Expression Of The Human Papillomavirus Type 16 E6 Protein In Eukaryocyte And Its Effects On Cytokines Secretion And Apoptosis Of Macrophages

Objective: To provide the experimental basis for studying further effects on oncogenic mechanism of Human papillomavirus type 16 (HPV16) E6 protein, the eukaryotic expression vector of which was constructed and expressed in THP-1 macrophages, and to study the effects of E6 protein on lipopolysaccharide (LPS) and Dexamethasone-induced change of THP-1 macrophages secretion activities and apoptosis.Methods: Polymerase chain reaction(PCR) was used to amplify the HPV16E6-encoding gene and isolated 477bp E6 gene. The fragment of E6 gene was directly ligated into pUCm-T vector, after identified by PCR , restriction enzyme analysis and sequenced, the fragment of E6 was subcloned into eukaryotic expression vector pcDNA3.1 (-). The recombined plasmid of pcDNA3.1 (-) /E6 was transfected into THP-1 macrophages. The expression of E6 protein was analyzed by SDS-PAGE and Western-blotting. TNF-α and IL—1 β ELISA kits were respectively used to determine the effect of E6 protein transient expression on cytokines secretion induced by lipopolysaccharide of THP-1 macrophages. Microscopic count technology by Giemsa stain was used to determine the effect of E6 protein transient expression on Dexamethasone-induced apoptosis of THP-1 macrophages.Results: (1) The recombinant pcDNA3.1 (-)/E6 was digested by BamH I and HindIII, a approximate 500bp DNA fragment could be seen in agrose electrophoresis, it indicated that E6 fragment was already subcloned into pcDNA3.1(-) plasmid, namely the eukaryotic expression vector pcDNA3.1 (-)/E6 has been constructed...