CN107012146B - 基于位点特异性重组的四膜虫表达载体及其构建和应用 - Google Patents
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Abstract
本发明公布了一种基于位点特异性重组的四膜虫表达载体及其构建和应用。该载体以质粒pNeo4为骨架，组合串联的HA标签序列、限制性内切酶酶切位点及四膜虫ACTIN1基因的转录终止序列为模块，将模块合成、酶切位点消化插入到pNeo4的5'端多克隆酶切位点中，构建出表达载体pNeo4‑3HA。使用该载体时，分别扩增获得重组同源臂—目的基因编码区C端序列及其3'侧翼序列，经酶切连接克隆到pNeo4‑3HA载体的5'上游和3'下游多克隆酶切位点中。得到的构建体经转化四膜虫、抗性筛选获得表达突变细胞株。本载体实现了在自身启动子调控下C端表达融合标签的内源基因的目的，操作简单快捷，3个HA标签提高了免疫荧光定位灵敏度，还可用于蛋白亲和纯化和蛋白相互作用研究。
Description
技术领域
本发明涉及分子生物学技术领域，特别涉及一种含串联的3个HA标签的位点特异性重组表达载体pNeo4-3HA及其构建方法和应用。
背景技术
目的蛋白融合标签的表达载体广泛地应用于蛋白定位、相互作用和功能研究中。在模式生物—纤毛类嗜热四膜虫中，融合标签可以通过同源重组的方式有效地重组到内源基因位点上，所以融合标签的蛋白表达载体在四膜虫中是一种极其有用的分子工具(Cassidy-Hanley et al,1997)。但目前能使用的该类构建载体还很少。现在已有的目的基因融合标签的构建体的构建方式可分为两种：一种方法是构建内源基因启动子调控下的目的基因融合标签的表达构建体。该方法可以在目的基因自身启动子调控下将目的基因融合标签一起表达出来，可以得到内源基因在细胞中的真实定位模式。但目前获得该类构建体的方法或者是通过使用多次重叠PCR和巢式PCR的方法得到构建体(Xu et al,2012)，或者是使用重叠PCR与多个载体的酶切、连接的方法获得构建体(Kataoka et al,2010)。这些方法在构建重组质粒时过程复杂，往往需要使用多对引物和较长的重叠PCR引物，导致经济成本上升并因此无法增加较多的标签数目，从而可能由于许多目的基因在自身启动子调控下表达的水平不高而标签数目又少，使其抗体亲和量较少，最终导致无法获得清晰的定位信号；另一种方法是构建目的基因的过表达构建体，该构建体可使目的基因在金属离子Cd2+诱导下，在MTT1的启动子调控下过量表达，从而借以观察目的基因在四膜虫细胞中的定位模式(Shang et al,2002；Zweifel et al,2009)，该构建方法简单，易于操作，通过过量表达目的基因能将表达量低的目的基因的定位信号的清晰度提高，发现定位细节，但也可能由于基因的过量表达而造成细胞发育受阻或出现非特异性定位。
为实现既能简便经济地获得在目的基因自身启动子调控下的内源基因融合标签表达构建体，又能将标签序列增加以提高目的基因的定位信号清晰度，本发明构建了一种基于特异性位点重组的目的基因C端含3HA标签的内源基因表达载体，pNeo4-3HA。该载体是利用敲除载体pNeo4为构建骨架(Mochizuki,2008)，在其5'端的多克隆酶切位点的Sac1和Pst1之间将模块(3HA-motif)插入其中构建而成。动力蛋白基因ACTIN1的3'-UTR及转录终止子为融合3HA标签的目的基因的转录提供终止信号。该表达载体可用于目的蛋白的细胞定位、纯化和蛋白相互作用分析。
发明内容
本发明的目的在于提供一种含串联的3个HA标签的基于位点特异性重组的表达载体pNeo4-3HA及其构建方法；以及该表达载体pNeo4-3HA在基因自身启动子调控下的表达、定位、蛋白纯化和蛋白相互作用分析中的应用。
为实现上述目的，本发明提供如下技术方案：
一种3HA-motif，其核苷酸序列为SEQ ID NO：1。
一种表达载体pNeo4-3HA，含有如权利要求1所述的3HA-motif。
一种表达载体pNeo4-3HA的构建方法，包括以下步骤：
(1)设计并合成3HA-motif：在Sac1和Not1酶切位点之间加入18个随机碱基；之后，在Not1后加一个A碱基，紧跟其后为3个优化密码子的HA序列和TGA三联体密码子；每两个HA序列之间加三个碱基做linker；之后在酶切位点Sal1和Pst1之间是嗜热四膜虫ACTIN1基因的3'-UTR和转录终止子序列；
(2)将3HA-motif克隆到pUC18-T载体上，获得质粒pUC18-3HA，酶切、测序鉴定正确；
(3)将质粒pUC18-3HA和载体pNeo4分别通过Sac1和Pst1消化后，将3HA-motif经连接插入到载体pNeo4的5'多克隆酶切位点中，从而获得了特异性位点重组表达载体pNeo4-3HA。
本发明的有益效果：用特异性位点重组表达载体pNeo4-3HA去构建的表达构建体，由于在目的基因的C端带有3个HA标签，大大增加了使抗体对HA标签的结合量，获得的目的基因定位信号将更加清晰；并由于是在目的基因自身启动子调控下的定位，消除了过量表达目的基因所导致的非特异性定位，所以获得的定位信号也更加真实可靠。该表达载体为今后四膜虫内源基因在自身启动子调控下的融合标签表达构建提供了更为快捷、经济的方法，提高了构建效率，也为今后该载体在蛋白纯化和蛋白间相互作用中的应用奠定了基础。
附图说明
图1：含3个HA标签的3HA-motif模块序列。
图2：质粒pUC18-3HA的酶切鉴定电泳图。
图3：质粒pUC-3HA的测序结果。
图4：特异性位点重组表达载体pNeo4-3HA结构示意图和酶切鉴定。
图5：重组表达质粒pNeo4-3HA-ZFR2的构建和鉴定。
图6：pNeo4-3HA-ZFR2转化嗜热四膜虫筛选图。
图7：ZFR2的定位图比较。
具体实施方式
实施例1
(1)3HA-motif的序列设计、优化和合成。根据四膜虫密码子偏爱性先对3个HA标签的碱基序列进行优化并在每2个HA之间加入3个碱基作为linker序列，然后在嗜热四膜虫基因组数据库网站中找到ACTIN1基因的cDNA序列，并找出其终止密码子后的3'-UTR和转录终止子序列；然后根据载体骨架pNeo4的质粒图谱设计模块3HA-motif序列(SEQ ID NO：1)，全长266bp。(图1)：其中在Sac1和Not1酶切位点之间加入了18个随机碱基；之后，用一个A碱基跟在Not1位点后将该酶切位点的碱基数补足为9个；紧跟其后的是3HA序列和终止密码子的碱基序列TGA，并且每两个HA序列之间加三个碱基做linker；TGA后是Sal1酶切位点，在Sal1和Pst1之间是嗜热四膜虫ACTIN1基因的3'-UTR和转录终止子序列。设计完成后，交上海生工生物工程技术有限公司人工合成并克隆在载体pUC18-T中，获得pUC18-3HA质粒。将pUC18-3HA质粒转化大肠杆菌DH5α大量扩增，提取质粒并进行酶切鉴定(图2)和测序(图3)。图1：为设计的3HA-motif模块。其中上面图为结构示意图，下面为设计序列。上下两图之间不同的阴影对应不同的酶切位点。图2：M为DNA标准分子量；1：pUC18-3HA质粒经Sac1和Pst1酶切鉴定。切下266bp的3HA-motif模块片段。图3：pUC18-3HA质粒测序结果。其中测序序列与预测序列完全一致。
(2)特异性位点重组表达载体pNeo4-3HA的获得。将pUC18-3HA质粒和骨架载体pNeo4分别转化感受态大肠杆菌DH5α，大量扩增后，提取质粒后分别经Sac1和Pst1消化、胶回收，获得纯化的3HA-motif片段和线性的pNeo4。按一定比例将它们混合后，在16℃过夜连接，连接产物转化感受态大肠杆菌DH5α，涂在含氨苄青霉素的LB固体培养基上，37℃过夜培养，挑取单菌落在LB液体培养基中大量培养，筛选并提取质粒做PCR鉴定和酶切鉴定，从而获得了特异性位点重组表达载体pNeo4-3HA(图4中A)。图4中：A图为表达载体pNeo4-3HA的质粒示意图；B图为表达载体pNeo4-3HA质粒酶切鉴定图。M：DNA标准分子量；1：pNeo4-3HA质粒，大小为5266bp；2：pNeo4-3HA质粒经Sac1和Pst1酶切鉴定切下266bp的3HA-motif模块片段。3：pNeo4-3HA质粒经Not1酶切鉴定；4：pNeo4-3HA质粒经Sal1酶切鉴定。因除了在3HA-motif模块中含有Sal1酶切位点外，在pNeo4-3HA质粒的3'多克隆酶切位点中也含有Sal1位点。所以，切出了两条带。
(3)构建重组表达质粒pNeo4-3HA-ZFR2。将ZFR2基因的编码区的C端序列(ZFR2_C，995bp)用引物ZFR2-5'-F和ZFR2-5'-R及3'侧翼序列(ZFR2_3'，885bp)用引物ZFR2-3'-F和ZFR2-3'-R分别用进行扩增获得两段重组同源臂，并在ZFR2_C的两端带Sac1和Not1位点；在ZFR2_3'的两端带Xho1和Kpn1位点。经胶回收纯化后，先将ZFR2_C和pNeo4-3HA-ZFR2用Sac1和Not1酶切，按一定比例将它们混合后连接，转化感受态大肠杆菌DH5α，涂在含氨苄青霉素的LB固体培养基上，37℃过夜培养，挑取单菌落在LB液体培养基中大量培养，筛选并提取质粒pNeo4-3HA-ZFR2_C,PCR和酶切鉴定，ZFR2_C重组在了pNeo4-3HA的5'多克隆酶切位点中(图5中A)；然后，将ZFR2_3'和pNeo4-3HA-ZFR2_C分别再经Xho1和Kpn1酶切，胶回收后连接，如上述方法，提取重组质粒pNeo4-3HA-ZFR2，PCR和酶切鉴定，ZFR2_3'重组在了pNeo4-3HA的3'多克隆酶切位点中(图5中B)。图5中：A图为pNeo4-3HA-ZFR2_C构建体鉴定结果。M：DNA标准分子量；1：pNeo4-3HA-ZFR2_C质粒；2：以pNeo4-3HA-ZFR2_C为模板PCR鉴定，得到的片段长度～1000bp；3：pNeo4-3HA-ZFR2_C经Sac1和Not1酶切鉴定，得到的片段长度～1000bp；B图为将ZFR2-3'片段插入到pNeo4-3HA-ZFR2_C的3'多克隆位点中获得重组质粒pNeo4-3HA-ZFR2构建体的鉴定结果。M：DNA标准分子量；1：pNeo4-3HA-ZFR2质粒；2：以pNeo4-3HA-ZFR2为模板PCR鉴定，得到的片段长度～890bp；3：pNeo4-3HA-ZFR2经Xho1和Kpn1酶切鉴定，得到的片段长度～890bp。
(4)重组表达质粒pNeo4-3HA-ZFR2转化嗜热四膜虫细胞，获得表达突变细胞株。质粒pNeo4-3HA-ZFR2经Sac1和Kpn1酶切后，纯化、浓缩获得线性化的表达构建体，分别转化嗜热四膜虫野生型细胞B2086和CU428；在Cd2+存在的情况下，经梯度浓度的巴龙霉素筛选，用鉴定引物ZFR2-F-JD和ZFR2-R-JD进行PCR鉴定，获得部分内源ZFR2基因被构建体替代的表达突变细胞株3HA-ZFR2-B(2株)和3HA-ZFR2-C(3株)(图6)。图6中：M：DNA标准分子量；1，3：依次为突变细胞株3HA-ZFR2-B1和3HA-ZFR2-B1；2：为野生型嗜热四膜虫细胞；4,5,6：依次为突变细胞株3HA-ZFR2-C1、3HA-ZFR2-C2和3HA-ZFR2-C3。WT：野生型未替代基因扩增的条带，1672bp；3HA-ZFR2：替代基因扩增的条带，2931bp。
(5)选取重组较好的3HA-ZFR2-B1和3HA-ZFR2-C2扩大培养、饥饿后，在细胞浓度为2.5×105cells/ml的条件下，混合细胞，诱导配对；同时将实验室之前获得的过表达突变细胞株OE-Zfr2p也在相同条件下诱导配对；然后，在配对8-10小时时取样、固定细胞，进行间接免疫荧光定位实验。将固定细胞用封闭液室温下封闭2小时后，用鼠抗anti-HA作为一抗，以1:200的比例在4℃下温浴过夜；然后用1:400的比例的藕联TRITC的羊抗鼠二抗室温下温浴细胞1小时；最后用1μg/ml DAPI染细胞核10分钟后防淬灭剂、盖玻片、封片，在DeltaVision活体细胞成像仪下进行荧光观察、拍照(图7)。图中：3HA-Zfr2p是特异位点重组表达突变细胞株的间接免疫荧光定位；OE-Zfr2p是过表达ZFR2突变细胞株的免疫荧光定位。a,DAPI染alignment时期HA-Zfr2突变细胞株；e，TRITC对a图中获得的HA-Zfr2突变细胞株的定位；b，DAPI染macronuclear elimination时期HA-Zfr2突变细胞株；f，TRITC对b图中获得的HA-Zfr2突变细胞株的定位；c，DAPI染alignment时期OE-Zfr2p突变细胞株；g,i，TRITC对c图中获得的OE-Zfr2突变细胞株的定位；d，DAPI染macronuclear elimination时期的OE-Zfr2p突变细胞株；h,j，TRITC对d图中获得的OE-Zfr2突变细胞株的定位。
本发明用特异性位点重组表达载体pNeo4-3HA构建获得的ZFR2表达构建体pNeo4-3HA-ZFR2转化得到的表达突变细胞株在大核上的定位比同样浓度抗体孵育所得到的过表达突变细胞株的定位信号更清晰，并且消除了过量表达ZFR2所导致的在类基体上的非特异性定位，得到更为准确的定位信号。该发明为今后目的基因在自身启动子调控下获得准确清晰的定位信号提供了条件，也可将其用于蛋白纯化和蛋白相互作用的研究中。
本发明中使用的引物：
ZFR2-5'-F:TCGAGCTCGGGAATTCTTAGCCAACAACAAGC(SEQ ID NO：2)
ZFR2-5'-R:ATAAGAATGCGGCCGCTTATTCTTTTAATTCACTAAATCCTTC(SEQ ID NO：3)
ZFR2-3'-F:CCGCTCGAGGGGCGGTTTGGTGTTTTCTAAAAG(SEQ ID NO：4)
ZFR2-3'-R:CGGGGTACCGGATTCATAGCTAAATTAATCACCTG(SEQ ID NO：5)
ZFR2-F-JD:GCTCAAAGACTATTAAGATATGTTAAG(SEQ ID NO：6)
ZFR2-R-JD:CTGAGAAACTAAATTGGACATTAAAGC(SEQ ID NO：7)。
SEQUENCE LISTING
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Claims (3)
1.一种表达载体pNeo4-3HA，含有核苷酸序列为SEQ ID NO：1的3HA-motif；所述3HA-motif通过以下步骤设计合成：在Sac1和Not1酶切位点之间加入18个随机碱基；之后，在Not1后加一个A碱基，紧跟其后为3个优化密码子的HA序列和TGA三联体密码子；每两个HA序列之间加三个碱基做linker；之后在酶切位点Sal1和Pst1之间是嗜热四膜虫ACTIN1基因的3'-UTR和转录终止子序列。
2.如权利要求1所述的表达载体pNeo4-3HA的构建方法，包括如下步骤：
(1)设计并合成3HA-motif：在Sac1和Not1酶切位点之间加入18个随机碱基；之后，在Not1后加一个A碱基，紧跟其后为3个优化密码子的HA序列和TGA三联体密码子；每两个HA序列之间加三个碱基做linker；之后在酶切位点Sal1和Pst1之间是嗜热四膜虫ACTIN1基因的3 '-UTR和转录终止子序列；
(2)将3HA-motif克隆到pUC18-T载体上，获得质粒pUC18-3HA，酶切、测序鉴定正确；
(3)将质粒pUC18-3HA和载体pNeo4分别通过Sac1和Pst1消化后，将3HA-motif经连接插入到载体pNeo4的5 '多克隆酶切位点中，从而获得了特异性位点重组表达载体pNeo4-3HA。
3.如权利要求1所述的表达载体pNeo4-3HA在基因自身启动子调控下的表达、定位、蛋白纯化和蛋白相互作用分析中的应用。
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| Localization and functional analysis of HmgB3p, a novel protein containing high-mobility-group-box domain from Tetrahymena thermophila;Jing Xu等;《Gene》;20130515;第526卷(第2期);第87-95页 * |
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