CAJ | 학술논문

应用噬菌体展示和重组抗体技术制备抗氧氟沙星单链抗体(scFv)库,筛选获得氧氟沙星特异性噬菌体 scFv 以及同源模拟其三维结构。将从氧氟沙星杂交瘤细胞提取的总 RNA,用 RT-PCR 反转录合成 cDNA,以针对鼠源重链可变区(VH)及轻链可变区(VL)基因的兼并引物,扩增获得 VH 和 VL 可变区基因,通过SOE-PCR 法将 VH 基因和 VL 基因通过柔性多肽 Linker (Gly4 Ser)3拼接成全长 scFv 基因片段,将双酶切后的scFv 基因片段插入 T7噬菌体,经体外包装后转化宿主菌 BLT5403,成功构建库容量为3×105 pfu/ mL 的抗体库,经4轮吸附-洗脱-扩增的富集,采用直接竞争 ELISA 筛选到4个特异性噬菌体 scFv,运用 Expasy 软件模拟特异性 scFv 的三维结构。为进一步大量表达氧氟沙星单链抗体奠定了基础。
응용서균체전시화중조항체기술제비항양불사성단련항체(scFv)고,사선획득양불사성특이성서균체 scFv 이급동원모의기삼유결구。장종양불사성잡교류세포제취적총 RNA,용 RT-PCR 반전록합성 cDNA,이침대서원중련가변구(VH)급경련가변구(VL)기인적겸병인물,확증획득 VH 화 VL 가변구기인,통과SOE-PCR 법장 VH 기인화 VL 기인통과유성다태 Linker (Gly4 Ser)3병접성전장 scFv 기인편단,장쌍매절후적scFv 기인편단삽입 T7서균체,경체외포장후전화숙주균 BLT5403,성공구건고용량위3×105 pfu/ mL 적항체고,경4륜흡부-세탈-확증적부집,채용직접경쟁 ELISA 사선도4개특이성서균체 scFv,운용 Expasy 연건모의특이성 scFv 적삼유결구。위진일보대량표체양불사성단련항체전정료기출。
To construct a library of mouse single chain variable fragment (scFv) antibody against ofloxacin using phage display and recombinant antibody technique, specific anti-ofloxacin scFv was screened and 3D structure was homology modeling. Total RNA was extracted from hybridoma cell of ofloxacin mAb, and was used to amplify VH and VL gene by RT-PCR using random primer. Then they were linked by a DNA linker encoding (G1y4 Ser) 3 as VH-linker-VL sequence forming scFv by SOE(splicing by overlap extension) PCR. These fragments were inserted into phage T7 after double digestion and transformed with host bacteria BLT5403. 3 ×105 pfu / ml single chain antibody phage libraries were successfully constructed. Four positive phage scFv clones were screened by direct competitive ELISA after four times of enriched procedure in the order of adsorption-elution-amplificatio, 3D structure of specific scFv was homology modeling finally. This research lays a foundation for further massive expression of anti-ofloxacin scFv.