HPV+18+E2蛋白及其删除突变体对MΦ分泌与凋亡的影响.pdfVIP

Affected of HPV18 E2 protein or delete mutant on apoptosis and cytokines secretion of MΦ ABSTRACT Objective: To study the effect of over-expression of GFP-E2, GFP-TAD (N-extremity domain of HPV18 E2) and GFP-DBD (C-extremity domain of HPV18 E2) fusion proteins on the apoptosis and cytokines secretion of MΦ, and to furtherly explore the contribution of E2 gene to the uterine cervix cancer. Methods: (1) Primers of TAD or DBD were designed by Primer5.0 software. Then TAD or DBD gene was amplified by PCR from pEGFP-C1/HPV18 E2 respectively, and cloned into pEGFP-C1 vector. After transformed into E.coli JM109, the positive clones were screened by endonuclease restriction digestions and sequenced . (2) Plasids of pEGFP-C1/TAD, pEGFP-C1/DBD, pEGFP-C1/E2 and pEGFP-C1 were taken as four griups and named as GFP-TAD, GFP-DBD, GFP-E2 and GFP respectively. Every group plasid was transfected into MΦ with 0.5 μg/ml respectively and control group was set at the same time. At 48 hours after transfection, the expression and localization of GFP or GFP-HPV18 E2 fusion protein and its mutants were observed under fluorescent microscope. (3) The cytokine contents of TNF-α or IL-1β in the supernatant of culture medium were tested quantitatively with ELISA kit and the mRNA expression of them was detected by RT-PCR respectively. The apoptosis rates of MΦ were tested by flow cytometry or stained. All results were analyzed by SPSS 13.0. Results: (1) The result of agarose electrophoresis DNA sequencing indicated that the sequence of the recombinant pEGFP-C1/TAD and pEGFP-C1/DBD were correct. Forty-eight hours after transfection of recombinant plasmids or pEGFP-C1 into MΦ, Western blot showed that the lanes which relative molecular weight about 29.4 kDa, 53.4 kDa, 70.7 kDa or 38