【摘要】 本文用含有HLA-A*2402重链胞外段基因的质粒为模板进行PCR,利用下游引物拼接上依赖BirA的可生物化序列后,构建融合基因sHLA-A*2402-BSP,与质粒pET-21d重组后,在宿主菌E.coliBL21(DE3)中诱导表达。表达的融合蛋白与β2m及HLA-A*2402限制性抗原肽EB病毒BRLF1蛋白中的九肽NH2-TYPVLEEMF-COOH进行复性折叠,形成HLA-A*2402-抗原肽复合物单体,并用Westernblot和夹心ELISA法鉴定。证实成功地制备出sHLA-A*2402-生物素化序列融合蛋白并使之正确折叠复性。为研究在原核系统中表达、纯化与复性及体外构建MHCI类分子四聚体,探讨免疫识别机制奠定了基础。更多还原

【Abstract】 With plasmid containing the extracellular gene fragment of the heavy chain of HLA-A*2402 as template and to add a 15 amino acid substrate peptide for BirA dependent biotinylation to the COOH-terminus of the human HLA heavy chain, the fusion gene of sHLA-A*2402-BSP was constructed by PCR, cloned into pET-21d and then expressed through induction in E.coli BL21(DE3) cells. The fusion protein expressed was purified and refolded in vitro by limiting dilution with β2m and HLA-A*2402 binding peptide (NH2-TYPVLEEMF-COOH of EB virus BRLF1) to produce the sHLA-A*2402-peptide complex monomer. This product was detected by Western blot and ELISA. The experimental results showed that the fusion protein of sHLA-A*2402-BSP was successfully constructed and vefolded, and the highly efficient expression of this fusion protein lays the foundation for further studies on the expression and identification in prokaryotic expression system and the construction of tetrameric peptide-HLA complexes to explore the mechanism of immune recognition.更多还原