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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 anti-transferrin receptor single-chain antibody and viral peptide / HLA-A2. Methods Recombinant DNA technology was used to ligate HLA-A2 gene and TfRscFv gene into the prokaryotic expression vector pET28a (+) with a flexible linker (Gly-4-Ser) 3 to transform The Escherichia coli BL21 (λDE3) strain was harvested to obtain the recombinant protein. The target protein was induced by IPTG. The purified target protein and β2 microglobulin (β2m) and HLA-A2-restricted hepatitis B virus (HBV) core antigen peptide HBcAg18-27 dilution in vitro renaturation to form HBcAg18-27 / HLA-A2 / TfRscFv fusion protein . The spatial conformation of fusion protein was detected by ELISA and microsphere binding assay. Results The constructed fusion protein gene had the correct sequence. The protein expressed in the form of inclusion body after induced by IPTG had the correct molecular weight. The fusion protein diluted in vitro had the correct conformation of HLA-A2. Conclusion The HBcAg18-27 / HLA-A2 / TfRscFv fusion protein was successfully constructed and was used to further induce the hepatitis B virus peptide-specific cytotoxic T lymphocytes to kill tumor cells by loading the fusion protein onto the surface of tumor cells through its single chain antibody basis.