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目的 构建含HGVNS5A区基因的原核表达载体。方法 采用PCR方法从重组了HGV 6 6 72 - 72 92区段基因的pUC19中扩增出HGV 6 86 4- 72 77nt片段 ,定向克隆入pGEMEX1质粒 ,再经SacⅠ、HindⅢ酶切亚克隆入高效表达载体pRSETA。结果 重组pRSETA 经序列测定证实插入基因为目的基因 ,符合表达框架 ,经IPTG诱导 ,在BL2 1(DE3)菌株中表达连接 6个组氨酸的HGVNS5A融合蛋白。Westernblotting显示在Marker约 2 5kDa处可见明显的蛋白带 ,与预期结果一致。结论 成功构建了重组HGVNS5A区基因表达载体 ,在BL2 1(DE3)中能有效表达。
Objective To construct a prokaryotic expression vector containing HGVNS5A gene. Methods HGV 6 86 4-72 77nt fragment was amplified from pUC19 of HGV 6 722-72 92 gene by PCR and cloned into pGEMEX1 plasmid. The recombinant plasmid was subcloned into pGEMEX1 by SacⅠ and Hind Ⅲ, Vector pRSETA. Results Recombinant pRSETA was confirmed by sequence analysis. The inserted gene was confirmed by sequence analysis. The recombinant plasmid pRSETA was expressed in BL2 1 (DE3) strain. The recombinant fusion protein HGVNS5A was expressed in BL2 1 (DE3) strain. Western blotting showed that a significant protein band was observed at about 25 kDa of Marker, consistent with the expected results. Conclusion The recombinant HGVNS5A gene expression vector was successfully constructed and expressed efficiently in BL2 1 (DE3).