[公告]pPICHOLI 穿梭载体系统
<p class="MsoNormal" style="MARGIN: 0cm 0cm 0pt; TEXT-INDENT: 21.75pt;"><span style="FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-bidi-font-size: 10.5pt;">由于</span><span lang="EN-US" style="mso-bidi-font-size: 10.5pt;"><font face="Times New Roman">PARS</font></span><span style="FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-bidi-font-size: 10.5pt;">序列被整合到载体中，使从酵母中提取的质粒非常简单的实现复原。不需要将亚克隆转入一系列表达载体中，也不需对阳性菌株进行检测，而多克隆位点又使</span><span lang="EN-US" style="mso-bidi-font-size: 10.5pt;"><font face="Times New Roman">DNA</font></span><span style="FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-bidi-font-size: 10.5pt;">片段很方便的连接到载体中。</span><span lang="EN-US" style="mso-bidi-font-size: 10.5pt;"><p></p></span></p><p class="MsoNormal" style="MARGIN: 0cm 0cm 0pt;"><span style="FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-bidi-font-size: 10.5pt;">特点：</span><span lang="EN-US" style="mso-bidi-font-size: 10.5pt;"><p></p></span></p><p class="MsoNormal" style="MARGIN: 0cm 0cm 0pt 18pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo1; tab-stops: list 18.0pt;"><span lang="EN-US" style="mso-fareast-font-family: 'Times New Roman'; mso-bidi-font-size: 10.5pt;"><span style="mso-list: Ignore;"><font face="Times New Roman">1.<span style="FONT: 7pt 'Times New Roman';"> </span></font></span></span><span style="FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-bidi-font-size: 10.5pt;">原核表达系统是一个易于操作、花费少，并且可得到高水平异源蛋白产物；</span><span lang="EN-US" style="mso-bidi-font-size: 10.5pt;"><p></p></span></p><p class="MsoNormal" style="MARGIN: 0cm 0cm 0pt 18pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo1; tab-stops: list 18.0pt;"><span lang="EN-US" style="mso-fareast-font-family: 'Times New Roman'; mso-bidi-font-size: 10.5pt;"><span style="mso-list: Ignore;"><font face="Times New Roman">2.<span style="FONT: 7pt 'Times New Roman';"> </span></font></span></span><i><span lang="EN-US" style="COLOR: black; FONT-FAMILY: Arial;">P. pastoris</span></i><span lang="EN-US" style="COLOR: black; FONT-FAMILY: Arial;">/pPICHOLI </span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: Arial; mso-hansi-font-family: Arial; mso-bidi-font-family: Arial;">系统是一个强有力的真核表达系统，由于结合了</span><span lang="EN-US" style="COLOR: black; FONT-FAMILY: Arial;">AOX</span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: Arial; mso-hansi-font-family: Arial; mso-bidi-font-family: Arial;">或</span><span lang="EN-US" style="COLOR: black; FONT-FAMILY: Arial;">CUP1</span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: Arial; mso-hansi-font-family: Arial; mso-bidi-font-family: Arial;">启动子，使其可以快速生长。</span><span lang="EN-US" style="mso-bidi-font-size: 10.5pt;"><p></p></span></p><p class="MsoNormal" style="MARGIN: 0cm 0cm 0pt 18pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo1; tab-stops: list 18.0pt;"><span lang="EN-US" style="mso-fareast-font-family: 'Times New Roman'; mso-bidi-font-size: 10.5pt;"><span style="mso-list: Ignore;"><font face="Times New Roman">3.<span style="FONT: 7pt 'Times New Roman';"> </span></font></span></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: Arial; mso-hansi-font-family: Arial; mso-bidi-font-family: Arial; mso-bidi-font-style: italic;">适合表达翻译后修饰的可溶性蛋白和不适宜在大肠杆菌中表达的蛋白（真核）（例如一些毒素蛋白）；</span><span lang="EN-US" style="mso-bidi-font-size: 10.5pt;"><p></p></span></p><p class="MsoNormal" style="MARGIN: 0cm 0cm 0pt 18pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo1; tab-stops: list 18.0pt;"><span lang="EN-US" style="mso-fareast-font-family: 'Times New Roman'; mso-bidi-font-size: 10.5pt;"><span style="mso-list: Ignore;"><font face="Times New Roman">4.<span style="FONT: 7pt 'Times New Roman';"> </span></font></span></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: Arial; mso-hansi-font-family: Arial; mso-bidi-font-family: Arial; mso-bidi-font-style: italic;">容易克隆和高的转化效率；</span><span lang="EN-US" style="mso-bidi-font-size: 10.5pt;"><p></p></span></p><p class="MsoNormal" style="MARGIN: 0cm 0cm 0pt 18pt; TEXT-INDENT: -18pt; mso-list: l0 level1 lfo1; tab-stops: list 18.0pt;"><span lang="EN-US" style="mso-fareast-font-family: 'Times New Roman'; mso-bidi-font-size: 10.5pt;"><span style="mso-list: Ignore;"><font face="Times New Roman">5.<span style="FONT: 7pt 'Times New Roman';"> </span></font></span></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: Arial; mso-hansi-font-family: Arial; mso-bidi-font-family: Arial; mso-bidi-font-style: italic;">方便的亲和纯化和重组蛋白的检测。</span><span lang="EN-US" style="mso-bidi-font-size: 10.5pt;"><p></p></span></p><p class="MsoNormal" style="MARGIN: 0cm 0cm 0pt;"></p><p class="MsoNormal" style="MARGIN: 0cm 0cm 0pt;"><b><span lang="EN-US"><font face="Times New Roman"><p></p></font></span></b></p><span style="FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';"> 穿梭表达载体</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">pPICHOLI</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">和</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">pPICHOLI-HA</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">结合了真核和原核启动子元件。噬菌体</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">T7</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">启动子，含有主要衣壳蛋白的核糖体结合位点，促进细菌的表达，它被放在</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">P. pastoris</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">启动子的下游。强的乙醇脱氢酶启动子（</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">AOX</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">）是被紧紧调控的，在含有葡萄糖的培养基中，蛋白表达是完全被抑制的，而生长在甲醇中，则可被最大限度的诱导。</span><span lang="EN-US" style="COLOR: black;"><p></p></span><p></p><p class="MsoNormal" style="MARGIN: 0cm 0cm 0pt; TEXT-INDENT: 21.75pt;"><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">pPICHOLI-1</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">（</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">3579bp</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">）有两个</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">G bases</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">直接位于</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">Sal</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">位点的上游，</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">pPICHOLI-2</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">（</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">3578bp</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">）和</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">pPICHOLI-3</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">（</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">3577bp</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">）分别敲除一个和两个</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">G bases</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">。</span><span lang="EN-US" style="COLOR: black;"><p></p></span></p><p class="MsoNormal" style="MARGIN: 0cm 0cm 0pt; TEXT-INDENT: 21.75pt;"><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">pPICHOLI-HA</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">载体含有一个</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">HA</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">（血球凝集素）抗原决定簇代替了生物素酰化序列。</span><span lang="EN-US" style="COLOR: black;"><p></p></span></p><p class="MsoNormal" style="MARGIN: 0cm 0cm 0pt; TEXT-INDENT: 21.75pt;"><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">pPICHOLI-C</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">加进了</span><span style="FONT-FAMILY: 宋体; mso-hansi-font-family: 'Times New Roman'; mso-bidi-font-family: 宋体; mso-font-kerning: 0pt; mso-ansi-language: ZH-CN; mso-bidi-font-size: 10.5pt;">酿酒酵母</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">CUP1<span style="mso-tab-count: 1;"> </span></font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">启动子（代替了</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">AOX</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">启动子）。这个载体没有</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">T7</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">启动子，不适合原核蛋白的表达。</span><span lang="EN-US" style="COLOR: black;"><p></p></span></p><p class="MsoNormal" style="MARGIN: 0cm 0cm 0pt; TEXT-INDENT: 21.75pt;"><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">由于使用了共同的选择标记</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">zeocin</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">，这个穿梭载体仍然是小的（</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">~3.6</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">和</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">3.2kb</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">），因此，操作、克隆和转化都很方便。通过将</span><i style="mso-bidi-font-style: normal;"><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">Pichia</font></span></i><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">特异的自主复制序列（</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">PARS1</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">）整合到</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">pPICHOLI</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">载体中，线性化不再是必需的，转化效率增加到</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">10<sup>5</sup></font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">转化体</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">/ug DNA</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">。</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">pPICHOLI</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">双重表达载体包括一个</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">RGS</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">（</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">His</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">）</span><span lang="EN-US" style="COLOR: black;"><font face="Times New Roman">6tag</font></span><span style="COLOR: black; FONT-FAMILY: 宋体; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman';">和一个生物素化序列，这样就使表达蛋白易于检测和快速的纯化。</span><span lang="EN-US" style="COLOR: black;"><p></p></span></p>
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