全人源食管癌噬菌体单链抗体库的筛选及特异性鉴定

Screening and Identification of Human Single-chain Variable Fragment Using a Phage-Display Antibody Library Constructed from Esophageal Cancer Cells

目的:我们应用噬菌体抗体库技术构建了食管癌相关的人源单链抗体文库,本文目的在于对该抗体库进行鉴定,筛选食管癌抗体,同时对抗体的活性进行检测。方法:PCR鉴定TG1中食管癌单链抗体scFv的插入率;1.5%琼脂糖凝胶电泳鉴定SfiI和NotI双酶切质粒的结果;先以人正常食管上皮细胞吸附后再以食管癌细胞Eca109为抗原对所建抗体库进行4轮的亲和筛选;将阳性重组噬菌体克隆感染Ecoli HB2151进行可溶性抗体表达及经亲和柱层析纯化;用SDS-PAGE测定该抗体的相对分子量;用Western blot鉴定该抗体;用ELISA法、免疫组化法鉴定该抗体与人食管癌细胞的结合的特异性。结果:scFv基因插入率为91.7%;酶切后检测到目的基因片段。在亲和筛选过程中,食管癌噬菌体单链抗体得到富集,收获率逐轮得到提高,第4轮为第1轮的141倍;SDS-PAGE与Western blot结果显示抗体的相对分子量为左右和30kd条带染色;在Ecoli HB2151中实现了单链抗体的可溶性表达。免疫组织化学结果提示可溶性抗体仅染食管癌组织,而肝癌组织和胃癌组织不染色。免疫细胞化学结果表明此可溶性抗体可使Eca109细胞染色。ELISA测定结果显示可溶性抗体具有较高的免疫活性,能与Eca109细胞结合,而不与胃癌BGC-823和NHEEC结合。结论:利用噬菌体抗体库技术的阴性、阳性筛选得到了食管癌噬菌体单链抗体,且筛选后的抗体片段与人食管癌细胞有特异性的结合活性。

Objective： A human single-chain antibody library was constructed from lymph nodes of esophageal cancer patients using phage display techniques. The aim of this study was to screen and identify the human single-chain variable fragment（scFv） associated with esophageal cancer and to detect the activity of the antibody. Methods： The insert ratio of the scFv antibody library was identified by PCR. Positive insert clones were identified by Sfil/Notl double digestion followed by 1.5% agarose gel electrophoresis. Normal human esophageal epithelial celIs（NHEEC） and esophageal cancer cell line Eca109 were used to screen the phage antibody library. Strongly positive recombinant phage clones were used to infect E. coil HB2151 and induce expression of soluble scFv. Soluble scFv from periplasm was purified by affinity chromatography. The relative molecular weight of the scFv was detected by SDS-PAGE. Western blot, immunohistochemistry, immunocytochemistry and ELISA were used to characterize the activity of the antibody. Results： Enzymatic digestions showed that the positive insert ratio was 91.7%（22/24）. Agarose gel electrophoresis showed that target fragments were released as products of Sfil/Notl double digestion of positive insert clones. Recombinant scFv phage had a titer of 3.3×10^11 cfu/mL. The fourth phage harvest yielded 141 times as much as that of the first one. The results of SDS-PAGE and Western blot showed that the MW of the soluble scFv was about 30kD. Tissues from esophageal cancer showed strong immunohistochemical staining and the esophageal cancer cell line Eca109 showed strong immunocytochemical staining with the recombinant scFv. ELISA results revealed that soluble scFv was highly active and could bind Eca109 cells, but not BGC-823 or NHEE cells. Conclusion： A human phage-display antibody library was successfully produced using esophageal cancer cells found in lymph nodes. The scFv antibody fragment specifically binds esophageal cancer cells.