目的:在成功制备小鼠抗人IL-13Rα2高亲和力单克隆抗体(mAb)的基础上,通过分子克隆方法获得该抗体可变区基因序列。方法:从1株小鼠抗人IL-13Rα2 mAb杂交瘤细胞LX147-7中提取总RNA,以此为模板反转录获得cDNA,用针对小鼠mAb重链和轻链可...目的:在成功制备小鼠抗人IL-13Rα2高亲和力单克隆抗体(mAb)的基础上,通过分子克隆方法获得该抗体可变区基因序列。方法:从1株小鼠抗人IL-13Rα2 mAb杂交瘤细胞LX147-7中提取总RNA,以此为模板反转录获得cDNA,用针对小鼠mAb重链和轻链可变区基因序列的特异性引物分别进行PCR反应。将PCR产物连入克隆载体,经筛选阳性克隆,PCR和酶切鉴定正确后送测序,测序结果进行生物信息学分析。结果:成功克隆了抗人IL-13Rα2 mAb LX147-7重链和轻链的可变区基因。结论:获得了抗人IL-13Rα2 mAb LX147-7重链和轻链的可变区基因序列,为构建相关基因工程抗体打下了良好基础。展开更多
A murine phage antibody library was constructed.First,the total RNA was extracted from fresh spleens of nonimmunized mice,then the cDNA library was achieved via reverse transcription PCR.Gene fragments encoding V_H an...A murine phage antibody library was constructed.First,the total RNA was extracted from fresh spleens of nonimmunized mice,then the cDNA library was achieved via reverse transcription PCR.Gene fragments encoding V_H and V_L were amplified and assembled into a single gene using a DNA linker encoding a polypeptide of 15 amino acid residues(Gly4Ser)3 through PCR.And finally the recombinant DNA fragments were cloned into the phagemid pCANTAB5E vector and introduced into E.coli TG1.The phagemid particles displaying functional ScFv were rescued by reinfection of helper phage M13K07,thus a murine antibody library was obtained.展开更多
文摘目的:在成功制备小鼠抗人IL-13Rα2高亲和力单克隆抗体(mAb)的基础上,通过分子克隆方法获得该抗体可变区基因序列。方法:从1株小鼠抗人IL-13Rα2 mAb杂交瘤细胞LX147-7中提取总RNA,以此为模板反转录获得cDNA,用针对小鼠mAb重链和轻链可变区基因序列的特异性引物分别进行PCR反应。将PCR产物连入克隆载体,经筛选阳性克隆,PCR和酶切鉴定正确后送测序,测序结果进行生物信息学分析。结果:成功克隆了抗人IL-13Rα2 mAb LX147-7重链和轻链的可变区基因。结论:获得了抗人IL-13Rα2 mAb LX147-7重链和轻链的可变区基因序列,为构建相关基因工程抗体打下了良好基础。
文摘A murine phage antibody library was constructed.First,the total RNA was extracted from fresh spleens of nonimmunized mice,then the cDNA library was achieved via reverse transcription PCR.Gene fragments encoding V_H and V_L were amplified and assembled into a single gene using a DNA linker encoding a polypeptide of 15 amino acid residues(Gly4Ser)3 through PCR.And finally the recombinant DNA fragments were cloned into the phagemid pCANTAB5E vector and introduced into E.coli TG1.The phagemid particles displaying functional ScFv were rescued by reinfection of helper phage M13K07,thus a murine antibody library was obtained.
