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HCV NS3特异的 CD4+T细胞反应与 HCV感染的良性转归相关 .为了筛选其 CD4+T细胞表位 ,构建了 HCV NS3基因片段酵母展示文库 .首先 DNase 不完全酶切 HCV NS3基因产生长度为 1 0 0～ 30 0 bp的随机片段 ,然后在它们两端加上含限制性内切酶 Bam H 作用位点的接头 ,再以接头序列作引物进行 PCR扩增 .最后扩增产物用 Bam H 酶切后与 Bam H 线性化的穿梭载体 p YD1连接 ,转化大肠杆菌 (E.coli) DH5α,共得到 2× 1 0 6个转化子 .转化菌落的 PCR扩增结果表明 ,约 50 %转化子含插入片段 .随机选择 5个插入片段测序 ,然后与 DNA序列数据库中的序列比较 .结果显示它们分别与 HCV NS3序列有 96%～ 99%的同源性 .用从转化菌落中提取的质粒转化酵母 (S.cerevisiae)菌株 EBY1 0 0 ,得到含 2× 1 0 5个插入片段的 HCV NS3基因片段酵母展示文库 .半乳糖诱导的酵母细胞通过和 FITC标记的抗体结合 ,用 FACS可以在 2 0 %的细胞表面检测到融合蛋白的表达 .
The HCV NS3-specific CD4 + T cell response is associated with a benign prognosis of HCV infection. To screen for CD4 + T cell epitopes, a HCV NS3 gene fragment yeast display library was constructed.First DNase incompletely digested the HCV NS3 gene to a length of 1 0 0 ~ 30 0 bp random fragment, and then add a linker containing restriction endonuclease Bam H site at both ends, and then use the linker sequence as a primer to perform PCR amplification.Finally, the amplified product is treated with Bam H After digested with Bam H, the linearized shuttle vector pYD1 was ligated and transformed into E. coli DH5α to obtain 2 × 10 6 transformants.The PCR amplification results of transformed colonies showed that about 50% of transformants Containing 5 inserts were sequenced and then compared with those in the DNA sequence database.The results showed that they shared 96% -99% homology with the HCV NS3 sequence, respectively.The plasmids extracted from the transformed colonies Yeast (S. cerevisiae) strain EBY1 0 0, obtained with 2 × 105 inserts of HCV NS3 gene fragment yeast display library galactose-induced yeast cells by binding to FITC-labeled antibodies by FACS in 20 %of Detect cell surface expression of the fusion protein.