| Hepatitis virus B(HBV) infection is epidemic throughout much of the world. In our country, it has been established that about 10% of the populations are HBsAg carriers. HBV infection is associated with a wide spectrum of clinical representation, ranging from the healthy carrier state to acute or chronic hepatitis, as well as cirrhosis and hepatocellular carcinoma. Therapy of chronic HBV infection still poses a major problem even after a countrywide usage of LIBV vaccine. Currently, interferon a is the most valuable choice of treatment, yielding long term suppression of viral replication in 30% to 40% of selected patients. Hence, other therapeutic strategies such as gene therapy are clearly needed. HBV infection can be considered as an acquired genetic disease, which makes it possible for some modified genes to inhibit HBV DNA replication after the expression of the transgenes. And this strategy termed gene therapy has great value for the virus infection especially for HBV invasion as well as some other diseases such as malignant tumors. Recent experimental approaches include interference with viral replication at the level of gene expression. Besides oligodeoxynucleotides and ribozymes, dominant negative (DN) mutants have been engaged in this area. DN mutants, based on the functional inactivation of viral proteins by modified viral gene productsm, have been described for HSV, HIV and hepadnavirues including HBV as well. DN mutants are able to interact and or disrupt the function of their native counterparts. The expression of such mutants has led to the concept of ntracellular immunization? That is to say, the infected cells can inhibit the viral replication after the expression of the mutants, as for the naive cells, they will be able to defend the viral invasion. So the mutant peptides play a negative role in the interaction with the counterparts. HBV encodes X,C,S,P ORFs and its nucleocapsid consists of 120 viral core protein dimmers that are arranged in a highly symmetrical structure. HBV replication can only take place inside intact nucleotides. The core protein consists of 183 amino acids that self-assemble into an icosahedral structure of 240 monomers within the cytoplasm of infected cells. There are two functional domains including the N-terminus from aa 1 to 139 which has been shown to be essential for core assembly, and a second C-terminal arginine rich region from aa 140 to aa 183 with several defined functions that include nucleic acid binding required for positive-strand DNA synthesis and structural stabilization of core particles to allow complete assembly of the complex into an enveloped viral particles. The core protein has been found to form dimmers when a threshold concentration within the cell is reached. Therefore, it should be possible to create molecular variants that retain the ability to assemble into core particles but fail to support the replication of the wild type viral genome. The core protein variant represents an attractive molecule acting as DN mutant which is capable of enforcing the intracellular immunization. Characterization of HBV genome and proteins, as well as the understanding of intracellular immunization of dominant negative mutants has led to our construction and expression of an eukaryotic vector pcDNA3. 1k-CS, which carries a core gene infused in frame to a surface gene of the Chinese dominant subtype HBV adr1. To fulfill this purpose, at the first, we amplified HBV core gene and surface gene respectively from pHBV-A1 by PCR, and these two lineared fragments were subcloned into the vector of...
|
PDF Full Text Request