Construction Of The HBV-TR Recombinant Adenovirus Vector And Its Expression In Mouse Liver

The infection of hepatitis B virus(HBV) is still a serious public health problem around the world. Currently, the most effective drugs used to treat the hepatitis B are interferon-a (IFN-a ) , lamivudine and adefovir dipivoxil. But all of them can not eradicate the virus completely in most of the patients. The remarkable achievements in the gene therapy bring us wishes to cure this disease.The fusion protein gene of HBV core protein and human eosinophil derived neurotoxin, hereafter called HBV targeted ribonuclease gene(HBV-TR gene), was constructed based on the theory of capsid-targeted viral inactivation(CTVI ) .This fusion protein consists of two parts ,one is the targeted molecule-HBV core protein(HBc) which is a necessary element during the encapsidation of HBV ;the other is effector molecule-human eosinophil derived neurotoxin(hEDN) which is a ribonuclease. HBV-TR gene can inhibit the replication of HBV effectively in vitro.To further study the expression of HBV-TR gene in vivo, the recombinant adenoviral vector of HBV-TR was constructed and usedto infect normal mice after being amplified in HEK 293 cells. Then the expression of this recombinant adenoviral vector in vivo, especially in mouse liver, was studied by immunohistochemistry in order to lay the foundation for its anti-HBV function study in hepatitis B animal model.The system of AdMax Kit D was used to complete the construction of recombinant adenovirus. Firstly, the shuttle plasmid was constructed. BamH I and HindIII were used to digest pcDNA3. l(-)/TR to get TR gene fragment, Bgl II and HindIII were used to digest pDC316 at the same time. Then the TR gene fragment was inserted into digested pDC316 by the T4 DNA ligase to obtain the shuttle plasmid pDC316/TR. Other control shuttle plasmids were constructed by the same way.All the shuttle plasmids and rescue plasmid were purified by phenol-chloroform extraction and subsequent ethanol precipitation, and then resolved in appropriate volume of 0. 1 XTE(pH8. 0) to adjust their concentration to about 40 ug/mL. Then the shuttle plasmids pDC316/TR, pDC316/TRm, pDC316/hEDN, pDC316/HBc and empty pDC316 respectively were cotransfected with rescue plasmid pBHGlox(delta)El, 3Cre into HEK293 cells to acquire the recombinant adenovirus AdTR and other control adenoviral vectors. When most of the cells show typical cytopathic effect(CPE), the cells were collected by low speed centrifugation and then resuspend in PBS. The pelleted cells were frozen, molten and vortexed repeatedly, then the cell lysates were centrifugated and the supernatants were collected and stored at -70C.Using the genomic DNA extracted from the recombinant adenoviruses as the template, all the inserted DNA fragments were amplified by PCR reaction. Thus, all the recombinant adenoviruses including AdTR AdTRm AdhEDN and AdHBc wrer constructed successfully.AdTR and AdhEDN were amplified in HEK 293 cells to 1010pfu/mL and then injected via tail vein into normal mice once every day, while the normal control mice was only injected with normal saline. Four days later, all mice were killed and their livers were excised to make the sections for subsequent immunohistochemistry and pathological analysis.The results of immunohistochemistry showed that AdTR and AdhEDN successfully express TR fusion protein and hEDN protein, respectively in mouse liver. Furthermore, The HE staining showed slight pathological changes such as lymphocyte infiltration in the liver of mice infected by AdTR and AdhEDN.In summary, AdTR, the recombinant adenoviral vector of HBV-TR gene, and other control recombinant adenoviruses including AdTRm, AdhEDN, AdHBc all were constructed successfully. TR and hEDN all were expressed in mouse liver by the corresponding recombinant adenoviral vectors, which lays the foundation for anti-HBV function study of TR in hepatitis B animal model.