)碼：172
- 紙張：
- 版次：1
- 開(kāi)本：16開(kāi)
GSPT(G1 to S phase transition)是一種翻譯終止因子,它通過(guò)結(jié)合eRF1識(shí)別終止密碼子，參與蛋白質(zhì)翻譯終止過(guò)程中肽鏈的釋放，對(duì)細(xì)胞周期有調(diào)控作用。Survivin是凋亡抑制蛋白IAP（inhibitors of apoptosis）家族的最小成員，結(jié)構(gòu)獨(dú)特，功能多樣，并在細(xì)胞分化、細(xì)胞周期G2/M以及細(xì)胞凋亡等途徑中起調(diào)控作用。miRNA是內(nèi)源性非編碼小RNA，參與細(xì)胞分化、增殖、凋亡等一系列生理活動(dòng)。由于miRNA存在的普遍性、功能的多樣性，可以作為研究多功能蛋白的一種新型干擾手段。
本書(shū)利用miRNA干擾技術(shù)首次研究了eRF3a/ GSPT1對(duì)細(xì)胞增殖、遷移的調(diào)控作用，同時(shí)分析了Survivin突變體對(duì)細(xì)胞增殖、遷移的分子調(diào)控機(jī)制，又對(duì)eRF3a/ GSPT1、eRF3b/ GSPT2與Survivin的相互作用位點(diǎn)進(jìn)行系統(tǒng)的細(xì)化分析，這為今后研究GSPT與Survivin相互作用的功能研究提供理論支持。
肖瑞琳，博士，畢業(yè)于山西大學(xué)生物化學(xué)與分子生物學(xué)專(zhuān)業(yè)，現(xiàn)就職于太原理工大學(xué)。一直致力于miRNA功能及其相關(guān)分子靶點(diǎn)相互作用的研究，運(yùn)用基因編輯技術(shù)、蛋白質(zhì)組學(xué)分析等生物技術(shù)和實(shí)驗(yàn)方法，深入探究 miRNA 調(diào)控分子靶點(diǎn)的具體機(jī)制，進(jìn)一步揭示 miRNA 調(diào)控細(xì)胞功能的分子基礎(chǔ)。在該領(lǐng)域成果豐碩，已發(fā)表相關(guān) SCI 論文十余篇，主持多項(xiàng)省級(jí)科研項(xiàng)目，并參與多項(xiàng)國(guó)家級(jí)科研項(xiàng)目。
第 1 章 國(guó)內(nèi)外研究概覽································································· 1
1.1 eRF3/GSPT 蛋白 ······························································· 1
1.1.1 eRF3 的來(lái)源 ···························································· 4
1.1.2 eRF3 的結(jié)構(gòu)特征 ······················································ 7
1.1.3 eRF3a 的功能 ·························································12
1.2 Survivin 蛋白 ··································································24
1.2.1 Survivin 的來(lái)源及其家族 ···········································24
1.2.2 Survivin 的定位和結(jié)構(gòu)特征 ········································25
1.2.3 Survivin 的功能 ·······················································28
1.3 miRNAs ·········································································34
1.3.1 miRNAs 的來(lái)源 ······················································34
1.3.2 miRNAs 的形成及作用原理 ·······································39
1.3.3 miRNAs 的功能 ······················································41
1.4 本書(shū)研究目的、意義及創(chuàng)新點(diǎn) ············································56
1.4.1 研究目的及意義 ······················································56
1.4.2 研究創(chuàng)新點(diǎn) ····························································59
第 2 章 miR-144 通過(guò)調(diào)控 eRF3a/GSPT1 對(duì)細(xì)胞增殖、遷移的
影響研究 ········································································63
2.1 實(shí)驗(yàn)材料 ·······································································65
2.1.1 實(shí)驗(yàn)細(xì)胞及菌株 ······················································65
miRNA 介導(dǎo) GSPT 與 Survivin 蛋白互作及功能調(diào)控
II
2.1.2 細(xì)胞與菌株的質(zhì)量控制 ············································ 65
2.1.3 質(zhì)粒及 microRNA 模擬物 ········································· 65
2.1.4 主要的酶及試劑 ····················································· 67
2.2 實(shí)驗(yàn)方法 ······································································· 68
2.2.1 細(xì)胞培養(yǎng) ······························································ 68
2.2.2 雙熒光素酶報(bào)告質(zhì)粒的構(gòu)建 ······································ 70
2.2.3 質(zhì)粒轉(zhuǎn)染 ······························································ 71
2.2.4 RNA 干擾 ····························································· 71
2.2.5 RNA 提取及實(shí)時(shí)熒光定量 qPCR ································ 72
2.2.6 miRNA 的提取及實(shí)時(shí)熒光定量 qPCR ·························· 73
2.2.7 細(xì)胞增殖活性 MTT 檢測(cè) ·········································· 74
2.2.8 劃痕實(shí)驗(yàn) ······························································ 76
2.2.9 Western blot ··························································· 77
2.3 結(jié)果 ············································································· 78
2.3.1 GSPT1 和 miR-144 在結(jié)腸癌細(xì)胞 HCT116 細(xì)胞中的
表達(dá)水平檢測(cè) ························································ 78
2.3.2 GSPT1 3′UTR 是 miR-144 的靶物 ······························· 79
2.3.3 miR-144 通過(guò)下調(diào) GSPT1 抑制細(xì)胞增殖 ······················ 81
2.3.4 miR-144 通過(guò)下調(diào) GSPT1 抑制細(xì)胞遷移 ······················ 83
2.4 討論 ············································································· 85
第 3 章 Survivin 70 、71 位氨基酸突變體對(duì)結(jié)腸癌細(xì)胞增殖、
遷移的分子調(diào)控 ······························································· 87
3.1 材料 ············································································· 89
3.1.1 實(shí)驗(yàn)細(xì)胞株、菌株與質(zhì)粒 ········································· 89
3.1.2 試劑及工具酶 ························································ 90
3.2 方法 ············································································· 91
3.2.1 點(diǎn)突變 ································································· 91
3.2.2 pull-down 技術(shù) ······················································· 91
3.2.3 Survivin 突變體與 eRF3 的共定位 ······························· 92
3.3 結(jié)果 ············································································· 93
目 錄
III
3.3.1 Survivin 70、71 位氨基酸突變測(cè)序圖 ···························93
3.3.2 Survivin 70、71 位氨基酸的突變體重組蛋白的表達(dá) ·········94
3.3.3 Survivin 70、71 位氨基酸的突變對(duì)與 eRF3a/GSPT1
相互作用的影響 ······················································95
3.3.4 Survivin 70、71 位氨基酸的突變對(duì) Survivin/Survivin
二聚體形成的影響 ···················································96
3.3.5 Survivin 及其 70、71 位氨基酸突變體與 GSPT1 的
定位 ·····································································97
3.3.6 Survivin 70、71 位氨基酸的突變抑制了結(jié)腸癌細(xì)胞
HCT116 增殖 ··························································98
3.3.7 Survivin 70、71 位氨基酸突變抑制了結(jié)腸癌細(xì)胞
HCT116 遷移 ··························································99
3.3.8 Survivin 70、71 位氨基酸的突變促進(jìn)了細(xì)胞凋亡 ·········· 100
3.3.9 Survivin 70、71 位氨基酸的突變提高了其對(duì)順鉑藥物
的敏感性 ····························································· 101
3.4 討論 ··········································································· 102
3.5 結(jié)論與展望 ·································································· 106
第 4 章 人類(lèi)截短型 GSPT1 蛋白與生存素 Survivin 在體內(nèi)
相互作用及區(qū)域分析 ························································ 111
4.1 材料 ··········································································· 112
4.1.1 菌株與質(zhì)粒 ·························································· 112
4.1.2 寡聚核苷酸 ·························································· 112
4.1.3 工具酶及生化試劑 ················································· 113
4.1.4 主要試劑的配制 ···················································· 113
4.2 方法 ··········································································· 114
4.2.1 eRF3 截短體的酵母雙雜交重組質(zhì)粒構(gòu)建 ···················· 114
4.2.2 eRF3 截短體酵母雙雜交重組質(zhì)粒的共轉(zhuǎn)化 ················· 114
4.2.3 β-半乳糖苷酶活性分析 ··········································· 115
4.2.4 陽(yáng)性克隆的嚴(yán)謹(jǐn)型篩選 ··········································· 118
4.2.5 熒光共定位 ·························································· 120
miRNA 介導(dǎo) GSPT 與 Survivin 蛋白互作及功能調(diào)控
IV
4.3 結(jié)果 ············································································ 123
4.3.1 eRF3 N 末端區(qū)域截短設(shè)計(jì) ······································· 123
4.3.2 截短體構(gòu)建方案 ···················································· 123
4.3.3 實(shí)驗(yàn)設(shè)計(jì)原理 ······················································· 125
4.3.4 截短體功能預(yù)測(cè) ···················································· 125
4.3.5 截短型 eRF3a/GSPT1、eRF3b/GSPT2 酵母雙雜交重
組質(zhì)粒的構(gòu)建和鑒定 ·············································· 125
4.3.6 β-半乳糖苷酶活性分析 ············································ 129
4.3.7 eRF3 與 Survivin 共定位 ·········································· 130
4.4 討論 ············································································ 133
第 5 章 人類(lèi)肽鏈釋放因子 eRF3 與生存素 Survivin 在體外
相互作用及區(qū)域分析 ························································· 136
5.1 材料 ············································································ 136
5.1.1 菌株與質(zhì)粒 ·························································· 138
5.1.2 寡聚核苷酸 ·························································· 139
5.1.3 主要酶及生化試劑 ················································· 139
5.1.4 主要試劑的配制 ···················································· 140
5.2 方法 ············································································ 141
5.2.1 原核重組表達(dá)質(zhì)粒的構(gòu)建 ········································ 141
5.2.2 eRF3a 截短體原核表達(dá)重組質(zhì)粒的轉(zhuǎn)化與量誘導(dǎo)表達(dá) ···· 143
5.2.3 重組蛋白的大量表達(dá) ·············································· 145
5.2.4 pull-down 分析 ······················································ 148
5.2.5 Western blot 分析 ··················································· 150
5.3 結(jié)果 ············································································ 150
5.3.1 eRF3 截短體原核表達(dá)重組質(zhì)粒的構(gòu)建 ························ 150
5.3.2 eRF3 不同截短體蛋白的原核表達(dá) ······························ 152
5.3.3 體外相互作用的 pull-down 分析 ································ 153
5.4 討論 ············································································ 156
5.4.1 相互作用研究的系統(tǒng)驗(yàn)證 ········································ 156
5.4.2 細(xì)胞周期調(diào)控的協(xié)同作用假說(shuō) ·································· 156
目 錄
V
5.4.3 待解決的關(guān)鍵科學(xué)問(wèn)題 ··········································· 157
5.4.4 未來(lái)研究方向 ······················································· 157
5.5 總結(jié)與展望 ·································································· 158
5.5.1 總結(jié) ··································································· 158
5.5.2 展望 ··································································· 160
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