The Study Of Inhibition Effects Produced By Dominant Negative Mutant Fusion Protein PreS2-TLM-ScFv-HBcDN On HBV Replication In Vitro

BackgroundDiseases caused by viruses have been threatening the health of Humans. Nowadays, some old viruses occasionally reoccurred and induced infectious diseases, besides, a few new viruses with fatal virulence and infectivity arised. People have no resistance to these newly emerging infectious diseases. Furthermore, no effective prevention, treatment or control measures can be found in short term. Although some drugs are effective in preventing from viruses, they often have some side effects. Gene therapy can inhibit the replication of virus effectively in vitro. To make full use of the advantages of gene therapy in resisting viruses, it is significant to establish a method capable of promoting the therapeutic drugs to efficiently and specifically enter the infected cells. However, the strategy of gene therapy for preventing viruses has not been widely studied for the lack of the efficient and specific delivering vector.Cell-penetrating peptides (CPP) are able to penetrate the cell membrane and deliver the bioactive molecules into the cells efficiently. There is a translocation motif (TLM) with characteristic of CPP in the HBV PreS2domain. PreS2-TLM is located at41-52aa and can carry the fusion molecules into plasma independently of receptors or transporting proteins on the surface of cells. Small single chain Fv (ScFv) possesses the binding activity with antigen. ScFv can guide the therapeutic molecules to the antigen expressing cells once it is conjugated with the therapeutic molecules. In the strategy of gene therapy, CPP is often fused with ScFv (transbody) to make the therapeutic molecules enter the target cells efficiently and specifically. HBsAg is expressed on the surface of hepatocytes infected with HBV and can be bound with the specific antibodies of HBsAg.Dominant negative mutant of HBV core protein is most effective in inhibiting the replication of virus among the present gene therapies for HBV infection. Thus, in our study, we choose HBV as the object to explore a new way to specifically inhibit the replication of virus and accordingly to explore a new path to deal with the intractable infectious diseases existing or possibly occurring in the future.MethodsA eukaryotic expressing plasmid pPreS2-TLM-ScFv-EGFP was constructed with the techniques of molecular cloning and DNA recombination. The plasmid pPreS2-TLM-ScFv-EGFP was identified with restrictive endonuclease and sequencing. pPreS2-TLM-ScFv-EGFP was extracted and purified with the plasmid extraction kit and then transfected into Chinese hamster ovary cell (CHO) with lipofectamine2000. The stably transfected cell line was screened by Zeocin. The expression of the PreS2-TLM-ScFv-EGFP fusion protein in cells was observed with fluorescence microscope. Frequency of cells expressing the PreS2-TLM-ScFv-EGFP fusion protein was measured by flow cytometer. The fusion protein PreS2-TLM-ScFv-EGFP in the supernatant of cellular culture was detected by western blot assay. The supernatant containing the PreS2-TLM-ScFv-EGFP fusion protein was added into HBsAg-positive HepG2.2.15cells, HepG2and HeLa cells cultures, respectively. The location of the fusion protein PreS2-TLM-ScFv-EGFP in HepG2.2.15cells was determined with a Texas Red-conjugated anti-cGFP monoclonal antibody by immunofluorescence staining. Dominant negative mutant of hepatitis B virus core protein gene (HBcDN) and pPreS2-TLM-ScFv-HBcDN were constructed also with PCR and DNA recombination. pPreS2-TLM-ScFv-HBcDN was identified with restrictive endonuclease and sequencing. The plasmid pPreS2-TLM-ScFv-HBcDN was purified and transfected into CHO cells with lipofectamine2000, Zeocin and limited dilution method were used to screen the stable cell line of expressing the fusion protein PreS2-TLM-ScFv-HBcDN. Total DNA and RNA were extracted with the DNA and RNA extraction kits from the CHO-HBcDN cell line and used as the PCR and RT-PCR templates for amplification of the inserted segments HBcDN, PreS2-TLM-ScFv and PreS2-TLM-ScFv-HBcDN, respectively. PCR-amplified products were detected by agarose gel electrophoresis. The expression of the PreS2-TLM-ScFv-HBcDN fusion protein was determined by western blot assay. The supernatant with the fusion protein PreS2-TLM-ScFv-HBcDN was prepared from CHO-HBcDN cellular cultures, which was added into HepG2.2.15cells cultures. The viral core particles were extracted from HepG2.2.15cells according to the method reported. The encapsidated HBV pregenomic RNA was separated and purified with the RNA extraction kit. The relative quantitative RT-PCR was utilized to analyze the encapsidated HBV pregenomic RNA level.ResultsSequence of the PreS2-TLM-ScFv-EGFP fusion gene was completely correct for encoding the PreS2-TLM-ScFv-EGFP fusion protein after alignments with the known sequences by Editseq software. The observation result with fluorescence microscope indicated that the PreS2-TLM-ScFv-EGFP fusion protein was mainly located in the cytoplasm of CHO cells. The result determined by flow cytometer showed that the frequency of cells expressing stably the PreS2-TLM-ScFv-EGFP fusion protein was83.47±6.05%. The PreS2-TLM-ScFv-EGFP fusion protein expression was detected in the supernatant by western blot. Texas Red-conjugated anti-cGFP monoclonal antibodies preferentially bound to HepG2.2.15cells incubated with the supernatant containing PreS2-TLM-ScFv-EGFP fusion protein. The integrated optical density (IOD) in HepG2.2.15cells was significantly higher than the IOD in HepG2or in HeLa cells.Sequence encoding the PreS2-TLM-ScFv-HBcDN fusion protein was completely correct. The cell line stably expressing the PreS2-TLM-ScFv-HBcDN fusion protein was designated as CHO-HBcDN. Bands with specific sizes (bp) of the inserted segments were obtained by PCR and RT-PCR analyse in which total DNA and RNA extracted from CHO-HBcDN cells were used as the templates. The PreS2-TLM-ScFv-HBcDN fusion protein was detected in the culture supernatant. The package of HBV pregenomic RNA in HepG2.2.15cells was significantly inhibited, when HepG2.2.15cells were exposed to25%supernatant of CHO-HBcDN cultures for24,48and72h or to12.5%supernatant of CHO-HBcDN cultures for72h.ConclusionIn our study, PreS2-TLM-ScFv-EGFP fusion protein was expressed in CHO cells and secreted into the supernatant. PreS2-TLM-ScFv-EGFP fusion protein in the supernatant could be specifically internalized into HepG2.2.15cells and located in the cytoplasm. PreS2-TLM-ScFv-HBcDN fusion protein was expressed in CHO cells and secreted into the supernatant. PreS2-TLM-ScFv-HBcDN fusion protein in the supernatant can inhibit the package of pregenomic RNA in the viral core particle. The inhibition effects of the supernatant containing the fusion protein PreS2-TLM-ScFv-HBcDN on HBV replication in HepG2.2.15cells were related to the concentration and time exposed to the supernatant containing the fusion protein PreS2-TLM-ScFv-HBcDN.25%was the optimal concentration of inhibiting the replication of virus.