新型抗IL-1β和IL-17A双特异抗体的构建及特性鉴定

目的 双特异性抗体(bispecific antibody,BsAb)具有双重的生物学功能.本实验旨在设计并原核表达抗人IL-1β和抗人IL-17A的双特异性抗体(BsAbl/17),获得具有生物活性的BsAb1/17,为深入研究和利用双特异性抗体奠定基础.方法 利用重叠PCR方法构建VH1VL17-CL和VL1VH17-CH1基因片段,并且在所用引物的5'和3'端附加Nco Ⅰ和BamH Ⅰ的酶切位点.将重叠PCR产物进行胶回收后用Nco Ⅰ/BamH Ⅰ进行双酶切,酶切产物再次胶回收,将其连接到用Nco I/BamH I消化的pET-27b载体上.将重组质粒pET-27b-VH1VL17-CL(petA)和pET-27b-VL1VH17-CH1(petB)转化到E. coli Rosetta中.SDS-PAGE和Western blot进行鉴定,用real-time PCR检测其阻断IL-1 β刺激人T细胞表达细胞因子IL-18的活性,人IL-6定量酶联检测试剂盒检测其阻断IL-17A刺激HeLa细胞表达人IL-6的活性.结果 DNA测序结果证明成功构建了pET-27b-VH1VL17-CL(petA)和pET-27b-VL1VH17-CH1(petB)表达质粒.petA、petB诱导产物主要以包涵体形式存在,成熟蛋白纯化产物纯度超过90%以上,经SDS-PAGE分析表明表达产物的相对分子质量约为38×103,与理论值相符.Western blot和ELISA结果证实双特异抗体BsAb1/17对IL-1 β和IL-17A均具有很好的亲和力.通过RT-PCR检测证明其具有阻断IL-1 β刺激人T细胞大量表达细胞因子IL-18的活性,并且具有阻断IL-17A刺激HeLa细胞产生IL-6的活性.结论 成功构建了同时抗IL-1β和IL-17A的双特异抗体,并利用大肠杆菌表达系统高效表达较高纯度的具有生物活性的双特异抗体.

Abstract：

Objective To construct bispecific antibody BsAb1/17 against both IL-1β and IL-17A,express and purify the biologically active BsAbl/17 protein in prokaryotic system for further studies and applications. Methods VH1VL17-CL and VL1VH17-CH1 gene segments were constructed by overlap-PCR.Restriction enzyme sites Nco Ⅰ and BamH Ⅰ were designed at the 5'and 3' end primers respectively. The products of overlap-PCR were ligated to the Nco Ⅰ/BamH Ⅰ -prepared pET-27b vector. The recombinant plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 ( petB ) were transformed into E. coliRosetta separately. The expressing products were analyzed by SDS-PAGE and Western blot. Neutralization activity of the bispecific antibody for blocking the induction of IL-18 expression by IL-1β in human T cells was determined by real-time PCR. Neutralization activity of the bispecific antibody for blocking the induction of IL-6 expression by IL-17A in HeLa cells was determined by ELISA assay. Results The structure of the plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 (petB)was confirmed by DNA sequencing. After induction, the fusion proteins were expressed mainly as inclusion bodies. The purity of the both proteins exceeded 90%. SDS-PAGE analysis suggests the relative molecular mass of both products expressed by petA and petB were approximately 38× 103, which is in accordance with the theoretical value. The results of Western blot and ELISA test demonstrated that BsAb1/17 molecule had binding ability to both IL-1β and IL-17A. The BsAb1/17 could block IL-1β to stimulate human T cell to express IL-18 and block IL-17A to stimulate HeLa cell to express IL-6. Conclusion We successfully constructed a novel bispecific antibody BsAb1/17 against both IL-1 β and IL-17A, and expressed biologically active BsAb1/17 protein in prokaryotic system.