定点突变和随机突变PCR构建噬菌体突变次级抗体库方法的比较  

Comparison of site-mutated and error-prone PCR methods for constructing the secondary phage antibody libraries

作　　者：刘洋[1] 余洋[1] 王岩[1] 赵炜疆[1] 

机构地区：[1]汕头大学医学院神经科学中心,广东汕头515041

出　　处：《细胞与分子免疫学杂志》2013年第10期1064-1067,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81171138)

摘　　要：目的以突变Tomlinson I库中筛选的抗细胞黏附分子L1的胞外区单链抗体为例,对定点突变和随机突变两种构建噬菌体抗体次级库的方法进行比较。方法设计定点突变和随机PCR引物在基因水平经PCR、酶切、连接将已有噬菌粒引入突变后转染大肠杆菌构建噬菌体次级抗体库;之后使用噬菌体ELISA确定阳性单克隆比例,采用皮尔森χ2检验进行比较。结果两种方法构建的次级抗体库库容分别为1.4×106pfu/mL和2.5×106pfu/mL,两库阳性单克隆比例分别为32.5%和35.5%,二者相比差异无统计学意义(P>0.05)。结论两种突变方法都有望获得高亲和力的抗体。Objective To compare site-mutated PCR and error-prone PCR methods in constructing the secondary phage antibody libraries derived from a primary extracellular domain of cell adhesion molecule L1 （L1-ecd） binding singlechain variable fragment （scFv） antibody. Methods Secondary mutant phage libraries were established by transfecting the mutated phage at the DNA level to E. coli TGI with designed site-mutated PCR or error-prone PCR primers. Using the selected phagemid as the template, the mutated plasmid was amplified by PCR and then constructed with restriction enzyme cutting and ligation. Phage-based ELISA was used to calculate the ratios of the positive monoclones from the two libraries and the results were statistically compared using the Pearson X2 method. Results The size of the two libraries were 1.4×10^6 pfu/mL （ site-mutated library） and 2.5×10^6 pfu/mL （ error-prone library）, respectively. The ratios of positive clones were 32.5% and 35.5%, respectively. The P value was 0.67, showing no significant difference. Conclusion These two methods can be widely used to obtain antibodies with a high affinity on the basis of the existing phage antibody.

关 键 词：定点突变PCR 随机突变PCR 噬菌体次级抗体库 单克隆 

分 类 号：R392.11[医药卫生—免疫学] Q754[医药卫生—基础医学]