鼠疫耶尔森菌F1-V融合蛋白改构体的构建、原核表达及纯化

Reconstruction,Prokaryotic Expression,Purification of Recombinant F1-V Mutant from Yersinia pestis

目的：通过基于结构的基因突变获得鼠疫耶尔森菌F1抗原突变体（F1mut）,克隆、表达并纯化F1mut-V融合蛋白。方法：通过3轮PCR,将编码F1抗原分子N端1~14位氨基酸的基因序列移到3＇端,测序无误后将F1mut基因与V基因的5＇端连接,构建改构的融合基因F1mut-V,将其克隆到原核表达载体p ET-32a后转化大肠杆菌BL21（DE3）,经IPTG诱导后,目的蛋白为可溶性表达,通过硫酸铵分级沉淀、阴离子交换层析、疏水相互作用层析和凝胶过滤层析纯化,用SDS-PAGE和Western印迹分析纯化产物。结果：重组F1mut-V在大肠杆菌中为可溶性表达,表达量占全菌蛋白的25%以上,纯化后目的蛋白的纯度达95%,经Western印迹检测,与抗V、F1抗体均有特异性结合。结论：重组F1mut-V有望成为新一代亚单位疫苗的有效成分。

Objective：To clone,express and purify F1mut-V fusion protein from Yersinia pestis by structure-based immunogen design.Methods：In order to transplant the NH2-terminal first fourteen amino acid residues ofF1 to the COOH-terminus,the F1 mut gene was amplified by three-step PCR and fused to the NH2-terminal of Vantigen.Then the F1mut-V gene was cloned into prokaryotic expression vector p ET-32 a and transformed into E.coli BL21（DE3）.The soluble r F1mut-V was purified with ammonium sulfate precipitation,ion exchange chromatogra-phy,hydrophobic chromatography and gel filter chromatography and characterrized by SDS-PAGE and Western blot-ting.Results：The recombinant F1mut-V expressed in soluble form in E.coli and the target protein expressed ac-counts for 25% of the total protein of the bacteria.After sequential four purification steps,the purity of r F1mut-Vfusion protein achieved 95% and could specifically react with the antibody against F1 and V by Western blotting.Conclusion：The r F1mut-V would be the alternative active pharmaceutical ingredient of the next plague vaccine.