抗γ-精浆蛋白人-鼠杂合Fab噬菌体抗体库的构建及导向筛选

Construction and guided-selection of mouse-human hybrid Fab phage antibody library against γ-seminoprotein

目的:建立人-鼠杂合Fab噬菌体抗体库,筛选抗γ-精浆蛋白(γ-sm)人源性抗体轻链。方法:PCR扩增前列腺癌患者外周血淋巴细胞人抗体全套轻链基因,克隆人含鼠源抗γ-sm抗体Fd基因的pComb3X噬粒中,建立人-鼠杂合Fab噬菌体抗体库。通过稀释滴定、限制性酶切等分别对库容、基因重组率和多样性进行鉴定。以M13K07辅助噬菌体超感染,利用纯化的γ-sm对杂合库进行筛选,对筛出的阳性克隆进行功能检测。结果:构建了库容量为1．2×107CFU、重组率为90％、多样性好的人-鼠杂合噬菌体抗体库。ELISA检测出2个强阳性克隆,Western blot确证为抗γ--sm的人-鼠杂合Fab,竞争ELISA显示其表观亲和力约为亲本鼠抗体亲和力的71．8％。DNA测序显示筛到的人源轻链可变区基因属IGKV4-1*01胚系家族。结论:成功得到人源性抗γ-sm抗体轻链,为进一步获得全人源化抗体奠定了基础。

Objective： To construct a hybrid Fab antibody library and to select human Lc fragment against γ-semino- protein （γ-sm）. Methods： Human Lc repertoric gene was amplified by RT-PCR from PBMC of prostate cancer patients and was cloned into pComb3X vector containing murine Fd gene to construct the human-mouse hybrid Fab phage library. Purified γ--sm was used as antigen to screen for 3 rounds and the positive clones were analyzed by Western blot, inhibition ELISA and DNA sequencing. Results： A human-mouse Fab antibody library with good diversity was constructed, with a 1.2 × 10^7 CFU size and 90% Lc gene recombinant frequency. After 3 rounds of panning, two hybrid Fab clones specifically against γ-sm were selected, and their apparent binding constant was 71.8% that of the parental mouse antibody E4B7. These two positive clones contained the same light chain with high VL homology to human germline gene IGKV4-1 ＊ 01. Conclusion： Specific human Lc fragment against γ-sm can be successfully selected through construction of mouse-human hybrid Fab phage antibody library.