Zhang Qing, Zhang Si-he, Yi Jing, Su Ming-quan, Bao Guo-qiang, Hao Xiao-ke
Department of Clinical Laboratory, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2006 Mar;22(2):196-9.
To screen human anti-gamma-sm (gamma-seminoprotein) light chain (Lc) with guided selection of murine Fd fragment.
The human Lc repertorie genes were amplified by RT-PCR from PBMC in patients with prostate cancer, and cloned into the phagemid vector pComb3X with murine Fd gene against gamma-seminoprotein to construct the human-mouse hybrid Fab antibody library. The size of the library, antibody gene recombinant percentage and diversity were identified by colony counting, plasmid digestion and colony sequence analysis, respectively. Purified gamma-sm was used as antigen to screen the displayed phage hybrid antibody library rescued by helper phage M13K07 for three rounds. The positive clones were selected by ELISA with pIII-fusion antibody and then the sequences of light gene in the positive clones were analyzed by IMGT-VQUEST.
A 1.2x10(7) CPU human-mouse Fab antibody library was constructed with 90% Lc gene recombinant and great diversity. After 3 rounds' panning with gamma-sm, 5 positive clones were selected by ELISA and 2 clones with higher affinity were selected. Sequence analysis suggested these two positive clones contained the same light gene with high V(L) homology to human germline gene IGKV4-1*01.
Human anti-gamma-sm light chain was successfully screened by constructing mouse-human hybrid Fab phage antibody library with murine Fd-guided selection.
通过鼠源Fd片段的导向选择筛选人抗γ-精蛋白（γ-半胱氨酸蛋白酶抑制剂）轻链（Lc）。
采用RT-PCR从前列腺癌患者外周血单个核细胞（PBMC）中扩增人Lc repertoire基因，并与针对γ-精蛋白的鼠源Fd基因一起克隆到噬菌粒载体pComb3X中，构建人-鼠杂交Fab抗体库。分别通过菌落计数、质粒酶切和菌落序列分析鉴定文库大小、抗体基因重组率和多样性。以纯化的γ-精蛋白为抗原，对辅助噬菌体M13K07拯救的展示型噬菌体杂交抗体库进行三轮筛选。用pIII融合抗体通过ELISA筛选阳性克隆，然后用IMGT-VQUEST分析阳性克隆中轻链基因的序列。
构建了库容为1.2×10⁷菌落形成单位（CFU）的人-鼠Fab抗体库，Lc基因重组率为90%，具有高度多样性。用γ-精蛋白进行三轮淘选后，通过ELISA筛选出5个阳性克隆，并挑选出2个亲和力较高的克隆。序列分析表明，这两个阳性克隆含有相同的轻链基因，其V（L）与人种系基因IGKV4-1*01具有高度同源性。
通过构建鼠源Fd导向选择的人-鼠杂交Fab噬菌体抗体库，成功筛选出人抗γ-精蛋白轻链。
