Construction And Application Of A New Type HBV Vector

Hepatitis B virus (HBV) vector is a new type of viral vector found in recent years, which is different from viral vector commonly used such as adenovirus, adeno-associated virus, retrovirus, lentivirus, etc. It has great advantages over other vectors for its specifically targeting to the hepatocytes and its basic condition for reconstructing into hepa-target gene therapy vector. The advantages of the HBV vector include self-replicating continuously in liver cells, infecting repeatedly and no conspicuous cytotoxicity, packing viral particle, expressing, transferring and carrying exogenous genes. The disadvantage of HBV vector is that the gene structure is too complicated to content enough exogenous genes. It possesses low copy packaging efficiency and low security, low efficiency of infection. In addition, it is difficult for preparation. In order to solve these difficulties, it is very necessary to associate with other viral vectors such as adenovirus, adeno-associated virus, retrovirus, lentivirus, etc. Only by doing so, can high carry capability, transfection efficiency and replication be realized. As HBV vector is hepatotropic, it is thoroughly educed, enveloped by utilizing wild type HBV in the human body and forms reconstruction HBV particles with anti-HBV effects, or carrying therapeutic target genes and further infects other liver cells to realize amplification effectiveness. Therefore, by reconstruction among the viral vectors, the application domain of HBV vector is widened deeply. Therefore it may be used not only for anti-HBV gene therapy, but also for HBV molecular biological research by expressing special exogenous gene.On this study a stable cell line with permanent secretion of recombinant hepatitis B virus (HBV) was constructed through the construction of HBV vector with expression of different target gene, which is useful for molecular biological study or screening of HBV susceptible cell line. It can also be used together with adenovirus to construct target gene plasmids which express MMP8 and can be used for anti-cirrhosis gene therapy. Therefore, this study developed a new way for molecular biological research on HBV and biological treatment of cirrhosis. This study includes three parts.Part one Construction of a new type HBV Vectorobjective To explore an effective ways constructing and making HBV vector plasimids, which cannot express any structural genes ,by molecular biology technique. The new type HBV vector may be more security and applicable as a gene therapy vector.Method1 The original genes (Core, Polymerase, pre-S1, pre-S2, S and X) of HBV vector (pCH-S-GFP) were deleted by altering the start codon or adding stop codons, pCH-M5-GFP was successfuly constructed.2 Deleting the GFP genes of pCH-S-GFP and pCH-M5-GFP, then inserting the hRLuc gene into the S region, pCH-S-hRLuc and pCH-M5-hRLuc were successfuly constructed.3. To determine exogenous gene expression levels, after hepatocyte was cotransfected by the new plasimids and pCH3142, GFP and hRLuc were observed by fluorescence microscope or Luminometer.4 To observed the ability of recombinant HBV replication, HBV DNA in cell lysate was extracted and southern blot was done to detect HBV replication intermediates, after plasmid cotransfection.5 To detect the infectious capacity of the recombinant HBV on tree shrews hepatocyte tree shrews hepatocytes were infected by concentration and purification of reHBV particles from cell supernatant. The expression level of GFP would be observed after 1W.Results1 The three HBV vector plasmids were successfully constructed by gene recombination technology.2 After the cells cotransfected by the mutation plasmid, expression level of GFP and hRLuc was stronger than the original plasmid.3 Cotransfected by the mutation plasmid and pCH3142, the formation of HBV replication intermediates can be observed in Cell lysate by southern blot.4 Expresstion of GFP can be observed after reHBV infected tree shrews hepatocytes.Conclusion After the start codon and terminal codons of the HBV plasmids were mutated, HBV structure protein can not be expressed;Some report genes can be inserted into the S region of the new HBV vector plasmids, and stronger expression than the original plasmid;ReHBV can infect tree shrews hepatocytes.Part two The first aspect application of the new type HBV vector---Stable cell line for secretion of replication-defective hepatitis B virus vector expressing blasticidin resistant geneObjective:To construct a stable cell line with permanent secretion of recombinant hepatitis B virus(HBV)vector which express blasticidin resistant gene.Method1 Construction of HBV helper plasmid pcDNA3.1-CH3142 with G418-resistent non packing signal and HBV vector plasmids which express blasticidin resistant gene.2 HepG2 cells were cotransfected with both the HBV vector expressing blasticidin resistant gene and the helper plasmids with G418-resistant gene. Cell clones were selected by the addition of both blasticidin and G418. Positive cell clones were gained.3 Screening cell clones with high secretion of the HBV-Bsd recombinant hepatitis B virus particles by ELISA and dot blot hybridization4 After culturing by the addition of both G418 and Bsd, regaining the higher level recombinant HBV cell line for secretion of replication-defective hepatitis B virus by Southernblot and Native Westernblot technology, and test the expression level of recombinant HBV through PCR5 Application of PCR technology, wild-type hepatitis B virus and recombinant hepatitis B virus were identified and confirmed wild-type hepatitis B virus can not be formed in the new cell lines.6 The complete recombinated HBV particles were tested by immune electron microscopy.Result1 Over 50 cell clones were formed through G418 screening after transfection.2 36 cell clones were picked and expanded. HBV DNA in the supernatant was tested by dot blot hybridization with isotope labeled probe. Afterward the best 9 clones were selected3 After maintaining for 15 generations (about 3 months), HBV DNA was proved by southern blot with isotope labeled probe. Confirmed the formation of RC DNA, According to the results, 3 cell lines of HBV-Bsd25,HBV-Bsd7, HBV-Bsd27 were chosen.4 The quantity of HBV DNA of the three best cell lines (HBV-Bsd25, HBV-Bsd7, HBV-Bsd27) were 4.1*106, 3.6*106 and 1.2*106 copies/mL respectively.5 No wild type HBV was detected.6 Cesium chloride density gradient analysis in HBV-Bsd25 cell lines was done with the concentrated supernatant HBV virion, and enveloped recombinant HBV were proved by southern blot and HBsAg western blot.7 Complete HBV particles were observed by electronic microscope.Conclusion By the methods of HBV vector and HBV helper plasmids cotransfection, we could obtain the stable cell lines with permanent secretion of HBV virions and realized large preparation of recombinant HBV virions. This will contribute to the use of HBV vector for gene therapy. The recombinant HBV secretion stable cell line will also useful for the ongoing selection of HBV susceptible cell line.Part three The second aspect application of the new type HBV vector plasmids---An experimental study on rat liver cirrhosis model administrated with Adenovirus-HBV chimeric vector expressing collagenase IINo. 1 : construction and expression of adenovirus chimeric HBV vector in vitroObjective To construct 3 adenovirus chimeric HBV vector plasmids which expressing different purpose gene, then observe the expressing levels of tMMP8 mRNA and RFP in vitro. To detect the expression of GFP in ECV304 and HepG2 with a CMV promoter and the HBV S promoter-driven GFP plasmids.Method1 Three new plasmids expressing purpose gene were successfully constructed.2 To confirm the correctness of recombined adenovirus vector by PCR technology, the ideal gene fragments were obtained.3 After three new correct plasmids were amplificated, extracted and purified, concentration determination and plaque-forming ability were detected.4 After the three recombinant adenovirus infected HepaG2, RFP was detected by fluorescence microscopy, tMMP mRNA detected by RT-PCR technique.2 After ECV304 and HepG2 were transfected with the plasmids of CMV promoter and the HBV S promoter-driven GFP, the expression of GFP was detected with fluorescence microscopy.Result1 HBV-adenovirus chimeric vector carrying different purpose genes were successfully constructed.2 After recombinant adenovirus carrying MMP8 and tMMP8 infected hepatocytes, MMP8 and tMMP8 mRNA expression can be observed, recombinant adenovirus carrying RFP2 in hepatocytes can express RFP.3 Defferent promoter-driven GFP plasmids transfected defferent cell lines, the level of GFP expression is defferent.Conclusion Adenovirus vector can packaged HBV vector and expressing different purpose genes and formation HBV-adenovirus chimeric vector. After transfeced HepG2 cell lines, recombinated adenovirus vector can be successful expressing different purpose gene. HBV carrier s promoter-driven GFP in the vascular epithelial cells (ECV304) is low expression, in liver cell lines showed high expression. And CMV promoter-driven GFP in the vascular epithelial cells (ECV304) and liver cell lines showed high expression, suggesting that S promoter plasmid posseses hepatotropic.No.2: An experimental study on rat liver cirrhosis model induced by TAAObjective To explore the level of HGF/C-met, Liver histology, liver function and liver hydroxyproline on TAA induced rat liver cirrhosis model.Method 42 male Wistar rats were fed with thioacetamide drinking water, preparation of liver fibrosis model. After the success of the model preparation, the rats were randomly divided into 7 groups of 6. Every two weeks killed a group of rats, collecting serum for liver function testing and detection of liver tissue hydroxyproline content, for HE and Sirius Red staining, and RT-PCR detected HGF and its receptor c-Met, and the other six normal rats were set as control group.Result HGF/ c-Met mRNA expression was significantly higher than the normal control group. With the time to stop the extension of the application of TAA, liver function, histological changes, hydroxyproline content and HGF / c-Met mRNA expression level were returned to normal and a positive correlation was observed.Conclusion:Rat liver cirrhosis models can be induced successfuly by TAA administrated for 20 weeks. After ceasing TAA, Liver histology, liver function and liver hydroxyproline have varying degrees of self-healing trend. Self-healing in the liver tissue at the same time, HGF and its receptor c-Met expression levels in varying degrees downward trend, however, no significant difference between groups. The occurrence of liver injury, an increase in HGF / c-Met expression level may be adopted to promote liver regeneration and reverse liver fibrosisNo.3: An experimental study on rat liver cirrhosis model administrated with Adenovirus-HBV chimeric vector expressing collagenase IIObjective Neutrophil collagenase (MMP-8) can degrade typeⅠcollagen specifically. Introduction of MMP-8 gene into liver cells could be an advantageous tool to degrade collagen. In this study, the aim is to explore the effectiveness of the typeⅠcollagen degradation, and the promotion of liver cell regeneration and improvement of liver function, to reduce liver fibrosis through treating cirrhosis rat model in vivo with Adenovirus-HBV chimeric vector expressing collagenase IIMethod1 Rat liver fibrosis model was prepared by 0.3% thioacetamide-induced drinking water.2 Randomized and divided into groups and drug delivery 120 liver fibrosis model rats were randomly divided into four groups, were given Ad-CH-tMMP8, Ad-C-MMP8, Ad-CH-RFP2, tail vein injection of recombinant adenovirus, 1.5×1011 viral particles/Kg dose, one administration. Selecting another 30 rats as normal control group, administrated the normal water.3 Ten rats in each group were sacrificed in 2W, 4W, 8W, respectively, and separate the serum, -20℃preservation. part of liver tissue were put in 10% of the formalin-fixed for histopathological examination, the remainder of the liver tissue were stored in liquid nitrogen.4 Testing liver function by automatic biochemical analyzer, Hydroxyproline in liver tissue by acid hydrolysis method, the liver histopathological changes by HE, sirius red staining and immunohistochemical method, MMP8, HGF and c-Met and GAPDH mRNA by RT-PCR.5 Statistical analysis. Statistical analysis was conducted by SAS statistical analysis software.Result:1 The expression level of MMP8 mRNA. MMP8 mRNA in the treatment group could be expressed in rat liver, but no MMP8 mRNA expression in the control group. The expression level of MMP8 mRNA was downward trend, with the extension of treatment time.2 Liver function. As compared with two control groups, ALT in the two treatment groups were significantly decreased at 2 W, 4W and 8W(P <0.05), ALT in the two treatment groups was near to the normal group at 8W.3 Indicators of liver fibrosis. HA and LN in the two treatment groups were significantly decreased at 2 W, 4W and 8W (P <0.05), HA and LN in the two treatment groups was near normal group at 8W.4 Liver histology pathology Compared with the control group,pathology in the treatment group improve significantly,Inflammatory cell infiltration and the proliferation of tissue were significantly reduced. Hepatic lobules is damaged to a lesser extent. There is the phenomenon of liver regeneration5 The quantity of Hydroxyproline. Hydroxyproline is an amino acid derived from collagen and elastin hydrolysis, accounting for about 14% of the weight of collagen. It is rare in other proteins. The quantity of hydroxyproline is varied with the severity of liver cirrhosis. At 2W application of the treatment of adenovirus, in the liver tissue, the hydroxyproline quantity was significantly lower than that of the control group6 The expression level of HGF/cMet mRNA. HGF/cMet mRNA in the two treatment groups were significantly increased at 2 W, 4W and 8W(P <0.05) compared with control groups.Conclusion:After application of recombinant adenovirus, liver tissue can express MMP8. The latter can effectively degrade collagen typeⅠ, reduce liver fibrosis, liver cells regenerate. The expression level of HGF / cMet mRNA was increased. This innovation is expected that MMP-8 gene therapy is brought into the clinical application of liver cirrhosis, it is a new strategy of treatment for reversing liver cirrhosis.