AIM: To investigate the biological impact of hepatitis B virus X- hepatitis C virus core (HBV X-HCV C) fusion gene on hepatoma cells. METHODS: The recombinant adenoviruses Ad- XC, Ad-X and Ad-C expressing HBV X-HCV C ...AIM: To investigate the biological impact of hepatitis B virus X- hepatitis C virus core (HBV X-HCV C) fusion gene on hepatoma cells. METHODS: The recombinant adenoviruses Ad- XC, Ad-X and Ad-C expressing HBV X-HCV C fusion gene, HBV X gene and HCV C gene were constructed, respectively. Hepatoma cells were infected with different recombinant adenoviruses. MTT, colonyforming experiment, FCM, TUNEL assay were performed to observe the biological impact of the HBV X-HCV C fusion gene on liver cells. RESULTS: MTT showed that the Ad-XC group cells grew faster than the other group cells. Colony-forming experiment showed that the colony-forming rate for the Ad-XC group cells was significantly higher than that for the other group cells. FCM analysis showed that Ad-XC/Ad-X/Ad-C infection enhanced the progression of G1→S phase in the HepG2 cell cycle. The apoptosis index of the Ad-XC, Ad-X, Ad-C group cells was significantly lower than that of the Ad0 and control group cells. Semi-quantitative RT-PCR showed that the expression level of c-myc was the highest in Ad- XC infected cells. Tumor formation was found at the injected site of mice inoculated with Ad-XC-infected LO2 cells, but not in control mice. CONCLUSION: Ad-XC, Ad-X and Ad-C facilitate the proliferation activity of HepG2 cells and inhibit their apoptosis in vitro. The effect of Ad-XC is significantly stronger than that of Ad-X and Ad-C. Up-regulation of c-myc may be one of the mechanisms underlying the synergism of HBV X and HCV C genes on hepatocarcinogenesis in athymic nude mice.展开更多
AIM: To investigate the immunogenicity of a novel DNA vaccine,pSW3891/HBc, based on HBV core gene in Balb/c mice.METHODS: A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid...AIM: To investigate the immunogenicity of a novel DNA vaccine,pSW3891/HBc, based on HBV core gene in Balb/c mice.METHODS: A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes (CTLs) of mice was quantitatively measured by lactate dehydrogenase release assay.RESULTS: HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1:97 200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector:target ratio of 20:1 in mice that received pSW3891/HBc DNA vaccine.CONCLUSION: pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice.展开更多
Plant germplasm underpins much of crop development. Millions of germplasm accessions have been collected and conserved ex situ, and the major challenge is now how to exploit and utilize this abundant resource.
AIM:To examine whether 2'-5'oligoadenylate synthetase (OAS) gene promoter can be specifically activated by hepatitis C virus (HCV)-core protein.METHODS: Human embryo hepatic cell line L02 wa transfected with p...AIM:To examine whether 2'-5'oligoadenylate synthetase (OAS) gene promoter can be specifically activated by hepatitis C virus (HCV)-core protein.METHODS: Human embryo hepatic cell line L02 wa transfected with pcDNA3.1-core plasmid and selected by G418. Expression of HCV-core was detected by reverse transcription polymerase chain reaction and Western blotting. The OAS promoter sequence wa amplified from the genomic DNA and inserted into pGL3-basic vector. The resultant pGL3-OAS-Luc plasmid was transiently transfected into L02/core cell and luciferase activity was assayed. RESULTS: L02/core cell line stably expressing HCV core protein was established. The pGL3-OAS-Luc construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells.CONCLUSION: HCV-core protein activates the OAS gene promoter specifically and effectively. Utilization of OAS gene promoter would be an ideal strategy for developing HCV-specific gene therapy.展开更多
A hepatitis C virus(HCV)cDNA fragment,534bp in length and designated asQ534,was obtained by PCR amplification with self-designed primers.Q534 was cloned in-to Hinc Ⅱ site of pUC18 and the recombinant plasmid pQ534 wa...A hepatitis C virus(HCV)cDNA fragment,534bp in length and designated asQ534,was obtained by PCR amplification with self-designed primers.Q534 was cloned in-to Hinc Ⅱ site of pUC18 and the recombinant plasmid pQ534 was then selected from thebacterial transformants.The sequence analysis indicated that Q534 was a cDNA fragmentof HCV core gene,and located in HCV genome from positions 320 to 853 incorrespondence with Chiron’s prototype sequence.The homologies between Q534 and theprototype at the levels of nucleotides and amino acids were 90.0% and 97.6%,respectively.The homologies of Q534 with Japanese HCV-J and HCV-BK strains were 96.6% and97.0% at the nucleotide level,and 98.2% and 98.8% at the amino acid level.In terms ofthe sequence,this Chinese HCV isolate should belong to HCV group Ⅱ.展开更多
Objective: To clone core gene cDNA of Chinese hepatitis C virus (HCV) into eukaryotic expression vector pTM3 and to express HCV core antigen in HepG2 cells. Methods: Core gene cDNA of HCV was introduced into eukaryoti...Objective: To clone core gene cDNA of Chinese hepatitis C virus (HCV) into eukaryotic expression vector pTM3 and to express HCV core antigen in HepG2 cells. Methods: Core gene cDNA of HCV was introduced into eukaryotic expression vector pTM3. Using vaccinia virus/bacteriophage T7 hybrid expression system, HepG2cells were trans feeted with the recombinant plasmid pTM3-Q534 by lipofectin. Results: From the transfected bacteria Top10F, 2pTM3-Q534 clones containing the recombinant plasmid were identified from randomly selected 10ampicillin-resistant colonies. By indirect immunofluorescence technique HCV core protein was identified in HepG2cells trans feeted with the recombinant plasmid. Conclusion: The construction of a recombinant plasmid and the expression of core gene cDNA of HCV in HepG2 were successful.展开更多
Objective To express the hepatitis C virus (HCV) core gene in E . coli on a high level. Methods The cDNA coding for HCV core protein was amplified by polymerase chain reaction (PCR). The PCR product was purified a...Objective To express the hepatitis C virus (HCV) core gene in E . coli on a high level. Methods The cDNA coding for HCV core protein was amplified by polymerase chain reaction (PCR). The PCR product was purified and digested with restriction enzymes and inserted into the downstream of P RP L promoter of a high level expression vector pBV220 . HCV core gene was expressed in E . coli in a non fused form. The expression protein was analysed by SDS PAGE , and its immunoactivity was tested by ELISA . Results Sequence analysis of the amplified PCR products confirmed that we have successfully cloned and expresssed the intact core protein of HCV. SDS PAGE showed that a specific protein with a molecular weight of 21kDa at a level of 14.0% of the total bacterial proteins appeared in bacteria harboring pBV/HCVCore, while this protein was absent in the control bacteria harboring pBV220. The results of enzyme immunoassay analysis showed that this protein could be specifically recognized by the HCV positive sera from patients with hepatitis C .Conclusion The intact HCV core protein was successfully expressed in E . coli in a non fused form on a high level, and its immunoactivity was high.展开更多
文摘AIM: To investigate the biological impact of hepatitis B virus X- hepatitis C virus core (HBV X-HCV C) fusion gene on hepatoma cells. METHODS: The recombinant adenoviruses Ad- XC, Ad-X and Ad-C expressing HBV X-HCV C fusion gene, HBV X gene and HCV C gene were constructed, respectively. Hepatoma cells were infected with different recombinant adenoviruses. MTT, colonyforming experiment, FCM, TUNEL assay were performed to observe the biological impact of the HBV X-HCV C fusion gene on liver cells. RESULTS: MTT showed that the Ad-XC group cells grew faster than the other group cells. Colony-forming experiment showed that the colony-forming rate for the Ad-XC group cells was significantly higher than that for the other group cells. FCM analysis showed that Ad-XC/Ad-X/Ad-C infection enhanced the progression of G1→S phase in the HepG2 cell cycle. The apoptosis index of the Ad-XC, Ad-X, Ad-C group cells was significantly lower than that of the Ad0 and control group cells. Semi-quantitative RT-PCR showed that the expression level of c-myc was the highest in Ad- XC infected cells. Tumor formation was found at the injected site of mice inoculated with Ad-XC-infected LO2 cells, but not in control mice. CONCLUSION: Ad-XC, Ad-X and Ad-C facilitate the proliferation activity of HepG2 cells and inhibit their apoptosis in vitro. The effect of Ad-XC is significantly stronger than that of Ad-X and Ad-C. Up-regulation of c-myc may be one of the mechanisms underlying the synergism of HBV X and HCV C genes on hepatocarcinogenesis in athymic nude mice.
基金Supported by the 135 Project of Jiangsu Province, No. 044
文摘AIM: To investigate the immunogenicity of a novel DNA vaccine,pSW3891/HBc, based on HBV core gene in Balb/c mice.METHODS: A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes (CTLs) of mice was quantitatively measured by lactate dehydrogenase release assay.RESULTS: HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1:97 200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector:target ratio of 20:1 in mice that received pSW3891/HBc DNA vaccine.CONCLUSION: pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice.
文摘Plant germplasm underpins much of crop development. Millions of germplasm accessions have been collected and conserved ex situ, and the major challenge is now how to exploit and utilize this abundant resource.
基金Supported by National Natural Science Foundation of China,No.30671846
文摘AIM:To examine whether 2'-5'oligoadenylate synthetase (OAS) gene promoter can be specifically activated by hepatitis C virus (HCV)-core protein.METHODS: Human embryo hepatic cell line L02 wa transfected with pcDNA3.1-core plasmid and selected by G418. Expression of HCV-core was detected by reverse transcription polymerase chain reaction and Western blotting. The OAS promoter sequence wa amplified from the genomic DNA and inserted into pGL3-basic vector. The resultant pGL3-OAS-Luc plasmid was transiently transfected into L02/core cell and luciferase activity was assayed. RESULTS: L02/core cell line stably expressing HCV core protein was established. The pGL3-OAS-Luc construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells.CONCLUSION: HCV-core protein activates the OAS gene promoter specifically and effectively. Utilization of OAS gene promoter would be an ideal strategy for developing HCV-specific gene therapy.
文摘A hepatitis C virus(HCV)cDNA fragment,534bp in length and designated asQ534,was obtained by PCR amplification with self-designed primers.Q534 was cloned in-to Hinc Ⅱ site of pUC18 and the recombinant plasmid pQ534 was then selected from thebacterial transformants.The sequence analysis indicated that Q534 was a cDNA fragmentof HCV core gene,and located in HCV genome from positions 320 to 853 incorrespondence with Chiron’s prototype sequence.The homologies between Q534 and theprototype at the levels of nucleotides and amino acids were 90.0% and 97.6%,respectively.The homologies of Q534 with Japanese HCV-J and HCV-BK strains were 96.6% and97.0% at the nucleotide level,and 98.2% and 98.8% at the amino acid level.In terms ofthe sequence,this Chinese HCV isolate should belong to HCV group Ⅱ.
文摘Objective: To clone core gene cDNA of Chinese hepatitis C virus (HCV) into eukaryotic expression vector pTM3 and to express HCV core antigen in HepG2 cells. Methods: Core gene cDNA of HCV was introduced into eukaryotic expression vector pTM3. Using vaccinia virus/bacteriophage T7 hybrid expression system, HepG2cells were trans feeted with the recombinant plasmid pTM3-Q534 by lipofectin. Results: From the transfected bacteria Top10F, 2pTM3-Q534 clones containing the recombinant plasmid were identified from randomly selected 10ampicillin-resistant colonies. By indirect immunofluorescence technique HCV core protein was identified in HepG2cells trans feeted with the recombinant plasmid. Conclusion: The construction of a recombinant plasmid and the expression of core gene cDNA of HCV in HepG2 were successful.
文摘Objective To express the hepatitis C virus (HCV) core gene in E . coli on a high level. Methods The cDNA coding for HCV core protein was amplified by polymerase chain reaction (PCR). The PCR product was purified and digested with restriction enzymes and inserted into the downstream of P RP L promoter of a high level expression vector pBV220 . HCV core gene was expressed in E . coli in a non fused form. The expression protein was analysed by SDS PAGE , and its immunoactivity was tested by ELISA . Results Sequence analysis of the amplified PCR products confirmed that we have successfully cloned and expresssed the intact core protein of HCV. SDS PAGE showed that a specific protein with a molecular weight of 21kDa at a level of 14.0% of the total bacterial proteins appeared in bacteria harboring pBV/HCVCore, while this protein was absent in the control bacteria harboring pBV220. The results of enzyme immunoassay analysis showed that this protein could be specifically recognized by the HCV positive sera from patients with hepatitis C .Conclusion The intact HCV core protein was successfully expressed in E . coli in a non fused form on a high level, and its immunoactivity was high.