TNF-α纳米抗体的筛选、表达及特异性检测

Screening,Expression and Specificity Detection of Anti-TNF-α Nanobody

目的:构建肿瘤坏死因子(tumor necrosis factor,TNF)纳米抗体的噬菌体文库,筛选并表达与TNF-α具有亲和特异性的纳米抗体。方法:(1)利用TNF-α免疫羊驼,提取外周血淋巴细胞总RNA,构建噬菌体文库;多次淘洗筛选到与TNF-α有亲和力的克隆。(2)通过ExPASy分析其分子量和亲疏水性等理化性质,并将筛选得到的VHH基因在大肠杆菌E.coli DH5α中表达。(3)表达的NbTNF-α蛋白质经过Ni金属螯合亲和层析纯化,采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测蛋白质的抗原特异性和亲和力。结果:(1)通过噬菌体文库的构建和淘洗,筛选到8个与TNF-α有亲和力的VHH基因片段。(2)通过软件预测,这8个NbTNF-α蛋白均为亲水性蛋白,其分子量为19.6~20.1kDa;在大肠杆菌中重组表达这8个抗体蛋白。(3)ELISA检测结果表明,有5株纳米抗体NbTNF-α-1、NbTNF-α-2、NbTNF-α-3、NbTNF-α-4和NbTNF-α-5能与TNF-α特异性结合。结论:成功筛选并表达了5株具有TNF-α特异性的纳米抗体,可能成为抗TNF-α的候选药物。

Objective:To construct the phage library of anti-TNF-α(vascular endothelial growth factor)andto screen and express the nanobodies which have specificity and affinity with TNF-α.Methods:(1)The llama was immunized with TNF-α,and the total RNA of peripheral blood lymphocytes was extracted to construc a phage library,the clones having affinity with TNF-αwere screened by multiple panning.(2)Then their molecular weight,p I and hydrophilicity were analyzed by ExPASy.And the VHH genes were cloned into the expression vector p NCS to construct the recombinant plasmids(pNCS-NbTNF-α)and to express these recombinan nanobodies(NbTNF-α)in E.coli DH5α.(3)The recombinant nanobodies were purified by Ni metal chelate affinity chromatography,followed by detection the specificity by enzyme linked immunosorbent assay(ELISA).Results:(1)Ten VHH gene fragments having affinity with TNF-αwere obtained after phage library construction and panning.(2)Based on the bioinformatics analysis,it was found that eight nanobodies were hydrophilic proteins with molecular weights of 19.6-20.1kDa.All of NbTNF-αwere expressed in E.coli DH5αin soluble form.(3)It was showed that five recombinant nanobodies,NbTNF-α-1,NbTNF-α-2,NbTNF-α-3,NbTNF-α-4 and NbTNF-α-5could specifically bind to TNF-α.Conclusion:Eight nanobodies with specificity to TNF-αwere screened and expressed in E.coli successfully,and five NbTNF-αshowed good affinity with TNF-α,which could be possible candidates for anti-TNF-αdrug.