Expression, Purification And Application Of DHBV Capsid Protein, And The Time Courses Of Transcriptional And Expressional Analysis Of DHBV PreC/C Gene

1. Sequence analysis of DHBV PreC/C gene and its cloning, expression, protein purification and the preparation of polyclonal antibody against DHBV capsid proteinIn order to developing a method that can currently and rapidly detect the DHBV infection in clinical and to explore the functions of DHBV capsid protein deeply, the PreC/C gene of DHBV CHv strain and its encoding protein was analyzed by bioinformatics methods. The PreC/C gene fragments were obtained through PCR amplication and then constructed a prokaryotic expression vector pET-32a(+)/PreC/C after its sequencing confirmation. The expression E.coli strains Rosetta (DE3) was used to expressed the DHBV capsid protein and the induction conditions were optimized in different temperatures, different induction time and different concentrations of IPTG. After the recombinant protein was expressed and purified, it was used to immune the rabbit to prepare the polyclonal antibody against DHBV capsid protein. The results showed that the whole length of DHBV genome was 3006 bp and the PreC/C gene was 789 bp, which encoded DHBV capsid protein composed of 262 amino acids. When the recombinant capsid protein was induced in different conditions, the expression level of the protein would reach the peak with 6 hours induction under 37℃ and 0.8mM IPTG. In the optimal conditions of induction, the expressed protein recombinant capsid protein account for about 80% of the total proteins of E. coli. After the recombinant DHBV capsid protein was purified through Ni-agarose by its His targets, it showed a single band after SDS-PAGE and the concentration was about 2mg/ml. The polyclonal antibody against capsid protein was prepared and the titer was detected through agar diffusion reaction which showed a result of 1:32.2. The development of indirect ELISA based on the recombinant DHBV capsid proteinTo develop stable and reliable serologic method for DHBV diagnosis, this study established an indirect ELISA methods for DHBV diagnosis based on DHBV recombinant capsid protein. Firstly, a checkerboard titration was performed to determine the optimal working concentrations of coating antigen, then the time and temperature of coating antigen, the time of block and the concentration of HRP labeled second antibody were optimized. Based on the optimal conditions, the cut-off value of the indirect ELISA was determined by detecting 27 DHBV negative serum samples. According to the cut-off value, the sensibility, specificity, repeatability and reliability of the ELISA were evaluated. The results showed that the ELISA works best when the antigen was coated 6.7μg/well for overnight at 4℃ after 2 hours at 37℃, blocked with 1% BSA for 2 hours at 37℃ and to use 1:1000 HRP labeled second antibody. The sensitivity assay showed when the positive serum was diluted to 1:128000, it could be detected as positive. The repeatability assay showed that the variable coefficient in inter-and intra-assay were both below than 10%. Known anti-sera samples of other duck common pathogens were tested by the developed ELISA and the results showed it was specific for DHBV anti-sera detection. Compared this method to routine PCR for DHBV detection, it showed a 95.45% coincidence rate when detected 75 clinical serum samples suspected of DHBV infection. In short, our developed indirect ELISA has the characteristics of high sensitivity, well repeatability and strong specificity. In the mean time, the goat anti-duck HRP antibodies were used in our study to replace the two antibodies of rabbit anti-duck and goat anti-rabbit which were used in the previous DHBcAg ELISA protocols. This improvement makes the method more simple and save the cost.3. Time courses of transcriptional and expressional analysis of DHBV PreC/C geneIn the process of DHBV replication, the whole viral genome will transcribe to a long chain RNA called preRNA, which has there functions as we know so far. One is that it can be the intermediate of the replication of DHBV genome, because it can reverse transcribe to DNA from RNA. The second role it plays is the mRNA of DHBV P protein, which can translated to DNA polymerase P proteins. The third role it plays is to translated to DHBV capsid proteins. In order to study whether the preRNA is used to translated to capsid protein at the early phase of infection, we firstly developed a infection model, which used the primary duck hepatocytes. Then the real-time quantitative PCR was used to detect the relative amount of preRNA at different time points after DHBV infection. At the same time points, the expression of DHBV capsid protein was analyzed by using indirect immunofluorescence and western blotting. The results showed the preRNA was appeared after 12 hours of the DHBV infection and reached its peak at 20 hours post-infection. However, the capsid protein was not expressed at 12 or 20 hours post-infection, it just can be detected at the 24 hours post-infection. This results indicated that the preRNA was not used to translation of the capsid protein in the early phase of viral infection. This study and data provided a novel idea of the assembly of virus particles during the infection course.