Résumé
Objective 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.
Résumé
Objective To construct the vector pET15b-Z-VP1 by inserting the Z fragment into amino-terminal of JCV VP1.Methods The VP1 and Z fragment were amplified by PCR from plasmid pET15b and pEZZ18 respectively,and then they were linked by recombinant PCR.The Z-VP1 fragment was inserted into plasmid pET15b by restriction enzyme BamHⅠ and NcoⅠ.Results The VP1 and Z fragment were obtained by PCR and gel purification.The Z-VP1 fragment,which was linked by recombinant PCR from VP1 and Z fragment,was inserted into plasmid pET15b between BamHⅠ and NcoⅠ sites,and confirmed by enzyme digestion and DNA sequencing.The expression of VP1-Z was confirmed by Western blotting.Conclusion The plasmid pET15b-Z-VP1 has been constructed successfully by inserting Z fragment into amino-terminal of VP1.