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<div class="x4vfcpe" id="ny_right"> <div class="x4vfcpe" id="ny_biaoti"><span>您現(xiàn)在的位置：<a href="/">首頁</a> > <a href="/article.html">技術(shù)文章</a> > 慢病毒包裝概述及步驟</span></div> <div id="x4vfcpe" class="about"> <div class="x4vfcpe" id="newscont"> <h1 class="newsname">慢病毒包裝概述及步驟</h1> <li id="x4vfcpe" class="time">發(fā)布日期：2013-11-19 瀏覽次數(shù)：6406 </li> <ul class="dcont"> <p><a ><strong><span style="font-size:16px"><span style="font-family:宋體">慢病毒包裝概述</span></span></strong></a></p><p><span style="font-size:16px"><span style="font-family:宋體"> 目前慢病毒也被廣泛地應(yīng)用于表達(dá) RNAi 的研究中。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體"> 由于有些類型細(xì)胞脂質(zhì)體轉(zhuǎn)染效果差，轉(zhuǎn)移到細(xì)胞內(nèi)的 siRNA 半衰期短，體外合成 siRNA 對基因表達(dá)的抑制作用通常是短暫的，因而使其應(yīng)用受到較大的限制。采用事先在體外構(gòu)建能夠表達(dá) siRNA 的載體，然后轉(zhuǎn)移到細(xì)胞內(nèi)轉(zhuǎn)錄 siRNA 的策略，不但使脂質(zhì)體有效轉(zhuǎn)染的細(xì)胞種類增加，而且對基因表達(dá)抑制效果也不遜色于體外合成 siRNA ，在長期穩(wěn)定表達(dá)載體的細(xì)胞中，甚至可以發(fā)揮長期阻斷基因表達(dá)的作用。在所構(gòu)建的 siRNA 表達(dá)載體中，是由 RNA 聚合酶Ⅲ啟動子來指導(dǎo) RNA 合成的，這是因為 RNA 聚合酶Ⅲ有明確的起始和終止序列，而且合成的 RNA 不會帶 poly A 尾。當(dāng) RNA 聚合酶Ⅲ遇到連續(xù) 4 個或 5 個 T 時，它指導(dǎo)的轉(zhuǎn)錄就會停止，在轉(zhuǎn)錄產(chǎn)物 3' 端形成 1~4 個U 。 U6 和 H1 RNA 啟動子是兩種 RNA 聚合酶Ⅲ依賴的啟動子，其特點是啟動子自身元素均位于轉(zhuǎn)錄區(qū)的上游，適合于表達(dá)～ 21ntRNA 和～ 50ntRNA 莖環(huán)結(jié)構(gòu)（ stem loop ）。在 siRNA 表達(dá)載體中，構(gòu)成 siRNA 的正義與反義鏈，可由各自的啟動子分別轉(zhuǎn)錄，然后兩條鏈互補結(jié)合形成 siRNA ；也可由載體直接表達(dá)小發(fā)卡狀 RNA（small hairpin RNA, shRNA），載體包含位于 RNA 聚合酶Ⅲ啟動子和 4 ～ 5 T轉(zhuǎn)錄終止位點之間的莖環(huán)結(jié)構(gòu)序列，轉(zhuǎn)錄后即可折疊成具有 1~4 個 U 3 ' 突出端的莖環(huán)結(jié)構(gòu)，在細(xì)胞內(nèi)進(jìn)一步加工成 siRNA 。構(gòu)建載體前通常要通過合成 siRNA 的方法，尋找的 siRNA ，然后從中挑選符合載體要求的序列，將其引入 siRNA 表達(dá)載體。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體"> <strong>慢病毒載體</strong>（ Lentiviral vector ）較逆轉(zhuǎn)錄病毒載體有更廣的宿主范圍，慢病毒能夠有效感染非周期性和有絲分裂后的細(xì)胞。慢病毒載體能夠產(chǎn)生表達(dá) shRNA 的高滴度的慢病毒，在周期性和非周期性細(xì)胞、干細(xì)胞、受精卵以及分化的后代細(xì)胞中表達(dá) shRNA ，實現(xiàn)在多種類型的細(xì)胞和轉(zhuǎn)基因小鼠中特異而穩(wěn)定的基因表達(dá)的功能性沉默，為在原代的人和動物細(xì)胞組織中快速而地研究基因功能，以及產(chǎn)生特定基因表達(dá)降低的動物提供了可能性。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體"> <strong>慢病毒</strong>表達(dá)載體包含了包裝、轉(zhuǎn)染、穩(wěn)定整合所需要的遺傳信息。慢病毒包裝質(zhì)粒可提供所有的轉(zhuǎn)錄并包裝 RNA 到重組的假病毒載體所需要的所有輔助蛋白。為產(chǎn)生高滴度的病毒顆粒，需要利用表達(dá)載體和包裝質(zhì)粒同時共轉(zhuǎn)染細(xì)胞，在細(xì)胞中進(jìn)行病毒的包裝，包裝好的假病毒顆粒分泌到細(xì)胞外的培養(yǎng)基中，離心取得上清液后，可以直接用于宿主細(xì)胞的感染，目的基因進(jìn)入到宿主細(xì)胞之后，經(jīng)過反轉(zhuǎn)錄，整合到基因組，從而高水平的表達(dá)效應(yīng)分子。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體"><img alt="" src="http://img62.chem17.com/9/20131119/635204593281193133344.jpg" style="height:400px; width:478px" /></span></span></p><p> </p><p><span style="font-size:16px"><span style="font-family:宋體"><a ><strong>慢病毒包裝作用</strong></a>：</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">1. 對于一些較難轉(zhuǎn)染的細(xì)胞，如原代細(xì)胞、干細(xì)胞、不分化的細(xì)胞等，能大大提高目的基因轉(zhuǎn)導(dǎo)效率，而且目的基因整合到宿主細(xì)胞基因組的幾率大大增加，這就為 RNAi，cDNA 克隆以及報告基因的研究提供了一個有利的途徑。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">2. 進(jìn)行穩(wěn)轉(zhuǎn)細(xì)胞株的篩選；</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">3. 為活體動物模型實驗提供高質(zhì)量的包含目的基因的病毒液；</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">在細(xì)胞相關(guān)的實驗操作中，對于一些按常規(guī)方法難以轉(zhuǎn)染甚至無法轉(zhuǎn)染的細(xì)胞，通過病毒介導(dǎo)的實驗?zāi)軌虼蟠筇岣呋虻霓D(zhuǎn)導(dǎo)效率，以達(dá)到目的基因的瞬時表達(dá)。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體"> </span></span></p><p><a ><strong><span style="font-size:16px"><span style="font-family:宋體">慢病毒包裝步驟</span></span></strong></a></p><p><span style="font-size:16px"><span style="font-family:宋體"> 以六孔板中的1孔為例，每個樣品需要1×106個293T細(xì)胞。</span></span></p><p><br /><span style="font-size:16px"><span style="font-family:宋體"> 1、取1.5ml滅菌EP管，加入1.5μg包裝混合質(zhì)粒和0.5μg表達(dá)質(zhì)粒以及250μl的無血清培養(yǎng)基。輕柔混勻，室溫孵育5min。</span></span></p><p abs_visibility="true"><br /><span abs_visibility="true" style="font-size:16px"><span abs_visibility="true" style="font-family:宋體"> 2、取1.5ml滅菌EP管，取9μl 脂質(zhì)體2000l溶于250μl無血清培養(yǎng)基中。輕柔混勻，室溫孵育5min。<br /><br /> 3、將DNA溶液和脂質(zhì)體溶液輕柔混勻。室溫孵育20min<br /><br /> 4、用胰酶消化并記數(shù)293T細(xì)胞。用含血清的培養(yǎng)基重懸細(xì)胞。<br /><br /> 5、在六孔板中每孔，加入1ml含血清的生長培養(yǎng)基，再加入DNA-脂質(zhì)體復(fù)合物。<br /><br /> 6、將1ml重懸的293T細(xì)胞（1×106個細(xì)胞/ml）加入到平板中。37℃CO2孵箱中孵育一晚。<br /><br /> 7、移除含有DNA-脂質(zhì)體復(fù)合物的培養(yǎng)基移除，代之以DMEM（含丙酮酸鈉和非必須氨基酸）。<br /><br /> 8、轉(zhuǎn)染后48-72h收獲含病毒的上清。3000 rpm 離心20min，去除沉淀。<br /><br /> 9、病毒上清-80°C貯存。<br /><br /> 包裝出來的慢病毒對NIH/3T3細(xì)胞達(dá)90%以上感染效率。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體"><strong>注意事項：</strong><br /><br /> 1.在慢病毒感染細(xì)胞前，為檢測病毒上清培養(yǎng)液內(nèi)病毒的活性，并確定病毒與轉(zhuǎn)染細(xì)胞之間的比率，需要確定病毒的滴度。病毒的滴度可以通過轉(zhuǎn)染Hela細(xì)胞，然后使用抗體篩選穩(wěn)定的細(xì)胞轉(zhuǎn)染株，進(jìn)行計數(shù)和數(shù)值統(tǒng)計。<br /><br /> 2. 在慢病毒轉(zhuǎn)染細(xì)胞的過程中zui重要的一步是融合，只有一些較小的慢病毒載體才能轉(zhuǎn)導(dǎo)進(jìn)細(xì)胞，因此，病毒顆粒的吸附是慢病毒轉(zhuǎn)染的限速步驟。</span></span></p> </ul> <div class="x4vfcpe" id="news_sx"> <li id="x4vfcpe" class="sx_left">上一篇：<a href="/Article-578111.html">簡述腺病毒包裝</a><br />下一篇：<a href="/Article-577238.html">淺析慢病毒載體</a></li> <li id="x4vfcpe" class="sx_right"><a href="/article.html">返回</a></li> </div> </div> </div> </div> </div> <div class="x4vfcpe" id="clear"></div> <div id="x4vfcpe" class="footer"> <div id="x4vfcpe" class="w1200"> <div id="x4vfcpe" class="footT"> <div id="x4vfcpe" class="footAddr"> <div id="x4vfcpe" class="footTit">上?；偕锟萍加邢薰?/div> <div id="x4vfcpe" class="dAddr"><img class="ico" src="/skins/270236/images/icon_foot1.png">地址：上海市浦東新區(qū)康新公路3399弄25號樓1201室</div> <div id="x4vfcpe" class="dAddr"><img 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