ISSN 1007-7626 CN 11-3870 / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology 2012 11 28 11 1026 ~ 1032 NIRF 1 1 1 1 2 * 1 2 400016 NIRF Np95 / ICBP-90 like RING finger protein NIRF HBc hepatitis B virus core protein HBV paav-hbv1 3 BALB / C NIRF ELISA HBsAg HBeAg Western HBcAg NIRF HBV NIRF R329 NIRF Can Significantly Inhibit the Expression of Hepatitis B Virus Antigens in vitro and in vivo HU Bin 1 ZHOU Dan-Lin 1 XUAN Yan-Yan 1 1 DUAN Chang-Zhu 2 * 1 Department of Cell Biology and Medical Genetics 2 Key Laboratory of Clinical Laboratory Diagnostics of Ministry of Education Chongqing Medical University Chongqing 400016 China Abstract NIRF Np95 / ICBP-90 like RING finger protein is a protein that has been suggested to function in carcinogenesis and epigenetic modifications from recent research Reports have shown that NIRF interacted with the hepatitis B virus core protein HBc however the effects of NIRF on the secretion of hepatitis B virus HBV antigens remained unclear Here we established both the cell culture and animal models of HBV infection by the transfection of paav-hbv1 3 plasmid into HepG2 cells or venous hydrodynamical injection of the plasmid in BALB / C mice to explore the connection between NIRF and HBV antigen expression in vitro and in vivo The secretion of HBsAg and HBeAg in the cultural supernatants of the transfected cells or the sera of the injected mice were analyzed by ELISA The HBcAg secretion was assayed by Western blot or immunohistochemical analysis The results demonstrated that the expression and secretion of HBV antigens were decreased following NIRF overexpression both in vitro and in vivo Our findings suggested that the inhibition of NIRF on HBV antigens might provide a better understanding of HBV infection for developing new therapeutic strategies against hepatitis B virus Key words hepatitis B virus NIRF Np95 / ICBP-90 like RING finger protein antigens 2012-08-16 2012-09-25 No 30872248 CSCT No 2008BB5400 No KJ080326 * Tel 023-68485804 E-mail duanchzhu@ cqmu edu cn Received August 16 2012 Accepted September 25 2012 Supported by National Natural Science Foundation of China No 30872248 Chongqing Science and Technology Commission Foundation CSTC No 2008BB5400 Chongqing Education Commission Foundation No KJ080326 * Corresponding author Tel 023-68485804 E-mail duanchzhu@ cqmu edu cn
11 NIRF 1027 3 5 5 min hepatitis B virus HBV 2 μg 250 μl DMEM 8 μl 1 2 Lipofectamine 2000 Invitrogen 100 3 5 min 20 11 6 6 h 24 h 45% 4 5 HBV 4 PBS 3 Np95 / ICBP-90 like RING finger protein NIRF ICBP90 Np95 2002 3p23-24 1 min 15 min 6 802 NIRF _ 1 3 Western N Tudor PHD SRA / YDG RING-finger 5 1 Rb 7-9 NIRF 0 2 6 100 5 min cyclin D1 E1 8 % 12 % 10 Cdk2-CyclinE2 PCNP PEST-containing nuclear protein 2 h FLAG 11 p53 HBcAg Millipore β-actin p53 4 12 13 NIRF TBST 8 min 3 IgG 1 h TBST 8 min 3 HBc A B 1 1 NIRF PVDF Quantity One NIRF HBc HBc 1 4 14 HBV 6 ~ 8 BALB / C 18 g ± 2 g 4 8 12 μg 0 2 ml PBS 8 s 24 h HBV 4 4 7 10 30 d 2 4 % 1 1 5 ELISA HBsAg HBeAg 1 1 paav-hbv1 3 HBsAg HBeAg BamH Ⅰ EcoRⅠ Promega 50 μl FLAG NIRF 50 μl 37 30 min 1 2 0 4 % 80 μl 30 min Ep 4 14 000 r / SDS-PAGE 5 % ELISA A B 50 μl 37 15 min 50 μl 450 nm HepG2 10 % A DMEM HyClone 1 6 6 1 h 2 3 % H 2 O 2 10 min ml 250 μl DMEM 4 μg PBS 5 min 3
1028 28 37 30 min 24 h ELISA HBsAg HBsAg HBcAg HBeAg 3 d 4 PBS A pflag-nirf paav- HBV1 3 A Fig 37 30 min PBS 5 min 3 DAB 70 % 80 % 95 % 100 % 3 min 3 min 1 7 x 珋 ± SD SPSS12 0 t P < 0 05 P < 0 01 2 2 1 paav-hbv1 3 Fig 1 BamH Ⅰ 7 007 1 504 912 766 kb 4 EcoR Ⅰ 10 187 kb 2A HepG2 NIRF Fig 1 Identify of recombinant plasmid paav- HBV1 3 M Marker DL10000 TaKaRa Japan 1 paav-hbv1 3 digested by BamH Ⅰ 2 paav-hbv1 3 digested by EcoR Ⅰ Fig 2 Inhibitory effect of NIRF on HBV antigens in vitro A HepG2 cells were co-transfected with paav- HBV1 3 pflag-nirf or pflag and the pflag expression plasmid was used as a control The cultural supernatants were collected every day Levels of HBsAg and HBeAg of the cells were determined by ELISA All data represent x 珋 ± SD n = 3 The statistical analysis was performed by t-test * P < 0 05 B HepG2 cells were 2 2 NIRF HBV transfected or co-transfected with the specific plasmid as the NIRF HBV graph showed The cells were harvested at day 3 Western paav-hbv1 3 pflag-nirf pflag blotting was performed with FLAG or HBcAg antibodies HepG2 while β-actin was used as the loading control
11 NIRF 1029 HBsAg HBeAg 3 d HBV 6 ~ 8 Western HBcAg BALB / C paav- HBcAg HBV1 3 4 d ELISA Fig 2B NIRF HBsAg HBeAg Fig 3A HBsAg HBeAg 500 7 HBcAg d HBsAg HBeAg ELISA 2 3 HBV 30 d NIRF HBV Fig 3B Fig 3 Establishment of HBV transfected immunosuppressed mouse A The plasmid paav-hbv1 3 was injected hydrodynamically into the tails vein of BALB / C mouse in 8 s The mouse sera was collected everyday The level of HBsAg and HBeAg were measured using ELISA and were positive at the 4 th day after hydrodynamically injection All data represent mean ± SD n = 3 The statistical analysis was performed by t-test ** P < 0 01 B Pathology detection for mouse livers tissue 200 1 30 days of control group 2 30 days after transfected with HBV plasmid 2 4 NIRF HBV Fig 4B HBcAg HBV Fig 4C pflag-nirf NIRF HBV HBsAg HBcAg 7 d 4 d ELISA HBsAg HBcAg HBsAg HBeAg pflag-nirf NIRF HBcAg HBsAg HBeAg Fig 4A HBsAg NIRF 5 d HBsAg HBeAg ELISA HBV NIRF 3 HBsAg HBeAg HBV NIRF 4 d paav-hbv1 3 NIRF HBc HBsAg HBcAg HBsAg 15 NIRF
1030 28 Fig 4 Inhibitory effect of NIRF on HBV antigens in vivo A The plasmid paav-hbv1 3 and pflag-nirf were co-injected hydrodynamically into the tails vein of BALB / C mouse in 8 s The sera were collected at indicated time and levels of HBsAg and HBeAg were measured using ELISA The pflag expressing plasmid was used as a control All data represent 珋 x ± SD n = 3 The statistical analysis was performed by t-test * P < 0 05 B Immunohistochemistry for HBsAg of mouse livers after HBV and NIRF plasmid injected DAB staining 400 1 4 days of control group 2 HBsAg expression on 4 days after plasmid paav-hbv1 3 injection 3 7 days of control group 4 Significantly reduce of HBsAg on 7 days after NIRF had co-injected with paav-hbv1 3 plasmid C Immunohistochemistry for HBcAg of mouse livers after HBV and NIRF plasmid injected DAB staining 400 1 4 days of control group 2 HBcAg expression on 4 days after plasmid paav-hbv1 3 injection 3 7 days of control group 4 Significantly reduce of HBcAg on 7 days after NIRF had co-injected with paav-hbv1 3 plasmid
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