病毒肽/HLA-A2复合物与抗转铁蛋白受体单链抗体融合蛋白的构建、表达及鉴定

Construction,Expression and Identification of the Fusion Protein of Peptide/HLA-A2 Complex and Transferrin Receptor Single Chain Antibody

目的制备抗转铁蛋白受体单链抗体与病毒肽/HLA-A2的融合蛋白。方法利用重组DNA技术将HLA-A2基因和转铁蛋白受体单链抗体(TfRscFv)基因,用柔性的linker(Gly-4-Ser)3连接并克隆到原核表达载体pET28a(+)上,转化大肠埃希菌BL21(λDE3)菌株,获取重组子,经IPTG诱导表达目的蛋白。将纯化的目的蛋白和β2微球蛋白(β2m)与HLA-A2限制性的乙肝病毒(HBV)核心抗原肽HBcAg18-27在体外进行稀释复性,形成HBcAg18-27/HLA-A2/TfRscFv融合蛋白。利用ELISA和微球结合试验检测融合蛋白的空间构象。结果构建的融合蛋白基因具有正确的序列,经IPTG诱导后以包涵体形式表达的蛋白分子量正确,体外稀释复性后的融合蛋白具有正确的HLA-A2空间构象。结论成功构建了HBcAg18-27/HLA-A2/TfRscFv融合蛋白,为进一步将该融合蛋白通过其单链抗体加载到肿瘤细胞表面从而诱导乙肝病毒肽特异性细胞毒性T淋巴细胞杀伤肿瘤细胞奠定了物质基础。

Objective To prepare a fusion protein of single chain antibody of transferrin receptor with peptide/HLA-A2 complex.Methods A hybrid gene encoding HLA-A2/TfRscFv fusion molecule was constructed by ligation of sequences coding for the extracellular part of HLA-A2 heavy chain with that for the TfRscFv,and the hybrid gene was recombined into pET28a（＋）to generate pET28a（＋）＋（HLA-A2/TfRscFv）recombinant plasmid.Upon induction with isopropyl β-D-thiogalactoside（IPTG）,the fusion molecule was expressed in E.coli BL21 cells,and large amounts of recombinant protein accumulated in intracellular inclusion bodies.Then fusion protein（HBcAg18-27/HLA-A2/TfRscFv）was produced by in vitro refolding in the presence of HLA-A2-restricted HBV-derived peptide HBcAg18-27 and human β2-microglobulin.The conformation of fusion protein was detected by enzyme-linked immunosorbent assay（ELISA）and binding of polystyrollatex beads.Results The sequence of the fusion gene was correct,and large amounts of recombinant protein accumulated in intracellular inclusion bodies induced by IPTG.The fusion protein had an intact conformation after refolding in vitro.Conclusion We have successfully constructed the fusion protein of single chain antibody of transferrin receptor with peptide/HLA-A2 complex.This study laid the foundation for the fusion protein attaching the active peptide/HLA-A2 complex to TfR-expressing tumor cells through binding of TfRscFv to TfR,and inducing tumor cell lysis by viral antigen-specific CTLs.