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Objective: To construct the human papillomavirus type 18 (HPV 18) the E2 protein N-terminal domain (TAD), and the C-terminal domain (DBD) and green fluorescent protein (EGFP) fusion protein eukaryotic expression vector pEGFP-C1/TAD and the pEGFP the -C1/DBD, with pEGFP-C1/E2 and pEGFP-C1 were transfected THP-1 macrophages (macrophage of MΦ) analysis groups medium supernatant TNF-α and IL-1β content The MΦ apoptosis rate provided the experimental basis for further study of the E2 protein in HPV 18 carcinogenic mechanism. Methods: (1) with Primer5.0 primer design software design primer polymerase chain reaction (PCR) amplification of the the E2 protein N-terminal and C-terminal gene. PCR products were purified with the pEGFP-C1 vector connection transform E.coli JM109 and plasmids were extracted, and positive clones were screened by restriction enzyme digestion and sequencing. (2) The plasmid pEGFP-C1/TAD, pEGFP-C1/DBD pEGFP-C1/E2, pEGFP-C1 were 4 groups, i.e. a GFP-TAD group, GFP-DBD group, GFP-E2 group, GFP group; plasmid 0.5μg/ml of each transfected MΦ, and a blank control group (Control). 48 h after transfection, Western-blot and fluorescence microscopy to detect their expression and positioning. (3) ELISA to detect the expression medium supernatant of TNF-α and IL-1β content, RT-PCR was used to detect mRNA expression; collection MΦ, staining and flow cytometry (FCM) observed detection MΦ apoptosis. . The statistical analysis of experimental data by the statistical software SPSS13.0. Results: (1) of the PCR product was connected to the pEGFP-C1 vector after digestion and sequencing results show that the target fragment with HPV18 E2 gene TAD DBD sequence is exactly the same. Eukaryotic expression vector pEGFP-C1/TAD, pEGFP-C1/DBD well pEGFP-C1/E2, pEGFP-C1 were transfected THP-1 MΦ48 h, GFP-DBD fusion protein localized in the nucleus, GFP- TAD located in the cytoplasm, GFP-E2, GFP expression in the nucleus, cytoplasm, but the expression of GFP-E2 group nucleus fluorescence intensity stronger than the cytoplasmic GFP in the nucleus pulp evenly expression. Western-blot results showed that the expression in THP-1 MΦ molecular weight of approximately 29.4 kD, 53.4 kD, 70.7 kD, 38.6 kD bands with the expected GFP, GFP-TAD, GFP-E2, GFP-DBD same size. (2) transfection of THP-1 MΦ48 h, GFP-E2, GFP-TAD group culture supernatant of TNF-α, IL-1β was significantly higher than the Control group (P lt; 0.001), and between the two groups TNF-α concentrations there is a statistically significant difference (P lt; 0.05); GFP-TAD group IL-1β level slightly higher than the GFP-E2 group, but no significant difference in sex (P gt; 0.05); while GFP-DBD, GFP group TNF-α and IL-1β content in comparison with the control group, no significant difference (P gt; 0.05). The expression levels of TNF-α and IL-1βmRNA consistent with the content of the supernatant of TNF-α and IL-1β. (3) GFP-E2, GFP-TAD group the MΦ apoptosis rate was significantly higher than the Control group (P lt; 0.001), and GFP-TAD group MΦ rate of apoptosis, GFP-E2 group (P lt; 0.05); but GFP-DBD GFP group and Control group comparison, the MΦ apoptosis was no significant difference (P gt; 0.05). Conclusion: (1) successfully amplified the HPV18 E2TAD and DBD gene, constructed the eukaryotic expression the carrier pEGFP-C1/TAD pEGFP-C1/DBD with plasmid pEGFP-C1/E2 and pEGFP-C1 could in THP-1 MΦ expression, GFP-DBD fusion protein localized in the nucleus, GFP-TAD is located in the cytoplasm. (2) HPV18 E2 TAD and GFP fusion protein transiently high expression of the up-regulation of THP-1 MΦ secretion of TNF-α and IL-1β. (3) HPV18 E2 TAD and GFP fusion protein instantaneous high expression induced THP-1 MΦ apoptosis.
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