摘要目的 利用先期重组的原核表达质粒pET15b-VP1-Z体外表达重组的蛋白VP1-Z;研究VP1-Z是否组装成VLP-Z,以及VLP-z是否具有与野生型VLP相同的生物学活性.方法 重组质粒pET15b-VP1-Z转染大肠杆菌BL21(DB),IPTG诱导重组蛋白VP1-Z表达;按照野生型VLP的制备方法纯化蛋白;Western blot和考马斯亮蓝染色确定纯化蛋白及其纯度;电镜观察VLP-Z是否形成病毒样颗粒;血凝抑制试验检测VLP-Z是否具有血凝抑制活性;细胞免疫荧光检测VLP-Z能否转运进入细胞.结果 超速离心法得到纯度及浓度较高的重组蛋白VLP-Z,其相对分子质量约50×10~3;电镜观察发现VLP-Z组装成病毒样颗粒,直径约45～50 nm,较野生型VLP直径稍大;血凝抑制试验证实VLP-Z可以抑制人"O"型红细胞聚集;细胞免疫荧光显示,感染后VLP-Z可以转运进入HeLa细胞质及细胞核,与野生型VLP感染后相同.结论 成功制备了VLP-Z,且VLP-Z具有与野生型VLP相同的生物学活性.

abstractsObjective To express the recombinant protein VP1-Z, and investigate whether VLP-Z has the physiological functions like as wild-type VLP. Methods The expression plasmid pET15b-VP1-Z was introduced into competent E. coil BL21 (DF3)/pLys cells by transformation, and the expression of re-combinant protein VP1-Z was induced by incubation of the cells with IPTG. The protein was prepared as pre-viously described for wild-type VLP. The morphous of VLP-Z were observed by electron microscopy, and the physiological functions of VLP-Z were investigated by hemagglutination test and by immunofluorescence. Re-sults The purified VLP-Z composed of VP1-Z possessed hemagglutination activity and yielded a prominent band of 50×10~3 on SDS-PAGE and staining with Coomassie Brilliant Blue. The VLP-Z exhibited virus-like particles structure like as wild-type VLP with a diameter of 45-50 nm, which was slightly bigger than that of wild-type VLP(42-45 nm). In immunofluorescence test, VP1-Z was detected within the cytoplasm and nu-cleus after HeLa cells were inoculated with VLP-Z. Conclusion The physiological functions of recombinat-ed protein VLP-Z were comparable with wild-type VLP.