摘要
目的优化诱导条件大批量表达生物素化酶BirA底物肽(BSP)与HLA-A0203重链胞外域的融合蛋白(HLA-A0203-BSP),并制备负载HLA-A0203限制性EB病毒抗原肽EBNA3596-604的四聚体(HLA-A0203/SVR)。方法以HLA-A0203-BSP原核表达载体转化E.coliBL21(DE3)菌株,优化诱导条件进行大批量重组蛋白的表达。通过稀释法复性可溶性HLA-A0203/SVR单体,然后以BirA对其进行生物素化,并以阴离子交换树脂纯化。将纯化的HLA-A0203/SVR单体与荧光素标记的链亲和素按4∶1的比例混合形成四聚体,通过对特异性CTL进行染色验证其结合活性。结果当IPTG的浓度为0.4mmol/L,于37℃诱导过夜后,融合蛋白的表达最多。该重组蛋白相对分子质量(Mr)为34000,与HLA-A0203-BSP的理论Mr相一致。该重组蛋白以包涵体形式存在于沉淀部分,约占菌体总蛋白的30%。负载抗原肽的可溶性HLA-A0203/SVR单体是在同时存在HLA-A0203-BSP、β2微球蛋白及HLA-A0203限制性抗原肽SVR的情况下通过稀释法复性而获得。该单体生物素化并纯化后与荧光素标记的链亲和素按4∶1的比例混合后即形成四聚体。流式细胞术(FCM)分析证实,该四聚体具有与HLA-A2+供者特异性CTL结合的活性。结论HLA-A0203-BSP融合蛋白在优化条件下获得高效表达。以此蛋白制备的HLA-A0203/SVR四聚体具有与HLA-A2+供者特异性CTL结合的活性,为研究HLA-A0203个体EB病毒特异性CTL的免疫应答打下了基础。
AIM: To optimize expression condition of HLA-A, 0203 heavy chain ectodomain fused with a BirA substrate peptide (BSP) ( HLA-A * 0203-BSP) for E. coil BL21 (DE3) transformant and to prepare a functional HLA-A * 0203 tetramer loaded with an antigenic peptide derived from EBNA3 596-604 of Epstein-Barr virus (EBV). METHODS. The temperature, IPTG concentration and inductive duration of HLA-A * 0203-BSP fusion protein expressed for E. coil BL21 (DE3) transformant were optimized. SDS-PAGE and Western blot analyses were employed to detect the expressed fusion protein. The monomer of soluble HLA-A * 0203-petide was generated from the fusion protein by in vitro refolding of washed inclusion bodies in the presence of 152-microglobulin (152m) and HLA-A, 0203 restricted EBV EBNA3 596-604 peptide (SVRDRLARL, SVR). Refolded and purified monomer was then biotinylated with BirA. Following the purification of the obtained biotinylated monomer, the tetramer was formed by incubation with streptavidin-PE at a ratio of 4: 1. Flow cytometry (FCM) analysis was performed to determine its binding activity with specific cytotoxic T lymphocytes (CTL). RESULTS= SDS-PAGE and Western blot showed that the optimized expression condition was overnight induction at 37℃ with 0.4 mmol/L IPTG. The expressed protein of about 34 kDa in the form of inclusion bodies accumulated up to about 30% of total bacterial protein under the optimized expression condition. The monomer of soluble HLA-A * 0203/SVR was successfully generated and purified. Non-reducing SDS-PAGE analysis showed that the biotinylation was above 85%. HLA-A * 0203/SVR tetramer was constructed by mixing the monomer with streptavidin-PE at a ratio of 4: 1. FCM analysis indicated that this tetramer could bind specific CTL from HLA-A2* donors. CONCLUSION: HLA-A * 0203-BSP fusion protein was overexpressed in E. coil under the optimized condition. The tetramers of HLA-A * 0203/SVR were prepared from this fusion protein and it possessed binding activity with specific CTL, which provided a powerful tool for direct visualization and quantification of specific CTL from HLA-A * 0203 donors.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2007年第2期97-101,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金重点项目资助(30230350)
国家自然科学基金面上项目资助(30371651
30572199)