CN109402096B - 一种aid酶突变体及其应用 - Google Patents
一种aid酶突变体及其应用 Download PDFInfo
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- CN109402096B CN109402096B CN201811384028.4A CN201811384028A CN109402096B CN 109402096 B CN109402096 B CN 109402096B CN 201811384028 A CN201811384028 A CN 201811384028A CN 109402096 B CN109402096 B CN 109402096B
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Abstract
本发明提供一种用于哺乳动物细胞蛋白(尤其是抗体)筛选、工程化改造/进化的AID酶突变体及其应用。本发明提供的AID酶突变体在CHO细胞中的突变能力显著增强，相对于野生型AID能够提供数量更多、突变类型更丰富的抗体突变体，并且不影响CHO细胞的存活率，有利于快速高效的构建抗体突变库和加速抗体亲和力成熟。
Description
技术领域
本发明属于基因工程、生物工程领域，具体涉及一种AID酶突变体及其应用。
背景技术
过去20多年中，蛋白、多肽、抗体药物的开发和临床治疗应用取得了长足进展。驱动治疗性抗体、蛋白发展的关键技术之一是抗体的亲和力成熟。目前用于抗体筛选和亲和力优化的技术有核糖体展示、噬菌体展示、酵母展示、细菌展示以及哺乳动物细胞展示。噬菌体展示、细菌展示和酵母展示系统进行抗体进化的基本流程均为：1)利用易错PCR、DNA改组、交错延伸、随机体外重组等方法获得抗体突变，2)通过酶切、连接构建抗体质粒库，转染相应的细菌或者酵母细胞，展示抗体，3)筛选出具有优良性状的抗体突变体。但是以上展示技术是以非细胞(噬菌体)或者微生物细胞(细菌、酵母)为平台建立的，在抗体蛋白的表达、折叠及修饰(如糖基化、甲基化等)等方面与动物细胞仍然具有明显的差别，在药用过程中常常会导致人体产生针对抗体的免疫反应，从而影响抗体的应用及药效。由于哺乳动物细胞展示技术筛选的抗体在结构和修饰方面更接近于人体自身的抗体，故该技术近年来受到了越来越多的关注。
基于近年来体细胞高频突变的理论研究成果，人们发现在B细胞的成熟过程中，激活诱导的胞嘧啶脱氨基酶(AID)诱导胞嘧啶脱氨基产生尿嘧啶使抗体基因发生高频突变,从而为改变抗体的特异性和亲和力提供了基础。因此，现阶段的哺乳动物细胞抗体进化平台将动物细胞抗体展示技术与体细胞高频突变(SHM)联系起来，模拟体内亲和力成熟过程，进行抗体亲和力成熟。这类抗体进化平台主要包括三部分：1)选择或者建立具有突变能力的细胞，2)将抗体稳定的展示在突变细胞表面，3)流式分选或者磁珠筛选富集优良的抗体突变体。该平台不仅能够用于筛选抗体，还能够应用于多肽或蛋白的进化，使其稳定性增加，或使多肽、蛋白与配体的亲和力得到提升，可应用于多肽或蛋白类药物的研发。
国内外的一些研究小组已经根据AID致突变作用在哺乳动物细胞中建立了各自的抗体亲和力成熟系统，并且成功的在体外对一些抗体进行了亲和力成熟优化。然而这些系统都存在以下缺陷：1)细胞突变效率不高，导致库容量相对偏小；效率低包括以下两个原因：a.AI D除了突变抗体基因也能突变别的细胞基因，细胞不能耐受太高水平的AID；b.AID也能突变AID自身，多轮扩增后，有的细胞会丢失AID活性；2)获得稳定的展示目标抗体的细胞相对困难，亲和力成熟所需时间长。在本课题组之前的工作中，筛选出一株稳定且高表达目的基因的CHO细胞(参见专利ZL201410803422.2)，对抗体亲和力成熟进化筛选效率较高，能够在较短的时间和筛选轮次内将抗体亲和力大幅度提高。
参考文献：
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发明内容
针对上述现有技术存在的缺陷，本发明所要解决的技术问题是：如何提高哺乳动物细胞突变效率，尽可能提供多样化的抗体突变体/变异体，且在此基础上不影响哺乳动物细胞的生存和扩增能力。
本发明深入研究AID和抗体基因对抗体库产生效率的影响，构建具有更高突变效率的AID酶突变体。有报道，如果去掉AID酶C端的核输出信号(NES)，AID酶可在细胞核中聚集，突变能力也会增加。此外，人们在大肠杆菌中筛选了具有更高催化活性的AID酶突变体(T82I，K10E/E156G/T82I，R119G和D118A/R119G/K120R/A121R)。这些AID酶突变体能否在CHO细胞中发挥作用，提高抗体基因库多样性产生效率，值得深入研究。本发明中，我们构建了AID酶突变体，将其应用到本课题组现有的CHO细胞抗体进化平台，检测以上突变及突变组合是否具有更高的突变效率。
为实现本发明目的，本发明提供如下技术方案：
本发明提供了一种AID酶突变体，其氨基酸序列如SEQ ID NO.5、9或11所示。
本发明还提供了一种AID酶突变体的编码基因，其能够编码产生SEQ ID NO.5、9或11所示氨基酸序列；优选的，所述编码基因的核苷酸序列为SEQ ID NO.6、10或12所示。
本发明提供了一种包含AID酶突变体的载体，所述载体包含编码SEQ ID NO.5、9或11所示氨基酸序列的核苷酸序列。
优选的，上述载体包含SEQ ID NO.6、10或12所示的核苷酸序列。
本发明提供了一种细菌或非人哺乳动物细胞，所述细胞包含具有SEQ ID NO.5、9或11所示氨基酸序列的AID酶突变体；所述非人哺乳动物细胞优选CHO细胞。
本发明提供了一种AID酶突变体、其编码核酸、包含其编码核酸的载体、细菌或细胞在蛋白或多肽筛选、抗体亲和力成熟、抗体工程化改造、抗体筛选、和/或抗体突变库建立中的应用，所述AID酶突变体的氨基酸序列如SEQ ID NO.5、9或11所示。
本发明提供了一种AID酶突变体在筛选进化TNF-α抗体中的应用，所述AID酶突变体的氨基酸序列如SEQ ID NO.5、9或11所示。
本发明提供了一种优化的AID酶的编码核苷酸，所述核苷酸序列如SEQ ID NO.16所示。
本发明提供了一种包含SEQ ID NO.16的载体、细菌或非人哺乳动物细胞；所述非人哺乳动物细胞优选CHO细胞。
上述优化的AID酶的编码核苷酸、载体、细菌或非人哺乳动物细胞在蛋白或多肽筛选、抗体亲和力成熟、抗体工程化改造、抗体突变库建立和/或抗体筛选中的应用。
优选的，本发明所述蛋白可选自抗体、单链抗体(ScFv)、Fab片段、F(ab’)2片段、重链抗体、纳米抗体、多肽、融合蛋白等。
本发明的有益效果：
1、本发明提供的AID酶突变体在CHO细胞中的突变能力显著增强，相对于野生型AID能够提供数量更多、突变类型更丰富的抗体突变体。
2、本发明提供的AID酶突变体不影响CHO细胞的存活率，有利于抗体突变库、蛋白突变库的快速高效的建立和抗体亲和力成熟。
附图说明
图1.AID及其突变体结构示意图。
A.野生型AID结构(对照)，mAID(小鼠源)和hAID(人源)的基本结构相同；
B.删除NES结构域的AID突变体结构，mAID-del(鼠源)，hAID-del(人源)；
C.删除NES结构域并包含K10E/T82I/E156G点突变的AID突变体结构示意，mAID-del-K10E/T82I/E156G(鼠源，简写mAID-plus)，hAID-del-K10E/T82I/E156G(人源，简写hAID-plus)。
AID蛋白包括核定位结构域(NLS)，核输出结构域(NES)，催化结构域(Catalyticdomain)和APOBEC蛋白样结构域(APOBEC protein-like domain)。
图2.使用GFP突变体(GFP*)作为报告基因检测AID突变效率的原理示意图。将pEGFP-C1(Clontech)载体中GFP基因中的一个RGYW高频突变热点通过点突变引入一个终止密码子TAG，构建GFP*。GFP*在高频突变作用下可能产生突变使TAG突变成其它密码子，从而让蛋白可以翻译完全恢复荧光。通过流式检测GFP荧光的恢复程度可以分析高频突变的水平。
图3.利用GFP报告基因检测AID突变体突变效率。FL1:检测绿色荧光，FL4：检测APC荧光，Q1区显示了突变体数量和百分比。A、未作任何转染的阴性细胞；B、仅转染了pCDNA3.1(+)-GFPm的阴性对照组；C、转染mAID，D、转染mAID-del，E、转染mAID-plus，F、转染hAID，G、转染hAID-del，H、转染hAID-plus(C-H同时转染了pCDNA3.1(+)-GFPm)。
图4.利用GFP报告基因检测不同AID突变体的突变效率比较。以mAID数据为标准，对mAID-del、mAID-plus、hAID-del、hAID-plus数据进行归一化处理，比较突变效率差异倍数。差异显著性分析(TTEST)在原始数据基础上计算得出，*表示P<0.05，**表示P<0.01，***表示P<0.001。
图5.利用AID突变体在CHO细胞中对TNF-α抗体进行亲和力成熟进化，不同AID对不同抗体序列诱导的突变频率比较。TNF-α表示未经序列优化的野生型抗体，TNF-α-mopti表示经核酸序列优化的抗体；mAID表示野生型AID，mAID-plus表示AID突变体。
图6.野生型mAID与抗突变mAID(mAID-decrease)的突变频率比较。
具体实施方式
通过以下实施例对本发明作进一步的详细描述，但应理解本发明并不受以下内容所限制。
实施例1AID突变体构建
本发明构建4种AID突变体，删除NES结构域的AID突变体：小鼠源mAID-del(SEQ IDNO.5)，人源hAID-del(SEQ ID NO.7)；删除NES结构域并包含K10E/T82I/E156G点突变的AID突变体：mAID-del-K10E/T82I/E156G(简写mAID-plus)(SEQ ID NO.9)，hAID-del-K10E/T82I/E156G(简写hAID-plus)(SEQ ID NO.11)。突变体结构示意图参见图1。
野生型AID：mAID(SEQ ID NO.1)，hAID(SEQ ID NO.3)作为对照。
1、引物设计
根据mAID(SEQ ID NO.2)，hAID(SEQ ID NO.4)，mAID-del(SEQ ID NO.6)，hAID-del(SEQ ID NO.8)，mAID-plus(SEQ ID NO.10)，hAID-plus(SEQ ID NO.12)的核苷酸序列，使用软件设计PCR扩增引物如下：
| 引物名称 | 引物序列 | 序列号 |
| HindⅢ-mAID-F | GTACATAAGCTTATGGACAGCCTTCTG | SEQ ID NO.17 |
| mAID-XhoI-R | GTACATCTCGAGTCAAAATCCCAAC | SEQ ID NO.18 |
| HindⅢ-hAID-F | GTACATAAGCTTGCCACCATGGACAGCCTC | SEQ ID NO.19 |
| hAID-XhoI-R | GTACATCTCGAGTCAAAGTCCCAAAGTACG | SEQ ID NO.20 |
| HindⅢ-mAID-del-F | GTACATAAGCTTATGGACAGCCTTCTG | SEQ ID NO.21 |
| mAID-del-XhoI-R | GTACATCTCGAGTCACAAGGGCAAAAGG | SEQ ID NO.22 |
| HindⅢ-hAID-del-F | GTACATAAGCTTATGGACAGCCTCTTG | SEQ ID NO.23 |
| hAID-del-XhoI-R | GTACATCTCGAGTCACAGGGGCAAAAGG | SEQ ID NO.24 |
| HindⅢ-mAID-plus-F | GTACATAAGCTTATGGACAGCCTTCTG | SEQ ID NO.25 |
| mAID-plus-XhoI-R | GTACATCTCGAGTCACAAGGGCAAAAGG | SEQ ID NO.26 |
| HindⅢ-hAID-plus-F | GTACATAAGCTTATGGACAGCCTCTTG | SEQ ID NO.27 |
| hAID-plus-XhoI-R | GTACATCTCGAGTCACAGGGGCAAAAGG | SEQ ID NO.28 |
2、表达载体构建
常规质粒可随机整合到细胞基因组中稳定存在，但由于整合到基因组上的位置不同，目的蛋白的表达量会有巨大的差异。而非整合质粒不会像随机整合质粒一样，它是以附加体的形式相对稳定地存在细胞内，不产生染色体位置效应，能高效地转录和表达目的蛋白。所以本发明选择非整合质粒(pCEP4)做为表达载体，同时将哺乳动物细胞基因组中发现的两种增强子(Ig和Ek增强子)克隆到非整合质粒上，建立了pCEP4-Ig-Ek质粒。
PCR扩增mAID，hAID，mAID-del，hAID-del，mAID-plus，hAID-plus核酸序列，将上述片段与pCEP4-Ig-Ek质粒通过HindⅢ和XhoⅠ限制性内切酶切开后连接起来，构建非整合AID质粒，用于后续细胞转染。测序鉴定插入序列均完全正确。
实施例2利用GFP报告基因检测AID突变效率
构建pCDNA3.1(+)-GFPm质粒，用于检测非整合AID质粒的突变能力。该质粒的GFP基因上含有一个终止密码子(TAG)，GFPm无法表达出完整的GFP蛋白，转染该质粒的正常细胞不会产生绿色荧光(参见图2)。如果将该质粒和非整合AID质粒共转入CHO细胞中，部分GFPm基因中的TAG终止密码子突变成编码氨基酸的密码子，从而表达完整的GFP蛋白使细胞产生绿色荧光，通过流式细胞仪检测荧光细胞的比例可间接的检测AID的突变能力。
实验步骤：
1、细胞培养
本发明所选用的细胞为CHO细胞，使用IMDM培养基(Hyclone)培养，其配方为基本培养基添加了10％胎牛血清(fatal bovine serum，FBS，Hyclone),100×HT,4‰终浓度为100U/mL的双抗(青霉素和链霉素)，于37℃含5％的CO2饱和湿度的培养箱中培养。
2、细胞转染
脂质体转染CHO细胞基本流程：
转染前一天用普通无双抗IMDM培养基以25万/孔的密度将细胞种于六孔板中，使其在转染时达到80％满度，平衡一定量的Opti-MEM无血清培养基备用。
配置转染复合物：取两个EP管，分别命名为A和B。将5μLLipofectamineTM 2000(INVITROGEN)缓慢加入A管中的150μLOpti-MEM无血清培养基，静置5min；将1μg的pCDNA3.1(+)-GFPm质粒和1μg的非整合AID质粒混匀至B管中的150μLOpti-MEM无血清培养基，最后将B管中的混合物缓慢加入至A管中混匀，室温放置25min。
将原有的细胞培养基吸走，用800-1000μL的Opti-MEM培养基洗两遍，然后将静置好的转染复合物均匀加到细胞培养板孔中，另补加500μL的Opti-MEM培养基培养。
5.5-6h后换成普通无双抗培养基继续培养。
3、流式细胞仪检测
处理细胞：48h后，吸掉旧培养基，用1mL1×PBS洗涤细胞1遍，洗去表面残留的血清。分别加入三滴胰酶，放在37℃细胞培养箱3min，于倒置显微镜下观察，当细胞将要分离而呈现圆粒状时，向培养皿中加入1mL培养基来中和胰酶，吹打细胞使其离开培养皿，850r/min 3min离心收集细胞。吸去上清，将细胞用1mL1×PBS洗一遍。整个过程避光操作。
分别用500ul的1×PBS重悬，然后移入流试管中，准备进行流式检测。
以上检测实验独立重复3次以上。
实验结果：图3展示了其中一次流式检测结果：A为未作任何转染的阴性细胞；B为仅转染了pCDNA3.1(+)-GFPm质粒的阴性对照组细胞；图C-H为共转染了pCDNA3.1(+)-GFPm质粒和非整合AID质粒的实验组细胞，其中C转染的是野生型的mAID质粒，D转染的是mAID-del质粒，E转染的是mAID-plus质粒，F转染的是hAID质粒，G转染的是hAID-del质粒，H转染的是hAID-plus质粒。其中纵坐标表示的是绿色荧光蛋白的信号，Q1区显示的是突变体数量和百分比。由此图可以看出鼠源的AID突变效率明显高于人源的AID；优化后的AID-plus，不论人源或鼠源，均远高于AID-del以及野生型的AID。三次检测结果趋势相同。
利用GFP报告基因检测不同AID突变体的突变效率比较。图4中，以mAID数据为标准，对mAID-del、mAID-plus、hAID-del、hAID-plus数据进行归一化处理，比较突变效率差异倍数。差异显著性分析(TTEST)在原始数据基础上计算得出，*表示P<0.05，**表示P<0.01，***表示P<0.001。可以看出，三种不同处理/结构的鼠源AID的突变效率明显高于人源AID；优化后的AID-plus也远高于AID-del以及野生型的AID，P<0.05。mAID-plus的突变效率是mAID的4.5倍以上，突变效率差异极显著P<0.001。hAID-plus的突变效率是mAID的3倍左右，突变效率差异显著P<0.05，说明经本发明改造后的AID突变体的突变效率要高于之前文献报道的突变体，更易于构建大数量级的抗体突变库。
综上，mAID-plus的突变能力最强，因此后续的抗体进化选用mAID-plus构建抗体库。
实施例3利用AID突变体在CHO细胞中对TNF-α抗体进行亲和力成熟进化
1、细胞培养
本实施例所选用的细胞为CHO-TNF-α细胞，源于本实验室保存，将CHO细胞中存在的Puro片段替换成anti-TNF-α片段，得到了能够稳定表达抗TNF-α抗体的细胞，命名CHO-TNF-α。本实施例是在CHO-TNF-α细胞的基础上对抗体片段进行突变建库。
以CHO(中国仓鼠卵巢细胞)细胞为基础构建的能够稳定表达抗TNF-α抗体的CHO-TNF-α细胞使用IMDM培养基(Hyclone)培养，其配方为基本培养基添加了10％胎牛血清(fatal bovine serum，FBS，Hyclone),100×HT,4‰终浓度为100U/mL的双抗(青霉素和链霉素)，于37℃含5％的CO2饱和湿度的培养箱中培养。
2、细胞转染
脂质体转染CHO细胞基本流程：
转染前一天用普通无双抗IMDM培养基以25万/孔的密度将细胞种于六孔板中，使其在转染时达到80％满度，平衡一定量的Opti-MEM无血清培养基备用。
配置转染复合物：取两个EP管，分别命名为A和B。将5μLLipofectamineTM 2000(INVITROGEN)缓慢加入A管中的150μLOpti-MEM无血清培养基，静置5min；将2μg的非整合AID质粒混匀至B管中的150μLOpti-MEM无血清培养基，最后将B管中的混合物缓慢加入至A管中混匀，室温放置25min。
将原有的细胞培养基吸走，用800-1000μL的Opti-MEM培养基洗两遍，然后将静置好的转染复合物均匀加到细胞培养板孔中，另补加500μL的Opti-MEM培养基培养。
5.5-6h后换成普通无双抗培养基继续培养。
3、流式细胞仪检测及分选
细胞从六孔板转移到10cm培养皿培养，并根据细胞生长情况进行传代，共扩增至12-15盘，当每盘细胞生长到80％的时候，即可进行流式细胞分选。
处理细胞：吸掉旧培养基，用3mL1×PBS洗涤细胞1遍，洗去表面残留的血清。
分别加入1.5mL的胰酶溶液，放在37℃细胞培养箱3min，于倒置显微镜下观察，当细胞将要分离而呈现圆粒状时，向培养皿中加入1.5mL培养基与胰酶中和，吹打细胞使其离开培养皿，850r/min 3min离心收集细胞。吸去上清，将细胞用2mL冷的Opti-MEM无血清培养基洗一遍。
配置抗原抗体复合标记溶液：整个过程需要避光。把anti-HA-PE antibody(Abcam,1:250稀释在Opti-MEM培养基)和hTNFα-GFP(1:10000稀释在Opti-MEM培养基)混合物按200μL/皿加入到细胞中，4℃标记细胞30min，之后用5mL预冷的Opti-MEM洗两遍，最后用1mL预冷的Opti-MEM重悬，吸入到分选用流式管中，准备进行流式分选。
标记后的细胞FACS AriaIII(BD)进行分选，用盛有10mL培养基的15mL离心管接收目的细胞。
4、细胞基因组提取
分选得到的细胞生长到10cm培养皿的80％时，即可进行细胞基因组的提取。基因组提取采用
Genomic DNA Purification Kit提取试剂盒(Promega)，具体操作如试剂盒说明书。提完基因组测定浓度并标记。
5、目的基因克隆鉴定
anti-TNFα抗体基因的克隆测序：提取细胞基因组。以基因组为模板PCR扩增抗体片段，经凝胶回收试剂盒回收，2×EasyTaq Super Mix加A，TA克隆连接于pMD-19T-vector上测序，经比对检测突变。
(1)PCR体系：
(2)核酸凝胶电泳：
配置1.5％的核酸凝胶(2.25g琼脂糖+150mL 1×TAE),倒入制胶槽中，等待凝胶凝固。将PCR产物加入到孔中，120V电泳45min左右。
(3)回收目的片段：
用凝胶回收试剂盒回收，具体操作如试剂盒说明书。
TA克隆
给片段加A：
连接T载体：
转化：
取5μL连接产物加入到50μL trans 10感受态细胞中，提前打开恒温水浴锅，在冰上放置30min，然后在42℃水浴中热激45s，然后立即放置冰上2min，在超净工作台中向管中加入500μL LB液体培养基，放入恒温振荡器中37℃200rmp恢复培养1h，将上述步骤得到的菌液均匀涂在含有Amp抗生素的SOB培养板上，放置在37℃CO2培养箱过夜培养。
挑克隆测序：
1)取50个玻璃管，在超净工作台中分别加入5mL的LB培养基，每个玻璃管中再加入10μL Amp抗生素，用10μL的枪头随机挑取50个单克隆菌落打入玻璃管中，盖好盖子放在振荡培养箱中振荡培养10h左右，待菌体变浑浊取出。
2)PCR进行菌批鉴定：
对PCR产物进行凝胶电泳，将阳性率高的菌液送去测序。
PCR引物为：
CMV-F:CGCAAATGGGCGGTAGGCGTG(SEQ ID NO.29)
TM-R:CTGCGTGTCCTGGCCCACAGC(SEQ ID NO.30)
已有研究发现，AID诱导的突变位点具有倾向性，分为“hot spot”和“cold spot”，在WRC(W＝A/T,R＝A/G)和WGCW(W＝A/T)序列下的胞嘧啶突变率更高，在SYC(S＝G or C；Y＝pyrimidine)序列下突变效率相对较低。
在之前的工作中，本课题组开发了遵循上述规则的计算机软件，在不改变目标蛋白氨基酸序列的基础上优化编码抗体的核苷酸序列，使抗体片段可变区核酸序列成为突变的热点序列。本发明中，在TNF-α抗体的编码核酸序列(SEQ ID NO.14)基础上，我们利用上述软件对抗体的编码核酸序列进行优化，并根据哺乳动物细胞表达偏好二次优化，获得了核酸序列优化的抗体编码基因(SEQ ID NO.15)。
本发明中，我们分别使用野生型的鼠源mAID酶和AID酶突变体mAID-plus来对野生型TNF-α抗体和经过序列优化的TNF-α抗体进行人工进化。收获细胞后，提取细胞的基因组DNA，通过PCR扩增TNF-α抗体序列，克隆到T载体中并送到公司进行测序。对每个样品测试50个克隆，并进行序列比较以比较突变的效率，结果如表1所示。
表1.在亲和力成熟过程中观察到的TNF-α抗体编码核酸突变位点
注：“突变克隆总计：2/48”表示在48个有效测序克隆中出现2个突变细胞；
“总碱基数”表示对应有效测序克隆中目标基因的总碱基数，例如48×768bp＝36864bp；
“突变频率”为“突变位点总计/总碱基数”。
根据表1，无论抗体中的突变类型或突变位点总数，AID突变体的突变能力都高于野生型AID。针对野生型TNF-α抗体，位点突变频率增高4.5倍以上；针对序列优化过的TNF-α抗体，AID突变体的突变频率也高于野生型AID。
图5展示了不同AID对不同抗体序列诱导的位点突变频率和突变类型频率比较。TNF-α表示未经序列优化的野生型抗体，TNF-α-mopti表示经核酸序列优化的抗体；mAID表示野生型AID，mAID-plus表示AID突变体。无论针对野生型TNF-α抗体或经核酸序列优化的TNF-α-mopti，mAID-plus所诱导的位点突变频率(Frequency per 104)和诱导的不同类型氨基酸转换的频率(Frequency per 104)均远高于mAID。
可见，AID突变体在CHO细胞中的突变能力显著增强，不仅提高了位点突变频率，也提高了突变类型频率，整体上提高了在CHO细胞中建立抗体突变库的效率，并且不影响CHO细胞的生存和正常扩增能力。
实施例4.野生型mAID序列优化
由于AID酶也可以突变AID本身，一些细胞可能在细胞扩增期间失去AID活性并且突变率降低。因此，我们通过软件设计和序列优化识别将野生型的AID序列优化为AID本身不易识别的序列，并通过实验验证以确定它们是否可以提高突变效率。
利用本课题组开发的软件，在不改变其氨基酸序列的基础上将野生型mAID(小鼠)核酸序列优化为AID本身不易识别的核酸序列，并根据哺乳动物细胞表达偏好二次优化，获得抗突变mAID(mAID-decrease)的编码核酸序列(SEQ ID NO.16)。
1、引物设计
HindⅢ-mAID-decrease-F:GTACATAAGCTTATGGACAGTCTTCTG(SEQ ID NO.31)
mAID-decrease-XhoI-R:GTACATCTCGAGTCAAAATCCCAACATACG(SEQ ID NO.32)
2、表达载体构建
将mAID，mAID-decrease基因片段与pCEP4-Ig-Ek质粒通过HindIII和XhoI限制性内切酶切开后连接起来，构建非整合AID质粒，用于转染。
在之前的工作中，我们已经获得了稳定展示TNF-α抗体的CHO细胞(CHO-TNF-α细胞)(Chuan Chen,et al.Coupling recombinase-mediated cassette exchange withsomatic hypermutation for antibody affinity maturation in CHOcells.Biotechnology&Bioengineering,2016,113(1):39-51)。本发明中，我们分别用pCEP-mAID和pCEP-mAID-decrease转染CHO-TNF-α细胞，完成三轮人工进化。收获细胞，提取每轮进化的细胞基因组DNA，通过PCR扩增anti-TNFα抗体序列，克隆到T载体中，并送到公司进行测序。测试每种TNFα抗体基因的50个克隆并进行序列比对以比较pCEP-mAID和pCEP-mAID-decrease作用下TNF-α抗体基因的突变率，结果参见表2。
表2.在亲和力成熟过程中观察到的TNF-α抗体编码核酸突变位点
注：“突变克隆总计：2/44”表示在44个有效测序克隆中出现2个突变细胞；
“总碱基数”表示对应有效测序克隆中目标基因的总碱基数，例如44×768bp＝33792bp；
“突变频率”为“突变位点总计/总碱基数”。
S1、S2、S3分别代表进化的3个轮次。
根据表2，抗突变AID的突变能力高于野生型AID，在前两个轮次的筛选中(S1、S2)，抗突变AID所诱导的突变频率均为野生型AID的约2倍左右；筛选至第3轮次(S3)，抗突变AID所诱导的突变频率也明显高于野生型AID。使用抗突变AID在S2轮筛选中的突变频率与野生型AID在S3轮筛选中的效率几近相当。
图6展示了mAID和抗突变mAID对TNF-α抗体序列诱导的位点突变频率和突变类型频率比较。右图中，筛选S1轮和S3轮，抗突变mAID所诱导的不同类型氨基酸转换的频率(Frequency per 104)高于mAID，S2轮则突变类型频率相当。
可见，使用抗突变mAID不仅能够提供数量更多、氨基酸种类多样化的抗体突变体，同时还可以减少筛选的轮次，极大提高在CHO细胞中建立抗体突变库的效率。
实施例5.AID突变体对细胞存活的影响
为了检测较高活性的AID是否会对细胞的生长造成影响，用不同的AID分别转染展示不同类型抗体的细胞。我们通过软件设计和序列优化识别将mAID-plus序列优化为AID本身不易识别的序列mAID-plus-decrease，并通过实验验证以确定它们是否可以提高突变效率。
1、引物设计
HindⅢ-mAID-plus-decrease-F:GTACATAAGCTTATGGACAGCCTGCTG(SEQ ID NO.33)
mAID-plus-decrease-XhoⅠ-R:GTACATCTCGAGTCACAGGGGCAGCAGG(SEQ ID NO.34)
mAID-plus-decrease核苷酸序列如SEQ ID NO.35所示。
2、表达载体构建
将mAID-plus-decrease基因片段与pCEP4-Ig-Ek质粒通过HindIII和XhoI限制性内切酶切开后连接起来，构建非整合AID质粒，用于转染。
3、细胞培养和脂质体转染CHO细胞的方法与实施例3相同。
四天后将转染不同AID的细胞分别计数，实验重复三次，取平均值。
发现在表达野生型抗TNF-α抗体的细胞中(TNF-α)，与野生型AID相比，mAID-plus、抗突变AID(mAID-decrease)和mAID-plus-decrease基本不影响CHO细胞的生存或扩增。
而在核酸序列优化后的抗TNF-α抗体的细胞中(TNF-a-mopti，优化方法同实施例3)，与野生型AID相比，mAID-plus、抗突变AID(mAID-decrease)对CHO细胞生存基本无影响；mAID-plus-decrease则导致大量CHO细胞死亡，说明AID的突变能力已超出了细胞耐受，将放弃对AID的进一步优化。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 中国科学院生物物理研究所
<120> 一种AID酶突变体及其应用
<130> 1
<160> 32
<170> PatentIn version 3.3
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<223> mAID氨基酸序列
Met Asp Ser Leu Leu Met Lys Gln Lys Lys Phe Leu Tyr His Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg His Glu Thr Tyr Leu Cys Tyr Val
20 25 30
Val Lys Arg Arg Asp Ser Ala Thr Ser Cys Ser Leu Asp Phe Gly His
35 40 45
Leu Arg Asn Lys Ser Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr
50 55 60
Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp
65 70 75 80
Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Glu
85 90 95
Phe Leu Arg Trp Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg
100 105 110
Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg
115 120 125
Leu His Arg Ala Gly Val Gln Ile Gly Ile Met Thr Phe Lys Asp Tyr
130 135 140
Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn Arg Glu Arg Thr Phe Lys
145 150 155 160
Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Thr Arg Gln Leu
165 170 175
Arg Arg Ile Leu Leu Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp Ala
180 185 190
Phe Arg Met Leu Gly Phe
195
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<223> mAID核苷酸序列
atggacagcc ttctgatgaa gcaaaagaag tttctttacc atttcaaaaa tgtccgctgg 60
gccaagggac ggcatgagac ctacctctgc tacgtggtga agaggagaga tagtgccacc 120
tcctgctcac tggacttcgg ccaccttcgc aacaagtctg gctgccacgt ggaattgttg 180
ttcctacgct acatctcaga ctgggacctg gacccgggcc ggtgttaccg cgtcacctgg 240
ttcacctcct ggagcccgtg ctatgactgt gcccggcacg tggctgagtt tctgagatgg 300
aaccctaacc tcagcctgag gattttcacc gcgcgcctct acttctgtga agaccgcaag 360
gctgagcctg aggggctgcg gagactgcac cgcgctgggg tccagatcgg gatcatgacc 420
ttcaaagact atttttactg ctggaataca tttgtagaaa atcgtgaaag aactttcaaa 480
gcctgggaag ggctacatga aaattctgtc cggctaacca gacaacttcg gcgcatcctt 540
ttgcccttgt acgaagtcga tgacttgcga gatgcatttc gtatgttggg attttga 597
<210> 3
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<223> hAID氨基酸序列
Met Asp Ser Leu Leu Met Asn Arg Arg Lys Phe Leu Tyr Gln Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Tyr Val
20 25 30
Val Lys Arg Arg Asp Ser Ala Thr Ser Phe Ser Leu Asp Phe Gly Tyr
35 40 45
Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr
50 55 60
Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp
65 70 75 80
Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp
85 90 95
Phe Leu Arg Gly Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg
100 105 110
Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg
115 120 125
Leu His Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys Asp Tyr
130 135 140
Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn His Glu Arg Thr Phe Lys
145 150 155 160
Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Arg Gln Ile
165 170 175
Arg Arg Ile Leu Leu Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp Ala
180 185 190
Phe Arg Thr Leu Gly Leu
195
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<223> hAID核苷酸序列
atggacagcc tcttgatgaa ccggaggaag tttctttacc aattcaaaaa tgtccgctgg 60
gctaagggtc ggcgtgagac ctacctgtgc tacgtagtga agaggcgtga cagtgctaca 120
tccttttcac tggactttgg ttatcttcgc aataagaacg gctgccacgt ggaattgctc 180
ttcctccgct acatctcgga ctgggaccta gaccctggcc gctgctaccg cgtcacctgg 240
ttcacctcct ggagcccctg ctacgactgt gcccgacatg tggccgactt tctgcgaggg 300
aaccccaacc tcagtctgag gatcttcacc gcgcgcctct acttctgtga ggaccgcaag 360
gctgagcccg aggggctgcg gcggctgcac cgcgccgggg tgcaaatagc catcatgacc 420
ttcaaagatt atttttactg ctggaatact tttgtagaaa accatgaaag aactttcaaa 480
gcctgggaag ggctgcatga aaattcagtt cgtctctcca gacagattcg gcgcatcctt 540
ttgcccctgt atgaggttga tgacttacga gacgcatttc gtactttggg actttga 597
<210> 5
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<223> mAID-del氨基酸序列
Met Asp Ser Leu Leu Met Lys Gln Lys Lys Phe Leu Tyr His Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg His Glu Thr Tyr Leu Cys Tyr Val
20 25 30
Val Lys Arg Arg Asp Ser Ala Thr Ser Cys Ser Leu Asp Phe Gly His
35 40 45
Leu Arg Asn Lys Ser Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr
50 55 60
Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp
65 70 75 80
Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Glu
85 90 95
Phe Leu Arg Trp Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg
100 105 110
Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg
115 120 125
Leu His Arg Ala Gly Val Gln Ile Gly Ile Met Thr Phe Lys Asp Tyr
130 135 140
Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn Arg Glu Arg Thr Phe Lys
145 150 155 160
Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Thr Arg Gln Leu
165 170 175
Arg Arg Ile Leu Leu Pro Leu
180
<210> 6
<211> 552
<223> mAID-del核苷酸序列
atggacagcc ttctgatgaa gcaaaagaag tttctttacc atttcaaaaa tgtccgctgg 60
gccaagggac ggcatgagac ctacctctgc tacgtggtga agaggagaga tagtgccacc 120
tcctgctcac tggacttcgg ccaccttcgc aacaagtctg gctgccacgt ggaattgttg 180
ttcctacgct acatctcaga ctgggacctg gacccgggcc ggtgttaccg cgtcacctgg 240
ttcacctcct ggagcccgtg ctatgactgt gcccggcacg tggctgagtt tctgagatgg 300
aaccctaacc tcagcctgag gattttcacc gcgcgcctct acttctgtga agaccgcaag 360
gctgagcctg aggggctgcg gagactgcac cgcgctgggg tccagatcgg gatcatgacc 420
ttcaaagact atttttactg ctggaataca tttgtagaaa atcgtgaaag aactttcaaa 480
gcctgggaag ggctacatga aaattctgtc cggctaacca gacaacttcg gcgcatcctt 540
ttgcccttgt ga 552
<210> 7
<211> 183
<223> hAID-del氨基酸序列
Met Asp Ser Leu Leu Met Asn Arg Arg Lys Phe Leu Tyr Gln Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Tyr Val
20 25 30
Val Lys Arg Arg Asp Ser Ala Thr Ser Phe Ser Leu Asp Phe Gly Tyr
35 40 45
Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr
50 55 60
Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp
65 70 75 80
Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp
85 90 95
Phe Leu Arg Gly Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg
100 105 110
Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg
115 120 125
Leu His Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys Asp Tyr
130 135 140
Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn His Glu Arg Thr Phe Lys
145 150 155 160
Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Arg Gln Ile
165 170 175
Arg Arg Ile Leu Leu Pro Leu
180
<210> 8
<211> 552
<223> hAID-del核苷酸序列
atggacagcc tcttgatgaa ccggaggaag tttctttacc aattcaaaaa tgtccgctgg 60
gctaagggtc ggcgtgagac ctacctgtgc tacgtagtga agaggcgtga cagtgctaca 120
tccttttcac tggactttgg ttatcttcgc aataagaacg gctgccacgt ggaattgctc 180
ttcctccgct acatctcgga ctgggaccta gaccctggcc gctgctaccg cgtcacctgg 240
ttcacctcct ggagcccctg ctacgactgt gcccgacatg tggccgactt tctgcgaggg 300
aaccccaacc tcagtctgag gatcttcacc gcgcgcctct acttctgtga ggaccgcaag 360
gctgagcccg aggggctgcg gcggctgcac cgcgccgggg tgcaaatagc catcatgacc 420
ttcaaagatt atttttactg ctggaatact tttgtagaaa accatgaaag aactttcaaa 480
gcctgggaag ggctgcatga aaattcagtt cgtctctcca gacagattcg gcgcatcctt 540
ttgcccctgt ga 552
<210> 9
<211> 183
<223> mAID-plus氨基酸序列
Met Asp Ser Leu Leu Met Lys Gln Lys Glu Phe Leu Tyr His Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg His Glu Thr Tyr Leu Cys Tyr Val
20 25 30
Val Lys Arg Arg Asp Ser Ala Thr Ser Cys Ser Leu Asp Phe Gly His
35 40 45
Leu Arg Asn Lys Ser Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr
50 55 60
Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp
65 70 75 80
Phe Ile Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Glu
85 90 95
Phe Leu Arg Trp Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg
100 105 110
Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg
115 120 125
Leu His Arg Ala Gly Val Gln Ile Gly Ile Met Thr Phe Lys Asp Tyr
130 135 140
Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn Arg Gly Arg Thr Phe Lys
145 150 155 160
Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Thr Arg Gln Leu
165 170 175
Arg Arg Ile Leu Leu Pro Leu
180
<210> 10
<211> 552
<223> mAID-plus核苷酸序列
atggacagcc ttctgatgaa gcaaaaggaa tttctttacc atttcaaaaa tgtccgctgg 60
gccaagggac ggcatgagac ctacctctgc tacgtggtga agaggagaga tagtgccacc 120
tcctgctcac tggacttcgg ccaccttcgc aacaagtctg gctgccacgt ggaattgttg 180
ttcctacgct acatctcaga ctgggacctg gacccgggcc ggtgttaccg cgtcacctgg 240
ttcatctcct ggagcccgtg ctatgactgt gcccggcacg tggctgagtt tctgagatgg 300
aaccctaacc tcagcctgag gattttcacc gcgcgcctct acttctgtga agaccgcaag 360
gctgagcctg aggggctgcg gagactgcac cgcgctgggg tccagatcgg gatcatgacc 420
ttcaaagact atttttactg ctggaataca tttgtagaaa atcgtggtag aactttcaaa 480
gcctgggaag ggctacatga aaattctgtc cggctaacca gacaacttcg gcgcatcctt 540
ttgcccttgt ga 552
<210> 11
<211> 183
<223> hAID-plus氨基酸序列
Met Asp Ser Leu Leu Met Asn Arg Arg Glu Phe Leu Tyr Gln Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Tyr Val
20 25 30
Val Lys Arg Arg Asp Ser Ala Thr Ser Phe Ser Leu Asp Phe Gly Tyr
35 40 45
Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr
50 55 60
Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp
65 70 75 80
Phe Ile Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp
85 90 95
Phe Leu Arg Gly Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg
100 105 110
Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg
115 120 125
Leu His Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys Asp Tyr
130 135 140
Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn His Gly Arg Thr Phe Lys
145 150 155 160
Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Arg Gln Ile
165 170 175
Arg Arg Ile Leu Leu Pro Leu
180
<210> 12
<211> 552
<223> hAID-plus核苷酸序列
atggacagcc tcttgatgaa ccggagggaa tttctttacc aattcaaaaa tgtccgctgg 60
gctaagggtc ggcgtgagac ctacctgtgc tacgtagtga agaggcgtga cagtgctaca 120
tccttttcac tggactttgg ttatcttcgc aataagaacg gctgccacgt ggaattgctc 180
ttcctccgct acatctcgga ctgggaccta gaccctggcc gctgctaccg cgtcacctgg 240
ttcatctcct ggagcccctg ctacgactgt gcccgacatg tggccgactt tctgcgaggg 300
aaccccaacc tcagtctgag gatcttcacc gcgcgcctct acttctgtga ggaccgcaag 360
gctgagcccg aggggctgcg gcggctgcac cgcgccgggg tgcaaatagc catcatgacc 420
ttcaaagatt atttttactg ctggaatact tttgtagaaa accatggtag aactttcaaa 480
gcctgggaag ggctgcatga aaattcagtt cgtctctcca gacagattcg gcgcatcctt 540
ttgcccctgt ga 552
<210> 13
<211> 256
<223> TNF-α抗体氨基酸序列
Gly Ile Asn Ser Asp Pro Asn Ser Gln Ile Gln Leu Val Gln Ser Gly
1 5 10 15
Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala
20 25 30
Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala
35 40 45
Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly
50 55 60
Glu Pro Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu
65 70 75 80
Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn
85 90 95
Glu Asp Ser Ala Thr Tyr Phe Cys Ala Gly Arg Arg Ser Tyr Asp Tyr
100 105 110
Asp Val Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Ile Ser
115 120 125
Ser Gly Ser Gly Ser Ser Gly Ser Gly Ser Ser Gly Ser Gly Ser Ser
130 135 140
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
145 150 155 160
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
165 170 175
Gly Asn Tyr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
180 185 190
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
195 200 205
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn
210 215 220
Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
225 230 235 240
Glu Glu Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg
245 250 255
<210> 14
<211> 768
<223> TNF-α抗体原始核苷酸序列
ggaattaatt cggatcccaa ttcgcagatc cagctggtgc agtctggacc tgagctgaag 60
aagcctggag agacagtcaa gatctcctgc aaggcttctg ggtatacctt cacaaactat 120
ggaatgaact gggtgaagca ggctccagga aagggtttaa agtggatggg ctggataaac 180
acctacactg gagagccaac atatgctgat gacttcaagg gacggtttgc cttctctttg 240
gaaacctctg ccagcactgc ctatttgcag atcaacaacc tcaaaaatga ggactcggct 300
acatatttct gtgcaggaag aagaagctat gattacgacg tggctatgga ctactggggt 360
caaggaacct cagtcaccat ctcctcagga tctggaagtt caggaagcgg ctcaagtggg 420
tctggaagct cagacattgt gctcacccag tctccagctt ctttggctgt gtctctaggg 480
cagagggcca ccatatcctg cagagccagt gaaagtgttg atagttatgg caattatttt 540
atgcactggt atcagcagaa accaggacag ccacccaaac tcctcatcta tcgtgcatcc 600
aacctagaat ctgggatccc tgccaggttc agtggcagtg ggtctgggac agacttcacc 660
ctcaccatta atcctgtgga ggctgatgat gttgcaacct attactgtca acaaagtaat 720
gaggagcctc tcacgttcgg ctcggggaca aagttggaaa taaaacgg 768
<210> 15
<211> 768
<223> TNF-α抗体优化核苷酸序列
ggaatcaata gcgacccaaa ctcacagatc cagctggtgc agagcggacc tgaactgaaa 60
aaacccggag aaaccgtcaa aattagttgc aaggcatccg ggtacacctt cacaaactat 120
ggaatgaatt gggtgaaaca ggctccaggc aaggggctga aatggatggg ctggattaat 180
acttacaccg gggagcccac ttatgctgac gatttcaagg ggcggttcgc attttctctg 240
gaaactagtg cctcaaccgc ttacctgcag atcaacaatc tgaaaaacga ggacagcgcc 300
acatattttt gcgctggccg gagatcctac gactatgatg tggccatgga ttactgggga 360
cagggcacca gcgtcacaat cagctccggg tcaggatcta gtggcagcgg gtcaagcgga 420
tccggctcct ctgacattgt gctgacacag agcccagcat ccctggccgt ctctctggga 480
cagagggcaa ctatcagttg tcgcgccagc gaatccgtgg atagctacgg aaactacttc 540
atgcactggt accagcagaa gcctggccag ccccctaaac tgctgatcta tcgagcctct 600
aatctggaaa gtggcattcc tgctagattc tctgggagtg gatcaggcac cgactttaca 660
ctgactatta acccagtgga ggctgacgat gtcgcaacat actattgtca gcagagcaac 720
gaggagcccc tgacattcgg cagcgggacc aaactggaaa tcaagcgg 768
<210> 16
<211> 597
<223> 抗突变mAID（mAID-decrease）核苷酸序列
atggacagtc ttctgatgaa acaaaagaaa tttctttatc atttcaaaaa tgtccgctgg 60
gccaagggaa gacatgagac ctatctctgt tacgtggtga agaggagaga tagcgcgacc 120
tcctgtagtc tggacttcgg ccaccttcgc aacaagtctg gatgtcacgt ggaattgttg 180
ttcctacgct acatctcaga ctgggacctg gacccgggcc ggtgttatag agtcacctgg 240
ttcacctcct ggagtccgtg ttatgactgc gcaagacacg tcgccgaatt tctgagatgg 300
aatcctaatc tcagtctgag gattttcacc gcgcgcctct atttctgtga agaccgcaag 360
gccgagcctg agggtctgcg gagactgcac cgcgctgggg tccagatcgg gatcatgacc 420
ttcaaagact atttttattg ttggaataca tttgtagaaa atcgtgaaag gacattcaag 480
gcgtgggaag gtctacatga aaattctgtc agactcacaa gacaacttcg gcgcatcctt 540
cttcccttgt acgaagtcga tgacttgcga gatgcatttc gtatgttggg attttga 597
<210> 17
<211> 27
<223> HindⅢ-mAID-F
gtacataagc ttatggacag ccttctg 27
<210> 18
<211> 25
<223> mAID-XhoⅠ-R
gtacatctcg agtcaaaatc ccaac 25
<210> 19
<211> 30
<223> HindⅢ-hAID-F
gtacataagc ttgccaccat ggacagcctc 30
<210> 20
<211> 30
<223> hAID-XhoⅠ-R
gtacatctcg agtcaaagtc ccaaagtacg 30
<210> 21
<211> 27
<223> HindⅢ-mAID-del-F
gtacataagc ttatggacag ccttctg 27
<210> 22
<211> 28
<223> mAID-del-XhoI-R
gtacatctcg agtcacaagg gcaaaagg 28
<210> 23
<211> 27
<223> HindⅢ-hAID-del-F
gtacataagc ttatggacag cctcttg 27
<210> 24
<211> 28
<223> hAID-del-XhoⅠ-R
gtacatctcg agtcacaggg gcaaaagg 28
<210> 25
<211> 27
<223> HindⅢ-mAID-plus-F
gtacataagc ttatggacag ccttctg 27
<210> 26
<211> 28
<223> mAID-plus-XhoI-R
gtacatctcg agtcacaagg gcaaaagg 28
<210> 27
<211> 27
<223> HindⅢ-hAID-plus-F
gtacataagc ttatggacag cctcttg 27
<210> 28
<211> 28
<223> hAID-plus-XhoI-R
gtacatctcg agtcacaggg gcaaaagg 28
<210> 29
<211> 21
<223> CMV-F
cgcaaatggg cggtaggcgt g 21
<210> 30
<211> 21
<223> TM-R
ctgcgtgtcc tggcccacag c 21
<210> 31
<211> 27
<223> HindⅢ-mAID-decrease-F
gtacataagc ttatggacag tcttctg 27
<210> 32
<211> 30
<223> mAID-decreasel-XhoI-R
gtacatctcg agtcaaaatc ccaacatacg 30
<210> 33
<211> 27
<223> HindⅢ-mAID-plus-decrease-F
gtacataagc ttatggacag cctgctg 27
<210> 34
<211> 28
<223> mAID-plus-decrease-XhoI-R
gtacatctcg agtcacaggg gcagcagg 28
<210> 35
<211> 552
<223> mAID-plus-decrease优化核苷酸序列
atggacagcc tgctgatgaa gcagaaagag ttcctgtacc acttcaagaa cgtgcgctgg 60
gccaagggca gacacgagac atacctgtgc tacgtcgtga agagaagaga cagcgccacc 120
agctgcagcc tggacttcgg ccacctgaga aacaagagcg gctgccacgt ggaactgctg 180
tttctgcggt acatcagcga ctgggacctg gaccccggca gatgctacag agtgacctgg 240
ttcatcagct ggtccccctg ctacgactgc gccagacacg tggccgagtt tctgagatgg 300
aaccccaacc tgagcctgag aatctttacc gccagactgt acttctgcga ggacagaaag 360
gccgagcctg agggcctgag aaggctgcat agagccggcg tgcagatcgg catcatgacc 420
ttcaaggact acttctactg ctggaacacc ttcgtggaaa acagaggcag aacattcaag 480
gcctgggagg gcctgcacga gaacagcgtg cgactgacca gacagctgag aagaatcctg 540
ctgcccctgt ga 552
Claims (14)
1.一种激活诱导的胞嘧啶脱氨基酶(AID)突变体，其特征在于，其氨基酸序列如SEQ IDNO.5或SEQ ID NO.9所示。
2.一种AID酶突变体的编码核酸，其特征在于，所示核酸能够编码产生SEQ ID NO.5或SEQ ID NO.9所示氨基酸序列。
3.权利要求2所述编码核酸，其特征在于，所述编码核酸的核苷酸序列如SEQ ID NO.6或SEQ ID NO.10所示。
4.一种包含AID酶突变体编码核酸的载体，其特征在于，所述载体包含编码SEQ IDNO.5或SEQ ID NO.9所示氨基酸序列的核苷酸序列。
5.权利要求4所述载体，其特征在于，所述载体包含SEQ ID NO.6或SEQ ID NO.10所示的核苷酸序列。
6.一种非人哺乳动物细胞，其特征在于，所述细胞包含权利要求1所述的胞嘧啶脱氨基酶(AID)突变体、权利要求2或3所述编码核酸、或权利要求4或5所述载体；所述非人哺乳动物细胞为CHO。
7.权利要求1所述胞嘧啶脱氨基酶(AID)突变体、权利要求2或3所述核酸、权利要求4或5所述载体、或权利要求6所述细胞在蛋白筛选、抗体亲和力成熟、抗体工程化改造、或抗体库建立中的应用，上述蛋白可选自抗体、多肽、融合蛋白。
8.根据权利要求7所述应用，所述抗体可选自单链抗体(ScFv)、Fab片段、F(ab’)2片段、重链抗体、纳米抗体。
9.一种AID酶突变体在筛选进化TNF-α抗体中的应用，其特征在于，所述AID酶突变体的氨基酸序列如SEQ ID NO.5或SEQ ID NO.9所示。
10.一种优化的AID酶的编码核酸，其特征在于，所述编码核酸的核苷酸序列如SEQ IDNO.16所示。
11.一种包含优化的AID酶编码核酸的载体，其特征在于，所述载体包含SEQ ID NO.16所示核苷酸序列。
12.一种非人哺乳动物细胞，其特征在于，所述细胞包含SEQ ID NO.16所示核苷酸序列，所述非人哺乳动物细胞为CHO。
13.权利要求10所述核酸、权利要求11所述载体、或权利要求12所述细胞在蛋白筛选、抗体亲和力成熟、抗体工程化改造、或抗体突变库建立中的应用，上述蛋白可选自抗体、多肽、融合蛋白。
14.根据权利要求13所述应用，所述抗体可选自单链抗体(ScFv)、Fab片段、F(ab’)2片段、重链抗体、纳米抗体。
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