The Construction Of Recombinant B And C Genotype Hepatitis B Virus And Relative Studies

Objective:1. To construct recombinant full length B and C genotype hepatitis B virus, examining the ability of replication and expression of them in Huh7 cells.2. Exploring whether the change of global gene expression pattern is similar or not in huh7 cells transfected with B or C genotype HBV.3. To assess the function of shRNAs inhibiting HBsAg and HBeAg expression, HBsAg mRNA production, and HBV DNA replicative intermediate in B and C genotype HBV under the condition of mismatching between shRNAs and HBV.Methods1. Designing the primers for full length HBV genome, exstracting the HBV DNA from two patients infected with B and C genotype hepatits B virus respectively as template, amplifying the full length HBV with high fidelity hot-start Tag DNA polymerase;2. full length HBV and pUC19 were cut with SacⅠ, then inserting HBV into the site of Sac I in pUC19 with T4 DNA ligase, named as pUC19-BHBV and pUC19-CHBV;3. pUC19-HBV and pHY106 were cut by Sap I , then inserting HBV into the site of Sap I in pHY106 with T4 DNA ligase, named as pHY106-BHBV and pHY106-CHBV;4. pHY106-BHBV and pHY106-CHBV were transfected into Huh7 cells, pHY106 as control;①Collected supernatant of culture at 24 h, 48 h, 72 h after transfection for examining HBsAg and HBeAg with an ELISA kit;②lysated the cells, exstracted the HBV replicative intermediate from HBV core for Southern blot at 72 h after transfection;③72 h after transfection, washed cells with PBS buffer for 5 times, then lysated the cells, examined the HBV DNA level by real-time PCR quantitatively;5. cDNA microarray: Huh7 cells were transfected with recombinant B or C genotype HBV, namely pHY106-BHBV and pHY106-CHBV, pHY106 as control; 48 h after transfection, mRNA of cells were extracted, and reverse transcripted to cDNA, then hybrided with chip; The fluorescence signal intensity of Cy3 and Cy5 was analyzed with the GenePix Pro 3.0 software; the ratio of Cy5/Cy3 represent the differential value, the ratio above 2 or below 0.5 was regarded as significant data;6. Real-time PCR: Huh7 cells were transfected with pHY106-BHBV or pHY106-CHBV, pHY106 as control; 48 h after transfection, the total RNA were extracted, and reverse transcripted to cDNA, validated the partial results of microarray with real-time PCR;7. shRNA and pHY106-BHBV or pHY106-CHBV cotransfected HepG2 cells, the supernatant of the culture at 24 h, 48 h, 72 h, 96 h and 120 h after cotransfection was collected and frozen in -20℃for examining HBsAg and HBeAg level by an ELISA kit;8. 72 h after shRNA and pHY106-BHBV or pHY106-CHBV cotransfed, the total RNA of cells was extracted and reverse-transcripted, then examining the level of HBsAg mRNA by RT-PCR (reverse transcript polymerase chain reaction); 72 h after cotransfection, cells were lysated, HBV DNA replicative intermediate from HBV core was extracted and detected by Southern blot.Results:1. Constructing the recombinant full length B and C genotype HBV successfully;2. The expressiong of HBsAg and HBeAg were detectable after pHY106-BHBV and pHY106-CHBV transfected into Huh7 cells, the peak time was 48 h for HBsAg expression, the peak time for HBeAg expression is about 24 h late compared with that of HBsAg;3. Southern blot detected the HBV replicative intermediate from HBV core, including rcDNA, dsDNA and ssDNA.4. pHY106-BHBV and pHY106-CHBV could replicate after transfected into Huh7 cells, the HBV DNA leve get the peak at 72 h after transfection, the peak was about 8 log 10.5. 48 h after pHY106-BHBV transfected Huh7 cells, 60 differentially expressed genes were screened from total 4097 genes, among of them, one was overexpressed, the other 59 genes were expressed at lower levels; 6. 122 differentially expressed genes were screened from total 4097 genes after pHY106-CHBV transfected Huh7 cells, among of them, 34 genes were expressed at higher levels, the other 88 genes were expressed at lower levels;7. IFNGR2, GPR125, DDEF2 and PI3K which were down-regulatory genes after pHY106-BHBV transfected Huh7 cells with microarray analysis were validated by real-time PCR, the relative expression ratio of them were 0.8, 0.5, 0.3 and 0.4 respectively, which was consistent with the results of microarray; EEF2 and INF592 which were down-regulatory genes after pHY106-CHBV transfected Huh7 cells with microarray analysis were validated by real- time PCR, the relative expression ratio of them were 0.8 and 0.6, respectively, which was consistent with the results of microarray; only DDEF2 are found down-regulated in both microarrays;8. HBsAg and HBeAg expression were inhibited obviously by shRNA-458 and shRNA·635, the inhibitory action was detectable at 48 h after cotransfection, the peak time was 72 h, the most inhibitory ratio was approximately 80% and 50% in HBsAg and HBeAg; there was no difference was observed about the the inhibitory action of shRNA-458 and shRNA-635 on B or C genotype HBV;9. shRNA-458 and shRNA-635 had the similar inhibitory action on the production of HBsAg mRNA from B and C genotype HBV after 72 h of cotransfection, the inhibitory ratio was 60%-70%;10. HBV DNA replicative intermediate from B and C genotype was inhibited obviously by the two shRNAs also.Conclusions:1. pHY106-BHBV and pHY106-CHBV can replicate and express HBsAg and HBeAg in Huh7 cells, which is suitable for studying the pathogenesis of B and C genotype HBV, the interaction between HBV and host, as well as exploiting new drugs againt HBV.2. B and C genotype HBV all can change the expression pattern of Huh7 cells; but the change of expression pattern of Huh7 cells by B and C genotype HBV are obviously different, only DDEF2 are found down-regulated in both; which demonstrate B and C genotype HBV may differently influence on host. shRNA·458 and shRNA·635 have the powerfully inhibitory action on the expression3. of HBsAg and HBeAg, the production HBsAg mRNA and HBV replicativeintermediate from B and C genotype HBV although 2-4 bases mismatch betweenshRNAs and HBV, so shRNA-458 and shRNA-635 are two useful tools for inhibitHBV.