靶向淋巴细胞激活基因3(LAG-3)纳米抗体的筛选与活性分析  

Screening and activity analysis of nanobodies against lymphocyte activation gene-3(LAG-3)

作　　者：文蕊鑫 陈玥雯 杨紫薇 闫凤英 李卿 武卫党[1,3] WEN Ruixin;CHEN Yuewen;YANG Ziwei;YAN Fengying;LI Qing;WU Weidang(National Key Laboratory of Druggability Evaluation and Systematic Translational Medicine,Tianjin Institute of Pharmaceutical Research(TIPR),Tianjin 300301;School of Pharmacy,Anhui Medical University,Hefei 230032;Tianjin Joint Innovation Biotechnology Co.,Ltd.,Tianjin 300301,China)

机构地区：[1]天津药物研究院药物成药性评价与系统转化全国重点实验室,天津300301 [2]安徽医科大学药学院,安徽合肥230032 [3]天津和创生物技术有限公司,天津300301

出　　处：《细胞与分子免疫学杂志》2023年第11期1024-1031,共8页Chinese Journal of Cellular and Molecular Immunology

基　　金：药物成药性评价与系统转化全国重点实验室(天津药物研究院)自主研究课题(010161002)。

摘　　要：目的建立淋巴细胞激活基因3(LAG-3)免疫噬菌体纳米抗体文库,获得高特异性和亲和力的抗LAG-3纳米抗体,并鉴定其功能活性。方法采用人LAG-3蛋白免疫羊驼,获取外周血cDNA;通过巢式PCR获得纳米抗体基因,构建至pComb3XSS质粒,电转至大肠杆菌XL1-Blue中构建纳米抗体噬菌体免疫文库,并对文库进行质量分析。通过噬菌体展示技术筛选抗LAG-3特异性纳米抗体,通过第二代基因测序技术获得纳米抗体的基因序列。将目的序列构建到大肠杆菌BL21(DE3)周质表达载体pET-22b(+)中进行抗体表达,Prism A柱进行纯化,通过高效液相色谱-质谱分析(HPLC-MS)、ELISA、Western blot法和表面等离子共振技术(SPR)进行抗体性质和功能分析。结果构建的纳米抗体噬菌体免疫文库的库容为7.20×10^(8)菌落形成单位(CFU),序列分析文库多样性良好,经过4轮淘筛后获得3条具有不同氨基酸序列的抗LAG-3纳米抗体,分别为重链抗体重链可变区结构域-L1-3(VHH-L1-3)、VHH-L3-2和VHH-L13-2,纳米抗体表达纯化后纯度在95%以上,三种抗体均可以与重组人LAG-3蛋白进行特异性结合;其中VHH-L13-2抗体的亲和力指数KD值为3.971×10^(-9)mol/L,且对于LAG-3和纤维蛋白原样蛋白1(FGL-1)的结合具有抑制作用,半数抑制浓度(IC_(50))值为15.58 nmol/L。结论成功构建了LAG-3纳米抗体噬菌体免疫文库,筛选获得了特异性好、亲和力高的LAG-3纳米抗体,且对于LAG-3与其配体的结合具有一定的抑制作用。Objective To generate the phage display nanobody library immunized by lymphocyte-activation gene 3(LAG-3)and to validate the functional activity of obtained anti-LAG-3 nanobodies.Methods The peripheral blood cDNA library was isolated from the adult llama which was immunized by human LAG-3 protein.The nanobodies sequences were obtained by nested PCR and cloned into the phagemid vector pComb3XSS,then transformed into Escherichia coli XL1-Blue cells for library generation and quality analysis.Anti-LAG-3 specific nanobodies were screened by phage display and sequenced by next-generation sequencing.Nanobodies were cloned into pET-22b(+)vector and Escherichia coli BL21(DE3)cells were used for protein expression.The proteins were purified by using the Prism A column,then HPLC-MS,ELISA,Western blot,and surface plasmon resonance technology(SPR)were performed to characterize the nanobodies.Results The library capacity of the nanobody phage immune library with great diversity was 7.20×10^(8)CFU/mL.After four rounds of biopanning,three individual nanobodies with distinct amino acid sequences VHH-L1-3,VHH-L3-2 and VHH-L13-2 were picked.The purity of the purified nanobodies was more than 95%.All of these three nanobodies exhibited high binding affinities with recombinant human LAG-3 specifically,among which the KD value of VHH-L13-2 was 3.971×10^(-9)mol/L.VHH-L13-2 exhibited the inhibitory effects on the association of LAG-3 and its ligand FGL-1,and the half maximal inhibitory concentration(IC_(50))value was 15.58 nmol/L.Conclusion The anti-LAG-3 phage display nanobody library is generated successfully.The anti-LAG-3 nanobodies possess high specificity and binding affinity and exhibit the inhibitory effects on the association of LAG-3 and its ligand.

关 键 词：纳米抗体 淋巴细胞激活基因3(LAG-3) 噬菌体展示技术 原核表达 

分 类 号：S852.4[农业科学—基础兽医学]