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style="font-family:宋體">腺病毒概述</span></span></strong></a></p><p><span style="font-size:16px"><span style="font-family:宋體"> <strong>腺病毒</strong>（Adenovirus，AdV）是一種無包膜的線性雙鏈DNA病毒，基因組長約35000 bp，理論上可編碼22~40個(gè)基因，兩端各有長約100~150bp的末端反向重復(fù)序列（ITR），在其左端ITR3，端為長約300bp的包裝信號(hào)（Ψ）。病毒顆粒直徑約80~110 nm，病毒衣殼有252個(gè)殼粒，呈規(guī)則的正二十面體面體結(jié)構(gòu)，其中包括240個(gè)六聯(lián)體和12個(gè)位于正二十面體頂部的五聯(lián)體構(gòu)成。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體"> <strong>腺病毒</strong>是一種非整合型雙鏈DNA病毒，在自然界中廣泛分布。盡管一些類型的腺病毒會(huì)引起人胃腸道、呼吸系統(tǒng)或眼部的急性感染，但是應(yīng)用于基因治療研究的腺病毒是安全的，對(duì)人無致病、致畸、致癌的潛在危害。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體"> 利用基因工程手段將腺病毒基因組DNA改造成多種基因操作表達(dá)載體，目前常用的腺病毒包裝系統(tǒng)有：AdMax系統(tǒng)和AdEasyTM系統(tǒng)。</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">AdEasyTM包裝系統(tǒng) </span></span></strong></a></p><p><span style="font-size:16px"><span style="font-family:宋體"> AdEasyTM 包裝系統(tǒng)主要的特點(diǎn)是利用Cre/loxP系統(tǒng)在大腸桿菌（BJ5183）中完成外源基因插入腺病毒基因組的過程，獲得環(huán)狀的重組腺病毒基因組，并且具有在細(xì)菌中復(fù)制的必需元件。將重組腺病毒基因組酶切線性化后轉(zhuǎn)染HEK293細(xì)胞，避免了雙質(zhì)粒共轉(zhuǎn)染得到重組腺病毒。</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">使用這個(gè)系統(tǒng)需要<strong>注意以下問題</strong>： </span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">（1）要用能表達(dá)Cre重組酶的特定的大腸桿菌，建議建立菌種庫以免細(xì)菌發(fā)生變化；</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">（2）由于重組是在細(xì)菌中發(fā)生的，微小的基因組變化不易發(fā)現(xiàn)； </span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">（3）要獲得高品質(zhì)的重組腺病毒毒種（高病毒產(chǎn)量、高目的基因表達(dá)）應(yīng)挑選多個(gè)重組腺病毒質(zhì)粒，分別轉(zhuǎn)染HEK293細(xì)胞，獲得多個(gè)重組病毒來篩選。</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">AdMax包裝系統(tǒng) </span></span></strong></a></p><p><span style="font-size:16px"><span style="font-family:宋體"> AdMax包裝系統(tǒng)是通過Cre/loxP 獲得重組病毒，這個(gè)過程發(fā)生在HEK293細(xì)胞中，從而避免在細(xì)菌中重組。將克隆了外源基因的腺病毒穿梭質(zhì)粒與攜帶了腺病毒大部分基因組的包裝質(zhì)粒共轉(zhuǎn)染HEK293細(xì)胞，利用Cre/loxP系統(tǒng)的作用實(shí)現(xiàn)重組，產(chǎn)生重組腺病毒。這個(gè)系統(tǒng)有多種優(yōu)勢(shì)，包括操作簡便、重組效率高、獲得的病毒產(chǎn)率高、目的基因的表達(dá)水平高（因?yàn)閙CMV啟動(dòng)子比hCMV啟動(dòng)子強(qiáng)若干倍）等。 </span></span></p><p><span style="font-size:16px"><span style="font-family:宋體"> 與AdEasy系統(tǒng)相比，AdMax系統(tǒng)有一定的優(yōu)勢(shì)：只需要3~4周就能完成從質(zhì)粒構(gòu)建到重組出毒，成功率大于98%（其中95%的克隆含有目的基因），因在真核細(xì)胞內(nèi)重組出毒，保持了對(duì)腺病毒的生存壓力，有助于重組腺病毒的基因組的完整性；而AdEasy系統(tǒng)的出毒成功率只有18~34%（Stratagene公司新版的AdEasy XL系統(tǒng)成功率在94%左右），且在細(xì)菌（BJ5183）中完成病毒基因組重組，理論上，失去了生存壓力的腺病毒基因組更容易發(fā)生突變，病毒遺傳背景及活性可能會(huì)受到影響。</span></span></p><p> </p><p><a ><strong><span style="font-size:16px"><span style="font-family:宋體">腺病毒載體有下列優(yōu)點(diǎn)：</span></span></strong></a></p><p><span style="font-size:16px"><span style="font-family:宋體">1、腺病毒的基因結(jié)構(gòu)與功能研究的比較清楚；</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">2、可裝入基因的容量較大（8K）；</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">3、轉(zhuǎn)染范圍廣，轉(zhuǎn)染率較高，所有腺病毒載體均有轉(zhuǎn)染分裂期和非分裂期細(xì)胞的能力；</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">4、不整合到靶細(xì)胞基因組，無插入突變，無致癌性，對(duì)人無致病、致畸、致癌的潛在危害；</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">5、可以復(fù)制出滴度高（可以達(dá)到1011 pfu/mL）、穩(wěn)定性好、基因轉(zhuǎn)移效率高的病毒；</span></span></p><p><span style="font-size:16px"><span style="font-family:宋體">6、適用于在難以轉(zhuǎn)染的細(xì)胞中進(jìn)行的RNA干擾、過表達(dá)特定基因或過表達(dá)特定microRNA研究和In Vivo實(shí)驗(yàn)。 </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">腺病毒包裝</span></span></strong></a></p><p><span style="font-size:16px"><span style="font-family:宋體"> 重組腺病毒包裝是以Ad5血清型E1／E3缺陷型腺病毒DNA為架，在克隆，包裝，擴(kuò)增等各部條件做了改進(jìn)和優(yōu)化，達(dá)到了克隆陽性率更高，質(zhì)量更可靠，包裝穩(wěn)定，出毒迅速，滴度高等效果。在此基礎(chǔ)上，還拓展了更廣泛的應(yīng)用，可以提供各種帶報(bào)告基因的，帶不同標(biāo)簽的腺病毒構(gòu)建和包裝服務(wù)。 </span></span></p><p><span style="font-size:16px"><span style="font-family:宋體"> 所包裝的腺病毒已成功應(yīng)用于信號(hào)轉(zhuǎn)導(dǎo)，基因轉(zhuǎn)位，Co-IP，干細(xì)胞研究等科研實(shí)驗(yàn)。并且可以完成較高難度的克隆構(gòu)建，包括具有細(xì)胞毒性作用基因的腺病毒構(gòu)建，在短時(shí)間內(nèi)完成從原始質(zhì)粒到病毒DNA的構(gòu)建工作。</span></span></p><p><strong><span style="font-size:16px"><span style="font-family:宋體">服務(wù)簡介：</span></span></strong></p><p><span style="font-size:16px"><span style="font-family:宋體">提供各種RNA干擾和基因過表達(dá)的腺病毒包裝服務(wù)。</span></span></p><p><strong><span style="font-size:16px"><span style="font-family:宋體">服務(wù)內(nèi)容：</span></span></strong></p><p><span style="font-size:16px"><span style="font-family:宋體"> 是以帶熒光或不帶熒光的pEC3.1（+）為穿梭載體，pAd/BL-Dest為骨架載體進(jìn)行的過表達(dá)病毒包裝；以pGensil為穿梭載體，pAd/BL-Dest為骨架載體進(jìn)行干擾病毒的包裝。</span></span></p> </ul> <div class="x4vfcpe" id="news_sx"> <li id="x4vfcpe" class="sx_left">上一篇：<a href="/Article-578556.html">細(xì)胞培養(yǎng)基類型及其特性</a><br />下一篇：<a href="/Article-577677.html">慢病毒包裝概述及步驟</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(hào)樓1201室</div> <div id="x4vfcpe" class="dAddr"><img class="ico" src="/skins/270236/images/icon_foot3.png">郵箱：shgegenetech@163.com</div> <div id="x4vfcpe" class="dAddr"><img class="ico" 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