5-Fluorouracil combined with apigenin enhances anticancer activity through mitochondrial membrane potential (ΔΨm)-mediated apoptosis in hepatocellular carcinoma.

The development of chemoresistance may reduce the efficacy of chemotherapeutic drugs for treating hepatocellular carcinoma (HCC). In the present study, the effects of apigenin on intensifying the chemosensitivity of HCC cells and an HCC xenograft model in response to 5-fluorouracil (5-FU) were investigated. Sub-toxic concentrations of apigenin (4 µmol/L) significantly enhanced the cytotoxicity of 5-FU (100 µg/mL) in HCC cells. In vivo, combined treatment with apigenin (20 mg/kg, five times/week for 3 weeks) and 5-FU (20 mg/kg for 5 consecutive days) significantly inhibited the growth of HCC xenograft tumours. Annexin V-propidium iodide dual staining assays, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling assays and western blotting analysis were used to confirm the synergistic effects of apigenin and 5-FU on HCC apoptosis. Coincubation of HCC cells with apigenin and 5-FU increased levels of reactive oxygen species (ROS), which was followed by a decrease in the mitochondrial membrane potential (ΔΨm). In addition, combined triggered the mitochondrial apoptotic pathway, as indicated by decreased Bcl-2 expression and loss of ΔΨm, with significant activation of caspase 3 and poly(ADP-ribose) polymerase. The present study is the first to demonstrate that apigenin may potentiate the cytotoxicity of 5-FU in HCC via inhibition of ROS-mediated drug resistance and concurrent activation of the mitochondrial pathways of apoptosis.

[Effect of ozonized saline on signaling passway of Keap1-Nrf2-ARE in rat hepatocytes].

OBJECTIVE: To study the effect of ozonized saline on the activation of the Keap1-Nrf2-ARE signaling pathway in rat liver cells. METHODS: Twenty male Sprague-Dawley rats were randomly divided into ozonized saline (OS) group, model group, ozonized saline control (OSC) group and normal control (NC) group. The rats in OS group and model group were intravenously administered with OS or oxygen saline (5 ml/kg) respectively, once a day for 15 days, and then intraperitoneally injected with CCl4 dissolved in oliver oil. The rats in OSC group were pretreated with OS for 15 days. The rats in NC group were fed normally for 15 days. On the 16th day, the rats in OSC group and NC group were intraperitoneally injected with oliver oil (2 ml/kg) without CCl4. After 24 hours of CCl4 or olive oil intraperitoneal injection, the serum levels of alanine transaminase (ALT) and aspertate aminotransferase (AST) were measured. The liver tissues were also collected for detection of total anti-oxygen capability (TAOC), glutathione (GSH), catalase (CAT), Glutathione peroxidase (GPx). Western Blot was used to detect Nrf2 and immunofluorescence staining assay to display intracelluar distribution of Nrf2. RESULTS: Compared with the rats in model group,the serum ALT and AST levels of rats in OS group were significantly lower (P < 0.01) ,which were (1240.4 ± 188.2) U/L and (1245.4 ± 176.9) U/L vs (539.8 ± 175.3) U/L and (546.0 ± 130.2) U/L, and the TAOC, CAT, GPx and GSH activity of rats in OS group were significantly higher, which were (0.72 ± 0.24) U/mg, (1.05 ± 0.21) mg/g, (676.9 ± 115.1) U/mg and (45.2 ± 14.3) U/mg vs (1.37 ± 0.19) U/mg, (2.23 ± 0.55) mg/g, (1024.6 ± 162.9) U/mg and (68.2 ± 9.9) U/mg, respectively. In contrast with NC group, pretreatment of OS in OSC group elevated TAOC, CAT, GPx and GSH activity (P < 0.01 or P < 0.05). Ozonized saline can strengthen the Nrf2 expression in liver cells. CONCLUSION: Preconditioning injection of ozonized saline can reduce rat's liver injury induced by CCl4. The ozonized saline, as a novel Nrf2 activator, can reduce the oxidative damage of radical oxygen species (ROS) and the deleterious substance by activating the Keap1-Nrf2-ARE signaling pathway and its downstream genes expression.

[Safety and efficacy of Entecavir combined with Adefovir in patients with chronic hepatitis B who fail to respond to nucleoside analog treatment].

OBJECTIVE: To evaluate the safety and efficacy of the combined therapy with entecavir (ETV) and adefovir (ADV) in patients with chronic hepatitis B (CHB) who experienced failure of treatment with single or multiple nucleoside analogs, and analyze the factors that affect the patients response to the treatment. METHODS: Forty-five CHB patients who experienced treatment failure with sequential or/and combined nucleoside analogs received the combined therapy with entecavir and adefovir lasting for at least 6 months. The viroloigcal response (VR), biochemical response (BR) and combined response (CR) at 24 and 48 weeks of the treatment were evaluated. Univairante analysis was used to identify the factors that affect the response to the anti-viral therapy. RESULTS: The VR, BR and CR were 67.7%, 77.8% and 57.8% at 24 weeks, as compared to 76.2%, 78.6% and 61.9% at 48 weeks, respectively. The VR differed significantly between patients with a baseline HBV DNA level [lg(copies/ml)] of 3-6 and those with a level over 6 (85.2% vs 40%, Z=-4.796, P=0.037) at 48 weeks. The presence and absence of cirrhosis at the initial treatment significantly affected the BR at 24 weeks (17.1% vs 82.9%, P=0.048) and at 48 weeks (23.8% vs 76.2%, P=0.023). CONCLUSION: Entecavir combined with adefovir is an effective rescue therapy in CHB patients after failure of treatment with nucleoside analogs. Patients with a lower baseline HBV DNA level without cirrhosis may have better response to the combined treatment.

[A novel method for hepatitis C virus genotyping using RT-PCR reverse dot blot hybridization technique].

OBJECTIVE: To develop a rapid and specific method for hepatitis C virus ( HCV) genotyping using reverse dot blot hybridization technique and investigate the distribution of HCV genotypes and subtypes in Guangdong. METHODS: The primers and the probes targeting the 5'untranslated region (5'UTR) and core region of HCV genotypes 1b, 2a, 3a, 3b and 6a were designed, and the RT-PCR reverse dot blot hybridization (PCR-RDH) method for HCV genotyping was established. A total of 115 patients with hepatitis C were genotyped using this method, and 38 of them were also genotyped by sequencing and phylogenetic analysis to evaluate the accuracy and specificity of the method. RESULTS: Of the 115 patients, 111 were successfully genotyped to be 1b, 2a, 3a, 3b, 6a and mix-infection of 1b/2a at frequencies of 56.8%, 8.1 %, 3.6%, 5.4%, 25.2% and 0.9% respectively, and all the 15 healthy control samples showed negative results. The accuracy and reliability of the genotyping method of PCR-RDH was confirmed in 38 cases by amplification of HCV core and NS5B regions followed by DNA sequencing and phylogenetic analysis. CONCLUSION: This method for HCV genotyping, with high reliability and specificity, is suitable for clinical and epidemiological investigations. The prevalence of HCV genotypes 1b and 2a decreases while 1b remains the dominant genotype in Guangdong, where the prevalence of 6a significantly increases as compared with that 10 years ago.

[Expression of IFN-alpha/beta receptor in the PBMCs and liver of patients with hepatitis B and its clinical significance].

OBJECTIVE: To explore the role of interferon (IFN)-alpha/beta receptor beta subunit (IFNAR2) in the patients' response to IFN-alpha therapy as influenced by the grade of chronic hepatic inflammation, and understand the relation of IFNAR2 expression in the peripheral blood mononuclear cells (PBMCs) with HBV infection. METHODS: Liver tissue specimens were obtained from 21 patients with chronic hepatitis B for examination of the hepatic inflammation, and PBMCs were isolated from another 16 patients with chronic hepatitis B and 15 health control subjects. Both the hepatic tissues and PBMCs were examined for IFNAR2 expression using immunohistochemistry. RESULTS: The 21 patients with chronic hepatitis B were divided into 3 groups according to the severity of hepatic inflammation, namely G(1) (n=3), G(2) (n=7) and G(3) (n=11) groups. The patients in G(3) group showed had significantly higher IFNAR2 expressions in liver (25.1307-/+7.0700) than those of the G(1) (5.6913-/+1.8422) and G(2) (7.4706-/+5.3572) groups (P=0.000). The IFNAR2 levels in the PBMCs, however, did not show significant difference between patients with chronic hepatitis B and the healthy control subjects. CONCLUSION: In patients with chronic hepatitis B, IFNAR2 expression level is positively correlated to the severity of hepatic inflammation, and increased IFNAR2 expression in severe hepatic inflammation is therefore likely to result in increased response rate to INF-alpha therapy. The expression of IFNAR2 in the PBMCs is not associated with HBV infection.

[Changes of serum HBsAg in HBeAg positive chronic hepatitis patients with sustained viral response to long-term lamivudine treatment].

OBJECTIVE: HBsAg loss is rare in chronic hepatitis B patients, even in the patients with long-term nucleos(t)ide analogue therapy; therefore information about serum HBsAg kinetics will be of value in understanding this unusual occurrence. METHODS: Forty-five consecutive patients were studied, which were all HBeAg positive and never had antiviral therapy prior to lamivudine treatment; they then achieved rapid and good viral responses (defined as undetectable HBV DNA [Roche Lightcycler, less than 1000 copies/ml] at treatment week 24 and they remained so until week 156). Abbott Architect HBsAg assay was used to quantify serum HBsAg and HBV genotypes were determined by direct sequencing. RESULTS: Twenty-six (57.8%) patients had HBeAg loss during the observation and one patient had HBsAg loss following his HBeAg seroconversion. Serum HBsAg levels decreased to 39.5% (median) of their baseline values at week 12, but no further significant reductions of serum HBsAg were found afterwards. Changes of serum HBsAg were comparable between patients with or without HBeAg loss. Serum HBsAg levels at their baselines were higher in HBV genotype B (HBV/B, n = 21) patients than in genotype C (HBV/C, n = 24) patients. HBV/B patients achieved many more HBsAg reductions than HBV/C ones (75.5 vs. 26.0%, median, P less than 0.05) in the first 12 treatment weeks, however HBsAg levels at week 156 were comparable between these two subgroups. HBsAg changes mainly showed two distinct patterns: a biphasic pattern (HBsAg levels were less than 60% of baseline ones at week 12 and 24, n = 25) and a maintaining pattern (HBsAg levels were greater than 80% of the baseline ones at week 12 and 24, n = 14). Logistic regression analysis showed that low serum HBsAg at baseline (odds ratio 0.020, 95% confidence interval 0.002-0.743, P less than 0.05) and HBV/C infection (odds ratio 8.206, 95% confidence interval 1.070-62.948, P less than 0.05) were the determinants of the occurrences of the maintaining pattern. CONCLUSION: In patients we examined, their HBsAg changes were mainly presented as either a biphasic pattern or a maintaining pattern, which were associated with HBV genotypes (B/C) but not with HBeAg loss. This might explain that why HBsAg loss is a rare occurrence even with long-term lamivudine therapy.

[Factors influencing the success rate and stability of transient elastography for liver fibrosis evaluation].

OBJECTIVE: To investigate the factors influencing the success rate and stability of transient elastography(FibroScan)for assessment of liver fibrosis. METHODS: Liver stiffness was assessed using transient elastography in totally 637 subjects including healthy subjects, asymptomatic hepatitis B virus (HBV) carriers, patients with chronic hepatitis B and patients with HBV-related cirrhosis. Of these subjects, 302 received 2 examinations and totalling 939 examinations were performed. In each case, one operator performed 2 consecutive series of 10 validated measurements, or 2 operators performed a series of 10 validated measurements. The factors including gender, age, body mass index (BMI) and the state of diseases were analyzed for their association with the success of the examination. Intraclass correlation coefficient (ICC) was used to evaluate the reproducibility of the operation. RESULTS: Failure of the measurement occurred in 14 cases (2.2%), which was not associated with the age of the subjects and the state of diseases. The success rate of measurement decreased as the BMI increased (t=3.112, P=0.002), and was lower in female subjects (t=-2.193, P=0.029). The intra- and inter-operator stability of liver stiffness measurement was satisfactory, with ICC of 0.970 and 0.847, respectively. But for healthy subjects and asymptomatic HBV carriers, the stability was lower, with ICC of 0.736 and 0.639, respectively. Liver stiffness in patients with liver cirrhosis was positively correlated to complications and Child-Turcotte-Pugh (CTP) score. CONCLUSION: Liver stiffness measurement has high stability with FibroScan, and high BMI could lower success rate of the measurement. Liver stiffness as measured by FibroScan allows prediction of the liver function and presence of complications in patients with liver cirrhosis.

Protective effects of medical ozone combined with traditional Chinese medicine against chemically-induced hepatic injury in dogs.

AIM: To investigate the protective effect of medical ozone (O(3)) combined with Traditional Chinese Medicine (TCM) Yigan Fuzheng Paidu Capsules (YC) against carbon tetrachloride (CCl(4))-induced hepatic injury in dogs. METHODS: Thirty healthy dogs were divided randomly into five groups (n = 6 in each group), namely control, oleanolic acid tablet (OAT), O(3), YC and O(3) + YC, given either no particular pre-treatment, oral OAT, medical ozone rectal insulfflation every other day, oral YC, or oral YC plus medical ozone rectal insulfflation every other day, respectively, for 30 consecutive days. After pre-treatment, acute hepatic injury was induced in all dogs with a single-dose intraperitoneal injection of CCl(4). General condition and survival time were recorded. The biochemical and hematological indexes of alanine aminotransferase (ALT), aspartate aminotransferase/alanine aminotransferase (AST/ALT), serum total bilirubin (TBIL), prothrombin time (PT), blood ammonia (AMMO), and blood urea nitrogen (BUN) were measured after CCl(4) injection. Hepatic pathological changes were also observed. RESULTS: Compared to the other four groups, the changes of group O(3) + YC dogs' general conditions (motoricity, mental state, eating, urination and defecation) could be better controlled. In group O(3) + YC the survival rates were higher (P < 0.05 vs group control). AST/ALT values were kept within a normal level in group O(3) + YC. Hepatic histopathology showed that hepatic injury in group O(3) + YC was less serious than those in the other four groups. CONCLUSION: Medical ozone combined with TCM YC could exert a protective effect on acute liver injury induced by CCl(4).

[Protective effect of Yigan Fuzheng Paidu capsules combined with ozone on CCl4-induced acute hepatic injury in dogs].

OBJECTIVE: To investigate the protective effect of Yigan Fuzheng Paidu Capsules (YC) combined with medical ozone against hepatic injury in dogs induced by hepatotoxic drug. METHODS: Twenty-four dogs were randomized equally into 4 groups (n=6), namely the model group, oleanolic acid tablet (OAT) group, YC group and YC+O(3) group, given either no particular treatment, oral OAT at 10 mg/day, oral YC at 0.2 g/day, or YC at 0.2 g/day plus 150 ml medical ozone transrectal insufflation every other day, respectively, for totally 30 consecutive days. Acute hepatic injury was induced after the treatment in the dogs with a sing-dose intraperitoneal injection of 0.9 ml/kg CCl(4) and peanut oil mixture (1:1, W/W). The general condition, survival time, alanine aminotransferase (ALT), aspartate aminotransferase/alanine aminotransferase (AST/ALT), serum total bilirubin (TBIL), prothrombin time (PT), blood ammonia (AMMO), and blood urea nitrogen (BUN) were recorded or measured. The hepatic pathological changes were observed upon death or on day 15 following CCl(4) injection. RESULTS: Compared with the other 3 treatment protocols, YC plus O(3) showed favorable effects on the activity, mental state, diet, urination and defecation of the dogs, which had significantly higher survival rate and higher levels of ALT, TBIL, PT, and AMMO than the model and OAT groups (P<0.05). AST/ALT remained normal in YC+O(3) group, which had also milder hepatic injury than the other 3 groups. CONCLUSIONS: YC combined with medical ozone may decrease transaminase and blood ammonia levels, relieve jaundice, prolong the survival time of dogs with CCl(4)-induced hepatic injury.

[A comparison of clinical and virological characteristics of 1686 cases of HBeAg-negative and HBeAg-positive chronic hepatitis B].

OBJECTIVE: To investigate the clinical characteristics of HBeAg-negative and HBeAg-positive chronic hepatitis B (CHB). METHODS: A total of 1686 hospitalized CHB cases were analyzed retrospectively. The serum ALT values, HBV DNA levels and hepatic inflammation and fibrosis were analyzed by their serum HBeAg status. RESULTS: Among the 1686 cases, 628 (37.3%) were HBeAg-negative and 1058 (62.7%) were HBeAg-positive. Compared with HBeAg-positive group, HBeAg-negative group had a lower serum ALT and HBV DNA levels. However, hepatic necroinflammation grading and fibrosis staging in HBeAg-negative group were more advanced than that of HBeAg-positive group. Irrespective to serum HBeAg status, patients with serum HBV DNA less than 10(5)copies/ml, had a lower hepatic necroinflammation activity. CONCLUSIONS: HBeAg-positive CHB is still the predominant form of CHB in Chinese patients. Compared with patients with low HBV replication, patients with active HBV replication had a higher hepatic necroinflammation activity. The liver histological grading and staging in HBeAg-negative CHB patients were more advanced than that in HBeAg-positive patients.

[Influences of the genotypes of HBV and HBeAg regarding their response to PEG-IFN in chronic hepatitis B patients].

OBJECTIVE: To study the effects of genotypes of HBV and HBeAg on the response to PEG-interferon alpha (PEG-IFN) in chronic hepatitis B (CHB) patients. METHODS: PCR-RFLP and S gene sequencing were conducted in 42 CHB patients. RESULTS: The sustained response (SR) rates were 66.7% in genotype B and 27.3% in genotype C group. The P value was 0.039 by the Pearson Chi-square test, while it was 0.06 by the Fisher's exact test. The results suggested a trend that patients with genotype B HBV compared to genotype C had better SR to PEG-IFN therapy, although the difference was not significant. Results also showed that SR rate in patients with HBeAg-negative CHB (7/8 87.5%) was significantly higher than that in HBe+ CHB patients (8/21 38.1%, P < 0.05). CONCLUSION: Our results indicate that HBV genotype and HBeAg, especially the later, are main factors for predicting PEG-IFN therapy response in CHB patients.

[Genotyping of hepatitis B virus and its clinical significance in patients with chronic hepatitis B].

OBJECTIVE: To investigate the distribution of hepatitis B virus (HBV) genotypes in Guangdong and explore its clinical significance. METHODS: Fifty-five patients with chronic active hepatitis (CAH) from Guangdong province were included in this study. HBV surface gene amplified by PCR was analyzed by restriction fragments length polymorphism (RFLP) for HBV genotyping, and the relationship of HBV genotype with clinical, serological and histological data of the patients was analyzed. RESULTS: Twenty-eight out of 55 patients were infected with HBV strains of genotype B (51.0%), 18 with genotype C (32.7%), 4 with genotype D (7.3%), 4 with HBV classified as genotype B+C (7.3%), and 1 (1.8%) with HBV that did not conform to any of the genotypes from A to G. No significant differences in clinical, histological, or serological data of HBV DNA loading were detected between genotypes B and C. But in patients older than 30 years, the genotype C was accompanied by significantly higher HBV DNA loading and serum HBeAg level than genotype B (Fisher's exact test, P=0.002). CONCLUSIONS: HBV genotype B and C are the major genotypes prevalent in Guangdong Province. Genotype C is associated with the longer duration of HBeAg and higher HBV DNA level in patients older than 30 years, suggesting the higher risk of HBV genotype C infection to progress into chronic liver disease.

[Efficacy of interferon-alpha therapy for HBeAg-negative chronic hepatitis B and its influencing factors].

OBJECTIVE: To investigate the efficacy of interferon-alpha (IFN-alpha) therapy for HBeAg-negative chronic hepatitis B. METHODS: Sixty-five Chinese HBeAg-negative chronic hepatitis B patients were treated with 5 MU recombinant rIFN-alpha 1b subcutaneously thrice weekly for 5 to 24 months, followed by 12 months of treatment-free follow-up; one hundred and eighty-eight Chinese HBeAg-positive patients served as controls. For each patient, serum alanine transaminase (ALT) was measured biochemically and serum HBV DNA level was detected with fluorescent-quantitative PCR, HBeAg with enzymoimmunoassay every 1 to 3 months during therapy and during the follow-up period. HBeAg loss (only for HBeAg-positive cases), HBV DNA undetectable, and ALT normalization: the three together were considered a combined response. RESULTS: Rates of combined response were similar in HBeAg-negative patients (58.5%, 38/65) or HBeAg-positive ones at the end of treatment (weighted chi square test, chi2 = 1.878, P<0.05), but were higher at the end of the follow-up period in the HBeAg-negative cases (75.4%, 49/65) (weighted chi square test, chi2 = 4.796, P<0.05). Furthermore, relapse rates at the end of the follow-up period, were also similar in HBeAg-negative patients (15.8%, 6/38) or HBeAg positive (chi2 = 0.205, P>0.05). Combined response was achieved at a median of 6.0 months (2-16 months) of treatment course in HBeAg-negative patients while at a median of 6.0 months (1-22 months) in HBeAg-positive cases (Z = -0.186, P>0.05, by the Wilcoxon rank sum test). The only factor predictive of combined response, by binary logistic regression analysis, was inflammatory activity in the liver biopsy. Gender, age, baseline ALT level, baseline HBV DNA level, and anti-HBe were not predictive factors. CONCLUSION: Interferon-alpha therapy induces a similar primary and sustained response in HBeAg-negative and in HBeAg-positive chronic hepatitis B patients.

[Efficient and constant expression of cloned human beta2-microglobulin gene in E.coli].

OBJECTIVE: To obtain human beta2-microglobulin (beta2m) gene that is to be efficiently expressed in E.coli. METHODS: beta2m cDNA including only the coding region for the protein was amplified from Raji cell line by reverse transcriptase-PCR via the primers 5'-GGTGGTCATATGGCTATCCAGCGTACTCCA-3' and 3'-GGTGGTTGCTCTTCCGACATGTCTCGATCC-3'. The product was subsequently cloned into a modified pBV220 vector after digestion with Nde I/Sap I. The recombinant plasmid pBV220-beta2m was transformed into E.coli BL21 after sequence analysis, and the fusion protein was then expressed via induction at 42 for 5 h at D(600) of 0.55-0.60, followed by purification through chitin beads. RESULTS: The beta2m cDNA was identical with those published in Genbank. The expressed fusion protein was identified in the form of inclusion body at the ratio more than 45 % of the E.coli proteins, and was denatured with 8 mol/L urea, followed by refold and purification to a high purity, displaying a relative molecular mass of 12 000 on 10 % SDS-PAGE gel. CONCLUSION: The human beta2m gene was cloned successfully and expressed efficiently and constantly in E.coli BL21,which lays the ground for engineering MHC-tetramers.

[Application of multiplex PCR in genotyping of hepatitis B virus prevailing in Guangdong Province of China].

OBJECTIVE: To establish a convenient method for the genotyping of hepatitis B virus (HBV) using multiplex PCR. METHOD: Based on the alignment of 114 complete nucleotide sequences of HBV DNA belonging to different genotypes, acquired from the GenBank, genotype-specific sequences were identified according to which 6 pairs of primers were designed corresponding to each genotype. Subsequent genotyping of HBV was performed using these primers that were added, either alone or in conjunction with others, into a multiplex PCR reaction tube, and HBV genotype was determined according to the length of amplified DNA. RESULT: The genotyping result of multiplex PCR was consistent with that produced by PCR- restriction fragment length polymorphism as established by Lindh. We found in this study that among the HBV carriers in the vicinities Guangzhou of City, about 45% belonged to B genotype, 38.75% to C genotype and 16.75% to D genotype. CONCLUSION: This multiplex PCR method is simple, convenient and more differential.

[Effects of partial deletion in the core promoter of hepatitis B virus genome on viral antigen expressions].

OBJECTIVE: To study the effects of 20/21 bp partial deletion mutation (from nt 1 748 or nt 1 747 to nt 1 767) in the core promoter (CP) region of hepatitis B virus (HBV) genome complicated by precore stop condon mutation at nt 1 896 on the expression of the viral antigens. METHODS: Eukaryotic expression vector containing full-length HBV genome with the above mutations was constructed. After transfection of the recombinant HBV plasmids into HepG2 cells, the expression of the viral antigens was examined with enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. RESULTS: As shown by ELISA and Western blotting analysis, the amount of extracellular secretion of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) along with intracellular hepatitis B core antigen (HBcAg) in the cells transfected with vectors containing HBV genomes with partial deletion in the CP region was markedly reduced compared with that produced by wild-type HBV. CONCLUSION: The mutations in question causes marked reduction in viral antigen production by HBV in comparison that by wild-type HBV.

Construction of recombinant eukaryotic expression plasmid containing hepatitis B virus genome with partial deletion of the core promoter.

OBJECTIVE: To construct an eukaryotic expression vector containing the full-length genome with partially deleted core promoter of hepatitis B virus (HBV). METHODS: A linearized genome containing the entire HBV 3.5 kb mRNA transcriptional units (P3.8 I plasmid) was used, which initiated from the upstream sequences of the basic core promoter. The objective eukaryotic expression vector was constructed by molecular cloning and PCR-based site-directed mutagensis in vitro, and identifcation was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by cloning and sequencing analysis. RESULTS: The eukaryotic expression vectors containing HBV genomes with 20/21 bp deletion (position 1 748/1 747 to 1 767) in the core promoter or with precore stop mutation at nucleotide 1896 as well were constructed successfully as confirmed by sequence analysis with RFLP. CONCLUSION: The recombinant expression vector may lay the foundation for further studies into the biological significance of the above mentioned mutations in vitro.