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[目的]选取狂犬病毒糖蛋白富集表位基因片段进行克隆、表达和纯化,以期获得能应用于血清学诊断的纯化重组蛋白。[方法]从狂犬病毒感染的Vero细胞上清液中提取病毒RNA,通过RT-PCR扩增CTN株糖蛋白表位富集区基因片段,与表达载体pPROEX HTb连接,构建重组表达载体,在BL21(DE3)中表达,重组蛋白经Western blot鉴定活性,最后用电洗脱法纯化重组蛋白。[结果]重组表达质粒在大肠杆菌中表达高产量的重组蛋白,Western blot结果显示,重组蛋白具有免疫活性,用电洗脱法纯化后的重组蛋白具有较高的纯度,测定蛋白含量为1.07 mg/ml。[结论]狂犬病毒糖蛋白富集表位基因片段能在大肠杆菌中成功表达,重组蛋白具有免疫活性,并可通过电洗脱法纯化,为狂犬病疫苗免疫血清抗体检测试剂的研制奠定了基础。
[Objective] The research aimed to clone, express and purify the epitope fragment of glycoprotein of rabies virus in order to obtain the purified recombinant protein which can be used in serodiagnosis. [Method] The viral RNA was extracted from rabies virus-infected Vero cell supernatant and amplified by RT-PCR. The amplified fragment was cloned into expression vector pPROEX HTb to construct recombinant expression vector. (DE3), and the recombinant protein was identified by Western blot. Finally, the recombinant protein was purified by electroelution. [Result] Recombinant expression plasmid expressed high yield of recombinant protein in E. coli. The result of Western blot showed that the recombinant protein was immunocompetent. The purity of the recombinant protein purified by electroelution was higher than that of the recombinant protein. The protein content was 1.07 mg / ml. [Conclusion] The rabies virus glycoprotein enriched epitope gene fragment can be successfully expressed in E. coli. The recombinant protein has immunogenicity and can be purified by electroelution method, which lays a foundation for the development of rabies vaccine immune serum antibody test reagent.