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制备大容量天然噬菌体抗体库。从健康成人外周血和新生儿脐带血中提取淋巴细胞总RNA,反转录后PCR分别扩增抗体重链和轻链基因。将获得的基因片段通过overlap PCR连接为单链抗体基因(scFv),限制性内切酶酶切后连入pDAN5a载体中,连接产物转化大肠杆菌XL2-blue MRF’。与XL1-blue菌株相比,初级库库容增加了3.9倍。再将初级库噬菌体感染含有Cre重组酶的大肠杆菌BS1365,抗体基因在重组酶作用下发生同源重组,最终得到库容为1.7×1011的噬菌体抗体库。经检测,该抗体库多样性良好,利用该库可获得针对6种抗原的抗体。以上结果表明,应用XL2-blue MRF’作为抗体基因的转化宿主,提高了噬菌体抗体库的库容,获得的抗体库可用于下一步抗体药物的筛选。
Preparation of large volumes of natural phage antibody library. Lymphocyte total RNA was extracted from healthy adult peripheral blood and neonatal umbilical cord blood, and the antibody heavy chain and light chain genes were amplified by reverse transcription PCR. The obtained gene fragment was connected to single chain antibody gene (scFv) by overlap PCR, and then ligated into pDAN5a vector after restriction enzyme digestion, and the ligation product was transformed into Escherichia coli XL2-blue MRF ’. Compared with XL1-blue strain, the primary pool capacity increased 3.9-fold. The primary phage was then infected with Cre recombinase-producing Escherichia coli BS1365 and the antibody gene was subjected to homologous recombination under the action of recombinase to finally obtain a phage antibody library with a volume of 1.7 × 1011. After testing, the antibody library diversity is good, the use of the library can be obtained against six antigens antibodies. The above results show that using XL2-blue MRF ’as a transforming host for the antibody gene improves the capacity of the phage antibody library, and the obtained antibody library can be used for further antibody screening.