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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2016, Vol. 42 Issue (1): 1-7    DOI: 10.3785/j.issn.1008-9209.2015.03.251
生物科学与技术     
基因突变技术在抗体亲和力体外成熟中的应用
刘媛, 林曼曼, 张霄, 徐重新, 焦凌霞, 仲建锋, 武爱华, 刘贤金
江苏省食品质量安全重点实验室和农业部食用农产品安全监控重点开放实验室,南京210014
Applications of mutagenesis methods on affinity maturation of antibodies in vitro
LIU Yuan, LIN Manman, ZHANG Xiao, XU Chongxin, JIAO Linxia, ZHONG Jianfeng, WU Aihua, LIU Xianjin
(Key Laboratory of Food Safety of Jiangsu Province and Key Laboratory of Food Safety Monitoring and Management of Ministry of Agriculture, Nanjing 210014, China)
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摘要： 抗体基因体外突变技术可以模拟自体高频突变过程,是抗体亲和力体外成熟的重要手段,常见的抗体基因体外突变技术可以分为易错聚合酶链式反应(polymerase chain reaction,PCR)、DNA改组、突变株、定点突变和链替换等方法。易错PCR可以在抗体基因全长或部分区域随机引入突变,但随着突变率的增加,阳性克隆数量呈指数性递减。DNA改组包括抗体片段随机化切割和重组步骤,可以加快抗体的体外进化速度。突变株法易于构建超大容量的抗体库,但有害突变和突变率难以控制。定点突变的区域通常选择与抗原直接接触的互补决定区(complementary determination region,CDR)或自体突变热点进行操作。链替换通常保留母体抗体的一条重链或轻链,而对另一条链进行随机化组合。
这些基因突变方法在提高抗体亲和力中获得了不同程度的应用,但也存在效率不高,且方法选择较为盲目等问题。可是,采用抗体X射线晶体衍射和计算机模拟等方法,却可以帮助预测抗体结合部位,为突变位点的理性设计和方法选择提供有效信息,因而成为亲和力体外成熟技术未来发展的趋势之一。
Abstract: Antibodies selected from naive antibody libraries usually have low affinity at the micromolar level. Meanwhile, the affinities of natural antibodies obtained by repeated immunization were also observed to have a limit around 100 picomole because of the inability to discriminate slower dissociation kinetics relative to intrinstric B cell receptor internalization rate. As an essential determinant for antibody function for therapeutic and diagnostics use, a lots of antibodies, either isolated from antibody libraries or cloned from monoclonal antibodies, have been targeted to generate high affinity by directed evolution. Affinity maturation in vitro could mimic the mutation and selection process of affinity maturation in vivo, it could help to improve the affinity of the antibodies derived from antibody libraries and make the natural antibodies to break through affinity ceiling. At the mutagenesis level, strategies to affinity maturation in vitro can be grouped in five categories: error prone PCR, DNA shuffling, mutator strains, site directed mutagenesis and chain shuffling. Error PCR could generate random mutations into antibodies throughout the whole antibodies gene. But it is notable that when the mutation rate increases, the active clones decreases exponentially. DNA shuffling includes random fragmentation and reassembly steps; it can be used with longer DNA sequences, and also allows for the selection of clones with mutations outside the binding or active site of the antibodies. By mutator strains, the mutations are produced after the transformation, which allows the randomly mutated libraries of even 1012-1014 clones  be produced. However, by mutator strains, mutations are also produced in the vector part to cause deleterious effects. In site-directed mutagenesis, selected residues are targeted to be mutated. The most common sites for mutation are the CDR regions which directly contact with antigen. However, mutations that do not directly contact antigen can also result in enhanced affinity. And the mutation hotspots in affinity maturation in vivo can also be targeted to the mutation sites. In chain shuffling, a variable heavy or light chain of a specific antibody is recombined with a complementary variable domain library. In a lot of experiments, light chains were used to be shuffling, because heavy chain is crucial for the antigen binding. But there are also some reports on producing  the affinity-matured antibodies by heavy chain shuffling. These mutagenesis methods have been used with various degrees of success. However, the efficiency of these methods was relatively low and there was a lack of a rational guidance to choose these methods. Knowledge gained on antibody structure through X-ray crystallography or computer-aided design could help to predict the paratope and mutation sites, which is one of the development trend of affinity maturation in vitro.
出版日期: 2016-01-20
CLC:  Q 789  
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刘媛
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引用本文:

刘媛, 林曼曼, 张霄, 徐重新, 焦凌霞, 仲建锋, 武爱华, 刘贤金. 基因突变技术在抗体亲和力体外成熟中的应用[J]. 浙江大学学报(农业与生命科学版), 2016, 42(1): 1-7.

LIU Yuan, LIN Manman, ZHANG Xiao, XU Chongxin, JIAO Linxia, ZHONG Jianfeng, WU Aihua, LIU Xianjin. Applications of mutagenesis methods on affinity maturation of antibodies in vitro. Journal of Zhejiang University (Agriculture and Life Sciences), 2016, 42(1): 1-7.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2015.03.251    或    http://www.zjujournals.com/agr/CN/Y2016/V42/I1/1

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