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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 two methods of constructing phage antibody secondary libraries by site-directed mutagenesis and random mutagenesis by mutating the extracellular domain single chain antibody of anti-cell adhesion molecule L1 screened in Tomlinson I database. Methods Site-directed mutagenesis and random PCR primers were designed to amplify the secondary phage antibody library by introducing the existing phagemid into the bacteriophage by PCR, restriction enzyme digestion and ligation. Then the phage ELISA was used to determine the percentage of positive monoclonal antibody. Sen χ2 test for comparison. Results The secondary antibody pools constructed by the two methods were 1.4 × 106pfu / mL and 2.5 × 106pfu / mL, respectively, and the positive ratios of the two libraries were 32.5% and 35.5%, respectively, with no significant difference (P > 0.05). Conclusion Both mutation methods are expected to obtain high affinity antibodies.