抗鹅细小病毒噬菌体单链抗体文库的构建研究.docVIP

  抗鹅细小病毒噬菌体单链抗体文库的构建 研究# 吕雪峰1，李志慧*2，任锐1，吉凤涛1，苏双1** 5 10 15 （1. 吉林省畜牧兽医科学研究院，长春 130062； 2. 吉林大学超硬材料国家重点实验室，长春 130052） 摘要：应用噬菌体展示制备抗鹅细小病毒单链抗体库,筛选获得高特异性、高亲和力的单链 抗体 scFv。将鹅细小病毒扩增,免疫 4 日龄雏鹅,18 日后提取脾细胞 RNA,反转录获得 cDNA。 以针对鹅源重链可变区(VH)及轻链可变区(VL)基因的特异性引物,扩增获得 VH 基因和 VL 基 因。通过 PCR 法将 VH 基因、VL 基因与柔性多肽 Linker((Gly4Ser)3)拼接成 VL-Linker-VH 片段,酶切(sfiI+NotI)处理后,插入噬粒载体 pCANTAB5E,并转化宿主菌 TG1,通过辅助噬菌 体 M13K07 拯救,构建抗鹅细小病毒噬菌体单链抗体库。经 GPV 包被抗原 96 孔板亲和富集法 三轮淘筛,检测重组 scFv 的特异性抗原结合活性。IgBlast 序列分析发现，所得到的 scFv 基因 Vλ与人免疫球蛋白胚系基因同源性达 69.3 %，而 VH 同源性为 76.4 %。成功构建噬菌 体单链抗体库,获得鹅源抗鹅细小病毒特异性的 scFv,噬菌体中 scFv 插入率为 90%,获得库容 约为 1.2×106 的抗鹅细小病毒噬菌体单链抗体库。为噬菌体展示抗体技术运用及进一步建 立 GPV 检测及体内定位诊断方法研究打基础。 关键词：鹅；细小病毒；单链抗体；噬菌体展示技术 中图分类号：S852.2 20 construction of recombinat monoclonal antibody against goose parvovirus using phage display technology Lv Xuefeng1, Li Zhihui2, Ren Rui1, Ji Fengtao1, Su Shuang1 (1. veterinary research science institute of jilin province, ChangChun 130062; 25 30 35 40 2. national laboratory of superhard materials,jilinuniversity, ChangChun 130052) Abstract: To construct a library of single chain variable fragment (scFv) antibody against goose parvovirus （GPV）using phage display and getting the scFv antibody against GPV with high speciality and affinity by bio-panning. Total RNA was extracted from the spleen cell of the immuned goose，followed RT-PCR using random primer. The heavy-chain and light-chain variable region genes (VH and VL) repertoire of immunoglobulin were amplified respectively by PCR. Then they were linked by a DNA linker encoding (G1y4Ser)3as VH-linker-VL sequence forming scFv by PCR. These fragments were cloned into the phagemid pCANTAB5E and the recombinant phagemids were transformed to susceptible Escherichia coli TGl by electroporation. After rescuing with helper phage M13KO7, a library of phage scFv antibody was got. GPV was coated on 96 well plate to perform affinity enrichment panning, and to test the bio-activity of the scFv. IgBlast results revealed that the Vλ gene shar