利用重叠延伸PCR和同源重组克隆技术制备HLA-A＊ 0201/HBc18-27单链三分子复合体

Preparation of HLA-A＊ 0201/HBc18-27 single-chain trimer with overlap extension PCR and homologous recombination techniques

目的：构建和表达HLA-A＊0201/HBc18-27单链三分子复合体。方法：利用重叠延伸PCR将HBc18-27、（Gly4Ser）3、β2m、（Gly4Ser）4和HLA-A＊0201胞外区依次串联为单链三聚体（single-chain trimer,SCT）融合基因,通过同源重组克隆技术插入到pET28a原核表达载体,用异丙基β-D-硫代吡喃半乳糖苷（IPTG）在大肠杆菌BL21（DE3）中诱导表达,采用金属螯合亲和层析法纯化目的蛋白,通过稀释复性法折叠成HLA-A＊0201/HBc18-27的复合单体,并生物素化,将单体包被到直径4.5μm的磁性微球,用构象特异性单抗（W6/32）进行荧光染色,流式细胞术分析其空间构象。结果：pET28a-HLA-A＊0201/HBc18-27基因序列和蛋白大小与预测一致;纯化后融合蛋白纯度约93%;流式细胞术分析显示pET28a-HLA-A＊0201/HBc18-27单体具有正确的空间构象。结论：利用重叠延伸PCR和同源重组克隆技术成功制备了HLA-A＊0201/HBc18-27的单链复合体,无需限制性内切酶和连接酶,发展了主要组织相容性单链复合体技术,有效简化了pMHCⅠ类分子的制备过程。

Objective： To construct and express the single-chain trimer（ SCT） gene of HLA-A＊0201 / HBc18-27 complex. Methods： The SCT gene of HBc18-27peptide-（ G4S）3-β2m-（ G4S）4-HLA-A＊0201（ α1,α2,α3） was constructed and inserted into plasmid p ET28 a with overlap extension PCR and homologous recombination techniques. The SCT protein of HLA-A＊0201 / HBc18-27 was expressed by the addition of IPTG in Escherichia coli BL21（ DE3）,then purified by passing through a Ni＋-chelating affinity column. Soluble inclusion bodies wererefolded by dilution refolding method using the redox-shuffling refolding buffers. The refolded SCT protein was biotinylated,and then coupled onto the cell-sized magnetic beads（ 4. 5 μm） which pre-coated with streptavidin. PElabeled anti-Human HLA-ABC mA b（ W6 /32） was used as a probe to monitor the structural conformations of HLAA＊0201 / HBc18-27 molecule by flow cytometric analyses. Results： The recombinant plasmid of p ET28a-HLA-A＊0201 / HBc18-27 was confirmed by nucleotide sequencing and SDS-PAGE analysis. The fractions containing HLA-A＊0201 / HBc18-27 protein was eluted using elution buffer（ 250 mmol·L-1imidazole） with a purity of about 93% as estimated by densitometry analysis of SDS-PAGE. The strong binding of mA b W6 /32 with HLA-A＊0201 / HBc18-27SCT-coated magnetic beads implied the correct structural conformation of the SCT molecules. Conclusion： The overlap extension PCR and homologous recombination technique simplifies the construction of single-chain MHC class I molecules bypass the requirement of restriction sites,and will facilitate the preparation of soluble MHC class I / peptide complexes.