利用重叠延伸PCR和同源重组克隆技术制备HLA-A<sup>*</sup>0201/HBc<sub>18-27</sub>单链三分子复合体-东南大学学报医学版

利用重叠延伸PCR和同源重组克隆技术制备HLA-A^*0201/HBc_18-27单链三分子复合体

单位：东南大学医学院病原生物学与免疫学系, 江苏南京 210009

分类号：Q784

出版年·卷·期（页码）：2016·35·第六期（825-831）

摘要：

目的：构建和表达HLA-A^*0201/HBc_18-27单链三分子复合体。方法：利用重叠延伸PCR将HBc_18-27、（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/HBc_18-27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/HBc_18-27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/HBc_18-27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/HBc_18-27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pMHC Ⅰ类分子的制备过程。

Objective: To construct and express the single-chain trimer(SCT) gene of HLA-A^*0201/HBc_18-27 complex. Methods: The SCT gene of HBc_18-27 peptide-(G₄S)₃-β2m-(G₄S)₄-HLA-A^*0201(α1,α2,α3) was constructed and inserted into plasmid pET28a with overlap extension PCR and homologous recombination techniques.The SCT protein of HLA-A^*0201/HBc_18-27 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 were refolded 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.PE-labeled anti-Human HLA-ABC mAb(W6/32) was used as a probe to monitor the structural conformations of HLA-A^*0201/HBc_18-27 molecule by flow cytometric analyses. Results: The recombinant plasmid of pET28a-HLA-A^*0201/HBc_18-27 was confirmed by nucleotide sequencing and SDS-PAGE analysis.The fractions containing HLA-A^*0201/HBc_18-27 protein was eluted using elution buffer(250 mmol·L^-1 imidazole) with a purity of about 93% as estimated by densitometry analysis of SDS-PAGE.The strong binding of mAb W6/32 with HLA-A^*0201/HBc_18-27 SCT-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.

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