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1.
Hepatitis B virus (HBV) has extremely restricted host and hepatocyte tropism. HBV-based vectors could form the basis of novel therapies for chronic hepatitis B and other liver diseases and would also be invaluable for the study of HBV infection. Previous attempts at developing HBV-based vectors encountered low yields of recombinant viruses and/or lack of sufficient infectivity/cargo gene expression in primary hepatocytes, which hampered follow-up applications. In this work, we constructed a novel vector based on a naturally occurring, highly replicative HBV mutant with a 207-bp deletion in the preS1/polymerase spacer region. By applying a novel insertion strategy that preserves the continuity of the polymerase open reading frame (ORF), recombinant HBV (rHBV) carrying protein or small interfering RNA (siRNA) genes were obtained that replicated and were packaged efficiently in cultured hepatocytes. We demonstrated that rHBV expressing a fluorescent reporter (DsRed) is highly infective in primary tree shrew hepatocytes, and rHBV expressing HBV-targeting siRNA successfully inhibited antigen expression from coinfected wild-type HBV. This novel HBV vector will be a powerful tool for hepatocyte-targeting gene delivery, as well as the study of HBV infection.  相似文献   

2.
目的:构建增强型绿色荧光蛋白(EGFP)标记的乙型肝炎病毒(HBV)真核表达载体,并研究其在真核细胞和小鼠体内的共表达。方法:以质粒pBR322-HBVadr2.0和pCX-EGFP为基础,构建含有双拷贝HBV全基因组DNA和EGFP基因的真核表达载体pCX-EGFP-HBVadr2.0,分别转染真核细胞和小鼠肝组织,建立体外、体内表达系统,研究GFP和HBV基因的表达。结果:构建了真核表达载体pCX-EGFP-HBVadr2.0,EGFP和HBV病毒蛋白在体内和体外均可表达。结论:构建的pCX-EGFP-HBVadr2.0真核表达载体可以GFP作为HBV存在与否的报告基因,提高了培育检测转基因小鼠的效率,为转基因小鼠的制备及后续研究奠定了基础。  相似文献   

3.
HBV具备改造作为肝靶向性基因治疗载体的基本条件---天然嗜肝特异性,能够在肝细胞内持续复制、反复感染,病毒本身对细胞没有明显的细胞毒性;能够携带外源基因并被包装成病毒颗粒;能够介导外源基因的转移和表达。但由于基因结构复杂,目前改造的HBV载体均存在载容量小、复制包装效率低及安全性差等缺点。就近年相关研究进展进行综述。  相似文献   

4.
刘晓曼  杨倬  冯涛 《微生物学报》2012,52(2):191-197
【目的】尝试构建表达小干扰RNA(small interfering RNA,siRNA)的小环载体,并初步鉴定其对乙肝病毒(hepatitis B virus,HBV)复制及其基因表达的抑制作用。【方法】设计并合成靶向HBV S区的siRNA,将其克隆到小环载体pMC.BESPX-MCS2上,测序正确后将重组体pMC-H1-siHBS-U6转化入感受态E.coliZYCY10P3S2T,然后在培养基中加入L-阿拉伯糖,诱导其降解细菌骨架,获取只含有目的基因表达盒的小环RNA干扰载体pmc-H1-siHBS-U6。将小环RNA干扰载体与HBV真核表达质粒pHBV1.3共转染Huh-7细胞,分别在转染后1-7天,ELISA法检测Huh-7细胞上清中的HBsAg、HBeAg,并且通过Real-time RT-PCR法分析干扰RNA对HBV DNA及mRNA的抑制效果。【结果】成功构建了靶向HBV S基因的siRNA小环表达载体pmc-H1-siHBS-U6。该载体能显著抑制Huh-7细胞HBsAg和HBeAg分泌,并且其抑制效果能够维持2-3周时间。Real-time PCR证实HBV的DNA与mRNA水平分别降低了71%和80%,而对照siRNA及空载体则无此作用。【结论】成功构建了靶向HBV的小环RNA干扰载体,并且其能稳定、高效、特异地抑制HBV基因的表达与复制,该研究不仅对探索HBV的基因治疗提供了重要线索,而且为RNA干扰的应用提供了新的运载体系。  相似文献   

5.
We have developed an adenovirus vector to express foreign proteins under the control of the adenovirus E1a promoter. Two recombinant plasmids, harbouring either the S gene or the pre-S2 region and the S gene of hepatitis B virus under the control of the E1a promoter, were used to construct two recombinant adenoviruses. These two viruses direct the synthesis of hepatitis B virus surface antigen (HBsAg) particles during the time course of an infectious cycle. When the pre-S2 region is present in the constructed virus, the synthesis of particles carrying the receptor for polymerized human serum albumin (pHSA) is observed. Moreover, the inoculation of rabbits with this latter purified recombinant adenovirus elicits the production of antibodies that react with both HBsAg and pHSA receptor.  相似文献   

6.
The hepatitis C virus (HCV) core protein is a multifunctional protein that can interfere with the induction of an immune response. It has been reported that the HCV core protein inhibits HBV replication in vitro. In this study, we test the effect of the HCV core gene on the priming of the immune response to hepatitis B surface antigen (HBsAg) and on the replication of HBV in vivo. Our results showed that the full-length HCV core gene inhibits the induction of an immune response to the heterogeneous antigen, HBsAg, at the site of inoculation when HCV core (pC191) and HBsAg (pHBsAg) expression plasmids are co-administered as DNA vaccines into BALB/c mice. The observed interference effect of the HCV core occurs in the priming stage and is limited to the DNA form of the HBsAg antigen, but not to the protein form. The HCV core reduces the protective effect of the HBsAg when the HBsAg and the HCV core are co-administered as vaccines in an HBV hydrodynamic mouse model because the HCV core induces immune tolerance to the heterogeneous HBsAg DNA antigen. These results suggest that HCV core may play an important role in viral persistence by the attenuation of host immune responses to different antigens. We further tested whether the HCV core interfered with the priming of the immune response in hepatocytes via the hydrodynamic co-injection of an HBV replication-competent plasmid and an HCV core plasmid. The HCV core inhibited HBV replication and antigen expression in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, the mouse models of acute and chronic hepatitis B virus infections. Thus, the HCV core inhibits the induction of a specific immune response to an HBsAg DNA vaccine. However, HCV C also interferes with HBV gene expression and replication in vivo, as observed in patients with coinfection.  相似文献   

7.
BALB/c nude mice were injected intrahepatically with hepatitis B virus (HBV) DNA prepared from recombinant plasmids. Hepatitis B surface antigen appeared in the circulation in 19 of 23 mice (82%) 3 to 20 weeks postinjection and persisted for more than 6 months in most animals. Hepatitis B e antigen appeared transiently in the circulation in 12 of the 23 mice (52%) within a few weeks after the appearance of hepatitis B surface antigen. Antibodies to the core, X, and/or polymerase gene products of HBV have also been observed in 14 (61%) of the mice. Histopathological examination of the livers at 7 months postinjection demonstrated that nearly half had characteristics consistent with chronic hepatitis. HBV DNA appeared to be integrated into host liver DNA. No evidence of viral replication was observed in sera or livers from these mice at 7 months postinjection. These results demonstrate that an HBV chronic carrierlike state can be established in mice and that such a model could be used to study host and virus factors important in the establishment and maintenance of HBV-associated chronic liver disease.  相似文献   

8.
We have shown previously that insulin suppresses the expression of hepatitis B surface antigen (HBsAg) gene from an endogenous integrated viral genome in cultured human hepatoma Hep3B cells. In this study, we demonstrated that insulin suppresses the viral mRNA transcribed from transiently transfected tandem repeat hepatitis B virus (HBV) dimer DNA or DNA fragment that contains only the major HBsAg gene. Insulin treatment also resulted in a decrease in HBV viral particles produced by the HBV-DNA-transfected cells in a dose-dependent manner. Furthermore, when insulin was simultaneously added with glucocorticoid, which stimulates HBV gene expression, the stimulatory effect of glucocorticoid was completely abolished. Our results suggest that insulin has a dominant negative effect on the HBV gene expression in cultured human liver cells.  相似文献   

9.
应用ELISA和PCR法检测502例乙肝病人血清,401例HBsAg阳性血清中,有114例(28.4%)抗-HCV和HCVRNA双项阳性,25例(6.2%)HCVRNA单项阳性;21例(5.2%)抗-HCV单项阳性。将HBsAg乙肝病人分成HBVDNA,HBeAg阳性组和HBVDNA,HBeAg阴性组。前者抗-HCV阳性率为11.6%~20.5%,HCVRNA阳性率为16.2%~20.5%。后者抗-HCV阳性率为20.2%~55.6%,HCVRNA阳性率为23%~60.3%。结果说明长期携带HBV者和慢性乙肝病人均可重叠HCV感染。HBVDNA阳性组抗-HCV和HCVRNA阳性率明显高于HBVDNA阳性组  相似文献   

10.
Antisense therapy of hepatitis B virus infection   总被引:2,自引:0,他引:2  
Chronic infection with the hepatitis B virus (HBV) is a major health problem worldwide. The only established therapy is interferon-a with an efficacy of only 30–40% in highly selected patients. The discovery of animal viruses closely related to the HBV has contributed to active research on antiviral therapy of chronic hepatitis B. The animal model tested and described in this article are Peking ducks infected with the duck hepatitis B virus (DHBV). Molecular therapeutic strategies aimed at blocking gene expression include antisense DNA. An antisense oligodeoxynucleotide directed against the 5′-region of the preS gene of DHBV inhibited viral replication and gene expression in vitro in primary duck hepatocytes and in vivo in Peking ducks. These results demonstrate the potential clinical use of antisense DNA as antiviral therapeutics.  相似文献   

11.
DNA extracted from human nonhepatic tissues (placenta and kidney) have been digested with restriction endonucleases and examined by the Southern procedure with cloned 32P-labelled DNA of hepatitis B virus (HBV). In placental DNAs of women with the history of a hepatitis B infection and in one out of four cases of patients with no known HBV exposure or manifestation, HBV-related chromosomal nucleotide sequences were detected. The integration of HBV-related sequences was observed also in human kidney DNA. Moreover, in the placenta of women who had hepatitis B infection prior to delivery, unusual unintegrated forms of HBV have been found. We conclude that HBV sequences can be found not only in hepatic tissue but also in placental and kidney DNA, both of HBV-exposed and in one case even of a nonexposed patient.  相似文献   

12.
Coexistence of hepatitis B surface antigen (HBsAg) and antibody against HBsAg (anti-HBs) comprises an atypical serological profile in patients with chronic hepatitis B virus (HBV) infection. In this study, in total 94 patients with coexisting HBsAg and anti-HBs and 94 age- and sex-matched patients with positive HBsAg were characterized by quantitatively measuring HBsAg and HBV DNA, sequencing large S genes, and observing clinical features. Compared with common hepatitis B patients, the patients with coexisting HBsAg and anti-HBs had lower HBsAg and HBV DNA levels. These two groups had similar rate of pre-S deletion mutations. However, in patients with coexisting HBsAg and anti-HBs, more amino acid substitutions in the a determinant of S gene were observed in HBV genotype C, but not in genotype B. Fourteen patients with coexisting HBsAg and anti-HBs were followed up for an average of 15.5 months. There were no significant changes in the levels of HBsAg, anti-HBs, HBV DNA and ALT over the follow-up period. Compared with the baseline sequences, amino acid substitutions in the MHR of HBsAg occurred in 14.3% (2/14) patients. In conclusion, coexistence of HBsAg and anti-HBs may be associated with higher frequency of mutations in the a determinant of HBV genotype C.  相似文献   

13.
We report a novel and simple method for mutation detection in DNA oligonucleotides using a double-stranded DNA specific dye (SYBR Green I) in nanostructured molecular assemblies, called reverse micelles. The intercalation of SYBR Green I into the duplex DNA exhibits fluorescent emission in a CTAB/isooctane reverse micellar system as well as in an aqueous solution. We found marked differences in the fluorescence intensity between perfectly matched and mismatched 52-mer synthetic oligonucleotides, which were designed to contain the YMDD motif of the hepatitis B virus (HBV) polymerase gene, in a reverse micellar solution. Using this method, we successfully detected a mutation in PCR-amplified oligonucleotides of the HBV polymerase gene in sera of four patients with chronic hepatitis B. This detection method does not require DNA immobilization, chemical modification of DNA, or any special apparatus; it only needs a normal fluorescence spectrophotometer, an inexpensive dye, and just 10 pmol of sample DNA.  相似文献   

14.
Hepatitis B virus (HBV) with X gene mutations has been a putative pathogen of chronic hepatitis without serological markers of known hepatitis viruses. The aim of this study was to reconfirm whether the HBV with the X gene mutation is associated with these serologically “silent” non-B, non-C (NBNC) chronic hepatitis, alcoholic liver disease (ALD) and autoimmune hepatitis (AIH). HBV DNA was amplified from serum and sequenced in 30 patients with NBNC chronic hepatitis in comparison with 20 patients with ALD and 5 patients with AIH. HBV DNA was identified in 21 patients (70%) in NBNC chronic hepatitis by nested polymerase chain reaction while only one patient (5%) in ALD and none in AIH showed HBV DNA. Eighteen (85.7%) of the 21 identified HBV DNAs had an identical 8-nucleotide deletion mutation at the distal part of the X region. This mutation affected the core promoter and the enhancer II sequence of HBV DNA and created a translational stop codon which truncated the X protein by 20 amino acids from the C-terminal end. All the HBV DNAs had a precore mutation at the 83rd nucleotide resulting in disruption of HBe antigen synthesis. These results indicate that HBV mutants are closely associated with the majority of serologically “silent” NBNC chronic hepatitis cases and the population of such mutant HBV DNAs is not uniform.  相似文献   

15.
A plasmid carrying the complete genome of hepatitis B virus (HBV) inserted into the BamHI site of the pBR322 plasmid vector has been constructed. The physical map of HBV DNA established for 13 restriction endonucleases allows to conclude that the cloned DNA is similar, but not identical to the HBV DNA of ayw subtype.  相似文献   

16.
17.
18.
Summary The hepatitis B virus (HBV), the causal agent of serum hepatitis, has a diameter of 42 nm and is comprised of an outer surface coat and a 27 nm core. A unique DNA-dependent DNA polymerase is associated with the core of the virus. The core also houses a circular DNA that contains both double-stranded and single-stranded regions. In the endogenous reaction, the DNA polymerase repairs the single-stranded gaps of the viral DNA. The surface protein of the virus, called hepatitis B surface antigen, contains both lipid and carbohydrate, and is often present in particulate form in the blood of infected patients. In Asia and Africa HBV infection is associated with subsequent development of primary hepatocellular carcinoma. Although most patients recover completely from acute illness, the hepatitis B virus may cause chronic infection. Recently, a virus similar to human HBV was discovered in woodchucks. HBV has not yet been propagated in a cell culture system and the mode of replication of this unusual virus in hepatocytes is still moot. Although reliable therapy has not yet been provided, the problem of this world-wide infection has led to many interesting approaches to both vaccine production and anti-viral chemotherapy.  相似文献   

19.
Tian Y  Sir D  Kuo CF  Ann DK  Ou JH 《Journal of virology》2011,85(24):13453-13456
Recent studies indicate that hepatitis B virus (HBV) may induce autophagy to enhance its replication in cell cultures. To understand whether autophagy can indeed enhance HBV replication in vivo, we generated HBV transgenic mice with liver-specific knockout of the Atg5 gene, a gene critical for the initiation of autophagy. Immunoblot analyses confirmed the inhibition of autophagy in the livers of Atg5 knockout mice. This inhibition of autophagy slightly reduced HBV gene expression and affected nuclear localization of the HBV core protein. It also reduced the HBV DNA level in sera by more than 90% and the level of the HBV DNA replicative intermediate in the mouse liver to an almost undetectable level. Our results thus demonstrate that autophagy is important for HBV replication in vivo and raise the possibility of targeting this pathway to treat HBV patients.  相似文献   

20.
The mutation of YMDD motif of hepatitis B virus (HBV) polymerase gene is the most frequent cause in HBV resistant to lamivudine. The aim of the study was to investigate variation features of HBV polymerase gene in chronic hepatitis B (CHB) patients before and after lamivudine treatment. From the serum samples of five CHB patients before and after 12 months of lamivudine treatment, HBV polymerase gene was amplificated and positive DNA fragments were cloned into JM105 competent cell. Twenty positive clones of every sample were checked with mismatched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and YMDD variants were sequenced. Among five patients after 12 months of lamivudine treatment, M552I mutations in two patients with HBV DNA rebounding and D553G mutation in one non-responder were detected except two patients with negative HBV DNA consecutively. In summary, D553G mutation is probably one of the reasons that caused non-responders during lamivudine treatment. The mutations of YMDD motif occurred after lamivudine treatment are caused by the induction of lamivudine.  相似文献   

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