酶聯(lián)生物--抗原性多肽選擇的原則

<p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋體">為了使生產(chǎn)抗體獲得</font>zui<font face="宋體">佳效果，仔細(xì)地設(shè)計(jì)抗原多肽是很有必要的，設(shè)計(jì)應(yīng)滿足一個(gè)基本條件：在免疫過(guò)程中，該抗原既不會(huì)產(chǎn)生過(guò)強(qiáng)的免疫反應(yīng)，同時(shí)又能產(chǎn)生出對(duì)感興趣的蛋白有結(jié)合能力的抗體。</font></span><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><strong><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">盡管抗原設(shè)計(jì)是一個(gè)很復(fù)雜的課題，有諸多需要注意的細(xì)節(jié)，根據(jù)我們所積累的經(jīng)驗(yàn)，有幾點(diǎn)關(guān)鍵的基本設(shè)計(jì)原則可以提供給大家參考：</span></strong><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋體; font-size: 9pt;">1<font face="宋體">、確定抗體的用途</font></span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">新開(kāi)展一個(gè)研究項(xiàng)目，弄清楚所感興趣的蛋白的一些基本特性是很有必要的，特別是如果知道蛋白的結(jié)構(gòu)會(huì)對(duì)選擇抗體易于接觸和識(shí)別的識(shí)別區(qū)域有很大的幫助。然而，在沒(méi)有這樣的結(jié)構(gòu)信息（多數(shù)是這種情況）的情況下，了解研究的用途會(huì)影響多肽設(shè)計(jì)的策略。</span><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋體">例如：如果研究重點(diǎn)是集中在蛋白的不同區(qū)域，如</font>C<font face="宋體">端或</font><font face="Calibri">N</font><font face="宋體">端，或在一種特定狀態(tài)下的蛋白，如磷酸化等，那么按照所需序列設(shè)計(jì)的多肽和產(chǎn)生的相應(yīng)的抗體在應(yīng)用上應(yīng)該沒(méi)有太大的困難，然而，蛋白的構(gòu)象將影響抗體與其識(shí)別區(qū)域之間的相互作用。這種情況下可能存在的問(wèn)題是如果在折疊的蛋白中，該識(shí)別區(qū)域被藏在蛋白的內(nèi)部，抗體將無(wú)法接觸到該區(qū)域。（無(wú)法產(chǎn)生相互作用）。</font></span><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋體; font-size: 9pt;">2<font face="宋體">、識(shí)別區(qū)域的選擇原則</font></span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">一般來(lái)說(shuō)理想的抗原性識(shí)別區(qū)域應(yīng)具備親水、位于蛋白表面和結(jié)構(gòu)上易變形性等特點(diǎn)。因?yàn)樵诖蠖鄶?shù)的天然環(huán)境中，親水區(qū)域傾向于集中在蛋白表面，而疏水區(qū)域常常被包裹在蛋白內(nèi)部，同樣道理，抗體只能與在蛋白表面發(fā)現(xiàn)的識(shí)別區(qū)域相互作用，而當(dāng)這些識(shí)別區(qū)域有足夠的結(jié)構(gòu)易變形性而轉(zhuǎn)移到抗體可接觸的位置時(shí)，將會(huì)與抗體間有很高的親和性。</span><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋體; font-size: 9pt;">3<font face="宋體">、連續(xù)的與不連續(xù)的識(shí)別區(qū)域</font></span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">連續(xù)的區(qū)域是指由連續(xù)的氨基酸序列構(gòu)成的識(shí)別區(qū)域。大多數(shù)抗體是針對(duì)連續(xù)識(shí)別區(qū)域的，抗體能與這類區(qū)域以很高的親和力相結(jié)合表明這段序列不在蛋白內(nèi)部。不連續(xù)的識(shí)別區(qū)域是代表有一定折疊的一段多肽序列，或是將兩段分離開(kāi)的多肽連在一起的抗體的識(shí)別區(qū)域。</span><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;">在某些情況下，針對(duì)這樣不連續(xù)識(shí)別區(qū)域的抗體也能產(chǎn)生，只是用來(lái)免疫的抗原多肽必須具備與該不連續(xù)識(shí)別區(qū)域相似的二級(jí)結(jié)構(gòu)，而序列的長(zhǎng)度需要符合相關(guān)的要求。</span><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋體; font-size: 9pt;">4<font face="宋體">、基本建議</font></span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋體">為了避免識(shí)別區(qū)域隱藏在蛋白內(nèi)部的風(fēng)險(xiǎn)，我們通常建議選擇蛋白的</font>N<font face="宋體">，</font><font face="Calibri">C</font><font face="宋體">兩端來(lái)產(chǎn)生的相應(yīng)的抗體。因?yàn)樵谕暾牡鞍字校?lt;/font><font face="Calibri">N</font><font face="宋體">，</font><font face="Calibri">C</font><font face="宋體">兩端通常是暴露在蛋白表面的。然而，一定要注意膜蛋白的</font><font face="Calibri">C</font><font face="宋體">端疏水性太強(qiáng)，不適合作為抗原。</font></span><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋體; font-size: 9pt;">5<font face="宋體">、序列的長(zhǎng)度</font></span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋體">通常我們建議抗原多肽的序列長(zhǎng)度在</font>8-20<font face="宋體">個(gè)氨基酸殘基之間，如果太短，就有多肽太特殊、所產(chǎn)生的抗體與天然蛋白之間的親和力（結(jié)合能力）不夠強(qiáng)的風(fēng)險(xiǎn)，同樣，如果序列長(zhǎng)度超過(guò)</font><font face="Calibri">20</font><font face="宋體">，將有可能引入二級(jí)結(jié)構(gòu)，所產(chǎn)生的抗體失去特異性的可能，而且肽鏈越長(zhǎng)，通常合成難度增大，不易獲得高純度的產(chǎn)品。</font></span><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="color: rgb(255, 0, 0);"><strong><span style="font-family: 宋體; font-size: 9pt;">6<font face="宋體">、載體蛋白交聯(lián)的選擇</font></span></strong></span><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><font face="宋體">基本原則：將載體蛋白加在遠(yuǎn)離抗體識(shí)別區(qū)域的一端，在序列中沒(méi)有</font>Cys<font face="宋體">的情況下在</font><font face="Calibri">N</font><font face="宋體">或</font><font face="Calibri">C</font><font face="宋體">端加上</font><font face="Calibri">Cys</font><font face="宋體">為交聯(lián)的方法。</font></span><span style="mso-spacerun:'yes';font-family:宋體;mso-ascii-font-family:Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:'Times New Roman';font-size:9.0000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p>