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构建一个半合成抗体库 ,不经免疫制备人源抗Tie2Fab抗体。通过RT PCR方法 ,从人脐带血淋巴细胞总RNA扩增轻链基因及重链VH段基因 ,将轻链基因插入pCOMb3载体中 ,得人轻链质粒库 ;从HBsAb的Fd段基因制备含有不同长度随机化CDR3的FR3 CDR3 J CH1片段 ,然后将VH段基因与随机化的CDR3融合 ,得到Fd基因片段 ,再将其插入轻链质粒库中 ,得半合成人Fab质粒库。通过多次建库 ,获得总容量为 2× 1 0 7的半合成抗体库。其Fd段和轻链基因的重组率为 5 0 %。经 3轮淘洗 ,从噬菌体抗体库中筛选到与Tie2抗原特异结合的噬菌体克隆。测序确定抗体基因序列
A semi-synthetic antibody library was constructed and human-derived anti-Tie2Fab antibody was prepared without immunization. The light chain gene and the heavy chain VH gene were amplified by RT PCR from the total RNA of human umbilical cord blood lymphocytes and the light chain gene was inserted into the pCOMb3 vector to obtain a human light chain plasmid library. Length of the FR3 CDR3 J CH1 fragment of the randomized CDR3, and then fused the VH segment gene with the randomized CDR3 to obtain the Fd gene fragment, which is then inserted into the light chain plasmid library to obtain a semi-synthetic human Fab plasmid library. Through multiple database construction, a total volume of 2 × 107 semi-synthetic antibody library was obtained. The Fd segment and light chain gene recombination rate of 50%. After 3 rounds of panning, phage clones that specifically bind Tie2 antigen were screened from the phage antibody library. Sequencing determines the antibody gene sequence