【摘要】 目的:制备抗粒细胞-巨噬细胞集落刺激因子（GM-CSF）纳米抗体,并测定其亲和力。方法:分离提取GM-CSF免疫后羊驼外周血淋巴细胞总RNA,PCR扩增得到纳米抗体基因片段,与载体pHEN1重组后克隆至大肠杆菌TG1以构建初始文库,经拯救构建得到噬菌体展示纳米抗体文库,并对其进行生物淘选;通过大肠杆菌BL21（DE3）原核表达阳性纳米抗体克隆,并测定其亲和力。结果:构建了多样性良好且库容量为1.37×10⁹cfu的纳米抗体初始文库,3轮淘选后共筛选得到5株氨基酸序列差异性较大的纳米抗体,并对其中一株纳米抗体G1进行表达纯化,SDS-PAGE分析表明纳米抗体G1纯度较高,且有较高的亲和力(K_D=2.95×10^-8mol/L)。结论:制备了高亲和力的抗GM-CSF纳米抗体,可应用于相关炎症抗体药物研制和疾病监测等方面。更多还原

【Abstract】 Objective:To prepare anti-GM-CSF nanobody and determine its affinity to granulocyte-macrophage colony stimulating factor（GM-CSF）.Methods:Total RNA was isolated and extracted from GM-CSF immunized alpaca’s peripheral blood lymphocytes.Then the nanobody gene fragments were amplified by PCR and recombined with phagemid p HEN1.And then,the recombinant vectors were transformed into Escherichia coli TG1 to construct the initial library.After being rescued by M13KO7 helper phage,the phage-displayed nanobody library was constructed and then screened by biopanning.The immunopositive nanobody was expressed by E.coli BL21（DE3）and its affinity was determined.Results:The well-diversified initial library was constructed with a capacity of 1.37×10⁹cfu.After 3 rounds of biopanning,5 strains of nanobody with different amino acid sequences were screened.Among the nanobodies,nanobody G1 showed high affinity to GM-CSF(K_D=2.95×10^-8mol/L)and high purity by SDS-PAGE analysis after prokaryotic expression and purification.Conclusion:In this study,we produced an anti-GM-CSF nanobody with high affinity,which has potential application in related disease surveillance and drug development.更多还原