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View additional product information for Bac-to-Bac™ HBM TOPO™ Cloning Kit - FAQs (A11338)
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庆大霉素浓度可能过高。尝试降低庆大霉素的浓度至5 µg/mL，并在培养基中加入更多菌落。
若出现蓝色菌落，则大肠杆菌含有杆粒和质粒，使细胞能在筛选过程中存活。但是，由于未发生转座，所以LacZ基因未被破坏。靶心菌落表示在菌落生长时发生转座。将从混合克隆白色部分中分离得到的克隆重新划线，应该能够得到发生过转座的菌落。
这是同源性重组较差的典型标志。应检查使用的质粒/线性DNA比例。但是，如果存在一些蓝色空斑，则对那些病毒进行扩增并检测它们的蛋白。根据我们的经验，它们应该是正确的，即使其丰度相对较低。
是的，细胞被单个野生型病毒感染后，会以不同速度生成多角体，直到培养瓶中所有的细胞都被感染。细胞中多角体的形成需要3-4天左右，其大小和数量各不相同，直至达到最大能力并发生细胞破裂，从而将微小的病毒颗粒释放到培养基中。
正常情况下，约5-7天会出现很小的白点，约10天出现1 mm空斑。空斑的大小范围为1 mm至4 mm。
在打算挑选空斑的那一天，制备含Bluo-gal的DMSO溶液，浓度为20 mg/mL。在每个板中加入50 µL，使用玻璃涂布器在无菌条件下涂布。等待30-60分钟，空斑应变为蓝色。
有几种原因可导致培养板变蓝：
•病毒接种量过多。可尝试使用更高的稀释度。
•加入热的融化琼脂糖时，将细胞烫死了。过热的琼脂糖会使细胞裂解，将lacZ释放到琼脂糖中，使其变蓝。应复查铺板温度。如果培养板过湿，蓝色会扩散。
琼脂糖覆层过热。加入琼脂糖覆层后，细胞应仍然保持圆形和健康状态。
是的，这表明空斑上存在吸液问题。琼脂糖覆层“漂浮”，是因为板中的培养基未被全部吸走。将琼脂糖覆盖到细胞上之前，培养板应完全干燥，特别是在将要挑选空斑时。为达到这样的效果，我们通常会将培养板稍微倾斜，用Pasteur移液管沿培养板边缘吸一圈，同时小心不要破坏细胞单层。如果培养板边缘有任何培养基残留（小液滴），则继续吸液。琼脂糖覆层“漂浮”，也可能导致野生型病毒污染。野生型病毒可以迁移到培养板的其他部分，从而污染重组型空斑。野生型病毒比重组型病毒复制得快的多，可迅速超过重组型病毒。
细胞接种过多；我们建议在6孔板的每孔中接种8 x 105个细胞。
通常使用的MOI在5-10之间。如果加入过多病毒，会导致细胞过早死亡，并使蛋白表达水平降低。
感染动力学可能比预期要慢。继续观察培养板，直至感染后第8-9天。如果未出现空斑，则检查下面各项：
•如果细胞不健康，会产生较差的空斑或无空斑产生。理想状态下，细胞应处于对数生长中期，并且存活率大于90%。在感染导致细胞停止生长前，细胞应至少倍增1次。应确保细胞接种量正确，且汇合度约为70%。
•细胞营养不良和健康状态较差，都会抑制病毒复制周期。
•琼脂糖温度也很关键。在琼脂糖覆层后，应将培养板静置1小时，使琼脂糖完全凝固。
•在27°C孵育时，过度冷凝会抑制空斑形成——一旦发生冷凝，就应移除纸巾或打开装有培养板的容器。
•病毒滴度过低：使用较高的病毒滴度。您可能需要再次感染细胞，并收集更高滴度的病毒储液。
请检验入门克隆的构建，应确保插入片段位于载体读码框内。在LR反应后，利用PCR法分析重组病毒DNA，确认插入片段的大小和方向均正确。对PCR产物进行测序，确认用于表位标签表达的读码框正确。
为得到高滴度储液，应使用P1储液再次感染细胞并生成P2高滴度储液。按照BaculoDirect使用手册第18页的说明，生成P2储液。
请查看以下建议：
•在转染到昆虫细胞中之前，利用PCR分析检验LR反应。
•在转染实验中，我们建议使用无添加的Grace’s昆虫细胞培养基代替无血清培养基，因为无血清培养基中的有些成分可能干扰转染。
•转染期间，应保证无FBS、无添加剂、无抗生素，因为这些物质中的蛋白质会干扰Cellfectin II试剂。
•LR重组反应中，以pENTR/CAT质粒为阳性对照，以Cellfectin II试剂（模拟转染）作为阴性对照。
•确保细胞处于对数生长期，存活率高于95%，细胞使用量应与使用手册的建议一致。
•若转染效率较低，细胞可能需要1周时间才能出现病毒感染迹象；继续培养，同时每日观察细胞的感染迹象。
在37°C水浴中温浴更昔洛韦5-10分钟，然后涡旋数分钟。沉淀应该重新溶解于溶液中。
用于培养昆虫细胞的培养基通常具有酸性pH（6.0-6.5）或含有电子供应基团，可阻止6xHis标签蛋白与Ni-NTA发生结合。昆虫细胞生长培养基所含谷氨酰胺、甘氨酸或组氨酸等氨基酸的浓度显著高于哺乳细胞生长培养基，这些氨基酸可与6xHis标签蛋白竞争Ni-NTA基质上的结合位点。例如，Grace’s培养基（Life Technologies）含有约10 mM谷氨酰胺、10 mM甘氨酸和15 mM组氨酸。
使用含适当成分和pH（8.0）的缓冲液对培养基进行透析，透析用缓冲液与在天然条件下进行纯化所使用的推荐裂解缓冲液相似，通常可恢复最佳的结合条件。应注意，选用的培养基可能导致出现白色沉淀（可能由不溶性盐组成），但6xHis标签蛋白通常留在溶液中。使用无蛋白质的培养基时，可通过蛋白质定量进行检测；或者利用免疫印迹分析法检测6xHis标签蛋白的含量。离心后，可从清澈的上清液中直接纯化6xHis标签蛋白。
请查看以下可能原因和解决方案：
•使用较高的起始MOI扩增病毒储液：使用低代数病毒储液，进行低MOI扩增。
•收获病毒上清液时，未离心和除去细胞：重新使用低代数病毒储液，进行低MOI扩增；如果该病毒储液代数为P2，则该储液可用于扩增。
•某些基因可使病毒变得非常不稳定：拿出分装的P2病毒储液，复苏后进行扩增。
蛋白得率较低可能是因为：
•病毒储液含有重组和非重组杆状病毒的混合物：通过空斑纯化，分离重组杆状病毒。
•杆状病毒不是重组的：利用pUC/M13正向和反向引物对杆粒DNA进行PCR分析，对转座进行验证；使用新的重组杆粒DNA再次转染昆虫细胞。
•病毒滴度过低或过高：改变MOI。
•收获细胞的时间点不理想：采用针对表达的时间进程实验，确定获得最高蛋白表达水平的最佳时间。
•细胞生长条件和培养基不理想：根据培养瓶的尺寸和表达条件，优化细胞培养条件；为获得最佳的细胞生长和蛋白表达，我们建议使用Sf-900 II SFM或Sf-900 III SFM。
•所用细胞系不理想；尝试使用其他昆虫细胞系。
•细胞收获过晚：做一个时间进程实验，在不同时间点收获细胞。
检查MOI。P1病毒的滴度低于估计值，可能导致MOI较低。
请查看以下可能原因和建议：
•Cellfectin II试剂与杆粒没有混合或孵育时间不足：通过敲打或轻轻涡旋，混匀Cellfectin II试剂与杆粒，孵育混合物15-45分钟。
•杆粒得率低于预期：使用不同剂量的杆粒进行优化。
•杆粒在纯化或冻融过程中被剪切：在凝胶上验证杆粒的完整性。
•孵育时间不足：将混合物在27°C孵育8小时。
•所用细胞代数过高或已过对数生长期：为获得最佳结果，使用代数在8-15代之间的细胞；接种时，细胞应处于对数生长期。
•Cellfectin II试剂被冷冻过：购买新的试剂。
•所用培养基含有血清：转染时，使用无添加的Grace’s培养基。
有多种可能原因：
•转染所用培养基含有抗生素。
•细胞接种密度过低：我们建议细胞汇合度至少达到70%。
•所用细胞代数过低：我们建议细胞用于转染前，至少生长5代。
•转染后未使用青霉素/链霉素，导致污染：加入转染混合物孵育5-8小时后，移除混合物，并在每孔中加入含有抗生素的培养基。
这可能是因为来自杆粒制备的污染或细胞毒性。应设置无Cellfectin II试剂的杆粒阴性对照组。此外，在制备杆粒时，应使用PureLink HiPure质粒制备试剂盒，而不是基于硅胶的小量提取试剂盒。
最可能是挑选了中心为灰色或黑色的菌落。尝试分析更多的白色DH10Bac转化子。通常，我们建议挑选直径大于2 mm的白色菌落。将白色菌落在含50 µg/mL卡那霉素、7 µg/mL庆大霉素、10 µg/mL四环素、100 µg/mL Bluo-gal和40 µg/mL IPTG的新鲜的培养板上再次划线。孵育培养板24小时。
以下是针对该问题的可能原因和建议：
•使用纯的校正聚合酶进行分析：使用Taq聚合酶进行分析。
•插入片段太长，导致PCR出现问题：使用与您的M13引物成对的基因特异性引物，代替使用M13正向和反向引物。
•在目的基因中，富含GC长片段：在PCR反应中，考虑使用DMSO（高达8%）。
请查看以下可能原因和建议：
•DNA保存不恰当：为避免反复冻融，应将纯化的杆粒DNA分装保存在–20°C。
•对高分子量杆粒DNA的处理不恰当：在分离杆粒DNA时，不要涡旋振荡DNA溶液；此外，不要机械地重悬DNA沉淀；将溶液置于管内，轻轻敲打几次。
这可能是因为：
•使用了错误的抗生素或旧的培养基：应使用新鲜的培养基。
•菌落太老或太小：从新划线的培养板上选取较大的白色菌落。
•目的基因的特殊性质导致插入片段不稳定；例如，直接重复序列：应在30°C培养24小时，而不是在37°C培养过夜。
菌落的颜色区别很小可能是因为：
•琼脂的pH不正确：调整LB琼脂的pH至7.0。
•蓝色强度太弱；应确保您使用的是Bluo-gal，而不是X-Gal。您也可尝试将Bluo-gal的浓度增加至300 µg/mL。
•培养板上的菌落过多或过少：调整细胞的连续稀释度，从而获得最佳的菌落数量。
•孵育时间过短或温度过低：接种后48小时，方可挑选菌落；在37°C孵育培养板。
•IPTG浓度不理想：IPTG浓度范围在20–60 µg/mL之间，通常可得到最佳的颜色区分。
我们建议取40μL样品的八分之一在0.5% TAE琼脂糖凝胶上电泳。以23伏缓慢电泳12小时。中提的重组bacmid的条带形态应该可以看见。
请查看以下可能原因和建议：
•复苏/表达期间使用了LB培养基：在4小时的生长期使用SOC培养基。
•复苏/表达时间过短：延长复苏时间，在37°C时为4小时以上，或在30°C时为6小时。
•IPTG浓度不理想：我们建议使用20–40 µg/mL IPTG。
请查看以下可能原因和建议：
•用于转化的pFastBac DNA质量较差：使用纯化的质粒DNA进行转化，检查质粒DNA的质量。
•培养板中缺少庆大霉素：制备含50 µg/mL卡那霉素、7 µg/mL庆大霉素、10 µg/mL四环素、100 µg/mL Bluo-gal和40 µg/mL IPTG的新鲜的选择性培养板。
尽管您将要挑选的是白色（重组）菌落，但是您也应该预期会看到一些蓝色（含非重组杆粒）菌落。以下是关于无蓝色菌落的可能原因和建议：
•颜色形成时间不足：在鉴定菌落表型前，至少等待48小时。
•在琼脂板中，使用Bluo-gal代替X-Gal：在板中使用Bluo-gal，可增强蓝白菌落的对比度。
•转座后，生长不充分：接种前，使转化后细胞在SOC培养基中生长至少4小时。
•培养板中缺少Bluo-gal和IPTG：制备含50 µg/mL卡那霉素、7 µg/mL庆大霉素、10 µg/mL四环素、100 µg/mL Bluo-gal和40 µg/mL IPTG的新鲜的选择性培养板。
•培养板上菌落过多：连续稀释转化混合物，从而获得间隔合适的菌落（建议稀释度为10-2至10-4）。
•培养板太久或在光照下保存：不要使用放置超过4周的培养板；培养板应避光保存。
•孵育时间过短或温度过低：挑选菌落前，至少等待48小时。在37°C孵育培养板。
如果该低滴度储液为P1或P2储液，则可用于病毒扩增实验方案。如果低滴度储液曾经是高滴度储液，但随着时间或繁殖多次而发生滴度降低，则需要重新制备高滴度储液。如果高滴度储液代数大于P5，则可能有过多的缺陷型干扰颗粒感染细胞，而不能进行适当复制或生成蛋白。如果将已有储液接种后，重新分离得到了新的空斑（DIP不会形成空斑），则该储液可用于制备高滴度储液。
可以，用于制备P2病毒储液的实验方案也可用于制备P3、P4或P5病毒储液。我们不建议制备代数高于P5的储液，因为这会产生更多的缺陷型感染颗粒，并会降低蛋白表达水平。
可以，杆状病毒可以感染哺乳细胞，但是需要非常高的滴度。杆状病毒在肝细胞中的感染能力最强。但是，只有在使用高滴度储液直接感染细胞时，才有交叉感染风险。
杆状病毒可以感染果蝇细胞，但不能在果蝇细胞中复制。普通杆状病毒系统中驱动目的基因表达的启动子都是晚期启动子，并且需要来自杆状病毒基因组的早期蛋白。因此，它们在S2细胞中无效，因为无法生成早期蛋白。
通常，每孔加入含0.5 x 106个细胞的培养基2.5–3 mL，是一个良好的开端。第3天开始发生裂解。在第3-7天（90%细胞死亡），可收获和扩增病毒。
我们的研发团队通常会将病毒加到含0.5 x 106细胞/孔的12孔培养板中，每孔总体积为2.5 mL。大约3天后，取出0.75 mL培养液用于制备PCR用DNA，将剩余培养基收集至Eppendorf管中，作为P1病毒储液。此外，也可以挑选一个空斑，保存在Grace’s培养基中。
我们建议在细胞裂解率达到90%时，收获高滴度病毒。大约需要5-7天。如果延长细胞感染时间，则裂解细胞释放的蛋白酶会降解病毒表面蛋白，导致传染性病毒量降低。
这取决于病毒的加入量。如果感染细胞的MOI为5，则细胞通常在24小时被感染，细胞在65小时左右开始裂解。病毒使用量越少，则所需时间越长，反之亦然。
繁殖病毒储液时，为避免缺陷性干扰颗粒（DIP）的影响，应使用低MOI（0.03–0.1）。低MOI可确保每个细胞的病毒颗粒感染量低于1，防止DIP扩增。当细胞存活率为15%时，是合适的病毒收获时间。
注意：DIP是接近正常的病毒衣壳，含缺陷性基因组，无法进行成功的复制。尽管该“颗粒”本身无感染性，但当它与正常病毒颗粒或一些其他类型的DI颗粒共感染时，可以进行复制。
您可使用中性红或MTT对单层细胞进行染色，使空斑更明显。或者，您可延长室温下的空斑形成时间（平均2-5天），增强重组空斑的对比度。但是，中性红空斑染色法不适用于空斑纯化和病毒扩增。
用于表达研究时，我们建议使用滴度为>1 x 108 pfu/mL的病毒储液。
请参见以下公式：
pfu/mL =空斑数量(pfu)/稀释系数x接种体积(mL)
例如，如果病毒稀释度为10-8的孔中含有18个白色空斑，则病毒滴度计算为：
X pfu/mL = 18 pfu/10-8 x 1 mL
X = 1.8 x 109 pfu/mL
以下是空斑实验法的主要步骤：
•在6孔板中接种细胞，并使细胞到达80%融合
•对P1病毒储液（1–10-5）进行连续稀释，并加到细胞中
•在27°C孵育1小时
•在培养基中混合1%融化的琼脂糖
•移除病毒上清液
•在细胞上覆盖含琼脂糖的培养基
•将培养板静置2-3小时，使琼脂糖完全凝固
•培养10-14天
•计算空斑数量
进行该实验时，我们建议：
•细胞应处于非常健康的状态，代数较低（10-20代），处于对数生长期，并且存活率高于95%
•确认病毒储液是无菌的（无污染）
•使用高质量、低熔点的琼脂糖
•琼脂糖培养基的温度非常重要——太热，则细胞会死亡；太冷，则琼脂糖凝固太快
•覆盖琼脂糖培养基后等待2-4小时，使琼脂糖达到100%凝固
•在稀释的板中，空斑数量按下述公式计算：
（1/稀释度）x 空斑数量 = pfu/mL
例如，如果在稀释度为10-6的板中形成50个空斑，则为1/(10-6) x 50 = 5 x 107 pfu/mL
我们建议您采用空斑实验法检测病毒储液的滴度。如果需要，您也可以利用空斑实验纯化单个病毒克隆。
在Bac-N-Blue系统中，转移载体和杆状病毒DNA的重组发生于昆虫细胞中。Bac-N-Blue载体是一种线性AcMNPV衍生物，含有不完整的(3’) lacZ片段。相应的转移载体含有一个5’ lacZ片段。在同源重组的基础上，重组Bac-N-Blue杆状病毒DNA将具有一个受PETL启动子控制的lacZ基因。因此，重组Bac-N-Blue杆状病毒在空斑实验中会形成蓝色空斑，易于识别。在Bac-to-Bac表达系统中，转移载体与杆状病毒DNA间的重组或位点特异性转座发生于大肠杆菌（DH10Bac）中。在Bac-to-Bac表达系统中，含重组杆状病毒DNA的菌落筛选发生于含50 μg/mL卡那霉素（杆状病毒质粒）、7 μg/mL庆大霉素（pFastBac）、10 μg/mL四环素（辅助性质粒）、100 μg/mL Bluo-gal和40 μg/mL IPTG的Luria琼脂板上。
以下是关于如何防止杆状病毒表达系统中发生蛋白质水解作用的一篇很好的文献：
Hom LG, Volkman LE (1998) Preventing proteolytic artifacts in the Baculovirus expression system. BioTechniques 25:18–20.
杆状病毒的杆会根据包装DNA的需要而持续延伸。因此，理论上，该系统能够容纳几百个Kb。在包装极限成为问题之前，标准克隆技术将限制插入片段大小。
昆虫信号肽和/或前序列无法总是增强杆状病毒系统中外源分泌途径蛋白的表达和/或分泌。请查看以下文献：
•Jarvis DL, Summers MD, Garcia A Jr, Bohlmeyer DA (1993) Influence of different signal peptides and prosequences on expression and secretion of human tissue plasminogen activator in the baculovirus system. J Biol Chem 268(22):16754–16762.
•Tessier DC, Thomas DY, Khouri HE, Laliberte F, Vernet T (1991) Secretion of a plant protein in the baculovirus system was enhanced when its signal peptide was replaced with an insect-derived signal peptide. Gene (Amst.) 98:177–183.
我们的研发团队已成功表达了分子量高达300 kDa的蛋白。如果在血清含量>2%的培养基中表达，可将降解程度降至最低。如果您不介意采用额外的纯化步骤，可使用10%血清。为了确定获得最佳表达所需的最短收获时间，我们强烈推荐采用感染复数为5-10的高滴度储液进行不同时间的感染。每24小时为一个时间点，持续5天。
多角体蛋白为30 kDa。
驱动目的基因的启动子是多角体启动子。尽管对于大多数蛋白，多角体启动子通常强3-5倍，但该启动子可被p10启动子取代。然而，经高度修饰或分泌的蛋白在p10启动子的驱动下可达到更高的表达效率，因为p10启动子在末期阶段的早期激活，而多角体启动子在非常晚的末期才激活，两者正好相反。蛋白复合物高效和正确加工所需的细胞蛋白合成，在非常晚的末期停止。这解释了为什么一些报告提到在分泌和修饰蛋白的表达中，p10启动子与多角体启动子同样有效或p10启动子比多角体启动子的效率高2倍。但是，在大多数情况下，多角体启动子也有效。至于哪种启动子更强，取决于多种因素，包括蛋白质的性质和感染后的收获时间。
尽管Kozak序列在哺乳细胞翻译起始中的重要性已被证明，但昆虫细胞中是否也严格遵循Kozak规则仍有争议。确定其重要性的唯一方法是，对相同蛋白从不同起始序列的表达进行直接对比。即使这样，一种蛋白实现最佳表达所使用的规则可能并不适合其它蛋白。以下四篇文献证明了Kozak序列对昆虫细胞中的表达效率无任何影响：
•Hills D, Crane-Robinson C (1995) Baculovirus expression of human basic fibroblast growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression. Biochim Biophys Acta 1260(1):14–20.
•Ranjan A, Hasnain SE (1995) Influence of codon usage and translational initiation codon context in the AcNPV-based expression system: computer analysis using homologous and heterologous genes. Virus Genes 9(2):149–153.
可以。多种五亚基蛋白，如人类复制因子C，已使用重组杆状病毒进行表达。为实现最佳的多亚基蛋白表达，我们建议生产出每个亚基的、独立的高滴度储液（HTS）。利用这种方法，可通过改变每个亚基HTS的感染复数，控制每个亚基的表达量。请参考以下文献，获取更多信息：
•Chen W and Bhal OP (1991) Recombinant carbohydrate and selnomethionyl variants of human choriogonadotropin. J Biol Chem 266(13):8192–8197.
•Chen WY and Bhal OP (1991) Selenomethionyl analog of recombinant human choriogonadotropin. J Biol Chem 266(15):9355–9358.
•Fabian JR, Kimball SR, Jefferson LS (1998) Reconstitution and purification of eukaryotic initiation factor 2B (eIF2B) expressed in Sf21 insect cells. Protein Expr Purif 13(1):16–22.
MOI或感染复数，是指某个实验中，感染单个细胞的平均病毒颗粒数量。您可使用以下公式计算MOI：
MOI (空斑形成单位(pfu)/细胞) = [滴度(pfu) x 接种物所用病毒储液体积(mL)] / [细胞密度(细胞/mL) x 培养物体积(mL)]
可以，可以进行大规模表达实验。下表描述了不同大规模实验方法、要求、优势和参考文献：
- Stirred bioreactor
- Airlift fermentor
- Insect larvae
如果培养基是无血清的，可加入血清至浓度为10%。血清蛋白可作为蛋白酶的底物，防止病毒外壳蛋白发生降解。将病毒储液置于4°C避光保存。分装储液可保存在–80°C，使用前应检测病毒滴度，因为冻融循环会导致病毒滴度降低10-100倍。
需要。重组DNA存在未切割（occ+）DNA污染，将导致重组病毒随时间而逐渐稀释，因为未切割（野生型，occ+）病毒的感染和复制效率高于重组病毒。同时，使用纯化的、单一病毒群体进行起始表达，将确保得到可重复的结果。
请查看以下关于病毒感染不同阶段的描述：
早期
细胞直径增加——细胞直径可能增加25–50%。
细胞核体积增加——细胞核可能“填满”细胞。
末期
细胞生长停止——与单纯的细胞对照相比，细胞停止生长。
颗粒状外观
病毒出芽迹象——细胞出现泡状外观
病毒包涵体——少量细胞会含有包涵体，表现为昆虫细胞核内出现折射晶体。
脱落——细胞从培养皿或培养瓶上脱落。
晚期的细胞裂解——少量细胞可能会充满包涵体病毒、发生死亡和裂解，留下单层清理迹象。
是的，我们提供利用蜂毒素（HBM）分泌信号的Bac-to-Bac HBM TOPO分泌性表达系统（货号A11338或A11339）。该系统非常适用于研究毒性蛋白和糖蛋白。
贴壁Sf9细胞呈聚集状，接触点较小。感染后Sf9细胞聚集悬浮于培养液中，细胞体积变大。
请遵循以下建议：
•细胞应处于非常健康的状态，代数较低（5-15代），处于对数生长期，并且存活率高于95%。
•DNA必须高度纯化，无内毒素
•转染时，不可使用抗生素
•Cellfectin试剂应完全重悬
•设置对照组（培养基对照、DNA对照和转染试剂对照），用于对比和问题排查
昆虫细胞中的蛋白表达高峰，取决于感染复数（MOI）、感染时间和目标蛋白。系统优化包括使用MOI为5-10，表达时间为48-72小时。72小时后表达的蛋白可能是经过异常加工的，因为较高的病毒载量可引起细胞进程故障。
是的，杆状病毒是表达毒性蛋白（即，膜蛋白）的良好候选方案。多角体启动子在感染后18-24小时才能表达至最高水平。多角体启动子在裂解晚期激活。也就是说，其在早期8小时的时候活性极低，所以，如果目的基因毒性很强，可能会造成问题。该问题的解决方法是转换到诱导型表达系统。跨膜蛋白通常难以在任何系统中表达。
ATG通常对于高效的翻译启始是足够的，尽管翻译效率要视目的基因而定。最佳的建议应是保持cDNA中天然起始位点，除非确定这一位点的功能性不理想。如果从表达的角度来考虑，推荐构建并测试两种载体，一个具有天然的起始位点，另一个具有保守的Kozak序列。通常情况下，所有N-端融合型表达载体都已包含了一个RBS或翻译起始位点。
原核生物mRNA含有Shine-Dalgarno序列，也称为核糖体结合位点(RBS)，它是由AUG起始密码子5’端的多嘌呤序列AGGAGG组成。该序列与16S rRNA 3’端的互补，有助于mRNA有效结合到核糖体上。同理，真核生物（特别是哺乳动物）mRNA也含有完成有效翻译所需的重要序列信息。然而，Kozak序列不是真正的核糖体结合位点，而是一种翻译起始增强子。Kozak共有序列是ACCAUGG，其中AUG是起始密码子。-3位的嘌呤(A/G)具有重要作用；若-3位是一个嘧啶(C/T)，翻译过程会对-1、-2和+4位的改变更敏感。当-3位从嘌呤变为嘧啶时，可使表达水平降低多达95%。+4位对表达水平的影响相对较小，可以使表达水平降低约50%。
注：果蝇的最佳Kozak序列稍有不同，酵母完全不遵循这些规则。见下列参考文献：
•Foreign Gene Expression in Yeast: a Review. Yeast, vol. 8, p. 423-488 (1992).
•Caveneer, Nucleic Acids Research, vol. 15, no. 4, p. 1353-1361 (1987).
The concentration of gentamicin might be too high. Try lowering the amount to 5 µg/mL and try adding more of the colony to the culture medium.
In the case of a blue colony, the E. coli has the bacmid and the plasmid in it, allowing the cells to survive the selection process. However, because the transposition has not occurred, the LacZ gene is not disrupted. For bulls-eye colonies, this indicates that the transposition took place when the colony was growing. Re-streaking for an isolated clone from the white portion of the mixed colony should yield some colonies where transposition occurred.
This is typically an indication of poor homologous recombination. Check the plasmid/linear DNA ratio you used. If there are some blue plaques, however, expand those viruses and check for their protein. In our experience, they are correct, even if they were in relatively low abundance.
Yes, cells are infected with wild-type virus individually and will develop polyhedra at different rates until all the cells in the flask are infected. The polyhedra in cells will form in approximately 3-4 days, differing in size and number until they reach their maximum capacity and burst the cell, releasing tiny particles of virus into the medium.
Normally, very small white dots show up about 5-7 days and 1 mm plaques show up around day 10. Plaques can vary in size from 1 mm to 4 mm.
On the day you intend to pick plaques, make a solution of Bluo-gal in DMSO at 20 mg/mL. Add 50 µL per plate and spread with a glass spreader under sterile conditions. Wait 30-60 min, and your plaques should turn blue.
There are a few things that can turn plates blue:
- Too much virus when plating. Try a higher dilution.
- Cells are being singed when plated with hot melted agarose. This lyses the cells and releases lacZ into the agarose, turning it blue. Double-check plating temperatures. If plates are too wet, the blue can diffuse.
The agarose overlay was too hot. After addition of the agarose overlay, cells should still be round and healthy.
Yes, this is indicative of an aspirating problem on the plaques. The agarose overlays were “floating” because the medium was not completely aspirated from the plates. The plates need to be completely dry before the agarose is placed over the cells, especially when plaques will be picked. To do this, we typically tip the plate slightly and keep going around the rim of the plate with the Pasteur pipette tip, being careful not to disturb the cell monolayer. If any medium pooling at the rims of the plates (they will be small pools) is seen, continue to aspirate. This “floating” agarose overlay problem may also result in wild-type contamination. The wild-type virus is able to migrate to other portions of the plates and contaminate recombinant plaques. Wild-type virus replicates much faster than recombinant virus, and can quickly overwhelm the recombinant virus.
Too many cells were seeded; we recommend seeding 8 x 10e5 cells per well for a 6-well plate.
An MOI of 5-10 is typically used. If too much virus is added, unfortunately the cells die too soon and the protein expression level goes down.
The kinetics of infection may be slower than expected. Observe plates until the 8-9th day after infection. If no plaques appear, investigate the following:
- If the cells are not healthy, then poor-quality or no plaques can result. Ideally, cells should be in mid-log phase and have a viability of greater than 90%. Cells should double at least once before infection stops growth. Ensure that the correct amount of cells was used at ~70% confluency.
- The viral replication cycle can be inhibited due to poor nutritional and physical conditions of the cell.
- The temperature of the agarose is also crucial. After overlaying the agarose, the plates should be left untouched for 1 hour for the agarose to completely solidify.
- Excessive condensation during incubation at 27 degrees C can inhibit plaque formation-remove paper towels or open the container containing plates as soon as condensation appears.
- The viral titer is too low: Use a higher viral titer. You may need to re-infect your cells and collect a higher titer of your viral stock.
Please check the construction of your entry clone, and ensure that the insert is in frame with the vector. Analyze the recombinant viral DNA by PCR to confirm the correct size and orientation of your insert after the LR reaction. Sequence your PCR product to verify the proper reading frame for expression of the epitope tag.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
To get a high-titer stock, reinfect cells with the P1 stock and generate a P2 high-titer stock. Follow the directions in the BaculoDirect manual on page 18 to generate your P2 stock.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Please see our recommendations below:
- Check the LR reaction by PCR analysis prior to transfection into insect cells.
- We recommend using Grace's Insect Cell Culture Medium, Unsupplemented during the transfection experiment instead of serum-free medium, as components in serum-free medium may interfere with transfection.
- Ensure that FBS, supplements, or antibiotics are not included during transfection, as the proteins in these materials can interfere with the Cellfectin II Reagent.
- Use the LR recombination reaction using the pENTR/CAT plasmid as a positive control and Cellfectin II Reagent only (mock transfection) as a negative control.
- Ensure that cells are in the log phase of growth with >95% viability, and the amount of cells are in accordance with the suggestions in the manual.
- Cells may not show signs of viral infection for up to a week depending on transfection efficiency; continue culturing and monitor cells daily for signs of infection.
Warm the ganciclovir solution in a water bath at 37 degrees C for 5-10 min, then vortex for a few minutes. The precipitate should go back into solution.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Media used to culture insect cells usually have an acidic pH (6.0-6.5) or contain electron-donating groups that can prevent binding of the 6xHis-tagged protein to Ni-NTA. Amino acids such as glutamine, glycine, or histidine are present at significantly higher concentrations in media for growing insect cells than in media for growing mammalian cells, and compete with the 6xHis-tagged protein for binding sites on Ni-NTA matrices. Grace's medium (Thermo Fisher Scientific), for example, contains approximately 10 mM glutamine, 10 mM glycine, and 15 mM histidine.
Dialysis of the medium against a buffer with the appropriate composition and pH (8.0) similar to the lysis buffer recommended for purification under native conditions usually restores optimal binding conditions. Note that depending on the medium used, a white precipitate (probably made up of insoluble salts) can occur, but normally the 6xHis-tagged protein remains in solution. This can be tested by either protein quantitation if using a protein-free medium or by monitoring the amount of 6xHis-tagged protein by western-blot analysis. After centrifugation, 6xHis-tagged protein can be directly purified from the cleared supernatant.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Please review the following possibilities and solutions:
- Viral stock was amplified using high MOI originally: Go back to the lower-passage viral stock and do a low-MOI amplification.
- Did not spin down and get rid of cells when harvesting viral supernatant: Go back to the lower-passage viral stock and do a low-MOI amplification; if this viral stock is P2, this stock can be used in amplification.
- For some genes, the virus can become very unstable: Free the aliquoted P2 viral stock and do one run of amplification after reviving.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Low protein yield may occur due to the following reasons:
- Viral stock contains a mixture of recombinant and non-recombinant baculovirus: Perform plaque purification to isolate recombinant baculovirus.
- Baculovirus is not recombinant: Verify transposition by PCR analysis of bacmid DNA using pUC/M13 forward and reverse primers; re-transfect insect cells with new recombinant bacmid DNA.
- Use too low or too high viral titer: Vary the MOI.
- Time of cell harvest is not optimal: perform a time course of expression to determine the optimal time to obtain maximal protein expression.
- Cell growth conditions and medium are not optimal: Optimize cell culture conditions based on the size of your culture vessel and expression conditions; we recommend using Sf-900 II SFM or Sf-900 III SFM for optimal cell growth and protein expression.
- Cell line is not optimal; try other insect cell lines.
- Cells were harvested too late: Do a time-course experiment and harvest cells at different time points.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Check the MOI. It may be low because the titer of the P1 virus is lower than what was estimated.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Please see the possible reasons and suggestions below:
- Mixture of Cellfectin II Reagent and bacmid was not performed or was not incubated long enough: Mix the Cellfectin II Reagent and bacmid well by tapping or gentle vortexing, and incubate the mixture for 15-45 min.
- Bacmid yield is lower than estimated: Set up an optimization with different amounts of bacmid.
- Bacmid is sheared during purification or freeze/thaw: Verify the integrity of bacmid on a gel.
- Incubation time is not long enough: Incubate mix for 8 hr at 27 degrees C.
- Cells used are of high passages or have passed log-phase growth: For best results, use cells between 8-15 passages; plate cells when they are in log-phase growth.
- Cellfectin II Reagent has been frozen: Purchase a new vial.
- Medium used contains serum: Use unsupplemented Grace's medium in transfection.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
There are several possibilities:
- Using media containing antibiotics during transfections.
- Plating cells at too low a density: We recommend at least 70% confluence.
- Using cells at too early a passage: We recommend growing cells for at least 5 passages before using them for transfection.
- Contamination because of no pen/strep after the transfection: After 5-8 hr incubation with the transfection mixture, remove the mixture and add antibiotics containing media/well.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
This may be due to contamination or cytotoxicity from the bacmid prep. Make sure to include a negative control that is the bacmid only without Cellfectin II Reagent. Additionally, use the PureLink HiPure plasmid prep kit, not the silica-based miniprep kit for bacmid prep.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Most likely, a colony that was gray or dark in the center was picked. Try to analyze more white DH10Bac transformants. Typically, we recommend picking a white colony whose diameter is >2 mm. Restreak the white colonies on a fresh plate with 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline, 100 µg/mL Bluo-gal and 40 µg/mL IPTG. Incubate plates for 24 hours.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Please see the possible causes and suggestions we have to alleviate this problem:
- Insert is very long and causes difficulties in PCR: Instead of using both M13 forward and reverse primers, use one gene-specific primer paired with the M13 primer of your choice.
- Long GC-rich stretches in the gene of interest: Consider using DMSO (up to 8%) in the PCR reaction.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Please see the possible reasons and suggestions below:
- DNA stored improperly: Ensure that purified bacmid DNA is stored at -20 degrees C in aliquots to avoid repeated free/thaws.
- High molecular weight bacmid DNA handled improperly: When isolating bacmid DNA, do not vortex the DNA solution; additionally, do not resuspend DNA pellets mechanically; allow solution to sit in the tube with occasional tapping.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
This could be caused by the following:
- Wrong antibiotic or old media: use fresh media.
- Colonies are too old or too small: Use large white colonies from freshly streaked plates.
- Unstable insert caused by special feature of the gene of interest; for example, direct repeats: Incubate the culture at 30 degrees C for 24 hours instead of 37 degrees C overnight.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Poor color differentiation for your colonies could be caused by the following:
- Agar is not at the correct pH: Adjust pH of LB agar to 7.0.
- Intensity of the blue color is too weak; ensure that you are using Bluo-gal, not X-Gal. You can also try increasing the concentration of Bluo-gal to 300 µg/mL.
- Too many or too few colonies on the plate: Adjust the serial dilutions of cells to obtain an optimal number of colonies.
- Incubation period too short or temperature too low: Do not pick colonies until 48 hours after plating; incubate plates at 37 degrees C.
- IPTG concentration is not optimal: A range of 20-60 µg/mL IPTG generally gives optimal color development.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We recommend running 1/8th of the 40 µL midiprep sample on a 0.5% TAE agarose gel. Electrophorese slowly at 23 V for 12 hr. The banding pattern of the recombinant bacmid midiprep should be seen.
Please review the following reasons and our recommendations:
- Use LB medium for recovery/expression period: Use SOC medium for the 4 hr growth time.
- Recovery/expression time too short: Increase the recovery time to > 4 hr at 37 degrees C or 6 hr at 30 degrees C.
- IPTG concentration is not optimal: We suggest using 20-40 µg/mL IPTG.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Please review the following possibilities and recommendations:
- pFastBac DNA used for transformation was of poor quality: Use purified plasmid DNA for transformation and check the quality of your plasmid DNA.
- Gentamicin omitted from plates: Prepare fresh selective plates containing 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline, 100 µg/mL Bluo-gal, and 40 µg/mL IPTG.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Although you will be picking white (recombinant) colonies, you should expect to see some blue (contain non-recombinant bacmid) colonies. Here are some possible causes for seeing no blue colonies and recommendations for the same:
- Insufficient time for color development: Wait at least 48 hours before identifying colony phenotypes.
- Use Bluo-gal instead of X-Gal in agar plates: Use Bluo-gal in plates to increase contrast between blue and white colonies.
- Insufficient growth after transposition: Grow transformed cells in SOC medium for a minimum of 4 hours before plating.
- Bluo-gal and IPTG omitted from plates: Prepare fresh selective plates containing 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline, 100 µg/mL Bluo-gal, and 40 µg/mL IPTG.
- There are too many colonies on the plate: Serially dilute the transformation mix to obtain well-spaced colonies (10-2 to 10-4 is suggested).
- Plates are too old or stored in light: Do not use plates that are more than 4 weeks old; store plates protected from light.
- Incubation period too short or temperature is too low: Wait at least 48 hours before picking colonies. Incubate plates at 37 degrees C.
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If this lower-titer stock is a P1 or P2 stock, a viral amplification protocol can be used. If the low-titer stock was once a high-titer stock, but has dropped titer due to age or the stock was propagated many generations, then it may be necessary to regenerate the high-titer stock. If the high titer stock is >P5, then there may be an excessive amount of defective interfering particles that infect cells but do not properly replicate or produce protein. If the existing stock is plated out and a fresh plaque is re-isolated (DIPs do not form plaques), a new high-titer stock can be established.
Yes, the same protocol used to make your P2 viral stock can be used to make a P3, P4, or P5 viral stock. We don't recommend making the stock higher than P5, as more defective interfering particles will be produced and a decrease in protein expression level will occur.
Yes, baculovirus can infect mammalian cells, although only at very high titers. Baculovirus works best in liver cells. However, there is no danger of cross-contamination unless the cells are directly infected with the high-titer stocks. Bacuolvirus can infect Drosophila cells; however, it will not replicate in these cells. The promoters used to drive expression of your gene in a typical baculovirus system are both late promoters and require earlier proteins from the baculovirus genome. Thus, they will not work in S2 cells since the early proteins are not made.
Typically, 0.5 x 106 cells per well in 2.5-3 mL is a good starting point. Lysis should begin by day 3. Virus may be harvested and amplified between 3 and 7 days (90% cell death).
Our R&D team will typically pick a plug and add it to a 12-well dish with 0.5 x 10e6 cells/well and 2.5 mL total volume per well. After approximately 3 days, remove 0.75 mL to make DNA for PCR and keep the remaining medium in an Eppendorf tube as your P1 viral stock. As an aside, it is okay to pick a plaque and store it in Grace's medium.
We recommend harvesting high-titer virus when there is 90% cell lysis. This takes approximately 5-7 days. If the cells go longer, the proteases released from the lysed cells will start to degrade viral surface proteins and result in less infectious virus.
This is dependent on how much virus is added. If cells are infected at an MOI of 5, usually cells are infected at 24 hours, and cells begin to lyse at around 65 hours. If less virus is used, this takes longer, and more virus takes less time.
When propagating virus stock, use a low MOI (0.03-0.1) in order to avoid effects of defective interfering particles (DIPs). A low MOI, which ensures no more than 1 virion per cell, prevents the amplification of DIPs. A harvest time based on 15% cell viability is appropriate. NOTE: DIPs are nearly normal virus capsids containing genomes that are defective and are unable to undergo successful replication. While this "particle" is not infectious by itself, it can replicate when co-infected with normal virion, or with some other types of DI particles.
You can stain the monolayer with neutral red or MTT to make the plaques more visible. Alternatively, you can allow the plates to develop for a few days longer (2-5 days on average) at room temperature to increase the contrast in recombinant plaques. However, the plaques stained with neutral red cannot be used for plaque purification and viral amplification.
We suggest using a viral stock with a titer of >1 x 10e8 pfu/mL for expression studies.
Please see the equation below:
pfu/mL = number of plaques (pfu)/dilution factor x mL of inocula
So, if you have a well with viral dilution of 10-8 containing 18 white plaques, the viral titer is calculated as followed:
X pfu/mL = 18 pfu/10-8 x 1 mL
X = 1.8 x 10e9 pfu/mL
Please see the method below for an outline of the main steps of performing a plaque assay:
- Plate cells at 80% confluency in a 6-well plate
- Make a serial dilution of the P1 viral stock (1-10-5) and add to cells
- Incubate for an hour at 27 degrees C
- Mix 1% melted agarose into the medium
- Remove the viral supernatant
- Overlay the cells with the medium containing agarose
- Leave the plates for 2-3 hours for agar to completely solidify
- Incubate plates for 10-14 days
- Count plaques
When performing this assay, we suggest:
- Use cells that are in excellent health, of low passage (10-20) in log-phase growth, and high viability (>95%)
- Check viral stock for sterility (free of contamination)
- Use high-quality, low melting point agarose
- The temperature of the medium with agarose is crucial-too hot, cells will die; but if too cold, it will solidify too quickly
- Wait 2-4 hours before removing the plate after overlay so that the agarose can 100% solidify
- Count plaques on a dilution plate where (1/dilution) x # of plaques = pfu/mL
e.g., if you have 50 plaques on the 10-6 plate, then you have 1(10-6) x 50 = 5 x 10e7 pfu/mL
We recommend you perform a plaque assay to determine the titer of your viral stock. You may also perform a plaque assay to purify a single viral clone, if desired.
In the Bac-N-Blue system, recombination between the transfer vector and the baculovirus DNA occurs in insect cells. The Bac-N-Blue vector is a linearized AcMNPV derivative that contains an incomplete (3') lacZ fragment. The corresponding transfer vector contains a 5' lacZ fragment. Upon homologous recombination, the recombinant Bac-N-Blue baculovirus DNA will have a complete lacZ gene that is under the control of the PETL promoter. Thus, recombinant Bac-N-Blue baculovirus will provide blue plaques in the plaque assay and can be easily identified. In the Bac-to-Bac expression system, recombination or site-specific transposition between transfer and baculovirus DNA occurs in E. coli (DH10Bac). In the Bac-to-Bac expression system, selection of colonies containing recombinant baculovirus DNA occurs in the presence of Luria Agar plates with 50 µg/mL kanamycin (bacmid), 7 µg/mL gentamycin (pFastBac), 10 µg/mL tetracycline (helper plasmid), 100 µg/mL Bluo-gal, and 40 µg/mL IPTG.
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The following is an excellent reference for how to prevent proteolytic artifacts in the baculovirus expression system:
Hom LG, Volkman LE (1998) Preventing proteolytic artifacts in the Baculovirus expression system. BioTechniques 25:18-20.
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The baculovirus rod will continue to elongate as required to package the DNA. Thus, the system could theoretically accommodate hundreds of Kb. Standard cloning techniques will limit the insert size before packaging limits become an issue.
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Insect-derived signal peptides and/or prosequences cannot always enhance the expression and/or secretion of foreign secretory pathway proteins in the baculovirus system. Please see the following references:
- Jarvis DL, Summers MD, Garcia A Jr, Bohlmeyer DA (1993) Influence of different signal peptides and prosequences on expression and secretion of human tissue plasminogen activator in the baculovirus system. J Biol Chem 268(22):16754-16762.
- Tessier DC, Thomas DY, Khouri HE, Laliberte F, Vernet T (1991) Secretion of a plant protein in the baculovirus system was enhanced when its signal peptide was replaced with an insect-derived signal peptide. Gene (Amst.) 98:177-183.
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Our R&D team has successfully expressed proteins up to 300 kDa. If they express in >2% serum, it should minimize degradation. If you don't mind the extra step of purification, 10% serum could be used. We highly recommend doing a time-course infection with high-titer stock, with a MOI of 5-10, to make an assessment of the minimum harvesting time necessary for the best expression. Time points should be taken every 24 hours for 5 days.
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The polyhedron protein is 30 kDa.
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The promoter that drives the gene of interest is the polyhedron promoter.
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While the importance of a Kozak consensus sequence in translation initiation has been demonstrated in mammalian cells, there seems to be some debate as to whether the Kozak rules are as stringent in insect cells. The only way to determine its importance would be a direct comparison of expression of the same protein from different initiation sequences. Even then, the rules for optimal expression of one protein may not hold for another. Here are two references which indicate that a Kozak consensus sequence does not have any effect on efficiency of expression in insect cells:
- Hills D, Crane-Robinson C (1995) Baculovirus expression of human basic fibroblast growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression.
- Biochim Biophys Acta 1260(1):14-20.
- Ranjan A, Hasnain SE (1995) Influence of codon usage and translational initiation codon context in the AcNPV-based expression system: computer analysis using homologous and heterologous genes. Virus Genes 9(2):149-153.
Yes, it is possible. Several five-subunit proteins, such as human replication factor C, have been expressed using recombinant baculovirus. We recommend that a separate high-titer stock (HTS) of each subunit be produced to optimally express the multi-subunit protein. This way, the amount of each subunit expressed can be controlled by varying the multiplicity of infection (MOI) of each subunit's HTS. Please refer to the following articles for more information:
- Chen W and Bhal OP (1991) Recombinant carbohydrate and selnomethionyl variants of human choriogonadotropin. J Biol Chem 266(13):8192-8197.
- Chen WY and Bhal OP (1991) Selenomethionyl analog of recombinant human choriogonadotropin. J Biol Chem 266(15):9355-9358.
- Fabian JR, Kimball SR, Jefferson LS (1998) Reconstitution and purification of eukaryotic initiation factor 2B (eIF2B) expressed in Sf21 insect cells. Protein Expr Purif 13(1):16-22.
The MOI, or multiplicity of infection, is the average number of viral particles that infect a single cell in a specific experiment. You can calculate the MOI with the following equation:
MOI (pfu/cell) = [titer (pfu) x viral stock volume (mL) used in inocula] / [cell density (cells/mL) x culture volume (mL)]
Yes, large-scale expression experiments can be performed. Please see below for different large-scale methods, requirements, added benefits, and references:
- Stirred bioreactor
- Airlift fermentor
- Insect larvae
If the medium is serum-free, add serum to 10%. Serum proteins act as substrates for proteases and therefore prevent degradation of viral coat proteins. Store viral stocks at 4 degrees C, and protect from light. Aliquots can be stored at -80 degrees C, but viral titer should be checked before use, as freeze/thaw cycles of the virus can result in a 10- to 100-fold decrease in viral titer.
Yes. Contamination of your recombinant DNA with uncut occlusion body positive (occ+) DNA will lead to dilution of your recombinant virus over time because, in general, uncut (wild-type, occ+) virus infects and replicates at higher efficiency than recombinant virus. Also, initiating expression studies with a pure, single virus population will ensure reproducible results.
Please see the description below of the different stages of viral infection:
Early
- Increased cell diameter-a 25-50% increase in the diameter of the cells may be observed.
- Increased size of cell nuclei-the nuclei may appear to "fill" the cells.
Late
- Cessation of cell growth-cells appear to stop growing when compared to a cell-only control.
- Granular appearance
- Signs of viral budding-vesicular appearance of cells.
- Viral occlusions-few cells will contain occlusion bodies, which appear as refractive crystals in the nucleus of the insect cell.
- Detachment-cells release from the dish or flask.
Very late
- Cell lysis-a few cells may fill with occluded virus, die, and burst, leaving signs of clearing in the monolayer.
Yes, we offer our Bac-to-Bac HBM TOPO Secreted Expression System (Cat. No. A11338 or A11339), which uses the honeybee melittin (HBM) secretion signal.
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Adherent Sf9 cells round up and show a smaller contact point. Infected Sf9 cells in suspension culture round up and look larger when infected.
Please follow the recommendations below:
- Cells should be in excellent health, of their low passages (5-15), in log-phase growth, with viability >95%
- DNA must be of high purity, free of endotoxin
- No antibiotics should be used during transfection
- Cellfectin reagent has to be completely resuspended
- Include controls (media control, DNA control, and transfection reagent control) for comparison and troubleshooting
Peak expression of protein in insect cells is dependent on the multiplicity of infection (MOI), expression time, and the protein being expressed. Guidelines to optimize your system include using an MOI of 5-10 and an expression time of 48-72 hours. Protein expressed at times later than 72 hours may be processed aberrantly, because the large virus load can cause a breakdown of cellular processes.
Yes, baculovirus is a good candidate for the problem of expressing toxic proteins (i.e., membrane proteins). The polyhedron promoter does not express at maximal levels until 18-24 hr after infection. The polyhedron promoter is active late in the lytic cycle. That being said, it is minimally active as early as 8 hours, so if the gene is very toxic, there may be a problem. The solution in that case would be to switch to an inducible expression system. Transmembrane proteins can often be difficult to express in any system.
ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.
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Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
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You will get the antisense strand with pProEXHT.
You will get the + or sense strand with pFastBac I of pFastBac HT.