摘要
目的:以鼠源抗γ-sm抗体重链Fd片段为模板,筛选人源性抗人γ-精浆蛋白抗体轻链。方法:利用RT-PCR从前列腺癌患者外周血淋巴细胞扩增出全套的人抗体轻链基因,克隆入含鼠源性抗γ-sm抗体重链Fd片段的噬粒载体pComb3X/mFd中,电转化大肠杆菌XL1-Blue后建立人-鼠杂合的Fab抗体库。通过稀释滴定、限制性酶切和随机测序,对所建杂合库的库容、重组率和多样性分别进行鉴定,以M13K07辅助噬菌体超感染,利用亲和纯化的γ-sm为抗原,对挽救展示的噬菌体抗体库进行3轮淘筛和克隆鉴定。最后对筛选出的杂合抗体进行初步的功能检测。结果:构建成功1.2×107CFU、重组率为90%、多样性好的杂合人-鼠噬菌体抗体库。利用纯化的γ-sm3轮亲和淘筛后,5个克隆成功诱导表达特异性的pIII融合抗体。ELISA进一步检测表明,5个克隆均呈特异性阳性反应,其中2个克隆亲和力较高,测序显示其所含轻链基因序列完全相同,可变区来源于IGKV4-101胚系基因家族。结论:利用鼠源Fd片段为模板,导向筛选杂合噬菌体库,成功筛选到人源性抗人γ-精浆蛋白抗体轻链。
AIM： To screen human anti-γ-sm （ γ-sminoprotein） light chain （Lc） with guided selection of murine Fd fragment. METHODS： 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 γ-sminoprotein 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 γ-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 p lll-fusion antibody and then the sequences of light gene in the positive clones were analyzed by IMGT-VQUEST. RESULTS： A 1.2 × 10^7 CPU human-mouse Fab antibody library was constructed with 90% Lc gene recombinant and great diversity. After 3 rounds＇ panning with γ-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 VL homology to human germline gene IGKV4-1 ＊ 01. CONCLUSION： Human anti- γ-sm light chain was successfully screened by constructing mouse-human hybrid Fab phage antibody library with murine Fd-guided selection.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第2期196-199,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家高技术研究发展计划(863)资助项目(No.2001AA215321)
国家十五重大科技专项项目(No.2002AA2Z3109)
关键词
噬菌体抗体库
导向筛选
前列腺癌
phage antibody library
guide selection
pros-tate cancer
