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为了运用噬菌体展示技术对抗白细胞介素-4受体(IL-4R)特异性单链抗体进行表达与鉴定,试验提取了前期工作中经富集与筛选抗IL-4R单链抗体库而得到的特异性pCANTAB5E-sc Fv重组质粒,用NotⅠ和SfiⅠ双酶切鉴定样本并将质粒送上海英骏生物公司测序;利用pCANTAB-5E噬菌体展示系统(pCANTAB-5E、TG1、M13K07、HB2151)表达抗IL-4R单链抗体重组蛋白;通过SDS-PAGE和Western-blot分析单链抗体的表达情况及抗体的活性。结果表明:pCANTAB-5E噬菌体展示系统表达出的单链抗体重组蛋白分子质量在25~35 ku之间,28 ku左右,与测序结果预测的蛋白分子质量接近。说明成功表达了抗IL-4R单链抗体重组蛋白。
In order to express and identify specific IL-4R-specific single-chain antibodies using phage display technology, we extracted the cDNA library obtained by enrichment and screening of anti-IL-4R single chain antibody library in the previous work Specific pCANTAB5E-sc Fv recombinant plasmid was digested with NotⅠ and SfiI to identify the samples and the plasmid was sent to the Hynix Biological Sequencing; pCANTAB-5E phage display system (pCANTAB-5E, TG1, M13K07, HB2151) expression of anti-IL -4R single chain antibody recombinant protein; SDS-PAGE and Western-blot analysis of single-chain antibody expression and antibody activity. The results showed that the molecular weight of the recombinant protein expressed by pCANTAB-5E phage display system was about 25-35 ku, about 28 ku, which was close to that predicted by sequencing. Indicating that the anti-IL-4R single chain antibody recombinant protein was successfully expressed.