Objective To define that human cytomegalovirus (HCMV) can cross the placenta of the Balb/c mice and induce liver damage in developing fetus. Methods HCMV AD169 (6.0 logTCID 50 , 3.0 logTCID 50 , 1.5 logTCID 50 per mouse respectively) was injected into the peritoneum of mice (half of mice were female) when they were about 10 weeks old (weight 25 30g). Then, these mice were paired to mate. Fetus on day about to give birth was removed from the uterus and liver was obtained for virus isolation, pathological studies, examination of the viral DNA positive cells by in situ hybridization using digoxigenin labelled HCMV DNA oligonucleotide probe. Results HCMV could be isolated from the supernatant of tissues; HCMV DNA was found by PCR in supernatant of cell culture with CPE; the presence of viral DNA sequence in hepatocytes was confirmed by in situ hybridization; pathological changes in liver consist of swollen cytoplasm and destroyed nuclei of hepatocytes and distinct intranuclear inclusion in hepatocytes with a cellular infiltrates of predominantly phagocytic cells. All above were found more obviously in fetal mouse liver tissues from the group inoculated with 6.0 log TCID 50 HCMV AD169 as compared with 3.0 log TCID 50 group. In contrast, these positive results couldn't be well found in 1.5 log TCID 50 group. Nothing could be found in normal controls. Conclusions Our research suggests that primary maternal HCMV infection during pregnancy could induce congenital infection in fetus by transplacental transmission and induce fetal liver damage. The mouse model will provide the basis for the study on pathogenesis of congenital HCMV infection in liver and the development of prevention, diagnosis and antiviral agents for congenital HCMV infection in human being.

Objective To construcr a phage display library of human single-chain Fv antibodies from peripheral blood lymphocytes of people with positive serum antibody against hepatitis B virus core protein. Methods The heavy chain and light chain variable region gene of human immunoglobulin were amplified individually by RT-PCR from peripheral blood Lymphocytes mRNA of patients with positive serum hepatitis B virus core protein antibody and randomly combined through a DNA linker encoding the peptide (Gly 4Ser) 3 to construct single-chain variable fragments (ScFv)gene. In the presence of helper phage M13KO7, the ScFv were displayed on the surface of recombinant phages. Results Repertoire antibody library was obtained with 10 6 clonies resistant to ampicillin and kanamycin. Conclusions A typical phage display library of human single-chain Fv antibodies has been constructed Using RT-polymerase chain reaction (RT-PCR) and phage display technique, human single-chain Fv antibodies can be screened from this library.

0bjective To observe the cell membrane penetration of protein transduction domain (PTD)-HBeAg fusion protein in vitro.Methods The sequence of trans-activator of transcription (Tat)-PTD was synthesized and the whole HBcAg gene was amplified by polymerase chain reaction (PCR).Overlap extension PCR was employed to fuse Tat-PTD and whole HBcAg gene.Then the fusion gene was cloned into prokaryotic expression vector pMAL-c2X.The correct vector was transformed into E.coli Rosetta-gamiTM 2(DE3),and the protein was induced by isopropyl β-D-1-thiogalactopyranoside(IPTG).Western blot was used tO identify the protein. Furthermore,the fusion protein PTD-HBcAg was purified by affinity chromatography.HBcAg protein expressed using the same methods was employed as eontr0l.The purified protein was added tO HuH-7 cell culture,then the transduction of PTD-HBcAg and HBcAg in cells were detected by indirect immunofluorescence assay (IFA).Results The fusion protein was effectively expressed in E. Coli and purified by affinity chromatography.Both purified PTD-HBcAg and HBcAg could be recognized by HBeAg monoclonal antibody in Western blot analysis.IFA visualization showed that PTD-HBeAg could be introduced into HUH-7 ceils while HBcAg only could not be detected in cells.Conclusions PTD-HBcAg fusion protein can be expressed effectively and purified in prokaryotic expression system.PTD could mediate HBcAg penetrating eell membrane into the cells.

Objective To observe the effects of cytoplasmic transduction peptide (CTP)-HBcAg18-27-Tapasin induced murine bone marrow-derived dendritic cell (DC) maturation on T lymphocyte proliferation in vitro,Methods Bone marrow derived DC isolated from BALB/c mice were cultured with recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleutin (IL)-4 for 5 days followed by lipopolysaccharide added to induce DC maturation.10 μg/L CTP-HBcAg18-27-Tapasin,50 μg/L CTP-HBcAg18-27-Tapasin,10 μg/L CTP-HBcAg18-27 or RPMI-1640 were added into culture medium to induce DC maturation.DC phenotypes were analyzed by flow cytometry.The level of IL-12p70 in the supernatant was detected by enzyme linked immunosorbent assay.The proliferation of.T lymphocytes was performed by using cell counting kit-8 and intracellular cytokine of proliferative T cells were analyzed by flow cytometry.The means among groups were compared using one-way ANOVA and those between two groups were compared by least significant difference test.Results DC were cultured and induced successfully.The molecules on DC surface,such as CD80,CD86 and major histocompatibility antigen-Ⅰ were upregulated by CTP-HBcAg18-27-Tapasin.IL-12p70 level induced by 50 μg/L CTP-HBcAg18-27-Tapasin was (61.12±10.25) pg/mL,which was higher than those induced by 10 μg/L CTP-HBcAg18-27-Tapasin (50.43±10.42) pg/mL,10μg/L CTP-HBcAg18-27 (40.17±8.54) pg/mL and medium control (30.51±8.03) pg/mL (F=15.85,P=0.030 and 0.037).The proliferation of T lymphocytes induced by CTP- HBcAg18-27 -Tapasin was higher than control groups.The amounts of cytotoxic T lymphocyte (CTL) induced by 50 μg/L CTP-HBcAg18-27-Tapasin [(2.05±0.41) ％] and 10 μg/L CTP-HBcAg18-27-Tapasin [(1.06 ±0.10 )％] were both significantly higher than the 10 μg/L CTP-HBcAg18-27 group [(0.45±0.11)％] and medium group [(0.09±0.02)％,F=60.22,P=0.003].Conclusions CTP HBcAg18- 27 Tapasin could promote the differentiation and maturation of DC,and enhance the ability of DC stimulating T lymphocytes proliferation and increase CTL expression effectively.

OBJECTIVE To investigate CD25 expression in peripheral blood T lymphocytes from patients with chronic hepatitis B,and explore its significance in the pathogenesis of chronic hepatitis B.METHODS To detect CD25 expression in peripheral blood T lymphocytes of patients with chronic hepatitis B by means of flow cytometry.CD25 expression was observed in chronic hepatitis B patients.In the meantime,CD25 expression in T cells from severe chronic hepatitis or acute hepatitis B patients and asymptomatic carriers of HBV was also observed.RESULTS Varying degrees of CD25 were expressed in T cells from hepatitis B patients.The expression in CD3+ T and CD8+T cells was higher than that in CD4+T cells.CD25 expression in CD4+T was lower.The average of CD25 expression in CD3+T cells from patients with chronic hepatitis B,acute hepatitis B,and chronic severe hepatitis B and asymptomatic carriers of HBV was(2.92?0.13)%,(0.51?0.36)%,(1.60?0.07)%,and(0.95?0.23)%,respectively.The average of CD25 expression in CD4+T cells from patients with chronic hepatitis B,acute hepatitis B and chronic severe hepatitis B and asymptomatic carriers of HBV was(2.58?0.50)%,(0.34?0.07)%,(1.45?0.02)%,and(0.83?0.13)%,respectively.CD3+T and CD4+T CD25 expression in patients with chronic hepatitis and,severe chronic hepatitis B was increased compared with that of acute hepatitis B patients and asymptomatic carriers of HBV.Compared with chronic severe hepatitis B,the expression of chronic hepatitis B was higher.CONCLUSIONS CD4+ CD25+T cells in chronic hepatitis B virus infection are increased compared with acute hepatitis,CD4+ CD25+T cells may be related to immune tolerance.

AIM: To construct an eukaryotic expression vector of human single-chain variable fragment ~against hepatitis B virus core protein (anti-HBc ScFv) and detect its expression in HepG2 cells. METHODS: Anti-HBc ScFv genes were amplified from the plasmids abstracted from positive clone and inserted into pEGFP-c1 vector that contained green fluorescent protein gene. The recombinant plasmids were transfected into HepG2 cells, and resistant clones were obtained by G418 selection. The expression of the gene of fusion protein was determined by fluorescent invert microscope and ELISA. RESULTS: Recombinant plasmids were successfully constructed. The plasmid transfected HepG2 cells were obtained by G418 selection. Specific fluorescence was observed in HepG2 cells 48 hours after transfection. ELISA analysis confirmed the expression of anti-HBc ScFv in the cells. CONCLUSION: The construction of human anti-HBc ScFv eukaryotic expression vector and its expression in HepG2 cells lay the foundation for advanced research of intracellular anti-HBc ScFv.

AIM: To Screen and identify human single-chain variable fragment (ScFv) specific to hepatitis B virus core protein and determine its gene sequence. METHODS: The recombinant phages were panned by HBcAg coated in a 96-pore plate and 48 clones were identified specific to HBc after three rounds of panning. The specificity of ScFv from the positive clone was determined by ELISA. Then, the soluble ScFv was expressed in E.coli. HB2151 and secreted in the supernatant. Subsequently, SDS-PAGE and dot blot were performed to identify the ScFv in the supernatant and cell lysate. The gene of ScFv specific to hepatitis B virus core protein was sequenced. RESULTS: The ScFv screened from phage antibodies has a specific combination character with hepatitis B virus core antigen. Soluble ScFv was confirmed to express in E.coli. HB2151 and secrete in the supernatant. The sequence of ScFv gene conformed to that of heavy chain and kappa chain of human immunoglubulin. CONCLUSION: Human ScFv specific to hepatitis B virus core protein has been identified by means of the phage display technology, and its gene sequence has been determined.

AIM: To observe the change of nitric oxide (NO) generation system in the vascular adventitia, media and intima in septic shock rats. METHODS: The septic shock model was made in rats by caecal ligation and puncture. The intima, media and adventitia of the rat aorta were separated. NO production (NO - 2) , nitric oxide synthase(NOS) activity and L-arginine (L-Arg) transport were measured, separately. Inducible NOS (iNOS) distribution was detected by immunohistochemistry. RESULTS: Both in early and late stage of septic shock, NO - 2 from the intima was decreased by 66.1% and 78.9%( P

Objective:To clarify the effect of ubiquitination hepatitis B virus core antigen (Ub-HBcAg) on dendritic cells (DC) autophagy, and to explore the mechanism of autophagy in enhancing DC antigen presentation and inducing hepatitis B virus-specific cytotoxic T lymphocyte (CTL) responses.Methods:Ub-HBcAg lentiviral vector (LV-Ub-HBcAg), lentiviral vector-hepatitis B virus core antigen (LV-HBcAg) and no-load plasmid LV (LV) were constructed and packaged. DC2.4 cells were divided into LV-Ub-HBcAg group, LV-HBcAg group and LV group. The blank control group (NC group) was also set. The protein expression of autophagy-related protein P62, microtubule associated protein 1 light chain 3 beta (LC3B), autophagy related 5(ATG5) and Beclin-1 were detected by Western blotting. The expressions of co-stimulatory molecules such as CD86, CD80 and major histocompatibility complex (MHC)-Ⅱ were detected by flow cytometry. Cell counting kit-8 (CCK-8) method was used to detect T lymphocytes proliferation. The non-radioactive lactic acid dehydrogenase (LDH) release method was applied to detect the killing ability of CTL. Statistical analysis was conducted by independent sample t test. Results:The relative protein expressions of LC3B-Ⅱ/LC3B-Ⅰ, Beclin-1 and ATG5 in NC group were 0.445±0.076, 0.522±0.026 and 0.761±0.038, respectively, which were all lower than those in LV-Ub-HBcAg group (0.926±0.021, 0.919±0.016 and 1.451±0.028, respectively). The relative protein expression of P62 in the NC group was higher than that in LV-Ub-HBcAg group ((1.875±0.016) vs (0.647±0.121)). The differences were all statistically significant ( t=6.102, 9.842, 17.490 and 10.590, respectively, all P<0.01). The expressions of CD86 (75.51%), CD80 (83.35%), MHC-Ⅱ (66.66%) in the LV-Ub-HBcAg group were high, and those in the NC group were 8.03%, 7.49%, 0.04%, respectively. The specific CTL killing rate ((65.310±2.091)%) of the LV-Ub-HBcAg group was significantly higher than both NC group ((14.400±0.497)%) and LV-HBcAg group ((54.870±1.443)%), and the differences were both statistically significant ( t=23.690 and 4.111, respectively, both P<0.05). Conclusion:Ub-HBcAg promotes the DC autophagy, up-regulates the expressions of costimulatory molecules on cell surface of DC to induce the maturation and activation, and then stimulates T lymphocyte to induce a stronger specific CTL response under the effort of ubiquitination.

OBJECTIVETo investigate the effect of protein transduction domain-hepatitis B virus core antigen (CTP-HBcAg18-27)-Tapasin fusion protein-induced specific cytotoxic T lymphocyte (CTL) response on hepatitis B virus (HBV) replication in HBV transgenic mice.

METHODSTwenty HBV-transgenic mice were randomly divided into two groups for a 3-week course of once weekly subcutaneous immunizations with either CTP-HBcAg18-27-Tapasin fusion protein or CTP-HBcAg18-27. Mice administered isotonic saline served as blank controls. Expressions of cytokines in splenocytes were analyzed by flow cytometry. Serum levels of hepatitis B surface antigen (HBsAg) and HBV DNA were determined by microparticle enzyme immunoassay and real-time fluorescent PCR assay, respectively. Expression of HBsAg in hepatic tissues was detected by immunohistochemistry.

RESULTSImmunization with 100 mug of CTP-HBcAg18-27-Tapasin fusion protein led to a significant increase in proportions of CTLs in spleen (2.70%+/-0.20% vs. 50 mug of CTP-HBcAg18-27-Tapasin: 1.66%+/-0.53%, 50 mug of CTP-HBcAg18-27: 1.26%+/-0.56%, and blank controls: 0.75%+/-0.71%; F = 741.45, P = 0.000) and up-regulation of inflammatory cells in hepatic tissue. In addition, both immunizations of CTP-HBcAg18-27-Tapasin led to significant decreases in serum HBsAg and HBV DNA levels compared to those in the CTP-HBcAg18-27 group.

CONCLUSIONHBV-related modification of the expression of the molecular chaperone Tapasin may affect its interaction with intracellular antigen peptides, thereby leading to increases the number of specific CTLs in the spleen, decreases in serum HBsAg and HBV DNA levels, and down-regulation of HBsAg expression in hepatic tissue. These results obtained in HBV-transgenic mice suggest that the CTP-HBcAg18-27-Tapasin fusion protein has anti-HBV activity.

Animals ; DNA, Viral ; blood ; Female ; Hepatitis B ; immunology ; Hepatitis B Core Antigens ; genetics ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; physiology ; Male ; Membrane Transport Proteins ; genetics ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Virus Replication