摘要
目的筛选对粒细胞-巨噬细胞集落刺激因子(GM-CSF)具有中和活性的重链抗体重链可变区(VHH),即纳米抗体。方法采用皮下单次注射人源GM-CSF 0.5 mg的方法免疫骆驼,免疫5次后采集外周血,分离单核细胞,抽提总mRNA,逆转录后PCR扩增获得VHH基因;将VHH克隆到pADSCFV-S噬菌体载体,电转化TG1感受态细胞,构建VHH免疫库;固相包被重组人源GM-CSF对免疫库进行筛选,将获得的特异性结合人源GM-CSF的VHH基因通过酶切连接克隆到pABG真核表达载体,通过人胚肾上皮细胞293F细胞表达制备VHH-Fc样品。采用ELISA检测候选VHH-Fc分子与GM-CSF的响应曲线探究其结合活性,检测候选VHH-Fc与不同抗原的结合显色值初步确定其特异性;采用生物膜干涉(BLI)技术测定候选VHH-Fc与GM-CSF结合的亲和力;采用细胞增殖实验检测候选VHH-Fc对GM-CSF的抑制率曲线,确定其中和活性;采用蛋白质稳定性分析系统Uncle检测其热稳定性;小鼠尾静脉注射VHH-Fc 100μg,ELISA定量检测血清VHH-Fc浓度考察其在小鼠体内的药动学曲线。结果免疫5次后骆驼血清抗人GM-CSF抗体滴度高于1∶800000,构建的VHH库库容量约为5.55×10^(7)。筛选获得5个特异性结合人源GM-CSF的VHH-Fc候选分子,其中22N10可有效中和GM-CSF的促细胞增殖活性半数抑制浓度为17.23 nmol·L^(-1),与人源GM-CSF相互作用的亲和力为1.97×10-8 mol·L^(-1),可阻断GM-CSF与其受体GM-CSFRα的结合,热稳定性良好(溶解温度Tm1=59.2℃),小鼠体内半衰期为87.2 h,体内稳定性良好。结论获得靶向人源GM-CSF的候选VHH新分子22N10,在小鼠体内稳定性良好。
OBJECTIVE To screen and obtain nanobodies with neutralizing activity against granulocyte-macrophage colony stimulating factor(GM-CSF)to contribute to investigations into the mechanism of inflammation interventions and the development new drugs.METHODS Recombinant human GM-CSF was subcutaneously injected to immunize camels.Peripheral blood was collected after five immunizations,and mononuclear cells were isolated.Total mRNA was extracted,and the variable domains of the heavy chain of heavy-chain antibody(VHH,also called nanobody)genes were obtained by PCR amplification after reverse transcription.The genes were cloned into the pADSCFV-S phage display vector and electrotransformed into TG1 competent cells to construct a nanobody immune library that was screened with recombinant human GM-CSF immobilized on a solid phase.The VHH genes specifically binding to human GM-CSF were cloned into the pABG eukaryotic expression vector before VHH-Fc samples were prepared by using the human embryonic kidney 293 fibroblast expression system.The binding activity of candidate VHH-Fc molecules to GM-CSF was investigated through ELISA response curves,and binding colorimetric values with different antigens were detected to determine their specificity.The binding affinity between VHH-Fc candidates and GM-CSF was measured using biolayer interferometry(BLI).The inhibition rate curve of VHH-Fc candidates on GM-CSF was detected through cell proliferation assays to determine its neutralization activity.The Uncle system was used to investigate its thermal stability.100μg of VHH-Fc was injected into mice via the tail vein,and the serum concentration of VHH-Fc was quantitatively detected by ELISA to examine its pharmacokinetic curve in mice.RESULTS The camel serum titer of anti-human GM-CSF antibodies was higher than 1:800000 after the fifth immunization,and the capacity of the constructed nanobody library was about 5.55×107.Following the screening process,five candidate VHH-Fc molecules specifically binding to human GM-CSF were obtained.Among these,22N10 effectively neutralized the cell proliferation-promoting activity of GM-CSF(the IC50 value was 17.23 nmol·L^(-1)).Subsequent studies revealed that 22N10 interacted with human GM-CSF with an affinity of 1.97×10^(-8)mol·L^(-1),blocked the binding of GM-CSF to its receptor CSF2Rα,exhibited good thermal stability(Tm1=59.2℃),and showed favorable metabolic characteristics in mice.CONCLUSION A new candidate nanobody molecule 22N10 targeting human GM-CSF is obtained which is expected to facilitate the drug development and mechanism investigations.
作者
刘娇
陈蕾
秦慧
康庆琳
李格格
杨志新
杜鹏
周春阳
LIU Jiao;CHEN Lei;QIN Hui;KANG Qinlin;LI Gege;YANG Zhixin;DU Peng;ZHOU Chunyang(School of Pharmacy,North Sichuan Medical College,Nanchong 637000,China;Laboratory of Advanced Biotechnology,Academy of Military Medical Sciences,Beijing 100071,China;School of Basic Medical Science,Zhejiang University,Hangzhou 310009,China;School of Life Sciences and Biopharmaceuticals,Shenyang Pharmaceutical University,Benxi 117004,China)
出处
《中国药理学与毒理学杂志》
北大核心
2025年第8期591-599,共9页
Chinese Journal of Pharmacology and Toxicology
关键词
粒细胞-巨噬细胞集落刺激因子
纳米抗体
噬菌体展示
中和活性
真核表达
特异性
集落刺激因子家族
granulocyte-macrophage colony-stimulating factor
nanobody
phage display
neutralizing activity
eukaryotic expression
specificity
colony-stimulating factor family
