Q1: 什么是噬菌体展示技术？
A1:噬菌体展示技术(phage display technology)是将多肽或蛋白质的编码基因或目的基因片段克隆入噬菌体外壳蛋白结构基因的适当位置，在阅读框正确且不影响其他外壳蛋白正常功能的情况下，使外源多肽或蛋白与外壳蛋白融合表达，融合蛋白随子代噬菌体的重新组装而展示在噬菌体表面。被展示的多肽或蛋白可以保持相对独立的空间结构和生物活性，以利于靶分子的识别和结合。肽库与固相上的靶蛋白分子经过一定时间孵育后，洗去未结合的游离噬菌体，然后以竞争受体或酸洗脱下与靶分子结合吸附的噬菌体，洗脱的噬菌体感染宿主细胞后经繁殖扩增，进行下一轮洗脱，经过3轮～5轮的“吸附-洗脱-扩增”后，与靶分子特异结合的噬菌体得到高度富集。所得的噬菌体制剂可用来做进一步富集有期望结合特性的目标噬菌体。
Q2: 噬菌体展示与其他的抗体开发技术相比有什么优点？
A2: 与杂交瘤技术相比，噬菌体展示技术具有很大的优势。杂交瘤法仅适用于小鼠、大鼠、仓鼠和豚鼠。另一方面，噬菌体展示可用于包括但不限于人、小鼠、大鼠、兔、鸡、骆驼、骆驼、羊驼、牛、狗、绵羊、猴和鲨鱼在内的所有流行的抗体产生物种产生高亲和力单克隆抗体。基于杂交瘤的单克隆抗体开发一次只能产生少量针对特定免疫原的抗体，而噬菌体展示技术可以呈现免疫动物的整个抗体库，其中几乎10%的抗体是免疫原特异性。使用噬菌体展示技术发现具有所需性质的抗体的机会要大得多。此外，使用杂交瘤技术，很难结合能够选择性分离具有所需功能的抗体的富集步骤。在大多数情况下，首先产生所有的杂交瘤克隆，然后逐个验证。相比之下，噬菌体展示技术允许各种富集策略：可以富集具有所需性质的抗体，从而可以将那些没有所需功能的抗体排除在进一步验证之外。例如，抗体库筛选可以使用靶捕获强结合物同时使用控制来阻断/耗尽交叉反应结合物。总之，这种免疫抗体库方法允许收集动物体内几乎所有的抗体，并从收集物中分离出最强的结合物。
Q3: M13、T7和T4噬菌体系列的区别是什么？
A3: M13丝状噬菌体一直是最流行的选择，广泛用于各种类型的研究。病毒外壳由五个不同的衣壳蛋白组成，包括一个主要衣壳pVIII（2,700拷贝）和四个次要衣壳（一端是pIII和pVI，另一端是pVII和pIX）。与T4和T7不同的是，M13是溶原性噬菌体，它在周质中组装，在不裂解宿主的情况下从细菌膜中分泌出来。M13噬菌体上的多重衣壳蛋白为具有独特特性的多种肽和蛋白质提供了全面的展示选择。这五种衣壳都已成功地用于具有独特载体的外域显示。PIII和PVIII是M13噬菌体展示的首要选择。
噬菌体T4在许多方面不同于M13，T4具有更大的尺寸，尾部结构和编码50种不同蛋白质的双链DNA（dsDNA）基因组，较大的基因组DNA允许更大的外来蛋白的插入。可以在HOC和SOC上显示两个不同的结构域，这两种非必需的外壳蛋白。可以使用N-和C-末端插入。在没有膜分泌过程的情况下，使用T4噬菌体可以避免宿主毒性。
与丝状噬菌体和λ噬菌体相比，T7噬菌体的生命周期较短。子代噬菌体的组装在细菌细胞质中，通过细胞膜裂解而释放，因此没有大小限制和分泌过程导致的宿主毒性。此外，T7噬菌体在其他噬菌体不能存活的极端条件下非常稳定。
Q4: 可以用M13噬菌体展示系统来构建cDNA文库吗？
A4: 通常M13不适合cDNA表达，因为M13噬菌体展示需要前导序列（分泌所需）和外壳蛋白pIII或pVIII的N末端之间的框内表达。为了将相应的蛋白质适当地融合到外壳蛋白中，插入物必须在两端的正确阅读框中并且不包含框内终止密码子。这导致M13 cDNA文库中产生的克隆数量极少。
Q5: 培养噬菌体可以用什么培养基？
A5: TSB / TSA可用于培养大部分噬菌体。然而，不同的噬菌体展示系统，适合的培养基可以是各种各样的。
Q6: 噬菌体M13可以感染哪种大肠杆菌？ 它是否有机会污染我们实验室中的所有细胞株？
A6: M13噬菌体是一种丝状噬菌体，它使用细菌F接合菌毛的尖端作为受体来促进感染过程。因此，它们仅对含有F质粒（F+）的大肠杆菌菌株具有特异性。对于实验室中的菌株，我们建议您检查它们是否含有F质粒。如果他们不具有，M13噬菌体就不能感染它们。
Q7: 大肠杆菌TG1宿主菌是否含有抗生素抗性？
A7: 大肠杆菌TG1菌株不含抗生素耐药性，而噬菌体感染的TG1菌株可通过2YT-AK培养基（2YT含100μg/mL氨苄青霉素、50μg/mL卡那霉素）筛选。
Q8: PFU和CFU的区别？
A8: pfu，指菌斑形成单位，是衡量单个感染性粒子数量的单位，通常用于计算噬菌体。cfu，指的是集落形成单位，是活细胞的量度，其中集落代表源自单个祖细胞的细胞集合，并且通常用于计数细菌（例如大肠杆菌）。
Q9: 怎么判断构建的噬菌体文库的优劣？
A9：从免疫文库中选择的抗体亲和力与文库的大小成正比，文库库容越大，抗体亲和力越好。通常，最终库容的大小应该达到108。它足够大以分离高亲和力抗体。此外，作为一个优质文库，它应该具有高度的多样性。最终文库的QC结果表明，所有随机挑选的克隆均未发现共同的序列，表明文库的多样性处于很高的水平。
来源：http://www.kmdbioscience.cn/nt-shijuntizhanshiFAQs
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